Estrogen Receptor Null Mice - Endocrine Reviews

0163-769X/99/$03.00/0 

Endocrine Reviews 20(3): 358–417 

Copyright © 1999 by The Endocrine Society 

Printed in U.S.A. 

Estrogen Receptor Null Mice: What Have We Learned 

and Where Will They Lead Us? 

JOHN F. COUSE AND KENNETH S. KORACH 

Receptor Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute 

of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North 

Carolina 27709 

I. Introduction—A Historical Perspective 

II. Estrogen Receptors (ER) 

A. Gene and mRNA structure 

B. Mechanism of ER action 

C. Generation of the estrogen receptor null mice 

III. Reproductive Tract Phenotypes of the Female 

A. Uterus 

B. Vagina 

C. Oviduct 

D. Ovary 

IV. Mammary Gland 

A. �ERKO phenotype 

B. �ERKO phenotype 

C. ER� and oncogene-induced tumorigenesis: Wnt- 

1/�ERKO mice 

V. Reproductive Tract Phenotypes of the Male 

A. Testicular function and spermatogenesis 

B. Accessory sex organs 

VI. Neuroendocrine System 

A. Hypothalamic-pituitary axis 

B. Behavior 

VII. Phenotypes in Peripheral Tissues 

A. Skeletal system 

B. Cardiovascular system 

C. Adipogenesis 

VIII. Comparison with Human Disease and Models of Deficient 

Estrogen Action 

A. Ovarian carcinogenesis 

B. Chronic anovulation 

C. ER� and aromatase deficiency 

IX. Summary 

I. Introduction—A Historical Perspective 

NE OF the challenging problems confronting bio- 

“O logical scientists has been the manner in which 

hormones serve as regulators of biochemical processes in 

tissues of higher animals” [E. V. Jensen and E. R DeSombre, 

1973 (1).] 

At the time of the above statement, the laboratories of 

Address reprint requests to: Dr. Kenneth S. Korach, Receptor Biology 

Section, Laboratory of Reproductive and Developmental Toxicology, 

National Institute of Environmental Health Sciences, National Institutes 

of Health, MD B3–02, P.O. Box 12233, Research Triangle Park, North 

Carolina 27709 USA. 

358 

Elwood Jensen and Jack Gorski had spent 10 yr providing 

experimental evidence to support the concept of an intracellular 

“receptor” protein for steroid hormones. Their combined 

work had even led to a proposed model by which the 

interactions of the receptor and the steroid were involved in 

mediating the cellular effects of the hormone (1). The first of 

these receptors to be characterized was for the female sex 

hormone, 17�-estradiol (E 2) (2, 3). Since this time, similar 

receptors for testosterone, progesterone, glucocorticoids, 

thyroid hormone, vitamin-D 3, and retinoids have been discovered 

and now form a portion of a large family of nuclear 

hormone receptors (4). Although significant strides have 

been made since, Jensen’s introductory statement in a review 

published more than 25 yr ago is still contemporary with the 

current research goals toward understanding the growing 

family of nuclear receptors. This is not to say that little 

progress has been made, which is quite to the contrary, but 

rather to state that although advances in technology and 

molecular biology have allowed for extensive insight, there 

is still a great deal to be learned as we move into the next 

century. 

Our current understanding of the various roles of the 

steroid, thyroid, and retinoid hormones in development and 

normal physiology and the mechanisms by which these actions 

are mediated is due, in large part, to the generation of 

a series of reagents and tools over the past 40 yr. The first of 

these was the synthesis and use of tritium-labeled E 2 with 

high specific activity, allowing for the first reports of the 

detection and simple characterization of an estrogen-binding 

component, or “estrophilin” (1, 3, 5). This protein exhibited 

a binding affinity for estradiol that was several fold higher 

compared with the other gonadal steroids and was found 

only in tissues previously shown to respond to estradiol in 

terms of growth and increased RNA synthesis (1, 3). These 

studies described the uptake and concentration of radiolabeled 

estradiol by a specific protein unique to the cells of the 

uterus, vagina, and pituitary and thereby disputed the current 

thought that estrogen action required enzymatic metabolism 

of the hormone (2, 3, 6). Thus, the concept of a 

hormone receptor protein in target tissues was initiated. Autoradiographic 

studies with radiolabeled ligand further 

demonstrated the strong association of estradiol with the 

nuclei of cells lining the rat uterus within 4 h after injection 

(7). These studies were significantly advanced by pharmacological 

approaches with early nonsteroidal estrogen antagonists, 

the first being the triphenylethylene, MER-25 (2),

June, 1999 ESTROGEN RECEPTOR NULL MICE 359 

followed soon after by a series of similar compounds, e.g., 

clomiphene, nafoxidine, CI-628, and tamoxifen (1, 8). These 

reagents were previously known to inhibit the uterotropic 

effects of estradiol in a dose-dependent manner but, more 

importantly, were later shown to block estradiol uptake and 

binding in target tissues. Still, at this time the exact role that 

the hormone-receptor complex played in the final manifestation 

of the hormonal effects remained unclear. It was suggested 

that the receptor may simply fulfill a “transport” role 

to move the steroid hormone from the cytoplasm to the 

nucleus of the cell (1). Nonetheless, from these early studies, 

the definition of an estrogen target tissue now included not 

only the exhibition of a measurable response to the natural 

hormone (E 2) but also one that possessed detectable levels of 

the estrogen receptor (ER). 

Much of the early characterization of the ER relied on the use 

of sucrose gradient analysis of nuclear and cytoplasmic cell 

fractions under varied salt concentrations (3). This procedure, 

along with gel electrophoresis and filtration using radiolabeled 

estradiol, allowed for the generation of semipure fractions of the 

estradiol-binding protein. These preparations led to the generation 

of antisera specific to the ER protein, another notable step 

in receptor research (9). The later development of monoclonal 

antibodies to the ER and the immunohistochemical techniques 

that soon followed provided evidence of the predominantly 

nuclear localization of the receptor protein in target cells (10, 11). 

The late 1970s brought numerous reports of the tissue distribution 

and localization of the ER in humans and laboratory 

animals, confirming much of the findings of earlier steroid 

autoradiography studies (7, 11). The antibodies were also used 

to further purify large amounts of the ER from target tissues, 

allowing for more detailed studies of the receptor structure and 

function. 

The 1980s witnessed the cloning and sequencing of the 

cDNAs for several of the steroid hormone receptors, which 

proved to be a seminal step toward understanding their 

mechanisms of action. The first cDNA to be cloned for a 

member of the nuclear receptor family was that for the human 

glucocorticoid receptor (12), followed soon after with 

the description of the human ER cDNA (13, 14). Since this 

time, the cDNAs encoding several other members of the 

nuclear receptor family have been described (4). The current 

list of isolated ER cDNAs includes those for the chicken (15), 

mouse (16), rat (17), Xenopus laevis (18), and rainbow trout 

(19). Sequence analysis of the various receptor cDNAs demonstrated 

a high degree of similarity and led to their inclusion 

in a superfamily of nuclear receptors possessing a defined 

motif of functional domains (4). 

A vital contribution from the cloning of the nuclear receptor 

cDNAs was the ability to express the receptor proteins 

in in vitro mammalian (20) or yeast (21) cell systems. These 

techniques, as well as cell-free in vitro transcription (22), 

allowed detailed characterizations of the different functional 

domains of the receptor. Recombinant DNA methodologies 

provided for the construction of receptor cDNAs possessing 

precise truncations, deletions, point mutations, or additional 

sequences. These powerful techniques led to the in vitro 

expression of chimeric and mutant receptors and great advances 

in the dissection and mapping of the specific domains 

and distinct residues critical to receptor function (21–25). The 

late 1980s witnessed multiple descriptions of naturally occurring 

variants and mutations of several of the nuclear 

receptor transcripts (26), including the ER (27–29). Their 

identification and functional characterization have led to 

further insight into the mechanisms of action of specific 

domains of the receptor proteins as well as the receptors as 

a whole. Furthermore, the detection of these nuclear receptor 

transcript variants in vivo has allowed speculation concerning 

their possible roles in alternate or abnormal hormonal 

signaling in normal and neoplastic tissues (27). 

By the 1990s, it was evident that the members of the steroid/ 

thyroid hormone superfamily of receptors were intracellular 

proteins that functioned in the nucleus to regulate transcription 

of target genes. This growing family of receptors now includes 

those for the sex and adrenal steroids, thyroid hormones, retinoids, 

vitamin D 3, and eicosinoids (reviewed in Refs. 4 and 30). 

The inclusion of nuclear receptor-like proteins with no known 

ligand, termed orphan receptors, such as those for the chicken 

ovalbumin upstream promoter (COUP), and steroidogenic factor-1 

(SF-1), has expanded the family to now include approximately 

150 distinct proteins (30). 

Our knowledge of the expression patterns and mechanism 

of action of the nuclear receptors has led to a greater appreciation 

of their involvement in normal physiology and disease. 

The current decade has witnessed several advances in 

our understanding of the molecular biology and overall 

physiological role of these proteins. Especially significant to 

these efforts has been the discovery and/or development of 

three distinct aspects. The first of these was the discovery of 

coregulators, a second group of nuclear proteins that further 

modulate the actions of unoccupied as well as ligand (agonist 

or antagonist)-bound receptors (reviewed in Ref. 31). The 

continued characterization of the coregulator proteins has 

provided some insight toward the long sought explanations 

for the cell- and tissue-specific mixed agonist/antagonist 

activity of certain ligands (31). The second of these advances 

was the generation of crystal structures for the ligand-binding 

domains of the retinoic acid receptor-� (32), retinoic X 

receptor-� (33), thyroid receptor-� (34), and ER� (35, 36). 

These new data continue to allow for the description of the 

long speculated conformational changes that result in the 

receptor after binding of the natural ligand, and how these 

changes may differ from those induced by natural or synthetic 

agonists and antagonists. 

The third development of great impact in this decade has 

been the use of gene-targeting technology and transgenic techniques 

to disrupt the genes encoding several members of the 

steroid/thyroid hormone receptor superfamily. This methodology 

has allowed for the generation of transgenic mice that lack 

a functional gene for a specific receptor, as well as germline 

passage of this mutation. Hormone resistance due to naturally 

occurring mutations in the genes encoding the receptors for 

androgen (37, 38), glucocorticoids (39), and thyroid hormones 

(40) had already been described in humans and laboratory 

animals and thereby provided great insight into the role of these 

hormones in development and normal physiology. However, 

similar mutations had not yet been reported for other members 

of the superfamily of nuclear receptors. The impact of the genetargeting 

technique is evident in the generation of several 

“knockout” mice for the nuclear receptors, including mice lack-

360 COUSE AND KORACH Vol. 20, No. 3 

ing multiple forms of the retinoic acid receptors (41–43), the 

progesterone receptor (44), the vitamin D 3 receptor (45), the ERs 

(46, 47), and the coregulator protein, steroid receptor coactivator-1 

(48). If nothing else, the naturally occurring mutants combined 

with the generation of the knockout models, have 

confirmed the basic hypotheses put forth almost 30 yr ago, 

i.e., that the receptor proteins found to tightly and specifically 

bind a hormone within a tissue are indeed critical 

for mediating the biological effect of the hormone within 

the target cell (1). 

This review will discuss one such advance originating 

from the gene-targeting boom that occurred in the 1990s, the 

ER knockout mice. The broad content of this review is a 

reflection of the current state of research in the expanding 

field of estrogen action, as illustrated by the discovery of a 

second ER, the ER�. A large extent of the discussion will 

focus on the mice lacking the classical ER, the ER� knockout 

mice (�ERKO). The reasons for this are 2-fold: 1) the �ERKO 

mouse has been available for more than 6 yr, compared with 

less than 1 yr for the �ERKO, and therefore has been more 

thoroughly studied and characterized; 2) the phenotypes of 

the �ERKO mice appear to be more broad in nature compared 

with those of the less well studied �ERKO mice, although 

continued research may lessen this disparity. The 

extent to which the ERKO models have been used in various 

fields of biological research is illustrated in Table 1. This 

review will discuss the phenotypes of the ERKO mice and the 

multiple ways in which these models continue to influence 

the field of estrogen research. Foremost, several of the hypotheses 

of estrogen and ER action put forth from the efforts 

of numerous investigators over the years were confirmed by 

our observations in the ERKO. Furthermore, certain phenotypes 

have introduced unforeseen critical roles of estrogen in 

some physiological systems, such as in male fertility. Later 

years have witnessed the use of the �ERKO as a research tool 

to investigate specific biochemical pathways and neoplasia 

in the absence of estrogen action. Where applicable, we will 

contrast the phenotypes of the two ERKO models, as well as 

compare the relevant phenotypes of knockout models for 

other hormone signaling systems. And finally, we will discuss 

how these models compare with the few cases of insufficient 

estrogen synthesis and the single reported case of 

estrogen insensitivity in humans. Before we continue, however, 

we wish to take this opportunity to establish consistency 

in the abbreviations used to refer to the ER-knockout 

(ERKO) mice in the literature. Although the terms ER�KO 

and ER�KO have appeared in previous reports from our own 

as well as other laboratories, we propose that the abbreviations 

used above and throughout the remainder of this review, 

i.e., �ERKO and �ERKO, be the consensus abbreviations 

used hereafter to refer to the two models. 

A. Gene and mRNA structure 

II. Estrogen Receptors 

For several years it was thought that only a single form of the 

nuclear ER existed. However, in 1996, multiple laboratories 

independently reported the discovery of a second type of ER in 

the rat (49), mouse (50), and human (51). This newly discovered 

receptor was termed ER�, resulting in the classical ER being 

referred to as ER�. The two receptors are not isoforms of each 

other, but rather distinct proteins encoded by separate genes 

located on different chromosomes. As a result of this discovery, 

we now know that the ER shares a phenomenon of multiple 

forms previously described for other members of the nuclear 

hormone superfamily, including the receptors for thyroid hormone, 

retinoids, mineralocorticoids, and progesterone (26, 30). 

A detailed description of the structure and mechanism of action 

of the ERs is beyond the scope of this review, and therefore only 

a brief discussion of the relevant points will appear here. For 

more detailed descriptions of the mechanism of steroid receptor-regulated 

gene transcription, readers are encouraged to 

seek several recent reviews (52–55). 

Transcription of the mouse ER� gene in vivo predominantly 

results in a single transcript of approximately 6.3 kb transcribed 

from 9 exons. This transcript encodes a protein of 599 amino 

acids with an approximate molecular mass of 66 kDa (16). The 

human ER� is slightly shorter at 595 amino acids but exhibits 

a similar molecular mass (13, 14). Whereas the human ER� gene 

has been mapped to chromosome 6 (56), the mouse ER� gene 

is located on chromosome 10 (57). The existence of multiple 

promoter and regulatory regions in the 5�-untranslated sequences 

of the human and rat ER� have been described, but 

only a single open reading frame appears to exist (58–60). 

Numerous reports have described the discovery and characterization 

of naturally occurring variants and mutations of the 

ER� mRNA in normal as well as neoplastic tissues of several 

species (reviewed in Refs. 27, 28, and 61). Although the existence 

of true protein products of these ER� mRNA variants in 

vivo remains controversial, their transactivational activities 

in in vitro cell culture systems have been intensely 

described and have furthered our understanding of the 

functional domains of the receptor (27, 61). 

Initial studies indicated that the rodent ER� was composed 

of 485 amino acids and an estimated molecular mass 

of 54 kDa and therefore was slightly smaller than the ER� (49, 

50, 62). The majority of this difference in size between the two 

ERs was due to a significantly shorter N� terminus in the 

deduced ER� protein. Unlike the ER� gene, Northern blot 

analyses of ovarian RNA from both the rat and mouse indicates 

the presence of multiple ER� transcripts (50, 63, 64). 

Furthermore, open reading frames initiating up-stream from 

those originally described have now been discovered in the 

mouse, rat, bovine, and human ER� mRNA (65–69). These 

studies have also provided evidence to support that the 

upstream start codons are the likely initiation sites of 

translation and therefore suggest the possibility of an ER� 

protein of 527–530 amino acids and a calculated molecular 

mass of approximately 60 kDa (65–67, 69). However, convincing 

Western blots from tissue extracts to indicate the 

true in vivo molecular mass of the ER� have remained 

difficult to produce with the antibody preparations currently 

available. Similar to ER�, a number of variants of the 

ER� mRNA have already been described. These include a 

conserved insertion of 18 amino acids in a C�-terminal 

region of the ER� in the rat (70, 71), human, and mouse 

(72), the deletion of one or more exons in these same 

species (70, 72, 73), and various isoforms in the extreme 

C�-terminus of the human ER� (68).


TABLE 1. Published reports involving the �ERKO and �ERKO models and the ER�-deficient human 

Author 

ER-� knockout mice: general 

Yr. Title Ref. 

Lubahn et al. 1993 Alteration of reproductive function but not prenatal sexual development after insertional 

disruption of the mouse estrogen receptor gene. 

46 

Couse et al. 1995 Analysis of transcription and estrogen insensitivity in the female mouse after targeted 

disruption of the estrogen receptor gene. 

123 

Couse et al. 

ER-� knockout mice: general 

1997 Tissue distribution and quantitative analysis of estrogen receptor-� (ER�) and estrogen-� 

(ER�) messenger ribonucleic acid in the wild-type and ER�-knockout mouse. 

93 

Krege et al. 

Female reproductive tract 

1998 Generation and reproductive phenotypes of mice lacking estrogen receptor-�. 47 

Curtis et al. 1996 Physiological coupling of growth factor and steroid receptor-signaling pathways: estrogen 

receptor knockout mice lack estrogen-like response to epidermal growth factor. 

170 

Lindzey et al. 1996 Uterotropic effects of dihydrotestosterone in estrogen receptor knockout and wild-type mice. a 

154 

Das et al. 1997 Estrogenic responses in estrogen receptor-� deficient mice reveal a distinct signaling pathway. 173 

Cooke et al. 1997 Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium. 171 

Buchanan et al. 1998 Role of stromal and epithelial estrogen receptors in vaginal epithelial proliferation, 

stratification, and cornification. 

198 

Ghosh et al. 1998 Methoxychlor acts in ER�-KO mice through an ER-� independent mechanism. a 

174 

Rosenfeld et al. 1998 Estrogen receptor-� knockout mice reveal a role for estrogen in mammalian female sexual 

development. a 

494 

Kurita et al. 1998 Estrogen induces progesterone receptors in uterine stroma of estrogen receptor � knockout 

mouse. a 

495 

Buchanan et al. 1999 Tissue compartment-specific estrogen receptor-� participation in the mouse uterine epithelial 

secretory response. 

172 

Curtis et al. 1999 Disruption of estrogen signaling does not prevent progesterone action in the estrogen receptor 

knockout mouse uterus. 

183 

Schomberg et al. 

Mammary gland 

1999 Targeted disruption of the estrogen receptor-� (ER�) gene in mice: characterization of ovarian 

responses and phenotypes. 

142 

Bocchinfuso and Korach 1997 Mammary gland development and tumorigenesis in estrogen receptor knockout mice. 269 

Cunha et al. 1997 Elucidation of a role of stromal steroid hormone receptors in mammary gland growth and 

development by tissue recombination experiments. 

278 

Kenney et al. 1997 The response of null estrogen receptor mammary parenchyma and mesenchyme in vivo. 496 

Bocchinfuso et al. 

Male reproductive tract 

1999 An MMTV-Wnt-1 transgene induces mammary gland hyperplasia and tumorigenesis in mice 

lacking estrogen receptor-�. 

297 

Eddy et al. 1996 Targeted disruption of the estrogen receptor gene in male mice causes alteration of 

spermatogenesis and infertility. 

315 

Donaldson et al. 1996 Morphometric study of the gubernaculum in male estrogen receptor mutant mice. 316 

Hess et al. 1997 A role for oestrogens in the male reproductive system. 327 

Rosenfeld et al. 

Neuroendocrine system 

1998 Transcription and translation of estrogen receptor-beta in the male reproductive tract of 

estrogen knockout and wild-type mice. 

121 

Scully et al. 1997 Role of estrogen receptor � in the anterior pituitary gland. 282 

Shughrue et al. 1997 Responses in the brain of estrogen receptor �-disrupted mice. 400 

Shughrue et al. 1997 The disruption of estrogen receptor-� mRNA in forebrain regions of the estrogen receptor-� 

knockout mouse. 

352 

Simerly et al. 1997 Estrogen receptor-dependent sexual differentiation of dopaminergic neurons in the preoptic 

region of the mouse. 

405 

Young et al. 1998 Estrogen receptor � is essential in the induction of oxytocin receptor by estrogen. 497 

De Vries et al. 1997 Steroid-responsive vasopressin innervation in the brain of estrogen receptor-�-minus mice. a 

498 

Lindzey et al. 1998 Effects of castration and chronic steroid treatments on hypothalamic gonadotropin-releasing 

hormone content and pituitary gonadotropins in male wild-type and estrogen receptor-� 

knockout mice. 

317 

Lindzey et al. 1998 Steroid regulation of gonadotrope function in female wild-type (WT) and estrogen receptor-� 

knockout (ERKO) mice. a 

252 

Ogawa et al. 1998 Forebrain steroid receptor immunoreactive cells in neonatal and prepubertal estrogen receptor- 

� gene deficient (ERKO) mice. a 

426 

Moffatt et al. 1998 Induction of progestin receptors by estradiol in the forebrain of estrogen receptor-� genedisrupted 

mice. 

423 

Wersinger et al. 1998 Estradiol decreases proGnRH immunoreactivity in female wild type but not estrogen receptor � 

knockout mice. a 

499 

B. Mechanism of ER action 

The ERs are classified as class I members of the superfamily 

of nuclear hormone receptors, defined as a ligandinducible 

transcription factor (30). Early studies indicated the 

ER was cytoplasmic and became localized to the nucleus only 

upon ligand binding, providing the basis of the initial “twostep 

mechanism” of hormone action (1, 3). However, it is now 

accepted that the ER is a predominantly nuclear protein 

regardless of whether or not it is complexed with ligand (74). 

The inactive ER exists in a complex consisting of several


TABLE 1. continued 

Author Yr. Title Ref. 

Gu et al. 

Behavior 

1999 Rapid action of 17�-estradiol on kainate-induced currents in hippocampal neurons lacking 

intracellular estrogen receptors. 

364 

Ogawa et al. 1996 Reversal of sex roles in genetic female mice by disruption of estrogen receptor gene. 417 

Ogawa et al. 1997 Behavioral effects of estrogen receptor gene disruption in male mice. 318 

Rissman et al. 1997 Estrogen receptors are essential for female sexual receptivity. 419 

Wersinger et al. 1997 Masculine sexual behavior is disrupted in male and female mice lacking a functional estrogen 

receptor � gene. 

427 

Fugger et al. 1998 Estrogen receptor � is not required for estradiol’s effects on inhibitory avoidance behavior in 

female mice. a 

500 

Fugger et al. 1998 Characterization of spatial behavior and hippocampal electrophysiology in male estrogen 

receptor-�-minus mice. a 

501 

Fugger et al. 1998 Sex differences in the activational effect of ER� on spatial learning. 502 

Ogawa et al. 1998 Roles of estrogen receptor-� gene expression in reproduction-related behaviors in female mice. 418 

Ogawa et al. 

Cardiovascular system 

1998 Modifications of testosterone-dependent behaviors by estrogen receptor-� gene disruption in 

male mice. 

428 

Johns et al. 1996 Disruption of estrogen receptor gene prevents 17�-estradiol-induced angiogenesis in transgenic 

mice. 

503 

Iafrati et al. 1997 Estrogen inhibits the vascular injury response in estrogen receptor-� deficient mice. 464 

Johnson et al. 1997 Increased expression of the cardiac L-type channel in estrogen receptor-deficient mice. 472 

Rubanyi et al. 1997 Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse 

aorta: gender difference and effect of estrogen receptor gene disruption. 

467 

Srivastava et al. 1997 Estrogen up-regulates apolipoprotein E (Apo E) gene expression by increasing Apo E mRNA in 

the translating pool via the estrogen receptor �-mediated pathway. 

460 

Freay et al. 1997 Mechanism of vascular smooth muscle relaxation by estrogen in depolarized rat and mouse 

aorta. 

504 

Cross et al. 1998 Estrogen receptor knock-out mice exhibit higher myocardial contractility than wild-type mice 

but are equally susceptible to ischemic injury. a 

Bone 

505 

Roemmich et al. 1997 Bone strain independently augments bone mineral in pubertal mice with the disrupted 

estrogen receptor gene. a 

506 

Pan et al. 1997 Estrogen receptor-alpha knockout (ERKO) mice lose trabecular and cortical bone following 

ovariectomy. a 

448 

Korach et al. 

Other 

1997 The effects of estrogen receptor gene disruption on bone. 447 

Smithson et al. 1997 The role of estrogen receptors and androgen receptors in sex steroid regulation of B 

lymphopoiesis. 

507 

Hurn et al. 1998 Stroke in mice deficient in classical estrogen receptors (ERKO�). a 

508 

Couse et al. 1998 Use of the estrogen receptor-� to investigate the role of ER� in the developmental and 

carcinogenic actions of diethylstilbestrol. a 

509 

Taylor and Lubahn 1998 Impaired glucose tolerance in the ER�KO mouse. a 

490 

Yellayi et al. 1998 Role of estrogen receptor-� (ER�) in the development and function of the thymus in male and 

female mice. a 

ER-deficient humans 

510 

Smith et al. 1994 Estrogen resistance caused by a mutation in the estrogen receptor gene in a man. 116 

Sudhir et al. 1997 Endothelial dysfunction in a man with disruptive mutation of the oestrogen-receptor gene. 492 

Sudhir et al. 1997 Premature coronary artery disease associated with a disruptive mutation in the estrogen 

receptor gene in a man. 

493 

Dieudonne et al. 1998 Immortalization and characterization of bone marrow stromal fibroblasts from a patient with a 

loss of function mutation in the estrogen receptor-� gene. 

511 

a Abstract. 

heat-shock and other proteins that appear to disassociate 

upon ligand binding, resulting in a “transformation” of the 

receptor to an active state (75). With continued research, the 

two-step mechanism model has evolved to state that upon 

binding of estradiol, or an estrogenic ligand, the transformed 

receptors form dimers that tightly associate with specific 

consensus DNA sequences, consisting of 15-bp inverted palindromes 

in the regulatory regions of target genes (52, 74– 

76). This complex then interacts with basal transcription factors, 

coregulator proteins, and other transcription factors to 

ultimately regulate transcription of the target gene (52, 55, 

76). However, in recent years, pathways of gene activation by 

the steroid receptors that deviate from this classical model 

have been described. These include gene activation by 

ligand-bound steroid receptors without evidence of direct 

DNA binding, but rather via interaction with other DNAbound 

transcription factors, such as an AP-1 complex (77, 78). 

In addition, ligand-independent activation of the receptor 

through pathways that alter the activity of cellular kinases 

and phosphatases has been demonstrated both in vitro and 

in vivo (reviewed in Ref. 55). The discovery of these pathways 

strongly supports the great importance of the ER and its 

ability to possibly provide diverse physiological functions 

even in the absence of ligand.


The ER� and ER� proteins are composed of six functional 

domains, labeled A–F, a signature characteristic of members 

of the superfamily of steroid/thyroid hormone nuclear receptors 

(Fig. 1). The N�-terminal A/B domain is the least 

conserved among all members and demonstrates only 17% 

identity between the human ER� and ER� (64). In contrast, 

the C domain is the most highly conserved among the different 

members of the family. It possesses two zinc fingers 

forming a helix-loop-helix motif and primarily functions in 

tightly binding the receptor to the DNA hormone response 

elements. The sequences encoding the two zinc fingers possess 

97% homology between the ER� and ER� genes and are 

located in separate exons (exons 3 and 4) in each (50, 64, 79). 

The E domain, or ligand-binding domain, confers ligand 

specificity to the receptor and is moderately conserved 

among the members of the superfamily. The ER� and ER� 

proteins possess 60% conservation of the residues in the E 

domain; however, each binds estradiol with nearly equal 

affinity and exhibits a very similar binding profile for a large 

number of natural and synthetic ligands (80). The D domain 

possesses signals for nuclear localization of the receptor and 

exhibits approximately 30% identity between the two human 

forms of ER (64). The C�-terminal F domain is unique to the 

ER among the nuclear receptors for the gonadal and adrenal 

hormones (6) but is not well conserved among the ERs of 

different species nor between the ER� and ER�, which share 

approximately 18% homology (64). Studies using forms of 

the ER� missing the C� terminus have indicated a role for the 

F domain in modulating transactivational activity of the ER� 

when complexed with mixed agonist/antagonist ligands, 

possibly via influencing coregulatory function and/or 

dimerization of the receptor (81, 82). 

There are also functional domains that span those boundaries 

described above. Residues involved in the dimerization 

of the receptors are located in the second zinc finger of the 

C domain as well as in the major dimerization surface in the 

E domain (83, 84). Furthermore, two domains critical to the 

transactivational function of the ER are the AF-1 in the N� 

terminus and AF-2 in the C� terminus. These two domains 

may function independently or interact during the process of 

transactivation, depending on the cell type, target promoter, 

and the presence and/or type of ligand (52). The AF-2 domain 

is critical to the ligand-dependent transactivational activity 

of the receptor and may be involved in the recruitment 

of coregulator proteins, whereas the AF-1 is thought to be a 

region of site-specific phosphorylation involved in ligandindependent 

activity of the receptor (reviewed in Refs. 31 

and 52). Recent studies have also suggested the presence of 

a third domain, AF-2a, within the ligand-binding domain of 

the human ER� (85). 

The discovery of the ER� has introduced a new level of 

complexity to the current model as well. To date, there exist 

no data indicating a physiological response solely mediated 

by ER�. In contrast, the �ERKO mouse has confirmed the 

requirement for ER� in mediating several actions of estradiol, 

as will be discussed in this review. Nevertheless, in vitro 

experiments from several laboratories have indicated the 

possibility of cooperative activity between the two receptors, 

FIG. 1. Drawing of the mouse ER proteins, cDNAs, and genes as well as the targeting scheme employed to generate the ERKO mice via 

homologous recombination. Shown are the common functional domains of the ER� and ER� receptor proteins, indicating the residues involved 

in DNA and ligand binding. The common structure of the cDNAs and genomic genes for the ERs is illustrated, indicating the exon sequences 

that encode the functional domains of the receptor. Generation of the �ERKO mouse involved the targeted insertion of a 1.8-kb NEO sequence 

into exon 2 of the ER� gene such that the translational reading frame (indicated by the direction of the arrow) of the genes was the same (see 

Ref. 46). Generation of the �ERKO mouse involved a similar scheme, in which a 1.8-kb NEO sequence was inserted into exon 3 of the ER� gene; 

however, in this case the NEO gene is in the reverse orientation (see Ref. 47). The schematic drawing of the genomic DNA was adapted from 

that of the human ER� gene (Ref. 79). Drawing is not to scale.


acting in the form of heterodimers (50, 62, 65, 86). These 

studies generally report a tendency of ER� to form homodimers 

whereas ER� prefers to heterodimerize with ER�. 

However, Giguere et al. report (87) that the heterodimer is the 

preferred state when both mouse ERs are present. The transactivational 

activity of the heterodimer when assayed in in 

vitro mammalian cell transfection assays appears to lie between 

that of the more active ER� homodimer and the less 

active ER� homodimer (50, 62, 86). A major consideration 

when evaluating the possible physiological functions of an 

ER�/ER� heterodimer is evidence of coexpression of the two 

receptors in the same cell, which has not yet been definitively 

reported (88). To this end, studies and reagents are only now 

becoming available to directly assess this question. 

Several functional characteristics of the two ERs are similar. 

The residues critical to function of the AF-2 domain 

appear to be identical in the mouse ER� and ER� (87). Tremblay 

et al. (50, 89) demonstrated that a tyrosine residue critical 

to the function of the AF-2 domain was conserved in both the 

ER� and ER� and that mutation of this amino acid resulted 

in similar constitutive, ligand-independent transactivational 

activity in both receptors. In contrast, the N�-terminal AF-1 

domain shows no significant regions of similarity between 

the two ERs (87). However, a potential activation site of the 

mitogen-activated protein (MAP)-kinase pathway previously 

shown for ER� is present and active in the ER� (50, 89). 

Additionally, when acting on a basal promoter linked to a 

consensus estrogen response element, both ER� and ER� 

were able to recruit the coactivator SRC-1 and were equally 

susceptible to inhibition by the antiestrogens raloxifene, ICI 

164,384, and EM-800 (50). 

However, as studies continue, distinct differences at the 

molecular level and in the transactivational capacities between 

ER� and ER� have been described. Two separate 

studies have demonstrated the specificity of the agonist activity 

of 4-hydroxytamoxifen to be unique to ER�, although 

this appears to be highly dependent on the cell and promoter 

context as well as experimental design (50, 90). Furthermore, 

Paech et al. (91) reported that when interacting with DNAbound 

AP-1 transcription factors, the in vitro transactivational 

activity of estrogen agonists and antagonists was quite 

different depending on which form of ER was present. 

Whereas antagonists, such as raloxifene, tamoxifen, and ICI 

164,384, were able to block the stimulatory activity of the 

ER�/AP-1 complex, these same compounds acted as potent 

agonists when bound to an ER�/AP-1 complex (91). Further 

experimental support for the existence of distinct structural 

and functional differences between ER� and ER� was recently 

provided by Sun et al., who showed that certain nonsteroidal 

ligands were receptor selective in their binding and 

agonist/antagonist activities (92). 

Perhaps the most significant disparity lies in the tissue 

distribution of the two receptors. Studies employing the techniques 

of RT-PCR and/or ribonuclease protection assay 

(RPA) have indicated that ER� mRNA is predominant in the 

uterus, mammary gland, testis, pituitary, liver, kidney, heart, 

and skeletal muscle, whereas ER� transcripts are significantly 

expressed in the ovary and prostate (Fig. 2) (63, 80, 93, 

94). These same studies have indicated relatively equal levels 

of mRNA for the two receptors in the epididymis, thyroid, 

adrenals, bone, and various regions of the brain (80, 93, 

95–97). However, as more studies are reported, several discrepancies 

in the expression patterns of ER� and ER� among 

different species are becoming apparent (80, 93, 96). For 

example, whereas ER� mRNA is easily detectable in the 

pituitary of the rat (70, 98, 99), human (100), and rhesus 

monkey (96), levels in the pituitary of the mouse appear low 

to undetectable (93). A similar difference in expression is 

apparent in the mammary gland, in which normal and neoplastic 

human tissue and cell lines express detectable ER� 

mRNA (64, 68, 73, 101, 102), although the mammary gland 

of the mouse appears to predominantly express ER� (93). 

Furthermore, even in those tissues expressing both ERs, there 

is often a distinct expression pattern within the heterogeneous 

cell types composing the tissue. In the ovary, ER� is 

apparently localized to the granulosa cells of maturing follicles, 

whereas ER� is detectable in the surrounding thecal 

cells (69, 103, 104). In the prostate of the rat, expression of ER� 

and ER� is detectable in the stroma and epithelium, respectively, 

but does not appear to be colocalized in any portion 

of the tissue (49). However, through the combined use of 

immunohistochemistry and in situ hybridization, Shughrue 

FIG. 2. RT-PCR for ER� and ER� mRNA in various tissues of the wild-type mouse. RT-PCR was carried out on 0.5 �g of total RNA pooled from 

adult wild-type mice using primers specific for the mouse ER� and ER� transcripts (see Refs. 93 and 123). Equal amounts of the individual 

RT-PCR reactions were then fractionated on an agarose gel. Note the broad tissue distribution of ER� mRNA, whereas ER� transcripts are 

primarily expressed in the ovary, hypothalamus, lung, and male reproductive tract. RT-PCR for �-actin was carried out as a positive control. 

(�RT) indicates a negative control, i.e., PCR on total ovarian RNA minus reverse transcriptase, indicating the specificity of the ER primers for cDNAs 

generated by the reverse transcriptase enzyme.


et al. (88) have demonstrated colocalization of ER� and ER� 

to select regions of the rat forebrain. 

C. Generation of the ER null mice 

The field of estrogen action has been and continues to be 

a dynamic one with broadening scope. Although estradiol is 

often called the female sex steroid, the list of tissues and 

organs in which estrogen actions appear to be critical to 

physiological function continues to expand, in both sexes. 

The crucial actions of estradiol in the female reproductive 

tract, breast, and hypothalamic-pituitary axis have been well 

described. However, we now know of significant functions 

fulfilled by estrogens in male reproduction. Furthermore, the 

effects of estrogens in the physiology, maintenance, and 

overall health of the cardiovascular, central nervous, bone, 

immune, skin, and adipogenesis systems have been an area 

of continued research. And finally, the role that estrogen and 

normal and variant ERs may play in carcinogenesis, especially 

in the breast and female reproductive tract, continues 

to receive great attention. 

The rationale for generating mice that possess no functional 

ER is multifaceted, but in its most simple terms, was 

founded on the classical ablation experiments of the early 

part of this century. In 1900, Knauer (105) described the 

ability of ovarian grafts to prevent uterine atrophy in the 

castrated guinea pig. Five years later, Marshall and Jolly (106) 

described the capacity of ovarian extracts to induce estrus 

when administered to ovariectomized dogs. Similar protocols 

were later elegantly employed by Jost (107) to substantiate 

the endocrine function of the testis and the importance 

of testosterone in sex determination. These studies were critical 

to establishing the following basic criteria required to 

verify an endocrine role for a particular organ or tissue: 1) 

removal or destruction of the synthesizing organ should 

result in predictable symptoms presumed to be related to the 

absence of the hormone; 2) administration of material prepared 

from the removed organ should relieve these symptoms; 

and 3) the hormone should be present in and extractable 

from both the organ and blood (108). In the spirit of these 

earlier studies, the later part of this century has witnessed the 

marriage of two relatively new methodologies to introduce 

a modern version of the ablation experiment. The combination 

of in vitro culture of mouse embryonic stem cells and 

targeted homologous recombination has generated a tool 

that allows for the precise disruption or knockout of a particular 

gene of study and the passage of this mutation to 

offspring. Although one can debate whether this new technique 

is more or less invasive than the previous surgical 

methods, it is obvious that the classic “ablation” experiment 

has been elevated to a molecular level. The function of a 

specific hormone can now be studied rather than the function 

of a whole endocrine organ that may produce multiple secretions. 

Furthermore, this new technology allows for the 

study of a particular cellular component, such as a receptor, 

that is intrinsic to one or more tissues. Such studies were 

previously impossible or relied on the use of chemical antagonists, 

which introduced their own inherent limitations. 

Additionally, gene targeting provides for in vivo methods to 

study the roles of a particular receptor throughout the life of 

the animal, including the early development stages. The current 

tools that allow for the generation of transgenic mice 

have already made significant contributions to our knowledge 

of particular genes, especially those involved in development 

and reproduction (reviewed in Refs. 109 and 110). 

At the time the ERKO mouse was envisioned, the ER� was 

the only form of ER known to exist. Furthermore, there were 

no reports of ER mutations in normal tissue that resulted in 

estrogen insensitivity in humans or laboratory animals. This 

was in contrast to the descriptions of syndromes of receptorbased 

insensitivity to androgens (37, 38) and thyroid hormones 

(40). Therefore, investigators were inclined to conclude 

that mutations that resulted in insufficient estrogen 

synthesis or resistance to the hormone at the level of the 

target organ were lethal at the earliest developmental stages 

(111, 112). This view was strengthened by reports of the 

detection of ER� mRNA in both human (113) and mouse 

(114) oocytes as well as in mouse blastocysts (115). Accordingly, 

the concept of generating a mouse devoid of ER� was 

initially met with skepticism but was pursued as a collaborative 

effort between our laboratory and that of Dr. Oliver 

Smithies of the University of North Carolina at Chapel Hill. 

If disruption of the mouse ER� gene did prove lethal, a model 

to study the precise time and locations of critical ER-mediated 

actions during early development would be available. 

However, providing the animal was viable, an in vivo model 

of estrogen insensitivity would now be accessible for continued 

study. Six years after the initial description of the 

�ERKO mouse, we now take for granted that disruption of 

the ER� gene proved not to be lethal, but rather the animal 

develops normally and exhibits a life span comparable to its 

wild-type litter mates (46). However, as will be elaborated in 

detail in this review, the adult �ERKO mice exhibit several 

abnormalities and deficiencies, most notable of which are the 

phenotypic syndromes that result in infertility in both sexes. 

Since this time, additional discoveries have been made to 

enhance the utility of the �ERKO model. Soon after the 

generation of the �ERKO mouse, Smith et al. (116) reported 

the first and only known case of clinical estrogen insensitivity 

due to an inactivating mutation of the ER� gene in a human 

patient. Later came multiple descriptions of aromatase and 

subsequent estradiol deficiency in humans (117–120). And 

finally, a second collaborative effort resulted in the successful 

generation of the �ERKO mice, which also survive to adulthood 

and exhibit phenotypes unique from those of the 

�ERKO (47). However, splicing variants of the disrupted 

genes that may encode for receptors with decreased functional 

activity have been detected in small amounts in each 

of the respective ERKO models (as discussed below) and 

therefore complicate the interpretation of the nonlethality of 

the gene targeting. Nonetheless, we now know that a loss of 

full function of any one of the two ERs is neither lethal nor 

detrimental to embryonic and fetal development in both 

mice and humans. Perhaps the survival of the ERKO mice, 

the aromatase-deficient humans, and the ER�-deficient male 

will prompt a renewed effort among clinicians to suspect and 

investigate the possibility of estrogen insufficiency or resistance 

in patients not responding to conventional therapies. 

The ERKO mice provide broad and multiple advantages 

to the research efforts toward understanding the function


and mechanism of estogen action. Much of what is known 

about estrogen action was inferred from in vivo studies involving 

castration or the administration of ER antagonists or 

inhibitors of estradiol synthesis. These findings have been 

complemented by the vast knowledge gained from in vitro 

cell culture studies, employing chimeric and mutant versions 

of the ER, varied cell types, multiple combinations of promoter-reporter 

gene constructs, as well as synthetic agonists 

and antagonists. However, there are distinct disadvantages 

to these experimental schemes. Studies using aromatase inhibitors 

and/or estrogen antagonists are complicated by several 

factors, including variability of the compound to block 

the action of the natural hormone or enzyme. The effectiveness 

of various antagonists is highly dependent upon the 

animal model, the tissue or cell of study, the bioavailability 

of the compound at different target tissues, and the class of 

antiestrogen used (8). This dilemma is further complicated 

by the discovery of the ER�, since no known ER-selective 

agonists or antagonists have been characterized at an in vivo 

level. The limitations of in vitro cell culture experimental 

approaches are obvious and mostly based on their finite 

application to the whole animal. Therefore, the ERKO models 

provide a unique tool to investigate the role of the ER in 

the context of the whole animal, and equally important, 

during the complete life span of the animal. At their most 

fundamental level, the ERKO mice address the role of the ER 

in the development and normal physiology of all organ systems, 

as well as in carcinogenesis, toxicity, and aging. Furthermore, 

unlike the “castrate” model in which several hormones 

are removed from the system, the ERKO mice retain 

the capability to synthesize the gonadal steroids, including 

the natural ER ligand, estradiol. Therefore, the biochemical 

functions of estradiol and the ER can be investigated in the 

presence of presumably intact pathways for the other gonadal 

hormones. The presence of estrogens in the absence of 

ER also provides for the possibility of discovering pathways 

of estrogen action that are independent of nuclear ER, or 

mediated via previously unknown forms of the receptor. 

Additionally, although the majority of in vitro studies indicate 

that ER� and ER� may have redundant functions, their 

differences in tissue distribution and response to certain ligands 

indicate the presence of distinct roles fulfilled by each. 

The fact that the �ERKO mice exhibit an unaltered pattern of 

ER� mRNA expression strengthens the usefulness of this 

model to dissect these potential ER�-mediated actions (93, 

121). Finally, consistent with those criteria discussed earlier 

for establishing an endocrine function to an organ, the ERKO 

animals now provide a null background available for transgenic 

reintroduction of the ER of other species, mutated ERs, 

or targeted ER expression to a specific tissue or cell type. 

A detailed description of the targeting scheme employed 

to disrupt the mouse ER� gene can be found in the initial 

description of the �ERKO mice (46). As shown in Fig. 1, a 

1.8-kb insert possessing the gene for neomycin (neo) resistance 

under the control of the phosphoglycerate kinase 

(PGK) promoter and including a PGK-polyadenylation signal 

was inserted into a NotI site in exon 2 of an ER� gene 

fragment subcloned from a genomic library of 129/J mouse 

DNA. The targeting insert was placed in a replacement � 

type targeting vector (122) with the appropriate ER� gene- 

flanking sequences. Upon successful targeting in mouse embryonic 

stem cells (129/J), the neo insert is placed approximately 

270 bp downstream of the ER� translation start site 

and thereby inhibits proper expression of the ER� gene. Since 

this was the current state of the technology, no portion of the 

ER� gene was removed during the targeting event. Standard 

protocols of clone selection and blastocyst (C57BL/6J) injection 

were used to generate chimeric mice possessing the 

disrupted gene, some of which demonstrated germ-line 

transmission of the mutation when bred with wild-type 

mates (122). Southern blot and PCR analysis of genomic 

DNA from mice of all three genotypes indicated the correct 

targeting of the ER� gene and the absence of any heterologous 

recombination in other regions of the genome. Inbreeding 

of mice heterozygous for the ER� disruption resulted in 

a Mendelian distribution of all three genotypes as well as a 

balanced sex ratio, indicating that the ER� is not critical to sex 

determination at the level of the external genitalia (46). 

The generation of mice homozygous for a disruption of the 

ER� gene was similar to that described above for the �ERKO 

and can be found in detail in the initial description (47). A 

genomic clone that spanned a 15-kb region possessing the 

first three exons of the mouse ER� gene was selected from 

a 129/SvJ mouse library. A replacement � type targeting 

construct was generated to include 5� and 3� homologous 

sequences of 1.3 and 7.4 kb, respectively (Fig. 1). A PGK 

promoter-regulated neo gene was inserted in the reverse 

orientation into a PstI site in exon 3 of the ER� clone. Therefore, 

correct targeting resulted in disruption of the sequences 

coding for the first zinc finger of the ER� protein, a domain 

critical to normal function of the receptor. Chimeric and 

heterozygous offspring were generated as described above 

for the �ERKO mice. Once again, mice possessing the targeted 

disruption of the ER� gene were identified by diagnostic 

Southern blotting and PCR of genomic DNA. As with 

the �ERKO, inbreeding of mice heterozygous for the disruption 

yielded a Mendelian distribution of all three genotypes 

as well as a balanced sex ratio (47). 

In both knockout models, RT-PCR on RNA from target 

tissues indicated the presence of multiple splicing variants of 

the respective ER transcripts (47, 123). In neither case has 

wild-type-like mRNA transcribed from the disrupted receptor 

gene been detected. The greater proportion of the variants 

detected in each ERKO model possessed frame shifts that 

would result in a severely truncated or mutated ER if translated. 

However, in the �ERKO, a single-splice variant capable 

of encoding a mutant ER� protein with significantly 

decreased transactivational capacity in vitro was detected at 

very low levels (123). A similar ER� splice variant, in which 

the reading frame was preserved although coding sequences 

were removed, was detected in ovaries of the �ERKO mice. 

This single variant, if translated, would encode a mutant ER� 

lacking the first zinc finger and therefore would be unlikely 

to transactivate due to an inability to tightly associate with 

the chromatin structure within the regulatory regions of target 

genes. 

This is not the first report of targeted insertions resulting 

in aberrant splicing of a disrupted gene. Mice possessing a 

targeted disruption of the transforming growth factor-� gene 

produce a transcript in which the entire exon possessing the


disrupting insert is accurately removed via the conventional 

donor and acceptor splice sites, preserving the normal reading 

frame of the gene (124). Studies in three different human 

genes have demonstrated that point mutations resulting in a 

premature stop codon can lead to complete excision of the 

exon possessing the mutation (125, 126). Furthermore, Reed 

and Maniatis (127) used artificial deletions and insertions 

within exons of genes that normally display alternative splicing 

to demonstrate that the proximity of the acceptor and 

donor splicing sequences to one another plays a role in splicing 

mechanisms. It is possible that insertion of sequences as 

large as those used in the ERKO mice may disrupt the spatial 

requirements necessary for proper mRNA splicing. Therefore, 

the above studies, along with our experiences, are relevant 

to the practice of targeting genes by insertion of a large 

disrupting sequence possessing internal stop codons. 

1. Interpretation of phenotypes in receptor null mice. The use of 

methodologies to target and disrupt individual genes has 

created numerous models available for study (reviewed in 

Refs. 109, 110, and 128). Furthermore, this impact has been 

felt in several facets of the biological sciences. Although it 

may be initially thought that a particular gene plays no role 

in the physiology of certain animal systems, such as reproduction 

or behavior, disruption of the gene and the subsequent 

phenotypes often prove otherwise. Therefore, transgenic 

and knockout technologies have spawned a number of 

collaborative efforts between investigators of varied disciplines 

that may have never occurred. 

An issue that has become apparent from the numerous 

gene disruption studies and interdisciplinary collaborative 

efforts is a collection of caveats to be considered when evaluating 

phenotypical data from a knockout model. These have 

arisen mostly from the behavioral sciences (reviewed in Refs. 

129 and 130), but have expanding application to all areas of 

study provided by transgenic animals. The first of these 

caveats is one that may be most relevant to the steroid receptor 

mutant models, i.e., when studying the target tissue of 

an adult receptor-knockout mouse, one must realize the tissue 

passed through all the stages of development and “organization” 

in the absence of the respective receptor. Therefore, 

this tissue, and in essence the whole animal, cannot be 

assumed to be truly identical to the wild type in all aspects 

except for the absence of the targeted gene product. Any 

genetic redundancies or compensatory mechanisms that 

took place during development cannot be readily detected or 

accounted for during most experiments. Therefore, the lack 

of a phenotype does not necessarily discount the function of 

the disrupted gene in the physiology being studied. Additionally, 

it is difficult to distinguish between an organizational 

vs. activational basis for an observed deficit in a particular 

physiology when studying the adult mutant. For 

example, observed resistance to a hormone due to alterations 

downstream of the function of the disrupted gene may be 

apparent during adulthood, but may have been imprinted 

during development. 

Other caveats of interpreting data from receptor null mice 

are founded in the methods used to generate and maintain 

a line of knockout mice. The standard protocol for generating 

a knockout mouse involves the incorporation of embryonic 

stem cells of a 129 strain of mice that carry the desired 

mutation into the blastocyst of a C57BL/6 strain. The resulting 

chimeric animals are then often back-crossed to the 

C57BL/6 strain once again until mice homozygous for the 

disruption are acquired. Therefore, early generations of 

knockout mice are composed of a somewhat chimeric genome, 

especially in the chromosomal regions closest to the 

targeted locus. This is of special importance in behavioral 

studies in light of the known variations in the sexual behavior 

of different strains of mice (131). However, most relevant to 

the ERKO, significant variations in estrogen responsiveness 

of the female reproductive tract among the different strains 

of mice are also known to exist (132, 133). A recent report by 

Roper et al. (134) has further defined the genetic basis for the 

variations that exist in the effects of estradiol on classical 

uterine parameters in mice. These limitations can be overcome 

to some degree by increasing experimental sample 

sizes and including parental strains as a control group in all 

experiments (135). In addition, the various models of hormone 

and steroid receptor deficiency that are now available 

no longer make necessary the complete interpretation of data 

from any one model. Therefore the data from these models, 

when interpreted as a whole, should prove invaluable to 

elucidating the roles of the different sex steroid receptors in 

both development and adult physiology. 

III. Reproductive Tract Phenotypes of the Female 

The most well characterized estrogen target tissues are 

those of the mammalian female reproductive tract, comprised 

of the ovaries, oviducts, uterus, cervix, and vagina. 

Reproductive capabilities in the female are dependent on the 

sequential processes of differentiation during the embryonic 

and prenatal periods and the maturation of these tissues 

during puberty. Differentiation of the fetal gonads to ovaries 

results from a lack of the Y chromosome, and therefore the 

testis-determining genes, and the presence of putative, yet 

unidentified, autosomal ovary-determining genes (111). The 

ductal organs of the tract subsequently result from differentiation 

of the fetal ambisexual precursor, the Müllerian 

ducts, due to the lack of testicular hormones. Early studies 

indicated that the female reproductive tract is the default 

phenotypical sex and will differentiate and develop normally 

in the absence of ovaries and adrenal glands (107, 136). Therefore, 

it appears that estrogens are not required for differentiation 

and initial development of the female reproductive 

tract, whereas testosterone is critical to differentiation of the 

male genitalia. This conclusion has been reconfirmed by the 

male-to-female sex reversal observed in male mice homozygous 

for a targeted disruption of the gene encoding SF-1, a 

transcription factor that regulates the steroid hydroxylases in 

steroidogenic cells (137, 138). Despite an inability to synthesize 

steroids and a complete lack of gonads, all SF-1 knockout 

mice develop female internal genitalia, regardless of genetic 

sex (137). It is therefore not surprising that the �ERKO and 

�ERKO female mice exhibit a properly differentiated female 

reproductive tract possessing the constituent structures (46, 

47). However, estrogen insensitivity has severely disrupted 

sexual maturation of the whole reproductive tract in the


�ERKO female and ovarian function in the �ERKO female. 

The consequences of ER gene disruption on the individual 

components of the female reproductive tract is the topic of 

this portion of the review. 

Before we continue, we believe it is necessary to briefly 

reiterate those studies carried out to verify successful targeting 

of the ER� gene in the �ERKO. This discussion is 

appropriate for this portion of the review because the majority 

of these experiments were performed on uterine tissue. 

To determine the effectiveness of the gene targeting, Western 

blots of adult �ERKO uterine nuclear and cytosolic extracts 

were probed with the H222 antibodies, a rat monoclonal 

antibody specific to the ligand-binding domain of the human 

ER� (10). Our studies, as well as those of others, have demonstrated 

that this antibody possesses high cross-reactivity to 

the mouse ER� (139–141). These assays detected no wildtype 

ER� or any other immunoreactive fragments unique to 

the �ERKO uterus. Similar results were obtained when blots 

were probed with the rabbit antiserum ER-21, directed toward 

the 21 amino-terminal residues of the rat ER� (141). 

However, binding assays using 3 H-E 2 on �ERKO uterine 

extracts indicated the presence of high-affinity binding of the 

hormone at levels approximately 3–9% of the wild type (123). 

In agreement with these data, sucrose gradient analysis with 

3 H-E2 on low-salt cytosol extracts from �ERKO uteri indicated 

a binding factor with an 8S sedimentation value, similar 

to that of the wild-type ER� (123). The discovery of the 

ER�, reported approximately 3 yr after the generation of the 

�ERKO, prompted a renewed assessment of this �ERKO 

estrogen-binding data in several publications. Unfortunately, 

in a number of these reports, the original datum 

discussed above is not evaluated in full, and the authors 

elude to ER� as the likely binding source in the �ERKO uteri. 

Certainly at the time of the initial characterization, concern 

over the residual level of binding in the �ERKO uteri was 

often mixed with the wonder of possibly discovering an 

unknown ER. However, during these studies we also demonstrated 

that when the H222 antibodies were included in 

the sucrose gradient assays, the estradiol binding peak in the 

�ERKO uterine extract was shifted accordingly (123). The 

H222 antibodies have been shown by us, as well as by other 

laboratories, to be ER� specific and unable to recognize ER� 

by Western blot analysis or immunohistochemistry (142). As 

described earlier in this review, our RT-PCR analysis on 

mRNA from �ERKO uteri demonstrated the presence of a 

splicing variant of the disrupted ER� gene that could encode 

a mutant ER� possessing both the ability to bind estradiol as 

well the H222 epitope (123). Furthermore, we have recently 

shown that ER� mRNA is undetectable in the uteri of adult 

wild-type as well as �ERKO mice when assayed by ribonuclease 

protection assay (93). Therefore, we believe that relatively 

conclusive data have been generated to indicate that 

the estradiol-binding factor present in �ERKO uteri is most 

likely not ER�. 

A. Uterus 

1. Uterine phenotype and estrogen insensitivity. The ER has been 

detected by steroid autoradiography and immunohistochemical 

methods in the ductal structures of the rodent fe- 

male reproductive tract during several stages of development, 

including the late fetal and neonatal stages through 

puberty and adulthood (reviewed in Refs. 112 and 143). 

Several reports describe the initial appearance of ER immunoreactivity 

in the developing uterus as early as fetal day 15 

(112, 143). ER immunoreactivity was first detectable in mesenchymal 

cells, whereas induction in the epithelial cells occurs 

during the late fetal stages and increases significantly 

during the neonatal period (112, 143). The fully developed 

uterus is composed of many heterogeneous cell types comprising 

three major anatomical compartments, the outer 

myometrium, endometrial stroma, and luminal/glandular 

epithelium. In the immature CD-1 mouse, ER� immunoreactivity 

is easily detectable in the stroma on day 1 and continues 

to rise to a maximal level on day 10, whereas the 

appearance of epithelial ER� is delayed and reaches a peak 

around day 16 (144). Other reports indicate variations in the 

exact timing of the appearance of ER� among different 

strains and species, most likely reflecting temporal deviations 

in development (112, 143). 

The presence of an intact estrogen-signaling system appears 

to coincide with the appearance of ER�. In several 

species, estrogen treatment of fetal and neonatal females 

results in the stimulation of increased uterine levels of nucleic 

acid (136, 145), protein synthesis (146), ornithine decarboxylase 

(147), progesterone receptor (148), and cellular 

proliferation (145, 149, 150). However, a full biological response 

to estradiol in terms of maximum increases in uterine 

weight is not possible in the neonatal uterus, and can be 

observed only after the animal approaches weaning age 

(146). Furthermore, significant differences in the uterine response 

to estradiol between the neonate and sexually mature 

rodent are known (151). For example, estrogen stimulates 

cellular proliferation in all tissues of the immature uterus, 

whereas this response becomes limited to the epithelial compartment 

during adulthood (151, 152). Therefore, sexual maturation 

of the uterus is not simply marked by the presence 

of ER�, but rather the acquisition of the capacity to undergo 

the correct synchronized phases of proliferation and differentiation 

elicited by the ovary-derived sex steroids. 

As shown in Fig. 3, the uteri of both adult �ERKO and 

�ERKO females possess all three definitive uterine compartments, 

the myometrium, endometrial stroma, and epithelium. 

However, in the �ERKO, each is hypoplastic and results 

in whole uterine weights that are approximately half 

that recorded for wild-type littermates (46). In contrast, the 

uteri of adult �ERKO females appear normal and able to 

undergo the cyclic changes associated with the ovarian steroid 

hormones (47). Therefore, perinatal development of the 

female reproductive tract in the mouse appears to be independent 

of ER� and ER� actions. However, estrogen responsiveness 

and subsequent sexual maturity in the uterus has 

been ablated by disruption of the ER� gene. The �ERKO 

endometrial stoma is characterized by a less organized structure 

and hypotrophy, with a sparse distribution of uterine 

glands compared with that of the wild type (153). Luminal 

and glandular epithelial cells in the �ERKO uterus most often 

appear healthy, but are consistently cuboidal and lack the 

normal “estrogenized” morphology of a tall columnar shape 

and basally located nucleus (Fig. 3). This phenotype is in-


FIG. 3. Histology of uterine and vaginal tissue of wild-type, �ERKO, and �ERKO females. Cross-sections of uterine tissue from (a) wild-type, 

(b) �ERKO, and (c) �ERKO adult females illustrating the presence of all three anatomical tissue compartments in the uteri of the wild-type 

and ERKO mice (33�; inset, 132�). The representative wild-type uterine section illustrates a normal myometrium (My), endometrial stroma 

(St), and epithelium (Ep) (inset). The representative �ERKO uterine section illustrates the characteristic hypoplasia of each compartment, a 

slightly disorganized endometrial stroma, and a lack of estrogenization of the luminal and glandular epithelium (inset). Note the dramatically 

smaller diameter of the �ERKO uterus, as indicated by the ability to fit the whole transverse section of the tissue in the field of view. The 

representative �ERKO uterine section is indistinguishable from that of the wild-type, including the presence of estrogen-stimulated luminal 

epithelium (inset). Below are cross-sections of vaginal tissue from representative (d) wild-type, (e) �ERKO, and (f) �ERKO female mice (66�). 

The representative wild-type vaginal section illustrates a normal stroma (St) and hypertrophied epithelium (Ep) showing estrogen-induced 

stratification and cornification. In contrast, these estrogen actions are lacking in the �ERKO vagina. Once again, the tissue of the �ERKO is 

indistinguishable from the wild-type control. Scale bar � 1 �m. 

teresting in light of the increased levels of estradiol found in 

the serum of adult �ERKO females (Table 2). However, 

Lindzey et al. (154) demonstrated that the concurrently elevated 

ovary-derived androgens in the �ERKO female (Table 

2) do provide for some maintenance of uterine weight, which 

can be further reduced upon ovariectomy. We recently reported 

that ER� mRNA is barely detectable in the adult 

mouse uterus, including those from �ERKO mice (93), making 

it unlikely that ER� could provide a compensatory role 

in the �ERKO uterus. Numerous immunohistochemical 

studies for ER� and the apparent loss of estrogen sensitivity 

in the �ERKO uterus indicate that the classical ER� is the 

predominant form responsible for mediating estrogen actions 

in the mouse uterus. 

The response of the ovariectomized rodent uterus to estradiol 

has been well documented and is often described as 

biphasic, with the initial phase consisting of effects that become 

apparent within the first 6hofasingle estrogen treatment 

(reviewed in Ref. 155). These include metabolic re- 

sponses in the form of increased water imbibition, vascular 

permeability and hyperemia, prostaglandin release, glucose 

metabolism, and eosinophil infiltration (155). A series of 

biosynthetic responses are also characteristic of the first 

phase and include increased RNA polymerase and chromatin 

activity, lipid and protein synthesis, and increased glucose-6-phosphate 

dehydrogenase (155). As several of the 

above processes continue, they are accompanied by dramatic 

increases in RNA and DNA synthesis, mitosis, and cellular 

hyperplasia and hypertrophy that peak 24–72 h after exposure 

(155). Initial studies suggested that this biphasic pattern 

may be the result of at least two types of acceptor sites for 

estradiol-receptor complexes in the uterus (156). However, 

the uteri of �ERKO females fail to exhibit components of both 

phases after estrogen treatment, providing strong evidence 

for the requirement of ER� in the full response (46, 123). In 

brief, when treated with 40 �g E 2 or diethylstilbestrol (DES) 

per kg body weight for three consecutive days, wild-type 

mice exhibited the expected 3- to 4-fold increase in uterine


TABLE 2. Serum hormone levels in adult wild-type and �ERKO mice 

Hormone 

Gonadal steroids 

Wild-type (SEM) 

Female 

�ERKO (SEM) Wild-type (SEM) 

Male 

�ERKO (SEM) 

Estradiol (pg/ml) b 

29.5 � 2.5 84.3 � 12.5 a 

11.8 � 3.4 12.9 � 3.4 

Progesterone (ng/ml) b 

2.3 � 0.6 4.0 � 1.1 0.5 � 0.3 0.3 � 0.1 

Testosterone (ng/ml) 

Anterior pituitary 

0.4 � 0.4 3.2 � 0.6 9.3 � 4.0 16.0 � 2.3 

LH (ng/ml) 0.3 � 0.04 1.7 � 0.3 a 

2.4 � 1.2 3.7 � 0.7 

FSH (ng/ml) 4.9 � 0.6 5.4 � 0.7 26.0 � 1.4 30.0 � 1.1 

PRL (ng/ml) 

nd, Not determined. 

a 

t test, wild-type vs. ERKO, P � 0.001. 

18.8 � 10.7 3.5 � 1.3 nd nd 

b 

These values in the female are different than those reported in Ref. 123, which were carried out on pooled sera. The values above are the 

means from assays on individual samples and therefore are more likely to reflect the true levels in the two genotypes. 

wet weight, whereas no such response was observed in the 

uteri of �ERKO mice (46, 157). It should be noted that this 

pharmacological dose of estrogen is well beyond that required 

to achieve a maximum response in the wild-type 

rodent. Nonetheless, estrogen-treated �ERKO uteri exhibited 

no apparent components of the initial phase of estrogen 

effects, including water imbibition and hyperemia. Histological 

analysis and [ 3 H]thymidine incorporation assays indicated 

a lack of significant cellular proliferation and DNA 

synthesis in uteri from the estrogen-treated �ERKO mice 

(123, 153). Interestingly, although the heterozygous females 

possess approximately one-half the normal complement of 

ER�, their uterine response to estrogens is equal to that of the 

wild-type females. In a similar study, wild-type and �ERKO 

mice treated with hydroxy-tamoxifen (1 mg/kg) produced 

comparable results (157), eliciting the expected estrogenic 

response in the wild-type and having no effect on the �ERKO 

uterus. These studies thereby confirm that the estrogen agonist 

activity of hydroxy-tamoxifen, which is somewhat 

unique to the mouse uterus (8), is mediated via the ER� 

pathway. 

The mitogenic and stimulatory action of estradiol in the 

uterus is a complex process involving increased RNA polymerase 

and ribosomal activity (158), resulting in the regulation 

of a plethora of genes. It is well accepted that the 

ligand-bound ER complex is not directly involved in the 

mediation of all responses elicited by estrogens in the uterus, 

but rather serves as a stimulus for a cascade of signaling 

pathways that act to amplify the estrogen action. However, 

certain genes appear to be directly regulated by the ER�estradiol 

complex and possess functional estrogen-responsive 

elements within their regulatory regions. Two such examples 

are the genes encoding the progesterone receptor 

(PR) (159, 160) and the secretory protein, lactoferrin (161). In 

fact, the regulation of the uterine PR and lactoferrin genes 

have often been used as assays for the estrogenic activity of 

experimental compounds. Therefore, with a similar intent, 

we used these estrogen markers to attest for estrogen insensitivity 

in the uteri of the �ERKO mouse. A single dose of 

estradiol, known to be effective in inducing the PR and lactoferrin 

genes within 24 h in uteri of wild-type mice, produced 

no such up-regulation in the uteri of the �ERKO mice, 

confirming the need for a direct action of the ER� in this 

mechanism (123). Interestingly, a recent report by Tibbetts et 

al. (162) demonstrated that the estrogen-stimulated increases 

in PR are localized to the stromal and myometrial compartments, 

whereas the increases in lactoferrin are isolated to the 

luminal and glandular epithelium in the mouse uterus. 

Therefore, disruption of the ER� gene has resulted in estrogen 

insensitivity in all three anatomical compartments of the 

uterus. However, it must be noted that constitutive levels of 

PR and lactoferrin mRNA are present in the �ERKO uteri, 

suggesting that these genes are also under the influence of 

pathways independent of ER�. A testimony to the complexity 

of estrogen action in the uterus is the finding that while 

estradiol up-regulates PR expression in the myometrium and 

stroma, it simultaneously abolishes PR levels in the luminal 

epithelium (162). This would indicate an inhibitory role of the 

estradiol-ER� complex on PR expression in this portion of the 

uterus. Speculating that this pathway may therefore be lacking 

in the �ERKO uterus, an investigation as to the source of 

the PR mRNA in the �ERKO uteri is warranted. 

2. Changes in growth factor functions. A component of the 

cascade of events that lead to the obvious changes in the 

physiology of the adult uterus after estrogen exposure are the 

auto- and paracrine actions of polypeptide growth factors. 

Several members of the epidermal growth factor family have 

been suggested as possible mediators of estrogen-induced 

mitogenesis in the uterus. This hypothesis is based on experiments 

demonstrating that estradiol up-regulates the 

uterine levels of epidermal growth factor and its receptor 

(EGF, EGF-R) (163, 164), transforming growth factor-� (165), 

and insulin-like growth factor-I (IGF-1) (166). Furthermore, 

mice homozygous for a targeted disruption of the EGF-R 

gene exhibit a hypoplastic uterus that is significantly reduced 

in size (167), similar to that of the �ERKO. Experimental data 

indicate that treatment of ovariectomized wild-type mice 

with EGF mimics the early effects of estradiol and DES in 

terms of inducing modified cell morphology and increases in 

the levels of ER, DNA synthesis, phosphatidylinositol turnover, 

PR, and lactoferrin in the uterus (168–170). Further 

studies have illustrated that cotreatment with anti-EGF antibodies 

was able to attenuate the uterine response to estradiol, 

presumably due to inactivation of the EGF-signaling 

pathway (168). In turn, cotreatment with the estrogen antagonist 

ICI-164,384 was able to reduce the uterine response 

to EGF (169). These in vivo studies suggest a cross-talk mechanism 

between the EGF and ligand-independent ER- signaling 

pathways. The results of the animal studies have been


supported by numerous in vitro experiments demonstrating 

ligand-independent activation of the nuclear signaling pathway 

of the ER�, possibly via altering the phosphorylation 

pattern of the ER� (reviewed in Ref. 55). The culmination of 

these and several other studies has led to the proposed model 

in which the mitogenic actions of estradiol in the rodent 

uterus appear to be at least partially mediated by EGF; however, 

in turn the mitogenic effects of EGF require the presence 

of ER�. 

Therefore, the �ERKO female provides an excellent in vivo 

model to study this cross-talk between the ER�- and EGFsignaling 

systems in the uterus. The uteri of �ERKO females 

possess wild-type levels of functional EGF and EGF-R (170). 

Nonetheless, the mitogenic actions and induction of estrogen-responsive 

genes elicited by EGF in the wild-type uterus 

have been ablated in the �ERKO, confirming the interaction 

of these two signaling systems (170). However, not all EGF 

responses are lacking in the uteri of �ERKO females, as this 

same study demonstrated that the mechanisms for EGFmediated 

up-regulation of the c-fos gene remained intact 

(170). These studies have thereby confirmed the need for 

functional ER� for the mitogenic actions of EGF in the uterus. 

Cunha et al. has extended the use of the �ERKO mouse to 

investigate the intersecting roles of ER�-mediated estrogen 

stimulation and growth factors in the uterus through a series 

of tissue recombination experiments. The observation of estrogenic 

effects in wild-type uterine epithelial cells that are 

apparently lacking ER� has prompted numerous investigations 

to illustrate a role for paracrine factors secreted by the 

underlying ER�-positive stromal compartment, and thereby 

mediating the epithelial response (143). These studies have 

been advanced by methods that provide for the delicate 

construction of tissue recombinants, in which uterine stoma 

and epithelium are enzymatically disassociated and recombined 

with similar tissue from uteri from animals of different 

treatments or models to ultimately regenerate a chimeric 

stromal-epithelial unit (reviewed in Ref. 143). These tissue 

recombinants are implanted under the kidney capsule of 

ovariectomized nude mice, which are then acutely treated 

with estrogen agonists or antagonists. Later removal of the 

recombinant grafts allows for the evaluation of certain end 

points of estrogen action in each portion of the recombinant. 

Cooke et al. (171) described experiments in which wild-type 

(ER��) uterine stroma were recombined with �ERKO 

(ER��) uterine epithelium and vice versa. The results of these 

studies illustrate that proliferation of the epithelial portion of 

the recombinant was possible only when ER�� stroma were 

present and did not require ER� in the epithelium (171). 

Interestingly, similar recombinant experiments using tissue 

from the EGF-R knockout mice illustrated that the estrogensignaling 

pathways required for stimulation of the stroma 

and subsequent induction of epithelial growth are intact in 

the absence of EGF-R (167). Aside from the proliferative 

effects of estradiol, previous studies suggested that estrogen 

stimulation of secretory products from uterine epithelium, 

e.g., lactoferrin, is directly mediated by the epithelial ER� 

(140, 162). However, Buchanan et al. (172) recently employed 

tissue recombinants similar to those described to demonstrate 

that both stomal and epithelial ER� are required for 

estrogen induction of the uterine epithelial secretory prod- 

ucts, lactoferrin and complement component C3. Therefore, 

estrogen-induced proliferation of the uterine epithelium requires 

the presence of ER� in the stromal compartment only, 

whereas induction of certain epithelial secretory products is 

dependent on the presence of ER� in both uterine compartments. 

3. Maintenance of selective estrogen actions in the �ERKO uterus. 

A distinct advantage of null receptor models, whether naturally 

existing or experimentally generated via molecular 

methodologies, is their use as an in vivo tool for discerning 

alternate pathways of hormone action. Recent studies by the 

Lubahn laboratory have indicated the preservation of a distinct 

estrogen-signaling pathway in the �ERKO uterus (173, 

174). Das et al. (173) reported that two consecutive treatments 

(over a period of 12 h) with the catecholestrogen, 4-hydroxyestradiol 

(4-OH-E 2)at10�g/kg body weight resulted 

in significant increases in water imbibition in the uteri of 

ovariectomized �ERKO mice. Induction of lactoferrin 

mRNA in the uterine epithelium of wild-type mice was 97and 

85-fold after treatment with 10 �g/kg body weight estradiol 

or 4-OH-E 2, respectively (173). In contrast, only the 

4-OH-E 2 was active in the �ERKO uterus, resulting in a 

60-fold increase in lactoferrin mRNA levels compared with 

a 1.4-fold induction by estradiol (173). A similar, yet more 

modest, response was reported in the wild-type and �ERKO 

uteri after treatment with the xenoestrogens, kepone (15 

mg/kg body weight) (173) and methoxychlor (15 mg/kg 

body weight) (174). 

Most interesting was the lack of inhibition of this response 

by the pure estrogen antagonist, ICI-182,780, in both the 

wild-type and �ERKO mice, indicating the possibility of a 

non-ER�-mediated signaling pathway for certain compounds 

exhibiting estrogenic activity. Additionally, the nature 

of the timing and type of lactoferrin response elicited by 

the 4-OH-E 2 and xenoestrogens in the �ERKO is quite distinct 

from that of the original descriptions by Teng et al. (175) 

concerning estrogen regulation of this gene in the mouse 

uterus. Given the knowledge of the low-to-absent expression 

of ER� in the uterus and the ability of the ICI compounds to 

antagonize ER� signaling in vitro, it is not likely that this 

receptor is involved in this phenomenon. The catecholestrogens 

are naturally synthesized and proposed to play a role 

in steroid regulation of the hypothalamus and pituitary (176, 

177), ovarian function (178), and embryo implantation (179). 

Furthermore, the discovery of local synthesis of these estrogens 

in mammary tissue has led to implications of their 

involvement in breast cancer (180). Therefore, further investigation 

into the alternate mechanisms by which these compounds 

may activate nuclear processes is needed. 

4. Maintenance of progesterone action in the �ERKO uterus. Like 

estradiol, ovarian derived progesterone, is an integral steroid 

hormone in the physiology and function of the uterus. The 

PR has been localized to cells composing all three anatomical 

compartments of the uterus and exhibits varied levels in each 

during the stages of the estrous cycle (reviewed in Ref. 181). 

The PR also exists in two forms, PR A and PR B, which differ 

only in the length of the N� terminus. In contrast to the ER, 

PR A and PR B are encoded by a single gene but transcribed


from two distinct promoters in both the rat (160) and human 

(159). 

As previously discussed, the PR gene is strongly regulated 

by the estradiol-ER� complex. This is evidenced by in vitro 

and in vivo studies showing inhibition of estrogen stimulation 

of the PR gene with antiestrogens as well as the presence 

of a single imperfect estrogen-response element in the proximal 

region of the PR gene promoter (160). However, in vitro 

assays indicate that both the proximal as well as a distal 

promoter appear to be at least partially ER� dependent and 

may also involve ligand-independent pathways of ER� action 

for full expression (160, 182). The lack of estradiol-induced 

increases in PR mRNA levels in the �ERKO uterus 

confirms a regulatory dependence of the PR gene on ER� 

action (123). Therefore, it was hypothesized that disruption 

of the ER� gene may subsequently result in abnormally low 

levels of PR in the �ERKO uterus, and thereby render this 

tissue refractory to progesterone as well. However, Northern 

blot and ribonuclease protection assays have indicated basal 

levels of PR mRNA in the �ERKO uterus that are equal to 

those in wild-type, although estrogen-stimulated increases in 

these levels are absent (123). Binding assays with a radiolabeled 

progestin indicate levels of PR protein in the �ERKO 

uteri are present but reduced to approximately 60% of that 

in wild type (183). A notable finding was the greater proportion 

of PR found to be tightly associated with the nuclei 

of cells from the �ERKO uteri (�25%), compared with the 

wild type (�5%) (183). It is possible that a lack of ER� has 

resulted in a more plentiful pool of available coregulator 

proteins, allowing the PR present in the �ERKO uterus to 

maintain a more “active” state. Western blots indicate no 

difference in the relative levels of PR A and PR B between the 

two genotypes, with PR A consistently present in greater 

amounts in both (183). Therefore, a loss of ER� action in the 

uterus has led neither to a complete loss of PR nor to altered 

and preferential transcription from one of the two PR gene 

promoters. 

Curtis et al. (183) carried out a series of studies demonstrating 

the preservation of PR-mediated progesterone actions 

in the �ERKO uteri. Stimulation of the genes encoding 

amphiregulin (184) and calcitonin (185) in the rodent uterus 

has been shown to be an early and late response to progesterone, 

respectively. A treatment regimen of progesterone 

shown to be effective in inducing these two genes in the 

ovariectomized wild-type uterus was equally effective in the 

uteri of ovariectomized �ERKO females (183). Furthermore, 

the documented ability of estradiol to inhibit the progesterone 

induction of the amphiregulin gene was absent in the 

�ERKO (183). These studies indicate that a sufficient level of 

PR and an active progesterone signaling pathway are present 

in the uteri of �ERKO mice. 

A well known physiological role for progesterone is the 

preparation of the uterine endometrium for the forthcoming 

pregnancy. Implantation of the embryo is a complex process 

that requires a high degree of synchronicity between the 

blastocyst and the hormone-induced changes of the uterine 

endometrium (reviewed in Ref. 186). In general, ovarian 

synthesis of estradiol, which peaks at ovulation, serves to 

“prime” the uterus by eliciting increased differentiation and 

proliferation of the luminal and glandular epithelium, and 

the induction of significant increases in PR in the endometrial 

stroma and myometrium (162, 186). Subsequent increases in 

progesterone released from the ovarian corpora lutea then 

cause decidualization, a complex process involving massive 

proliferation and differentiation of the endometrial stroma 

along with localized increases in vascular permeability and 

edema (186). This process involves the synthesis of specific 

hormones, such as PRL, cytokines, prostaglandins, extracellular 

matrix components, and various enzymes within the 

uterine tissues (186). The result is a remarkable swelling of 

the uterine stroma thought to be necessary for implantation 

by forcing the uterus to close down on the blastocyst (186). 

The final stage of apposition in the mouse is the grasping of 

the blastocyst and ultimate attachment to the uterine wall, a 

process thought to be dependent on the secondary rises in 

ovarian derived estradiol (186). Therefore, preparation of the 

uterus for blastocyst implantation is dependent on the multifunctional 

and sometimes opposing effects of estradiol and 

progesterone. 

A laboratory model for the decidualization process has 

been generated for several species and generally involves 

exogenous steroidal treatments designed to mimic those of 

the ovarian cycle, coupled with a mechanical stimulus or 

trauma of the uterine lining to simulate implantation (186, 

187). In brief, ovariectomized mice are treated with estradiol 

(100 ng) for 3 consecutive days, followed by 2 days of no 

treatment. The animals are then administered progesterone 

(1 mg) and a reduced dose of estradiol (7 ng) for 8 consecutive 

days, with mechanical stimulation of the uterus taking place 

on the third day. Using this treatment scheme and the progesterone 

receptor-knockout (PRKO) model, Lydon et al. (44) 

demonstrated the absolute dependence of the decidualization 

process on progesterone action and the PR by reporting 

the complete lack of decidualization in the PRKO mice. However, 

Curtis et al. (183) demonstrated that a progesteroneinduced 

decidual response is possible in the �ERKO uterus, 

despite its inability to respond to the estradiol priming meant 

to mimic estrus. Most interesting were investigations indicating 

that although the uterine decidualization in the wild 

type was dependent on estradiol, as evidenced by its inhibition 

with ICI-182,780, progesterone alone was sufficient to 

induce decidualization in the �ERKO uterus (183). 

The reasons for the apparent loss of estrogen dependence 

for successful decidualization in the �ERKO uterus can only 

be speculated upon at this time. One role of estrogen in 

uterine decidualization is thought to be the induction of 

significant increases in PR in the endometrial stroma (186). 

This is supported by recent immunohistochemical studies by 

Tibbetts et al. (162) demonstrating strong up-regulation of 

stromal PR and simultaneous down-regulation of epithelial 

PR in the mouse uterus after 4 days of estradiol treatment. 

In light of the puzzling results indicating the PR in the 

�ERKO uterus is strongly associated with the nucleus, and 

therefore possibly in a more “active” state, the ER�-independent 

decidualization observed in the �ERKO uterus may 

be due to an enhanced ability to respond to progesterone. 

However, a release of suprabasal levels of estradiol during 

the luteal phase of the ovarian cycle is synchronized with the 

time of implantation and thought to be critical to enhancing 

and maximizing the uterine decidualization process (186).


Nonetheless, the deciduomas induced in the �ERKO females 

are neither reduced in size nor appear less complex or differentiated 

than those observed in the wild type (183). It is 

also possible that a lack of ER� during development as well 

as adulthood has resulted in a uterus with a heightened 

tendency toward decidualization, caused by to the altered 

expression of other gene products. The complexity of the 

decidual process is illustrated by the several models that lack 

the ability to exhibit uterine decidualization, such as mice 

lacking leukemia-inhibitory factor (188), prostaglandin synthase-2 

(189), and Hoxa-10 (190). Furthermore, a process 

thought to be critical to implantation is the acquired ability 

of portions of the uterine epithelium to self-destruct and 

become detached from the uterine wall, possibly clearing a 

route by which the underlying swelling endometrium can 

breach and provide a site for implantation (186, 191). Histological 

analysis of �ERKO uteri indicates a uterine epithelium 

that may be less healthy and more often exhibits sloughing 

compared with wild-type uteri. It is therefore possible 

that the inherently impaired luminal epithelium of the 

�ERKO female has resulted in a lowering of the threshold 

required to induce decidualization. 

B. Vagina 

The fully developed adult vagina serves as both a copulatory 

receptacle and a birth canal in the female and may be 

divided into two distinct sections, the upper vagina and 

lower vagina. The cranial end of the upper vagina is attached 

to the cervix and is derived from the Müllerian ducts during 

differentiation of the female tract (143). The lower vagina, 

which connects the tract to the vulva and external genitalia, 

is differentiated from the urogenital sinus (143). As shown in 

Fig. 3, the wild-type vagina is a highly sensitive estrogen 

target tissue, composed of an inner mucosal layer of stroma 

and overlying epithelia, a middle layer of muscularis, and an 

outer sheath of connective tissue. Detectable levels of ER� are 

present in both the stromal and epithelial compartments 

making up the mucosa of the duct (7). Estradiol exposure 

during the ovarian cycle induces a series of effects in the 

vaginal mucosa that are often used to estimate serum gonadal 

hormone levels and approximate the current stage in 

the estrous cycle (107). These changes in the mucosa include 

cytodifferentiation of the stromal cells and a rapid proliferation 

and differentiation of the epithelial cells, resulting in a 

stratified and cornified epithelial layer closest to the lumen 

(192). This process also involves the estrogen stimulation of 

a series of keratins (193, 194). As shown in Fig. 3, despite the 

chronically elevated levels of estradiol in the serum of 

�ERKO females, histological analysis consistently indicates 

a complete lack of vaginal estrogenization. A similar effect on 

the vaginal mucosa has been produced in mice (195, 196) and 

rats (197) after prolonged ovariectomy or treatments with the 

antiestrogens, ZM-189,154, EM-800, and tamoxifen. Administration 

of exogenous estradiol, DES, or hydroxytamoxifen 

to �ERKO mice results in no discernible vaginal response 

(153). In contrast, the vaginal mucosa of the �ERKO female 

appears to undergo the normal cyclic changes associated 

with ovarian steroidogenesis (Fig. 3) (47), strongly indicating 

that this is an ER�-mediated process. 

Buchanan et al. (198) have used the stromal-epithelial tissue 

recombinant scheme described above for uterine tissue to dissect 

the contributions of the different tissue compartments in 

the estradiol response of vaginal tissue as well. As in the uterus, 

these studies demonstrated that stromal ER�, but not epithelial 

ER�, are required for estradiol-induced epithelial proliferation 

in the mouse vagina (198). Similar to the observations in the 

uterine recombinants, both vaginal stromal and epithelial ER� 

were required for estradiol-induced stratification and cornification 

of the epithelium, including the induction of the gene for 

the secretory protein cytokeratin 10 (198). All recombinations 

involving tissue from the �ERKO vagina became atrophied, 

even in the presence of estradiol (198). 

C. Oviduct 

The mouse oviduct is a coiled tubular organ connecting the 

uterus to the ovarian bursa and derived from the Müllerian 

duct during fetal development of the female reproductive 

tract (143). It functions as a route for passage of sperm to the 

ovulated oocyte and the subsequently fertilized blastocyst to 

the uterus. In the CD-1 mouse, ER� immunoreactivity is 

easily detectable in both the stroma and the epithelium of the 

oviduct as early as day 2 of life (144). The levels of ER� 

immunoreactivity in the epithelium continue to rise and 

plateau at approximately day 15 and remain high throughout 

adulthood (144). Furthermore, Newbold et al. (199, 200) described 

the high degree of sensitivity of the fetal and neonatal 

oviduct to the detrimental effects of developmental DES 

exposure. During adulthood, the levels of ER� in the oviductal 

epithelium fluctuate during the ovarian cycle, reaching 

peak levels during the proliferative phase (201). The 

ovarian sex hormones found in high concentrations in the 

oviductal fluid are thought to play a role in ovum and zygote 

transport through the oviduct (201). However, studies using 

ovariectomized laboratory animals have produced conflicting 

results in terms of what this role may be, depending on 

the animal model and hormone dosing regimen used (202). 

Despite the apparent ontogeny of ER� in the developing and 

adult mouse oviduct, no gross phenotypes in the oviduct of 

�ERKO females have been observed. Similar to the uterus, 

the epithelium of the �ERKO oviduct usually appears 

healthy yet unstimulated, despite the chronically high levels 

of serum estradiol. Due to the inability of the �ERKO to 

ovulate, possible defects in the transport functions of the 

oviduct are not easily studied. Assays for ER� mRNA in the 

mouse oviduct detect little if any ER� transcripts. Accordingly, 

in �ERKO females there appears to be no obvious 

defects in the structure and function of the oviduct that 

impede fertility. 

D. Ovary 

In most mammals, differentiation of the bipotential fetal 

gonad to an ovary in the genotypic female occurs later in 

gestation than differentiation of the testis in the male (111). 

The factors involved in development and differentiation of 

the ovary are not well understood, although the process does 

not appear to be dependent on the presence of primordial 

germ cells (111). The appearance of follicles and the onset of


estrogen synthesis in the fetal gonad may be the first indication 

of differentiation to an ovary, although the secreted 

estrogens do not appear to be critical to development of the 

ductal structures of the female reproductive tract. Still, fetal 

ovarian estradiol may play a role in development of the 

ovary itself, as evidenced by the complete lack of ovaries in 

SF-1 knockout mice (137, 138). Furthermore, a recent study 

has demonstrated immunohistochemical detection of ER� 

and ER� in the neonatal rat ovary (103). Interestingly, a lack 

of ER� or ER� during development appears to have no gross 

effect on ovarian differentiation, since individual knockouts 

of both respective receptors possess normal ovaries at birth 

and during prenatal development (47, 142). A study of ovarian 

development in a double-knockout lacking both forms of 

ER will therefore prove interesting in the future. Still, distinct 

ovarian phenotypes become apparent in the adult �ERKO 

and �ERKO females, resulting in infertility and subfertility 

in each, respectively (46, 47). 

1. Review of the physiology and function of the ovary. A brief 

description of ovarian morphology is necessary for a discussion 

of phenotypes that result from a lack of ER. The 

ovary may be conveniently divided into three broad functional 

units: the follicles, corpora lutea, and interstitial/stromal 

compartment (203, 204). All three possess the capacity to 

synthesize hormonal factors, especially steroids, in response 

to gonadotropins secreted from the anterior pituitary. The 

maturing follicle is a relatively ellipsoidal unit that can be 

further divided into three main cell types: the outermost 

thecal cells, which surround a single or multiple layers of 

granulosa cells, and together act to encase the germ cell 

(oocyte) at the approximate core. The overall size of the 

follicle and the number of cells composing the thecal and 

granulosa cell compartments are dependent on the stage of 

maturation (204). The corpora lutea are clearly defined and 

vascularized structures formed from the terminally differentiated 

thecal and granulosa cells that remain after ovulation 

(203). And finally, the interstitial and stromal tissue is 

composed of undifferentiated cells that may eventually be 

recruited for the thecal or granulosa units as well as dedifferentiated 

thecal and granulosa cells from past atretic follicles 

or regressed corpora lutea. This compartment also 

functions as the matrix within which the follicles are suspended 

(203). 

Ovarian function is often divided into two separate 

phases. The follicular phase refers to the period of follicle 

maturation and increased estradiol synthesis that leads up to 

and terminates with ovulation of the ovum. Ovulation marks 

the beginning of the luteal stage in which the developing 

corpora lutea secrete large amounts of progesterone as well 

as estradiol to allow for successful implantation of the blastocysts 

in the uterus. During the follicular stage of the ovarian 

cycle, the follicles may be categorized based on size, 

responsiveness to gonadotropins, and steroidogenic capabilities. 

These stages are most commonly referred to as the 

primordial, primary, secondary, tertiary or antral, atretic, 

and mature Graafian follicle (204). The primordial, or nongrowing 

follicles, are the most prevalent stage in the ovary 

at any one time and provide the pool from which follicles will 

be selected for maturation. These follicles consist of an oocyte 

arrested at the diplotene stage of the first meiotic division, 

surrounded by a single layer of cuboidal granulosa cells 

(204). Commencement of the follicular phase of the ovarian 

cycle involves the recruitment of primordial follicles to form 

the assembly of primary growing follicles to be prepared for 

ultimate ovulation. The factors required for this recruitment 

are not well understood. Henceforth, each stage is characterized 

by dramatic changes in the structure and functional 

capabilities of the follicle, which have been well characterized 

in several reviews (204–208). As the follicle progresses 

toward the secondary stage, rapid proliferation of the granulosa 

cells results in the formation of several concentric layers 

surrounding the oocyte (204). By this stage, stromal cells 

have differentiated to produce a defined stratum of thecal 

cells that encapsulate the granulosa cell/oocyte unit. A basement 

membrane acts to separate the heavily vascularized 

thecal layers from the granulosa cells and ovum. Since capillaries 

do not penetrate the basement membrane, the granulosa/oocyte 

compartment depends on the passive movement 

of hormonal factors through this extracellular structure 

(204). Oocyte and follicular growth are linear up to the tertiary 

stage, at which time the ooctye appears to reach a 

maximum size, while the follicle as a unit continues to enlarge. 

The tertiary follicle is characterized by a hypertrophied 

thecal layer, multiple layers of granulosa cells, and the appearance 

of an antrum, a space that separates the granulosa 

cells from the ooctye/cumulus complex. The process of follicular 

selection for ovulation, although not well understood, 

appears to occur at this stage of folliculogenesis, when several 

secondary-tertiary follicles will divert toward a pathway 

of atresia. Still under the influence of gonadotropins, the 

“selected” follicles will continue to enlarge, mostly due to 

increases in antrum size, to eventually reach the Graafian, or 

ovulatory stage. Follicular rupture and ovulation occur in 

response to a surge in gonadotropin levels, at which time 

cellular proliferation is ceased, and the remaining cells of the 

follicle terminally differentiate to form the corpus luteum 

(209). 

In consonance with its gametogenic function, the ovary 

fulfills a critical role as an endocrine organ, serving as the 

principal source of sex steroids in the female. Therefore, a 

normal functioning ovary is an essential prerequisite to the 

function and maintenance of the reproductive tract, mammary 

gland, and behavior of the female. The research efforts 

of several investigators during the past decades have generated 

the well accepted “two-cell, two-gonadotropin” 

model of ovarian estradiol synthesis. This model and the 

investigations leading to its description have been discussed 

in great detail in several recent reviews and therefore will be 

only summarized here (204, 206, 210). The two steroid-producing 

components of the maturing follicle are the thecal and 

granulosa cells, which predominantly produce androgens 

and estrogens, respectively (210). Ample evidence exists to 

indicate that thecal cells possess the full complement of steroidogenic 

enzymes necessary for estradiol synthesis. In contrast, 

estradiol synthesis by the granulosa cells is dependent 

on thecal-derived androgens as substrates for aromatization. 

The amount and activity of the expressed steroidogenic enzymes 

within the two cell types vary depending on the 

follicular stage, and thereby determine the predominant ste-


roid being produced. The cell-specific and temporal actions 

of the gonadotropins, LH and FSH, regulate the type and 

activity of the steroidogenic enzymes expressed within the 

granulosa and the thecal cells. The model states that LH 

acting via the constituitively expressed LH receptor on the 

cell surface of thecal cells stimulates the synthesis of androgens 

(androstenedione) in the growing follicle. This requires 

the initial conversion of cholesterol stores to pregnenolone by 

the cholesterol side-chain cleavage enzyme (P450 scc) and is 

thought to be a rate-limiting step in thecal cell steroidogenesis 

(204). Still within the thecum, pregnenolone is converted 

to progesterone and then to androstenedione via the enzymatic 

actions of 3�-hydroxysteroid dehydrogenase and 17�hydroxylase/C 

17–20 lyase (P450 17�), respectively (204, 206, 

210). Regulation by LH has been shown to occur at both the 

transcriptional and translational levels for the P450 scc and 

P450 17� genes (206). Granulosa cells lack expression of the 

P450 17� enzymes required to produce androgens, the precursor 

of estradiol, and therefore are dependent on the passage 

of the thecal-derived androgens through the basement 

membrane and into the granulosa compartment. This cellular 

cooperation provides the basis of the two-cell portion of 

the model. The second gonadotropin, FSH, acts solely upon 

the granulosa cells to stimulate the enzymatic conversion of 

the androstenedione and testosterone to estrone and estradiol, 

via P450-aromatase (P450 arom), and 17�-hydroxysteroid 

dehydrogenase, respectively (204, 206, 210). The estradiol is 

then released into the follicular fluid, whereupon the bulk 

passes back through the basement membrane and enters the 

circulation. Upon ovulation, the luteal phase begins with 

luteinization of the follicle and differentiation of the remaining 

granulosa and thecal cells to form the corpus luteum. The 

relative amounts and activities of the steroidogenic enzymes 

are altered once again and shift toward synthesis of large 

amounts of progesterone. 

Recent data have challenged the “two-cell” model to 

incorporate the descriptions of a role for the oocyte in regulating 

granulosa cell steroidogenesis. Elegant in vitro experiments 

involving the surgical removal of the oocyte from 

isolated growing follicles have demonstrated the existence of 

an ooctye-secreted factor that is able to inhibit granulosa cell 

estradiol and progesterone synthesis (211–213). 

2. Review of intraovarian estrogen actions. In 1940, both Pencharz 

(214) and Williams (215) independently reported a 

direct and specific ability of estradiol or DES to induce significant 

increases in ovarian weight in the hypophysectomized 

rat. These same seminal studies also described the 

synergistic effect of estradiol on gonadotropin-stimulated 

increases in ovarian weight (214, 215). Since then, numerous 

intraovarian effects of large amounts of locally synthesized 

estrogens have been described and postulated to be essential 

to normal follicular development and ovarian function. In 

granulosa cells of the growing follicle, estrogen has been 

reported to increase the levels of its own receptor (216), as 

well as induce DNA synthesis and proliferation (205, 217– 

220), increase the number and size of intercellular gap junctions 

(221), stimulate synthesis of IGF-I (222), and attenuate 

apoptosis and follicular atresia (223, 224). Estradiol is also 

known to augment the actions of FSH on granulosa cells, 

resulting in the maintenance of FSH-receptor levels (218, 

225–227) and the acquisition of LH-receptor (218, 228–231), 

an event critical to successful ovulation. 

Ultimately, the actions of estradiol act to enhance follicular 

responsiveness to gonadotropins and thereby result in increased 

aromatase activity and further estrogen synthesis 

(231, 232). Therefore, normal ovarian function appears to be 

dependent on a multitude of auto- and paracrine actions of 

estradiol that act in concert with the gonadotropins secreted 

from the anterior pituitary to provide for successful folliculogenesis 

and steroid production. Nonetheless, immunohistochemical 

detection and characterization of ER in the different 

ovarian compartments have proven difficult, although 

studies employing binding assays with radiolabeled ligands 

report the presence of ER in ovarian granulosa cells of the rat 

(216, 233–235), mouse (236), rabbit (236), and pig (235). 

The discovery of the ER� and its reportedly high mRNA 

levels in the ovary (49, 63, 93) reinforces the need for thorough 

immunohistochemical studies for the two distinct ERs 

in the ovary. Reports of localization of ER� and ER� transcripts 

in the rat ovary by in situ hybridization indicate the 

presence of low levels of ER� mRNA with no specific pattern 

(63), whereas ER� mRNA is easily detectable and predominantly 

localized to the granulosa cells of growing follicles 

(49, 63). Sar and Welsch (103) recently described immunohistochemical 

studies with ER�- and ER�-specific antibodies, 

reporting that ER� immunoreactivity is indeed highly 

expressed in and localized to the granulosa cells of growing 

follicles, whereas ER� staining appears limited to the interstitial/thecal 

cells in the rat ovary. Similar findings of immunohistochemical 

localization of the ER� to the ovarian 

granulosa cells were reported in the rat by Hiroi et al. (104) 

and in the cow by Rosenfeld et al. (69). Brandenberger et al. 

(94) reported the RT-PCR detection of ER� and ER� transcripts 

in normal and neoplastic human ovary and ovarian 

cell lines. This study further described the presence of easily 

detectable levels of ER� mRNA and very low levels of ER� 

mRNA in the granulosa cells, whereas the opposite was 

found in a cell-line derived from the ovarian outer surface 

epithelium (94). Misao et al. (237) also used RT-PCR to detect 

ER� and ER� transcripts in human corpus luteum. Both 

Misao et al. (237) and Byers et al. (63) demonstrated a downregulation 

of ER� mRNA during luteinization of the follicle 

and the differentiation of the corpus luteum in the human 

and rat, respectively. Interestingly, a report by Iwai et al., 

before the knowledge of ER�, described the detection of ER� 

immunoreactivity in the granulosa cells of the rabbit ovary 

(238), possibly illustrating another variation in the expression 

pattern of the two ERs among different species. Nonetheless, 

ER� and ER� are present in the adult rodent ovary. 

Therefore, disruption of the genes encoding these receptors 

may be expected to result in distinct ovarian phenotypes. In 

addition, the dissimilar expression patterns for ER� and ER� 

among the functional units of the follicle suggest that compensatory 

mechanisms fulfilled by the remaining functional 

gene in each respective ERKO may not be possible in the 

ovary. 

3. �ERKO ovary. The ovary of the neonatal and prepubertal 

�ERKO female does not exhibit any gross differences when


compared with those of wild-type littermates (142). The mature 

�ERKO ovary possesses a normal complement of primordial 

follicles, indicating no defects in germ cell generation 

or migration to the gonad during fetal development. 

However, at the commencement of sexual maturity, it becomes 

apparent that the �ERKO female is anovulatory and 

exhibits a distinct ovarian phenotype of enlarged, hemorrhagic, 

and cystic follicles as shown in Fig. 4. These cystic 

structures often accumulate in the ovary, making the gonad 

appear grossly as a bundle of dark grapes, a signature phenotype 

of the �ERKO female. However, growing follicles in 

the tertiary and pre- to small antral stage are present in the 

mature �ERKO ovary, indicating that ER� is not required for 

the recruitment of primordial follicles and the initial stages 

of the follicular phase. This is in contrast to the described 

phenotypes of mice possessing a mutation of the steel (Sl/Sl t ) 

locus (239), or a targeted disruption of the genes encoding 

growth differentiation factor-9 (240), or the vitamin D receptor 

(45), all of which exhibit phenotypes of follicular arrest 

at very early stages. The growing and cystic follicles in the 

�ERKO are often characterized by a hypertrophied thecum, 

indicating stimulation by LH. Induction of thecal cell function 

by the chronically elevated serum LH is illustrated by the 

similarly elevated levels of serum androgens exhibited in the 

adult �ERKO female (Table 2). Histological analysis of ovaries 

from numerous �ERKO females has revealed no corpora 

lutea, indicating an inability of the follicles to spontaneously 

ovulate and differentiate. Anovulation in the adult �ERKO 

could not be rescued even after appropriate stimulation with 

exogenous gonadotropins (142), strongly indicating that infertility 

in the �ERKO female is due in large degree to alterations 

in the hormonal milieu and ovarian responsiveness. 

A phenotype of anovulation and follicular arrest is also 

observed in the ovaries of other murine models of gene 

disruption. The ovaries of mice lacking the actions of FSH, 

either due to targeted disruption of the gene for the FSH�subunit 

(241) or the FSH-receptor (242), exhibit follicles that 

grow up to but not beyond the preantral stage. Therefore, 

FSH action is critical to completion of the follicular phase of 

the ovarian cycle. However, �ERKO female mice possess 

serum FSH levels within the normal range (Table 2) and 

actually show elevated levels of FSH-receptor mRNA in the 

ovary (142). Furthermore, since FSH signaling has been proposed 

to be a critical factor in estradiol synthesis (243, 244), 

the extremely elevated levels of serum estradiol in the 

�ERKO female indicate that this pathway is intact in the 

granulosa cells. Interestingly, neither report of mice lacking 

FSH action mention any significant differences in ovarian 

estradiol synthesis but describe a much smaller and thinner 

uterus in the female (241, 242). It is possible that the estradiol 

present in these models lacking FSH action is synthesized by 

the ovarian thecum. Alternatively, the presence of compensatory 

mechanisms that have allowed for granulosa cell aromatase 

activity that is independent of FSH stimulation must 

also be considered. Progression of the follicle to the more 

mature antral stage is also arrested in mice after targeted 

disruption of the gene for IGF-I (245), cyclin-D2 (246), connexin-37 

(247), activin type II receptor (248), and superoxide 

dismutase 1 (249), although low serum gonadotropin levels 

are thought to be the cause of this phenotype in the latter two 

models. 

Therefore, a definitive cause of the �ERKO ovarian phenotype, 

in addition to the loss of ER� action, was not obvious. 

In fact, some facets of ovarian physiology thought to be 

dependent on estrogen action, such as the attenuation of 

apoptosis and the induction of LH receptors in granulosa 

FIG. 4. Histology of a representative adult wild-type and �ERKO ovary. Shown are ovarian cross-sections from wild-type (a) and �ERKO (b) 

females at low magnification (13.2�). Note the presence of follicles at both the follicular and luteal (corpora lutea; CL) stages of the ovarian 

cycle in the wild-type ovary, whereas the �ERKO ovary is characterized by the presence of large, hemorrhagic, and cystic follicles, a sparse 

number of follicles at the early stages of proliferation, and a lack of corpora lutea. Panels c–d illustrate �ERKO ovarian tissue at high power 

magnification (132�): c, a healthy secondary follicle showing a normal oocyte and nucleolus; d, interface of two cystic follicles, demonstrating 

the heterogeneity in the number of granulosa cells that line the cysts, e.g., the left cyst has a single layer, whereas the right cyst has several 

layers of granulosa cells (indicated by arrows); e, hypertrophied thecal cells lining a hemorrhagic cyst in an �ERKO ovary. Scale bar � 0.5 �m.


cells of antral follicles, were apparently preserved in the 

follicles of the �ERKO ovary. Follicular atresia in the ovary 

is a hormonally controlled process that is critical to oocyte 

selection. Although the factors that trigger atresia are not 

well understood, it is characterized by apoptosis of the granulosa 

cells of the follicle (reviewed in Refs. 223 and 224). 

Estradiol has been shown to be one of the many factors 

reported to protect the follicle from becoming atretic (250). 

Furthermore, androgens reportedly accelerate the process, 

and it may be an altered estrogen/androgen synthesis ratio 

in the follicle that leads to atresia (250). However, despite 

elevated androgen production, the presence of androgen 

receptor (AR) mRNA, and a lack of ER� action in the mature 

�ERKO ovary, an inordinate amount of apoptosis is not 

observed in the follicles (142). Estradiol has also been shown 

to facilitate the FSH induction of LH receptors in the granulosa 

cells of the mature ovulatory follicle in both in vivo (225, 

231, 252) and in vitro experiments (229, 230). Nonetheless, the 

granulosa cells of the growing follicles, in addition to the 

enlarged cysts in the �ERKO ovary, possess significant levels 

of LH receptor mRNA when assayed by in situ hybridization 

(142). The most plausible explanation for these observations 

is that these estrogen actions are mediated by ER�, which has 

been shown to be expressed in a normal pattern in the granulosa 

cells of the �ERKO ovary, and concomitant with the 

expression of LH receptor (93, 142). 

Therefore, with data suggesting that disruption of the ER� 

gene did not result in an ovary completely refractory to estrogen, 

other aspects of ovarian physiology must be considered as 

possible factors in the etiology of the �ERKO ovarian phenotype. 

As previously discussed, ovarian function is tightly controlled 

by pituitary gonadotropins (see Section III.D.1). In turn, 

gonadotropin synthesis and secretion from the anterior pituitary 

are at least partially regulated by gonadal steroids acting 

via classical feedback mechanisms in the hypothalamic-pituitary 

axis (reviewed in Ref. 251). Indeed, disruption of the ER� 

gene has resulted in significant phenotypes in the hypothalamic-pituitary 

axis of the �ERKO female (see Section VI.A). Most 

notable is the increased and chronic secretion of LH in the 

�ERKO female that results in levels that are 4–7 times that 

found in wild-type females (Table 2) (252). As discussed above, 

synchronized increases in serum FSH and LH levels are critical 

to follicular maturation and ultimate ovulation in the ovary. 

However, it has been proposed that the follicular requirements 

for LH are finite, and the presence of abnormally high levels 

may force maturing follicles to either prematurely luteinize or 

become atretic (253). 

Therefore, the �ERKO ovarian phenotype is likely caused 

by the chronic exposure to abnormally high levels of LH. 

Support for this hypothesis can be drawn from a number of 

studies. Investigations involving prolonged treatment with 

antiestrogens over a period of at least 28 days have produced 

an ovarian phenotype in both mice (195, 196) and rats (197) 

that is similar to that of the �ERKO. Of course, interpretation 

of these studies is complicated by the ability of the antiestrogens 

to inhibit both ERs as well as estrogen action in both 

the ovary and hypothalamic-pituitary axis. However, these 

studies reported that the “�ERKO” phenotype of enlarged 

cystic follicles was produced only after prolonged treatments 

with those antiestrogens that possessed the ability to cross 

the blood-brain barrier and concurrently produce serum LH 

levels that were several fold higher than controls. Therefore, 

whereas the estrogen antagonists ZM-189,154 (197) and EM- 

800 (195, 196) produced chronically elevated LH levels and 

an ovarian phenotype strikingly similar to that of the 

�ERKO, tamoxifen did neither (195–197). More definitively, 

Risma et al. (254, 255) report that targeted transgenic overexpression 

of the LH�-subunit in the mouse that results in a 

15-fold increase in serum LH levels produces females that are 

anovulatory and exhibit an ovarian phenotype almost indistinguishable 

from that of the adult �ERKO female. 

Therefore, the similarity in the ovarian phenotypes described 

in the above studies, in which the models presumably 

possessed normal levels of ovarian ER�, combined with our 

observations in the �ERKO, strongly indicate that the ER� is 

not directly involved in the development of ovarian cysts due 

to hypergonadotropism. However, there are descriptions of 

at least two models in which serum LH is chronically elevated, 

yet do not manifest an ovarian phenotype similar to 

the �ERKO or those induced by antiestrogens or transgenics 

as described above. Female mice that are homozygous for a 

targeted disruption of the FSH�-subunit gene exhibit an 

approximate 5-fold increase in serum LH but do not show 

indications of enlarged cystic follicles in the ovary (241), 

indicating a role for FSH in this process as well. Bogovich 

(256) has provided supporting evidence by demonstrating 

that FSH is required along with prolonged exposure to LH 

(in the form of human CG) to induce follicular cysts in the 

rat. A likely role for FSH is the induction of LH receptor in 

the granulosa cells of the maturing follicles, thereby rendering 

the follicle responsive to the increased levels of LH. 

Another contrasting knockout model is that of the P450 arom 

gene (ArKO), in which the homozygous females possess 

significantly elevated serum LH and FSH levels but lack the 

capacity to synthesize estradiol (257). Although folliculogenesis 

is arrested at an antral stage in the ArKO ovary, no 

�ERKO-like cystic structures are reported (257). Therefore, 

assuming that a lack of estradiol synthesis would disrupt 

ligand-dependent activity of the ER� in the granulosa cells, 

the lack of ovarian cysts in the ArKO indicate an intraovarian 

role for estradiol in this phenotype. Therefore, although the 

�ERKO phenoytpe may be triggered by hyperstimulation of 

the follicles by LH, it is likely influenced by both FSH and 

ER� actions in the granulosa cells as well. Current studies 

utilizing prolonged treatment of �ERKO females with a 

GnRH antagonist to reduce serum gonadotropins are being 

carried out to further define the etiology of the ovarian 

phenotype (J. F. Couse, D. O. Bunch, J. Lindzey, D. W. 

Schomberg, and K. S. Korach, manuscript in preparation). 

Since the �ERKO ovarian phenotype develops and worsens 

only after sexual maturity, we have began studies to 

characterize ovarian function in the immature �ERKO female 

(J. F. Couse, D. O. Bunch, J. Lindzey, D. W. Schomberg, 

and K. S. Korach, manuscript in preparation). Although superovulation 

with exogenous gonadotropins was not successful 

in eliciting ovulation in the older �ERKO females, 

immature (�28 days) females do respond and produce viable 

oocytes that can be collected from the oviduct. However, the 

average number of oocytes collected from superovulated 

�ERKO females is significantly less than that yielded from


age-matched superovulated wild-type and heterozygous females. 

Therefore, intraovarian ER� action does not appear to 

be essential to ovulation when stimulated with exogenous 

gonadotropins. 

4. �ERKO ovary. As discussed above, the �ERKO has provided 

a number of indications to suggest that intraovarian 

ER� action is not critical to ovarian function. Furthermore, 

several presumed functions of estradiol in ovarian granulosa 

cells appear to be preserved in the �ERKO, such as the 

attenuation of apoptosis and the induction of LH receptors. 

Based on the reported localization of ER� mRNA (49, 50, 63) 

and protein (69, 103, 104) to the granulosa cells of growing 

follicles, as well as the maintenance of a normal expression 

pattern for ER� in the �ERKO ovary (93, 142), it is likely that 

the newly discovered ER is the predominant mediator of 

estrogen action in the follicle. In the rat ovary, ER� is easily 

detectable by immunohistochemistry and exhibits an almost 

ubiquitous expression pattern within the granulosa cells of 

follicles ranging from the primary up to the ovulatory or 

Graafian stage (69, 103, 104). Byers et al. (63) demonstrated 

that ER� mRNA levels remain relatively constant during the 

follicular stage of the ovarian cycle but are rapidly decreased 

after the gonadotropin surge-induced luteinization of the 

granulosa cells. Interestingly, in 1975, Richards (216) reported 

a similar profile for high-affinity [ 3 H]-E 2 binding in 

the granulosa cells of the rat ovary, showing increased binding 

levels as follicles matured followed by marked decrease 

after luteinization. Because the affinities of the two ERs for 

estradiol do not significantly differ, it was not possible at that 

time to realize that the receptor being detected in the ovary 

was distinctly different (i.e., ER�) from that which was being 

concurrently described in the uterus (i.e., ER�). 

As stated previously, female mice homozygous for the 

targeted disruption of the ER� gene possess no gross aberrant 

phenotypes as neonates or during adulthood (47). However, 

during a continuous mating study of 8 weeks in which 

sexually mature wild-type or �ERKO females were housed 

with a known fertile wild-type male, a significant deficit in 

fertility in the �ERKO became obvious. As shown in Table 

3, �ERKO females produced substantially fewer litters as 

well as significantly less numbers of pups per litter when 

compared with their wild-type littermates (47). Whereas the 

average litter size among wild-type females was 8.8 (�2.5) 

pups per litter, this number was reduced to 3.1 (�1.8) in 

�ERKO females (Table 3) (47). Furthermore, two of the tested 

�ERKO females yielded no litters, despite the observation of 

seminal plugs on multiple occasions, suggesting that abnormal 

sexual behavior was not a cause for the infertility. Gross 

TABLE 3. Fertility and superovulation data in the �ERKO female mice 

Genotype 

analysis of the uteri from the �ERKO females used in this 

study demonstrated no indication of embryo resorption during 

gestation. Therefore, the observed subfertility in the 

�ERKO females did not appear to be due to uterine dysfunction 

that results in premature termination of pregnancy. 

However, a possible defect in embryo implantation due to a 

lack of ER� could not be assessed in these studies. 

The nature of the subfertility in the �ERKO female described 

above strongly suggested an ovarian phenotype. Gross analysis 

of ovaries from �ERKO females indicated no distinct differences 

in size or morphology when compared with those of 

wild-type females. As shown in Fig. 5, histological analysis of 

ovaries from sexually mature �ERKO females illustrated the 

presence of a relatively normal interstitial compartment and 

follicles at various stages of the follicular cycle, ranging from 

primordial and primary to those with a clearly defined antrum, 

all possessing the expected thecal shell. Therefore, as demonstrated 

for ER� by the �ERKO, ER� does not appear to be 

essential for the establishment of germ cell number or ovarian 

development. Several of the speculated intraovarian roles of 

estrogen were discussed previously and include a proposed 

critical role in proliferation of the granulosa cells in the maturing 

follicle (217–220, 231). However, the multiple large follicles 

present in the �ERKO ovaries indicate no marked differences 

in granulosa cell number. Furthermore, serum estradiol levels 

in adult �ERKO females do not appear to differ from agematched 

wild-type females, at 24.2 (�3.3) and 30.5 (�2.8) pg/ 

ml, respectively. Biological evidence of estradiol synthesis in the 

�ERKO ovary is also illustrated by the apparently normal 

uterus and vagina, which exhibit the proper cyclic changes in 

morphology of a sexually mature wild-type mouse. Nonetheless, 

there were indications of an increased number of early 

atretic follicles and a sparse presence of corpora lutea in the 

�ERKO ovary when compared with the wild-type (47), suggesting 

that the observed subfertility in the �ERKO may be due 

to a reduction in completed folliculogenesis. 

A phenotype of compromised follicular maturation that 

more often terminates in atresia rather than ovulation, as 

described in the �ERKO female, is similar to that reported in 

a number of other mutant mice. As previously discussed, 

arrested folliculogenesis is described in knockout mice for 

other genes known to be expressed in granulosa cells, including 

the gene for FSH-receptor (242), P450 arom (ArKO) 

(257), IGF-I (245), and connexin-37 (247). It is therefore possible 

that a loss of ER� action has also resulted in alterations 

in the expression and/or function of one or more of these 

gene products. However, the normal serum estradiol levels 

and the appearance of estrogenic effects in the reproductive 

Continuous mating results Superovulation results 

n Litters per 

female (SEM) 

Pups per 

litter (SEM) 

n Oocytes per 

female (SEM) 

Wild-type 6 2.8 � 0.4 8.8 � 2.5 10 33.7 � 4.8 9–57 

Heterozygous nd nd nd 11 52.5 � 5.7 a 

20–77 

�ERKO 11 1.7 � 1.0 a 

3.1 � 1.8 b 

11 6.0 � 1.5 a 

nd, Not determined. 

0–13 

a 

P � 0.05, Student’s two tailed t-test vs. wild-type 

b 

P � 0.001, Student’s two tailed t-test vs. wild-type 

Range


FIG. 5. Histology of representative adult wild-type and �ERKO ovary. Shown are ovarian cross-sections from adult wild-type (a) and �ERKO 

(c) females and those from immature wild-type (b) and �ERKO (d) females after superovulation treatment at low power magnification (13.2�). 

Note the presence of follicles at various stages of the follicular phase in both the wild-type (a) and �ERKO (c) ovaries from the adult females, 

illustrating relatively little observable difference between the two genotypes. However, upon superovulation, a distinct phenotype becomes 

apparent in the �ERKO. Comparison of the superovulated wild-type (b) with the similarly treated �ERKO (d), indicates the presence of multiple 

corpora lutea (CL) in the wild-type whereas only two CL are obvious in the �ERKO. Most notable are the multiple numbers of unruptured 

ovulatory follicles present in the superovulated �ERKO ovary (indicated by arrows). High-power magnification (132�) of the adult �ERKO ovary 

illustrates follicles at progressive stages of maturation (e) and a healthy tertiary follicle showing an oocyte with nucleolus (f). High-power 

magnification (132�) of the superovulated immature �ERKO ovary illustrates a typical unruptured ovulatory follicle possessing several layers 

of granulosa cells and a centrally located oocyte (g), and a corpora lutea (h) indicating the successful luteinization and terminal differentiation 

of the follicle that occurs after ovulation. [Panels a–d reproduced with permission from Ref. 47.] Scale bar � 1 �m. 

tract of �ERKO females suggest that induction of the 

P450 arom gene by FSH in the granulosa cells appears intact. 

However, investigation of the expression of the above as well 

as other granulosa cell components is required to determine 

the cause of the ovulatory phenotype of the �ERKO female. 

To gain further insight into the ovarian phenotype of the 

�ERKO female, immature knockout and wild-type mice were 

stimulated to ovulate by administration of superphysiological 

levels of gonadotropins (PMSG and hCG) (47). After several 

independent trials, it became obvious that the ovulatory capacity 

of the �ERKO female was dramatically reduced, yielding 

an average of 6 (�1.5) oocytes per female vs. 33 (�4.8) and 52


(�5.7) oocytes per female in the wild-type and heterozygous 

animals, respectively (Table 3) (47). Additionally, the cumulus 

mass that surrounded the ovulated follicles from the �ERKO 

females was consistently composed of a decreased number of 

cells and a lessened integrity when compared with ova yielded 

from wild-type controls. Most interesting was the histology of 

the ovaries from the superovulated �ERKO females, which 

indicated the presence of numerous preovulatory but unruptured 

follicles (Fig. 5). It therefore appeared that the follicles of 

the �ERKO ovary were able to respond to the proliferative 

effects of PMSG in terms of increased size and antrum formation. 

However, a severe deficit in the response to the gonadotropin 

surge (hCG), required to induce luteinization and rupture 

of the follicle, was obvious in the �ERKO. A small number 

of the selected follicles were able to be expelled, as evidenced 

by the presence of ova in the oviduct and of corpora lutea in the 

corresponding �ERKO ovary. Therefore, a lack of ER� resulted 

in a drastic reduction in ovulatory capacity, yet with incomplete 

penetrance. Until �ERKO females are treated and tested in a 

similar manner to the �ERKO studies described, it will be difficult 

to determine the precise role for each ER in the ovary. 

The observation of numerous unruptured Graafian follicles 

in the ovaries of superovulated �ERKO females is strikingly 

similar to the phenotype reported for mice possessing 

a targeted disruption of the cyclin-D2 gene (246). Cyclin-D2 

is a positive regulator of cell cycle progression that is highly 

expressed in granulosa cells (reviewed in Ref. 258). Robker 

and Richards (259) demonstrated strong up-regulation of the 

cyclin-D2 gene by estradiol in rat granulosa cells and suggested 

that this protein may be a downstream mediator of the 

synergistic actions of FSH and estradiol that result in increased 

granulosa cell numbers in the maturing follicle. The 

dramatic increases in cyclin-D2 induced by estradiol are evident 

in vivo at both the mRNA and protein levels in the rat. 

Furthermore, assays on primary granulosa cell cultures from 

the rat indicate that estradiol stimulation of the cyclin-D2 

gene can be inhibited by the estrogen antagonist, ICI-164,384, 

strongly suggesting that it is an ER-mediated process (259). 

Therefore, given that ER� is the predominant form of ER in 

the granulosa cells, disruption of the ER� gene may likely 

result in significant deficits in cyclin-D2 expression in the 

granulosa cells of the growing follicles in the �ERKO female. 

However, FSH is also able to stimulate increases in cyclin-D2, 

although the temporal pattern of regulation by FSH is distinct 

from that elicited by estradiol (259). Nonetheless, it is 

possible that FSH action in the follicles of the �ERKO ovary 

have provided for some degree of cyclin-D2 expression and 

thereby may explain the incomplete penetrance of the 

�ERKO ovarian phenotype. 

The �ERKO ovarian phenotype that becomes apparent 

after superovulation is also similar to that reported for 

knockout models of the genes for the PR (PRKO) (44) and 

prostaglandin synthase-2 (189). The dramatic increases in 

both PR (260) and prostaglandin synthase-2 (261) in the granulosa 

cells of the ovulatory follicle shortly after the gonadotropin 

surge have been well documented. Furthermore, the 

lack of follicular rupture in the respective knockout models 

supports a critical role for each of these components in ovulation 

(reviewed in Ref. 262). Lydon et al. (44) reported infertility 

in the PRKO and a consistent inability of super- 

physiological doses of hCG to induce follicular rupture. 

Although regulation of the PR gene is strongly influenced by 

estradiol in the uterus (see Section III.A), sufficient evidence 

exists to indicate that this may not be the case in the ovary. 

For example, although the wild-type ovary possesses extremely 

high intraovarian levels of estradiol and the presence 

of ER�, levels of PR mRNA and protein remain at a modest 

basal level in granulosa cells of the pre- and antral follicle 

(260). However, within 4–6 h after the gonadotropin surge, 

transcription of the PR gene has peaked at levels several fold 

that before the surge, only to return to near-basal levels 

within 20 h (260). The mechanism of this strong and transient 

induction of the PR gene by LH is known to include significant 

increases in intracellular cAMP, but may also involve 

phosphorylation of ER� and/or a coactivator, which then 

combine to act in a synergistic nature (262). Therefore, the 

elevation in PR levels in the granulosa cells of the ovulatory 

follicle that is critical to follicular rupture may be attenuated 

in the �ERKO ovary. 

Another possibility for a lack of spontaneous ovulation in 

the �ERKO female may be not intaovarian in nature, but 

rather due to altered gonadotropin synthesis and secretion 

from the hypothalamic-pituitary axis. The sex steroids play 

an important role as a positive regulator of the preovulatory 

surge (reviewed in Refs. 263 and 264). Although the exact 

mechanism of action by which estrogens may be involved is 

not well defined, studies have shown that estradiol can induce 

GnRH release from the hypothalamus as well as cause 

increases in the level of GnRH receptors in the anterior pituitary 

(263). The ER� may be the predominant form of ER 

in the pituitary of the adult female mouse (93); however, both 

ER� and ER� have been detected in various regions of the 

hypothalamus (88, 97, 265). Preliminary data in the �ERKO 

female indicate that tonic levels of serum LH are within the 

normal range. However, a lack of hypothalamic ER� may 

have reduced the potential for positive regulation by estradiol 

in the hypothalamic-pituitary axis and thereby may 

result in a reduction in the frequency and/or amplitude of 

the preovulatory gonadotropin surge. Nonetheless, the results 

of the superovulation studies described above, in which 

an artificial bolus of gonadotropin is administered to induce 

ovulation, indicate a severe phenotype that can be localized 

to the ovary of the �ERKO female. 

IV. Mammary Gland 

In mammals, the mammary gland is essentially undeveloped 

at birth and does not undergo full growth until the 

completion of puberty and, in fact, remains undifferentiated 

until pregnancy and lactation. Development of the mammary 

gland may be divided into five distinct stages: embryonic 

and fetal, prepubertal, pubertal, sexually mature adult, 

and pregnancy/lactation (reviewed in Ref. 266). The influential 

factors involved at each stage differ in both type and 

magnitude. Although the developmental factors involved 

during the embryonic and fetal stages of the female mammary 

gland are poorly understood, estrogen action does not 

appear to be essential (266). However, studies have shown 

that the fetal and neonatal mammary gland of rodents is


responsive to the gonadal steroids, although distinct strain 

differences are evident (reviewed in Ref. 267). In male rodents, 

all or portions of the fetal glandular structure are 

destroyed via the “masculinization” effects of testicular androgens, 

an effect that can be reproduced in males by prenatal 

exposure to testosterone (267). Aberrant exposure of the 

neonatal female mouse to estradiol, testosterone, 5�-dihydrotestosterone, 

or PRL has been reported to result in an 

apparent increased sensitivity of the gland to mammotrophic 

hormones during adulthood, leading to varied degrees of 

excessive ductal growth and differentiation (267, 269). The 

later four stages of mammary gland development occur after 

birth and terminate in a gland capable of milk production. 

These stages are strongly regulated by the endogenous ovarian 

steroid hormones and are characterized by massive 

growth of the glandular ducts that emanate from the nipple 

until they have progressed through the fat pad composing 

the bulk of the breast. Upon pregnancy and the onset of 

lactation, the gland undergoes dramatic differentiation to 

produce milk- secreting structures, termed alveoli, throughout 

the ductal network. Therefore, since the majority of mammary 

gland growth and differentiation occurs after birth, this 

tissue provides a unique tool within which the interactions 

of gonadal and peptide hormone systems may be studied. 

A. �ERKO phenotype 

The mammary gland of an adult wild-type female mouse 

consists of a network of epithelial ducts originating from the 

nipple and forming a tree-like structure. The growth of the 

epithelial ducts begins during prepubertal development and 

continues during puberty until the branches of the gland 

have reached the limits of the fat pad (266). This ductal 

elongation is via cap cell proliferation at the terminal end 

buds of the individual ducts, as they maintain close contact 

with the stromal fat pad through which they are progressing. 

Studies using ovariectomized models have indicated that 

estradiol and GH, locally mediated as IGF-I, are required for 

the development of this ductal structure (266, 268). The mammary 

glands of adult �ERKO female mice exhibit a phenotype 

similar to the glands of a newborn female, confirming 

the need for ER�-mediated estradiol actions for ductal 

growth (269). However, the �ERKO gland does possess the 

component structures necessary for mammary gland development, 

i.e., the epithelial and stromal portions, connective 

tissue, and a small rudimentary ductal tree. Therefore, embryonic 

and fetal development of the mammary gland in the 

mouse occurs independently of ER� actions. However, this 

may be strain dependent, since studies in the C57BL mice, the 

background strain of the �ERKO, have also reported little 

effect of neonatal steroid exposure on the mammary gland 

(267). Nonetheless, despite apparently unaltered GH levels 

and the existence of significantly elevated levels of serum 

estradiol, the mammary gland of the �ERKO female has lost 

the capacity to commence the pre- and postpubertal stages 

of growth. 

Estradiol has been shown to directly stimulate the formation 

of terminal end buds and stimulate cellular proliferation of the 

mammary ductal epithelium (270). Furthermore, this physiological 

effect can be inhibited by antiestrogens (271), indicating 

a receptor-mediated pathway of estrogen action. Although ER 

has been detected in both the ductal epithelial cells as well as 

the stromal tissue of the mammary gland (270, 272), the mechanism 

by which estrogens directly regulate growth remain unclear. 

The lack of ER in the outermost proliferating cap cells of 

the terminal end buds suggests that the effects of estradiol may 

be indirect and may involve the regulation of cell cycle genes 

and/or paracrine-acting peptide growth factors and their cognate 

receptors (270). Both EGF and transforming growth factor-� 

are thought to be critical to proper mammary growth and 

development and are at least partially regulated by estradiol via 

the ER (273–275). Support for a role of estradiol-induced growth 

factor activity is provided by the study of Xie et al. (276), in 

which transgenic mice overexpressing a dominant-negative 

form of the EGF-R exhibited an attenuated response to EGF 

action in the mammary gland in vivo. The virgin transgenic mice 

of this study exhibited severe deficits in ductal growth and 

branching in the mammary gland, which were overcome only 

with pregnancy, when ovarian steroids increased endogenous 

EGF expression several fold that found in virgin females (276). 

Further evidence comes from Ankrapp et al., who recently reported 

that estradiol-releasing pellets implanted into the mammary 

fat pad of ovariectomized mice result in significantly 

increased levels of EGF and PR and induce terminal end bud 

formation in the gland (277). However, implantation of EGFreleasing 

pellets induced similar increases in PR and terminal 

end bud formation, suggesting that EGF may mediate the mitogenic 

actions of estradiol (277). Therefore, this study demonstrated 

a scenario of ER�-EGF cross-talk similar to that previously 

described in the uterus (see Section III.A.2). In brief, 

treatment of mice with pellets releasing both EGF and the antiestrogen 

ICI-182,780 exhibited inhibition of the mitogenic effects 

of the growth factor; in turn, an anti-EGF antibody was 

able to neutralize the growth effects induced by an implanted 

estradiol-releasing pellet (277). Therefore, as was confirmed in 

the uterus through the use of the �ERKO mice, EGF is also able 

to mimic the mitogenic effects of estradiol in the mammary 

gland, but once again appears to require the presence of functional 

ER� to complete this mechanism. 

It is well known that the mammary fat pad serves not only 

as a matrix, but also provides biochemical signals and/or 

factors necessary for normal ductal growth (reviewed in Ref. 

266). In the study by Ankrapp et al. (277) described above, it 

was found that the greatest induction of EGF by estradiol 

occurred in the stromal compartment of the gland. Confirmation 

of a requirement for ER� in the glandular stroma and 

the likely involvement of paracrine growth factors in estrogen 

regulation of mammary gland growth has been demonstrated 

by Cunha et al. using the tissue recombinant methodology 

described previously (see Section III.A.2). Tissue 

recombinations of mammary fat pad and epithelium from 

wild-type and �ERKO females were constructed and grafted 

under the kidney capsule of athymic nude mice for 4 weeks 

(278). Extensive ductal growth was observed only in recombinants 

involving wild-type (i.e., ER�) stroma, including 

those composed of �ERKO ductal epithelium (278). However, 

�ERKO (ER��) stroma was unable to induce growth 

in the overlying ductal epithelium from either genotype 

(278). The authors therefore concluded that stromal ER� is 

essential to the mitogenic actions of estradiol in the mam-


mary epithelium, a conclusion also reached from similar 

studies in the uterine and vaginal tissue. In light of these data 

produced by Ankrapp et al. (277) and Cunha et al. (278), 

current studies to determine whether the underdeveloped 

�ERKO mammary gland may be rescued by exogenous treatment 

with growth factors will prove interesting. 

Complete maturation of the mammary gland during pregnancy 

involves further ductal growth, extensive branching, 

and differentiation of the lobuloalveolar structures along the 

ducts, the hallmark of the lactating gland (266). These alveoli 

provide for milk production and will eventually fill the remaining 

spaces of the fat pad. This stage of mammary gland 

development is thought to require the combined actions of 

estrogen, progesterone, and PRL (266). The need for progesterone 

action in lobuloalveolar development was confirmed 

by studies of the PRKO mouse, which exhibited a normal 

pubertal ductal structure but was completely refractory to 

the induction of lobuloalveolar development after progesterone 

treatment (44). A similar phenotype is observed in the 

female PRL-receptor knockout (PRLR�/�) mice, which possess 

a normal virgin mammary gland as an adult, but a severe 

deficit in lobuloalveolar development and lactation after 

pregnancy (279). Interestingly, because the homozygous 

PRL-receptor (PRLR�/�) knockout females like others are 

infertile, this mammary gland phenotype was first detected 

in the heterozygous (PRLR�/�) females (279). Furthermore, 

the defects in lactation in the PRLR�/� mice were lessened 

after multiple pregnancies, due to the compensatory actions 

of other pregnancy-associated mammotrophic hormones 

that accumulated with each pregnancy (279). 

Due to the documented ability of the estradiol-ER� complex 

to increase PR expression in various tissues, including 

the mammary gland (280), a loss of ER� action may also 

result in a loss of PR-mediated progesterone functions. Although 

progesterone actions in the mammary gland were 

once thought to be strictly differentiative and may actually 

oppose the proliferative effects of estrogens, recent work has 

suggested that progesterone may also act as a mitogen in the 

breast (reviewed in Ref. 181). This is best illustrated by the 

less extensive ductal arborization of the mammary gland, as 

well as the lack of alveolar structures, in the PRKO mice (44). 

Furthermore, in vitro studies have indicated that PR-mediated 

progesterone actions can regulate the transcription of 

genes for cell cycle-related proteins, growth factors, and 

growth factor receptors (181). Therefore, the lack of ductal 

growth and differentiation in the �ERKO mammary gland 

may be largely due to a lack of physiologically sufficient PR 

and subsequent progesterone action. As shown in Table 2, 

the average serum progesterone levels in adult female 

�ERKOs are 4.0 (�1.1) ng/ml vs. 2.3 (�0.6) ng/ml in wildtype 

and are therefore within the normal range of cycling 

wild-type females (123). Preliminary analysis by ribonuclease 

protection assay indicates that the mammary glands of 

adult virgin �ERKO females possess slightly detectable levels 

of PR mRNA, but exhibit no increased regulation when 

treated with estrogen. Furthermore, current studies in our 

laboratory indicate that prolonged progesterone treatment of 

adult �ERKO females results in the formation of terminal end 

buds and differentiation in the mammary gland, indicating 

that the lack of progesterone action caused by disruption of 

the ER� gene can be partially overcome with treatment of 

superphysiological levels of exogenous progesterone. 

A similar secondary effect of targeted disruption of the 

ER� gene on the mammary gland may involve a lack of PRL 

stimulation. PRL is primarily secreted from the anterior pituitary, 

although local synthesis in the mammary epithelium 

has also been described (279). This peptide hormone plays a 

critical role in mammary gland physiology, especially in the 

induction of lobuloalveolar structures and milk production, 

as evidenced in the phenotype of the PRLR�/� mice (reviewed 

in Ref. 279). However, the synthesis and secretion of 

PRL from the anterior pituitary are strongly regulated by 

estradiol and ER� (281). Confirmation of the regulatory action 

of ER� on the PRL gene is provided by the �ERKO 

females, which exhibit a 20-fold decrease in PRL mRNA in 

the anterior pituitary (282) and a 5-fold decrease in serum 

PRL levels (Table 2) (see Section VI.A.4). Therefore, given the 

significant role of PRL in the differentiation to a lactating 

mammary gland, it is likely that the phenotype described in 

the �ERKO female is also at least partly due to a lack of PRL 

stimulation. Support for this hypothesis is provided by current 

studies in which elevated serum PRL levels are achieved 

in the �ERKO female via wild-type pituitary transplants 

placed under the kidney capsule and have resulted in significant 

ductal growth and differentiation in the �ERKO host 

mammary gland (W. P. Bocchinfuso, J. Lindzey, S. W. Curtis, 

J. E. Clark, P. H. Myers, and K. S. Korach, in preparation). 

However, preliminary analysis indicates that this partial rescue 

of the �ERKO mammary gland by the PRL secreting 

wild-type pituitary graft does not occur in an ovariectomized 

�ERKO host female, indicating a requirement for ovarian 

derived factors as well. Nonetheless, the �ERKO mammary 

gland appears to possess the intrinsic tissue components 

necessary for pubertal development and pregnancy-induced 

maturation, but fails to develop because of the loss of multiple 

stimuli that are downstream of ER� action. 

B. �ERKO phenotype 

Unlike the dramatic underdevelopment observed in the 

mammary gland of the �ERKO, no such phenotype is observed 

in adult �ERKO females. Virgin �ERKO females of 

4–5 months of age exhibit mammary glands that possess a 

normal ductal structure that fills the entire fat pad and are 

indistinguishable from those of age-matched wild-type females. 

This phenotype agrees with our description of minor 

amounts of ER� mRNA in the adult mouse mammary gland, 

whereas ER� transcripts are easily detectable (Fig. 2) (93). 

Furthermore, mammary glands from pregnant and nursing 

�ERKO females appear to have undergone normal differentiation 

and exhibit the lobuloalveolar structures required 

for lactation. Although litter sizes of the �ERKO females 

were reduced during the mating study described above, no 

marked abnormalities in nursing were observed. Therefore, 

the combined data from the �ERKO and �ERKO models 

indicate that ER� is the predominant receptor required to 

mediate the actions of estrogen in the mammary gland of the 

mouse.


C. ER� and oncogene-induced tumorigenesis: 

Wnt-1/�ERKO mice 

Several lines of evidence indicate that breast cancer in 

humans is strongly correlated with the extent of lifetime 

exposure to estrogen. Most notably, breast cancer almost 

exclusively occurs in females and is never seen before puberty 

but rather only after several years into the reproductive 

life span (283, 284). Furthermore, an increased length of a 

woman’s reproductive years, i.e., early menarche and late 

menopause, has been associated with an elevated risk of 

breast cancer (284), whereas women who have experienced 

premature menopause due to natural causes or castration 

appear to be at a much lower risk (283). Furthermore, studies 

have indicated that women who circulate higher levels of 

active estrogens may also be at greater risk of developing 

breast cancer (283). In apparent contrast, early pregnancy 

tends to provide a protective effect, although it is obviously 

associated with an increased level of steroid hormone exposure. 

In addition, several years of research have indicated 

little positive correlation with the risk of breast cancer and 

prolonged use of contraceptive pills composed of synthetic 

estrogens and progestins, although this issue remains controversial 

(reviewed in Refs. 285 and 286). Still, a large portion 

of chemotherapeutics for breast cancer are aimed at 

either blocking estrogen action or reducing estrogen levels 

(283). Therefore, although an association between estrogen 

action and breast cancer is apparent, it involves less understood 

yet critical mechanisms, including the periodicity and 

cyclicity of hormone exposure as well as the sensitivity of the 

end organ (283). This is further complicated by the influence 

that environmental exposures, geography, diet, body weight, 

and genetics also play in individual risk of developing breast 

cancer (283). 

Numerous studies have been carried out concerning the 

levels of ER and PR in neoplastic breast tissue and the prognostic 

value that these parameters may provide (reviewed in 

Refs. 287–291). Reports indicate that more than 70% of primary 

breast tumors are ER�-positive and exhibit estrogendependent 

growth (291). However, the most malignant 

mammary tumors are often ER�-negative and exhibit estrogen-independent 

aggressive growth, but are thought to 

progress from a once ER�-positive cell population (287). In 

addition, the role of local aromatase activity and estrogen 

production in breast cancer is receiving increased attention 

(reviewed in Ref. 292). Added complexity is introduced by 

the detection and description of numerous variants of the 

ER� transcript in breast cancer tissues, although their possible 

role in the etiology of the disease remains speculative 

(reviewed in Refs. 28 and 293). Recent reports also described 

the detection of ER� transcripts in multiple immortalized 

human breast cancer cell lines, normal human breast tissue, 

and human breast tumors (64, 73, 101, 102, 294). Vladusic et 

al. (73) also characterized an ER� mRNA variant detected in 

normal as well as malignant human breast tissue (73). 

It is clear from the severely underdeveloped mammary 

gland of the �ERKO that estradiol acting via the ER� is a 

potent mitogen in the breast. To gain insight into the potential 

role of ER� in the induction and promotion of mammary 

gland carcinogenesis, we crossed the �ERKO mice with the 

MMTV-Wnt-1 mice, a transgenic line that is highly susceptible 

to mammary adenocarcinoma. The family of Wnt genes 

encode a series of secretory glycoproteins that act in autoand 

paracrine pathways to stimulate cell proliferation and 

differentiation (reviewed in Ref. 295). The MMTV-Wnt-1 

mice possess a transgene designed for targeted overexpression 

of the Wnt-1 protooncogene in the mammary gland and 

exhibit a nearly 100% and 15% incidence of mammary hyperplasia 

and lobuloalveolar adenocarcinoma by 1 yr of age 

in females and males, respectively (295, 296). Therefore, 

breeding of the two transgenic lines allowed for the generation 

of animals that possessed the Wnt-1 transgene on either 

a wild-type or �ERKO background and thereby allowed for 

the assessment of the role of ER� in the initiation and promotion 

of protooncogene-induced mammary tumors (297). 

At 6 months of age, virgin wild-type Wnt-1 females exhibit 

extensive hyperplasia of the ductal epithelium and aberrant 

lobuloalveolar development that occupies the entire fat pad. 

This was the expected phenotype based on that described 

previously in the original line of Wnt-1 females derived from 

a different mouse strain (296). Interestingly, a similar phenotype 

of lobuloalveolar hyperplasia was observed in the 

rudimentary duct of the �ERKO-Wnt-1 females, although the 

extent of ductal growth was much reduced compared with 

that seen in the wild-type (269). Nonetheless, the rudimentary 

ductal structure previously described in the �ERKO 

female was obviously induced to proliferate by the presence 

of ectopic Wnt-1 expression. However, comparison of mammary 

glands from a series of age-matched animals indicated 

that the ductal growth observed in the mammary gland of a 

6-month-old �ERKO-Wnt-1 female remained confined to the 

nipple region and approximated that seen in a 2.5-month-old 

wild-type-Wnt-1 female, illustrating a significant delay in the 

proliferation of the �ERKO epithelium (269). Interestingly, 

the mammary hyperplasia in the �ERKO-Wnt-1 females did 

not markedly progress into the inguinal fat pad, but rather 

remained confined to the area of the nipple. Therefore, although 

the lobuloalveolar phenotype characteristic of Wnt-1 

overexpression was evident, ductal elongation did not occur 

in the �ERKO-Wnt-1 female, indicating that the hyperplastic 

action of ectopic Wnt-1 expression cannot substitute for ER�mediated 

terminal end bud formation and ductal morphogenesis 

(297). In the males, Wnt-1-induced epithelial hyperplasia 

was obvious in both the wild-type and �ERKO 

animals as well, but no distinct difference in growth rates 

between the two genotypes was evident (269). 

The incidence of lobuloalveolar carcinoma in the Wnt-1 

mice mirrored the observations described above for the epithelial 

hyperplasia. Wild-type-Wnt-1 and heterozygous- 

ER�/Wnt-1 females developed mammary tumors at a rapid 

rate, reaching an incidence of 50% by 6 months of age (297). 

Ectopic expression of the Wnt-1 gene was also able to induce 

tumorigenesis in the �ERKO females and therefore did not 

require the presence of functional ER� (297). However, a 50% 

incidence in tumors in the �ERKO-Wnt-1 females was not 

observed until 12 months of age, twice the time required for 

the wild-type-Wnt-1 colony (269). Ribonuclease protection 

assays indicated that the level of Wnt-1 transgene expression 

was relatively equal in the two ER� genotypes. Prepubertal 

ovariectomy had no effect on the overall incidence of tumors


in either genotype, although it resulted in a delayed incidence 

of palpable tumors in both, i.e., wild-type-Wnt-1 at 

50% at 10.5 momths and a further delay in the �ERKO-Wnt-1 

at 50% at 15 months (269). Therefore, ovarian factors clearly 

accelerated the growth rate of the Wnt-1-induced adenocarcinoma 

in both the wild-type and �ERKO females. However, 

multiple pregnancies had no significant effect on the time of 

tumor onset in the wild-type-Wnt-1 females (269). 

These studies indicate that ER� signaling is not involved 

in the induction of hyperplasia and tumorigenesis in the 

mammary gland that results from overexpression of the 

Wnt-1 protooncogene, but indeed plays a promotional role 

in these phenotypes. Similar findings of a promotional role 

for the ER� in uterine carcinogenesis were reported in transgenic 

mice that overexpressed an ER� gene (298). A possible 

explanation for the observed tumor latency may be drawn 

from an inherent difference between the wild-type and 

�ERKO mammary glands, i.e., because the �ERKO-Wnt-1 

glands possess a reduced amount of ductal morphogenesis 

and proliferating epithelia due to the lack of ER�, the number 

of cells most susceptible to neoplastic transformation may be 

decreased (297). Interestingly, the growth rates of the ductal 

hyperplasia and ultimate adenocarcinomas were also significantly 

reduced when the animals were ovariectomized, even 

in those lacking functional ER�. A possible explanation for 

this finding may be that ovarian estradiol is acting via non- 

ER�-mediated pathways, suggesting a possible role for ER�. 

However, in contrast to reports in other species, ER� mRNA 

remains difficult to detect by standard assays in the mouse 

mammary gland, including the glands of the �ERKO (93) and 

Wnt-1 females (297). Furthermore, ovariectomy also results 

in the loss of other gonadal hormones in addition to estrogen. 

Remarkably, overexpression of the Wnt-1 gene in the 

�ERKO mammary gland resulted in significant increases in 

PR mRNA levels compared with the low basal levels detected 

in control �ERKO glands, when assayed by ribonuclease 

protection assay (297). Recently, Shyamala et al. (299) 

showed that misregulation of the PR gene resulting in increased 

levels of PR A through transgenic methodologies may 

be tumorigenic in the mammary gland. Therefore, the ability 

of Wnt-1 to compensate for the reduced PR levels generated 

by the loss of ER� action may provide a pathway by which 

the role of ER� in tumor induction and promotion may be 

overridden. In addition, if the PR pathway was involved in 

the promotion of the Wnt-1 lobuloalveolar carcinomas, 

ovariectomy would be expected to result in a delay in their 

growth. 

In summary, the �ERKO/Wnt-1 mouse model has demonstrated 

that artificially induced hyperplasia and tumorigenesis 

of the mammary gland can take place in the absence 

of ER� signaling. These findings have potential importance 

in understanding the etiology of human breast cancer by 

illustrating that oncogenic induction of mammary neoplasia 

can occur in an ER�-negative tissue. Extending these observations 

further may indicate that hormone-independent 

breast tumors can originate from a cell population lacking 

ER� as well as those that are ER� positive. The characterization 

of mice produced from crosses of other overexpressing 

transgenic models, e.g., neu, with the �ERKO are cur- 

rently being carried out in a fashion similar to those studies 

described above. 

V. Reproductive Tract Phenotypes of the Male 

More than 40 yr ago, Jost employed a series of classical 

organ ablation studies to demonstrate that development of 

the male reproductive system was dependent on the secretion 

of testosterone and the peptide, anti-Müllerian hormone, 

by the fetal testes (107). We now know that development of 

the undifferentiated fetal gonad to a testis is determined by 

the presence of testis-determining factors or genes localized 

to the Y chromosome, e.g., the SRY gene (300). Testosterone 

produced from the Leydig cells of the fetal testes is crucial to 

the survival of the Wolffian duct, the primordial structure of 

the male reproductive tract, and its differentiation into the 

male reproductive structures (143, 192). In turn, secretion of 

anti-Müllerian hormone from the Sertoli cells of the fetal 

testis induces regression of the primordial female reproductive 

structures in the developing fetus (143, 192). Differentiation 

of the epididymis, ductus deferens, and seminal vesicle 

from the Wolffian duct is stimulated by testosterone, 

whereas development of the lower structures, the prostate, 

bulbourethral glands, urethra, scrotum, and penis, is primarily 

influenced by the more potent testosterone metabolite, 

5�-dihydrotestosterone (DHT) (143). The importance of 

androgen action in the development of the male reproductive 

tract is evident by the feminized phenotype that results in 

human males with complete androgen insensitivity syndrome 

(cAIS), caused by a naturally occurring mutation that 

results in the lack of functional AR. The phenotypes of the 

cAIS human male and the analogous testicular feminized 

male (Tfm) rodent are characterized by the complete absence 

of either male or female internal reproductive structures 

except for the presence of inguinal testes (37, 301). External 

genitalia in the cAIS human and Tfm mouse are indistinguishable 

from the normal wild-type female, but a short and 

often blunt-ended vagina is present (37, 301). During puberty 

and the later stages of sexual maturation in the male, virulization 

of the external genitalia is dependent on the actions 

of DHT (143, 302). This is illustrated in human males lacking 

sufficient 5�-reductase activity and DHT synthesis, who exhibit 

normal internal reproductive structures and testosterone 

levels but severely undervirulized external structures 

(reviewed in Ref. 37). Once again, the critical role of androgens 

in the development of the male reproductive tract was 

reiterated by the complete lack of gonadal development in 

mice homozygous for a disruption of the gene encoding SF-1 

(137, 138). Therefore, androgen action is critical to the development 

and function of the tissues of the male reproductive 

tract from fetal stages through adulthood, whereas a 

defined role for estrogen remains unclear. 

Although there is no apparent role for estrogen action in 

the development of the male reproductive tract, studies have 

reported significant effects of early exposure to estrogen 

agonists and/or antagonists on the adult male reproductive 

system. However, it was generally concluded that such effects 

were caused by estrogen actions at the hypothalamicpituitary 

level that ultimately resulted in decreased stimu-


lation of the testis and reduced androgen production (303, 

304). Still, a series of studies by McLachlan et al. demonstrated 

that perinatal exposure to the synthetic estrogen, 

DES, results in an assortment of apparently direct defects in 

the murine male reproductive tract, including undescended 

testes, epididymal cysts, aberrant expression of estrogeninducible 

genes, adenocarcinoma, and sterility (305–308). 

However, these studies indicated a toxic effect of developmental 

exposure to pharmacological levels of estrogens 

rather than a required physiological function of estrogen in 

the male reproductive tract. Investigators have reported the 

detection of ER by steroid autoradiography (309, 310) and 

immunohistochemistry (112) in the testes and accessory tissues 

of the male tract, even as early as day 16 of gestation in 

the mouse. In addition, the newly described ER� also appears 

in varied amounts in the testis, epididymis, and prostate 

of the rodent (93, 121, 311–314) and rhesus monkey (96). 

Hence, these studies suggest the presence of a functional 

estrogen- signaling system in the male reproductive tract and 

further demonstrate the direct detrimental effects that may 

occur when this system is aberrantly activated. 

A. Testicular function and spermatogenesis 

A defined role of ER and estrogen action in the function 

and maintenance of the male reproductive tissues remained 

elusive until the generation of the �ERKO mice. As expected 

in the presence of a functional androgen-signaling system, 

the reproductive tract of the �ERKO male mouse undergoes 

apparently normal prenatal development to produce internal 

and external structures that are indistinguishable from 

wild-type littermates. However, at approximately 20 weeks 

of age, significant decreases in the weight of the testis and 

epididymis/vas deferens are observed, whereas the seminal 

vesicle/coagulating glands and prostate appear normal in 

size (315, 316). As shown in Table 2, circulating gonadotropins 

in mature �ERKO males include levels of FSH within the 

wild-type range, whereas LH is slightly elevated (315, 317). 

In accordance with the increased LH secretion are observations 

of Leydig cell hyperplasia in the testis (121) and serum 

testosterone levels of approximately 2-fold in the �ERKO 

male compared with age-matched wild-type males (Table 2) 

(315, 317). Therefore, the decreased weights reported for the 

accessory organs of the male reproductive tract are not a 

secondary effect of any phenotype in the hypothalamic-pituitary 

axis, but rather a direct result of the loss of ER�mediated 

estrogen action within these tissues. 

One of the most striking initial observations in the �ERKO 

was complete infertility in the males when tested in a continuous 

mating study with wild-type, known-fertile females 

(315). No difference in fertility was observed in the heterozygous 

�ERKO males (315). Although the etiology of such 

infertility was unclear, initial experiments in which �ERKO 

males were placed in a mating environment with hormoneprimed 

wild-type females resulted in significantly fewer copulatory 

plugs, indicating a severe deficit in normal sexual 

behavior (315, 318). Further investigations have demonstrated 

that infertility in the �ERKO male is due to pleiotropic 

effects resulting from disruption of the ER� gene. In addition 

to a lack of normal sexual behavior, �ERKO male mice ex- 

hibit significantly lower sperm counts that further diminish 

with age compared with their wild-type and heterozygous 

litter mates (315). This is compounded by defects in the 

function of those sperm that are produced in the testis of the 

�ERKO male, including obvious deficits in motility and a 

complete inability to fertilize wild-type oocytes in an in vitro 

assay (315). 

The testes of the �ERKO male are slightly smaller than 

wild-type but do develop normally and possess the usual 

complement of seminferous tubules surrounded by interstitial 

tissue and Leydig cells (Figs. 6 and 7). Stimulation of the 

Leydig cells by LH and subsequent androgen synthesis in the 

�ERKO testes appears sufficient as mentioned earlier. However, 

a report by Hutson et al. (316) indicated a greater in- 

FIG. 6. Testes and seminal vesicle/coagulating gland weights in the 

wild-type and �ERKO males. Mean total testes weights (A) and seminal 

vesicle/coagulating gland weights (presented as % body weight) 

of age-matched wild-type and �ERKO males from 4–22 months of age. 

Sample number in each group was �9. Error bars indicate SEM. Statistical 

significance is indicated as follows (t test, wild-type vs. 

�ERKO, unequal variance): *, P � 0.05; **, P � 0.01; ***, P � 0.001.


FIG. 7. Histology of representative adult �ERKO testis. a, Longitudinal section of a testis and portions of the epididymis from an adult �ERKO 

mouse (79 days old). The cranial portion of the testis and part of the caput epididymis (Cp Ep) are in the upper portion of the panel, and the 

caudal pole of the testis and cauda epididymis (Ca Ep) are in the lower portion. As indicated, the rete testis (RT) is conspicuously dilated. The 

seminiferous tubules in the caudal pole of the testis have a thin seminiferous epithelium and a considerably dilated lumen (as indicated by the 

solid arrows), spermatogenesis is disrupted, and fluid is present in the interstitium surrounding these tubules. However, in the seminiferous 

tubules in the cranial pole of the testis, the seminiferous epithelium is thicker, the lumen is smaller, spermatogenesis is ongoing, and the tubules 

are closely situated. [Reproduced with permission from E. M. Eddy et al.: Endocrinology 137:4796–4805, 1996 (315). © The Endocrine Society.] 

b, High-power magnification (132�) of seminiferous tubules from an adult �ERKO male (110 days old) illustrating an apparently normal 

seminiferous tubule possessing an epithelium (SE) composed of Sertoli cells and spermatogonia (open arrows), juxtaposed with a tubule 

possessing a severely dilated lumen and a thinning seminiferous epithelium (solid arrow) with little active spermatogenesis occurring. Leydig 

cells (LC) are present in the interstitial space. 

cidence of retraction of the testes into the abdomen and a 

smaller yet more muscular cremaster sac in �ERKO males 

compared with wild-type counterparts. This anomaly was 

noticed only after excision and fixation of the urogenital 

system and was not observable upon external examination of 

the animals. Interestingly, this is one of the few phenotypes 

of the �ERKO that has also been observed in the heterozygous 

littermates as well (316). This same laboratory had 

earlier employed the Tfm mouse, a naturally occurring androgen-resistant 

mutant, to produce strong evidence in support 

of a biphasic model of testicular descent in which androgens 

are critical to the second step of testis migration, i.e., 

from the internal inguinal ring to the scrotum via action on 

the genito-inguinal ligament (or gubernaculum) (319). Previous 

studies had shown that exposure of fetal male mice to 

exogenous estrogens resulted in a failure in testicular descent, 

although it was concluded that this was probably an 

indirect effect due to inhibition of the hypothalamic-pituitary 

axis and anti-Müllerian hormone (319). However, the significant 

incidence of undescended or retracted testes in the 

�ERKO strongly suggests a previously unrecognized and 

possibly direct role for the ER� in development of the male 

reproductive tract. 

Spermatogenesis, the production of free spermatozoa, is 

the primary function of the testes. Similar to the process of 

gametogenesis in the females described earlier, spermatogenesis 

is an equally complex process involving several different 

cell types and hormone actions. This is reiterated by 

the severely impaired spermatogenesis observed in naturally 

occurring inactivating mutations of the AR gene (38, 301), as 

well as in a number of knockout mouse models (reviewed in 

Ref. 110), e.g., those for the genes encoding Hsp70–2 (320), 

CREM (321, 322), DAZLA (323), and HR6B (324). Although 

FSH was previously thought to be essential to spermatogenesis, 

mice possessing targeted disruptions for the FSH�-subunit 

gene (241) or the FSH receptor gene (242) are fertile and 

show only minor detriments in testicular and sperm function. 

Estradiol has been thought to play only a minor role if any 

in sperm production, although Sertoli cells lining the seminferous 

tubules produce estradiol (325) and express detectable 

levels of ER (326). Therefore, it was surprising to observe 

severe impairments in spermatogenesis at multiple levels in 

the �ERKO male. At 8 wk, the earliest age studied, �ERKO 

males possess epididymal sperm counts that are approximately 

55% of that found in wild-type littermates (315). This 

value continues to decrease with age, with �ERKO males 

possessing approximately 13% of wild-type sperm counts at


16 wk of age (315). Furthermore, the epididymal sperm collected 

from the �ERKO males are characterized by significantly 

decreased levels of motility and increased incidence of 

sperm heads separated from the flagellum (315). Even those 

sperm that possessed normal structure and motility were 

unable to fertilize wild-type oocytes in an in vitro fertilization 

assay (315). Therefore, despite levels of circulating gonadotropins 

and androgen within the normal range, disruption of 

the ER� gene has resulted in severe impairments in both 

spermatogenesis and sperm function. 

As shown in Fig. 7, histological analysis of testes from 

sexually mature �ERKO males indicated significant atrophy 

of the seminiferous epithelium and severe dilation of the 

tubule lumen. At 10–20 days of age, no morphological difference 

in the testis was apparent when comparing the 

�ERKO with wild-type. However, a distinct morphological 

phenotype becomes obvious by 40 days of age and 

progresses to produce a completely atrophied testis by 150 

days in the �ERKO male (315). Accordingly, sperm counts 

decrease as the testicular phenotype worsens, although immunohistochemical 

detection of Hsp70–2, a germ cell-specific 

protein, was possible even in the most severely disrupted 

tubules (315). 

Further characterization of testes from mature �ERKO 

males indicated a prominent rete testis that is dilated and 

protrudes into the interior of the organ as well as severely 

dilated efferent ductules (Fig. 7) (315, 327). The rete testes are 

composed of a network of intercommunicating channels located 

in the posterior-cranial portion of the testes and serve 

as a pathway by which suspended spermatozoa can pass 

from the testis to the epididymis. Connecting the rete testis 

to the epididymis are the efferent ducts, a series of multiple 

channels thought to play a significant role in reabsorption of 

much of the testicular fluid, and therefore act to also concentrate 

the sperm (303). Steroid autoradiography has indicated 

that the efferent ducts of the mouse possess the highest 

concentration of ER compared with any other region of the 

excurrent duct system (309). This expression pattern is evident 

in the mouse as early as neonatal day 3 (310). Immunohistochemical 

and RNA analyses have shown that ER� is 

the predominant form of ER in the efferent ducts and the 

cranial portion of the epididymis (311, 328), although ER� 

mRNA is also detectable (121, 311). 

Previous studies employing surgical ligation of the efferent 

ductules reported a severe dilation of the seminiferous 

tubules similar to that observed in the �ERKO male (329, 

330). Based on the similarity of phenotypes, it became clear 

that the testicular anomaly observed in the mature �ERKO 

male may be the result of a severe imbalance in the fluid 

equilibrium. However, was it due to hypersecretion of fluid 

from the testis or insufficient reabsorption of fluid by the 

epithelial cells lining the efferent ducts, or possibly a combination 

of both? Using surgical techniques to inhibit fluid 

transport at different points in the excurrent duct system, 

Hess et al. (327) demonstrated that the reabsorption abilities 

of the efferent ductules in the �ERKO male were lacking and, 

in fact, the secretory activity is actually reduced in the 

�ERKO testis. Further characterization indicated a reduction 

or often a complete lack of endocytotic vesicles and organelles 

common to fluid uptake in the epithelial cells lining 

the �ERKO efferent ducts (327). This study was the first 

report of a direct ER�-mediated estrogen function in the male 

reproductive tract. Interestingly, however, was the inability 

of the pure antiestrogen, ICI-182,780, to produce a similar 

phenotype in wild-type ductal fragments in in vitro experiments 

(327). Although the antagonist was able to cause some 

loss of fluid absorption in the wild-type ductal fragment, the 

resulting phenotype was not nearly as extreme as that observed 

in the �ERKO tissue fragments (327). The authors 

proposed that perhaps ER� was possibly mediating an agonistic 

effect of the ICI-182,780 and thereby may explain the 

lack of full corroboration with the in vivo �ERKO phenotype 

(327). This hypothesis was based on the work of Paech et al. 

(91), which demonstrated that estrogen antagonists, including 

ICI-164,384, may function as an agonist when interacting 

with AP-1 complexes in vitro. We, as well as others, have 

since shown that ER� mRNA expression in the �ERKO male 

reproductive tract is not altered, although its function remains 

unclear (93, 121). However, the preservation of ER� 

expression in the �ERKO strongly indicates that the reabsorption 

functions of the efferent ducts are indeed dependent 

on the presence of functional ER�. This view is strengthened 

by the lack of a similar testicular phenotype in �ERKO male 

mice observed at ages as old as 14 months (47). 

Interestingly, the luminal swelling, loss of germinal epithelium, 

and atrophy in the seminiferous tubules of the 

�ERKO testes appeared to commence at the caudal portion 

of the organ and progress toward the cranial region as the 

animal aged (Fig. 7) (315). This is thought to be due to a 

gradual increases in testicular pressure leading to restricted 

blood flow as the phenotype in the rete testis worsens and 

fluid accumulates within the encapsulated organ (315). The 

result of such decreased circulation is likely to become initially 

manifested in the less vascularized caudal region of the 

testis and eventually advance to affect the whole gonad. 

Despite the severe testicular phenotype that occurs in the 

�ERKO male with age, younger males do produce viable 

sperm. However, the motility and fertilization abilities of 

epididymal sperm collected from �ERKO males are severely 

compromised. ER (326) as well as P450 arom (331) have been 

reported in Sertoli cells and germ cells of the testis, respectively. 

Therefore, a loss of ER�-mediated estrogen action in 

the Sertoli cell may alter sperm function. It is also known that 

spermatozoa entering the epididymis are unable to fertilize, 

and undergo a critically active maturation process as they 

pass through the epididymal cords (303). Estradiol treatment 

of adult male mice has been reported to increase the rate at 

which spermatozoa pass through the epididymis (332). Furthermore, 

expression of ER� in the mouse epididymis appears 

to be highest in the caput epididymis, where sperm 

first enter after exiting the testis (309). Reports of ER� expression 

in the epididymis indicate an opposite distribution, 

i.e. highest levels are found in the cauda epididymis, in both 

the rat (311) and mouse (121). This pattern of epididymal ER� 

expression is preserved in the �ERKO male (121). Nonetheless, 

normal fertility in the �ERKO male indicates that any 

actions of estrogen required for sperm maturation and fertilization 

capacity appear dependent on the presence of ER�. 

The varied phenotypes leading to infertility in the �ERKO 

male have provided great insight into the role that ER� plays


in the development and function of the male reproductive 

tract. The importance of functional ER� is reiterated by the 

lack of phenotypes and apparent full fertility in the �ERKO 

male mice. It is certainly possible that there are overlapping 

functions of the two ERs and that compensatory mechanisms 

have reduced the observed phenotypes compared with those 

that might result if both ERs were lacking. The speculated 

phenotype of a double ER knockout, i.e., ��ERKO, might be 

similar to that reported in males homozygous for a disruption 

of the P450 arom gene (ArKO), and therefore lacking the 

capabilities to synthesize estradiol. However, intriguing inconsistencies 

are apparent when comparing the phenotypes 

of the �ERKO male with those of the ArKO. For example, the 

male ArKO mice exhibit no defects in fertility, at least at the 

younger ages examined (257). The testicular weight in adult 

ArKO males is within the normal range, and histology indicates 

normal testicular development with no indication of 

a phenotype similar to that of the �ERKO males (257). The 

possible existence of currently unknown ligands able to stimulate 

the ER pathways required for function of the male 

reproductive tract and therefore altering the phenotype in 

the ArKO must be considered. It is also possible that much 

of the actions of ER� in the male reproductive tract, as inferred 

from the �ERKO, may be ligand independent. Such a 

phenomenon may explain the findings of Hess et al. (327), in 

which an estrogen antagonist was unable to completely induce 

an �ERKO phenotype in the ligated wild-type efferent 

duct segments in vitro. Since the testicular phenotype in the 

�ERKO becomes more severe with age, it is possible that a 

similar, yet even further, delay in the manifestation of this 

anomaly may exist when the ligand is removed; however, 

there are currently no reports on older ArKO males. 

B. Accessory sex organs 

Certain accessory organs of the male reproductive tract 

that have not been already discussed, namely the prostate, 

bulbourethral glands, coagulating gland, and seminal vesicles, 

warrant mention. These glands have no known specific 

function other than to secrete components necessary to the 

volume of seminal plasma. All four tissues are dependent on 

androgen stimulation for growth and maintenance (reviewed 

in Ref. 333). However, ER has been detected in each 

during various stages of development in the rat (310). Furthermore, 

the prostate in various species appears to express 

significant mRNA levels for ER� as well as ER� (93, 96, 311, 

313). A series of studies by the Prins laboratory have described 

the toxic effects of neonatal DES exposure on the 

morphology and biochemistry of the rat prostate, including 

the regulation of AR, ER�, and ER� (314, 334–336). Nonetheless, 

no obvious abnormalities in the development of 

these glands have been observed in either the �ERKO or 

�ERKO mice (47, 315). However, categorical studies of these 

tissues have not been carried out to date on older animals of 

either model. One observation in �ERKO males is a significant 

increase in weight of the seminal vesicle/coagulating 

gland that becomes more apparent with age, as shown in Fig. 

6. This phenotype is most likely the result of continued stimulation 

of this tissue by the elevated levels of serum androgen 

that exist in the �ERKO (Table 2). 

VI. Neuroendocrine System 

The mammalian brain and pituitary are clearly target organs 

of steroid hormone action. Several studies have demonstrated 

a wide distribution of receptors for all three sex 

steroid hormones throughout the different regions of the 

brain (reviewed in Refs. 337 and 338) as well as in the heterogeneous 

cell types of the pituitary (reviewed in Ref. 339). 

The regulatory actions of the gonadal steroid and peptide 

hormones on the hypothalamic-pituitary axis have been 

characterized in a number of laboratory models and may be 

the most well understood area among a growing list of hypothesized 

actions of steroids in the central nervous system. 

In contrast, changes in sensorimotor, cognitive, and emotional 

functions that occur in many women during periods 

of peak steroid secretion in the ovarian cycle remain less well 

understood (340, 341). More recently, estrogen action has 

received great attention as a possible influential factor in 

several nonreproductive-related brain functions, including 

learning and memory, cognitive function, and pain sensitivity, 

as well as in the pathology of neurodegenerative diseases, 

such as Parkinson’s and Alzheimer’s (reviewed in Ref. 

342). 

Several reviews over the past 20 yr have thoroughly documented 

the known actions of steroid hormones in the central 

nervous system (see Refs. 337, 343–348). Sexual dimorphism 

has been described in multiple regions of the central 

nervous system (reviewed in Ref. 343). Analogous to the 

reproductive tract, the neuroendocrine system undergoes a 

process of sexual differentiation and maturation that is 

heavily influenced by the steroid receptor-signaling pathways. 

Briefly, sexual maturation of the neuroendocrine system 

may be defined as the acquisition of pituitary responsiveness 

to hypothalamic factors and ovarian steroids and 

the onset of steroid-induced sexual behavior. In turn, differentiation 

of the neuroendocrine system is demonstrated by 

the unique ability of the female hypothalamus to induce an 

LH surge in response to a rise in serum estradiol (346, 348). 

The “organizational” or differentiating effects of perinatal 

steroids are permanent and manifested as measurable structural 

changes or as subtle fixations in the system’s sensitivity 

to the “activational” effects of steroids during adulthood 

(343). 

Studies have indicated that the imprinting mechanisms 

controlled by the sex steroids in the brain are likely via 

multiple pathways, including the regulation of cell death, 

neuronal growth, and synaptogenesis (343, 347, 349). These 

effects are generally considered to be genomic events mediated 

via the nuclear receptor pathways (337). Indeed, the 

nuclear receptors for estrogen, androgen, and progesterone 

all have been detected in various amounts in the fetal, neonatal, 

and adult brain of several species (88, 337, 343, 350– 

351). Furthermore, the discovery of the ER� has introduced 

a new level of complexity to the neuroendocrine system as 

it relates to estrogen action. Recent studies have demonstrated 

an expression pattern for the ER� in the rat brain that 

is as broad as that for ER� as reviewed in Refs. 351 and 352. 

Studies in the primate have reported similar findings (96, 

354). Of particular relevance are the descriptions by Shughrue 

et al. (88) of colocalization of ER� immunoreactivity and


ER� mRNA in certain regions of the rat brain, including the 

medial nucleus of the amygdala and the periventricular preoptic 

nucleus. Therefore, with preliminary studies indicating 

distinct tissue localization of the two ERs in the reproductive 

tract, the brain may be the ideal tissue for the study of 

possible transactivational actions of ER�/ER� heterodimers. 

It is important to reiterate that studies have indicated a normal 

expression pattern for the ER� gene in the hypothalamus 

of the �ERKO mouse (93, 352). 

Aside from the receptor-mediated genomic actions of sex 

steroids that have been so well characterized, the possibility 

of nongenomic effects of gonadal hormones and their metabolites 

has also received increased attention. A number of 

rapid responses to gonadal steroids in various tissues have 

been reported and are believed to occur too soon after steroid 

exposure to be mediated by the classical mechanism of hormone 

nuclear receptors; therefore, they have been termed as 

being “nongenomic” (reviewed in Refs. 347, 355–358). These 

include the rapid activation of membrane calcium channels 

by progesterone in the maturing oocyte and spermatozoa, by 

estrogens in myometrial cells, and by androgens in rat osteoblast 

cells (347, 355, 356). Descriptions of similar nongenomic 

effects of steroids in the neuroendocrine system 

include rapid increases in cAMP levels in neurons, modulation 

of the GABA-GABA A receptor function, release of 

GnRH and dopmamine from nerve terminals, modulation of 

oxytocin receptors, and the release of PRL from GH3/B6 

pituitary cells (343, 356, 357). Supportive experimental findings 

indicate the presence of membrane steroid receptors, 

including those for estradiol, in various cell types (359–361). 

Evidence that a membrane ER is structurally similar to the 

nuclear ER� was provided by Pappas et al. (362) in which 

multiple ER�-specific antibodies were shown to detect and 

localize ER� immunoreactivity in the cell membrane. Furthermore, 

Blaustein describes findings of extranuclear ER� 

immunoreactivity in the cytoplasm, dendritic processes, and 

axon terminals of neurons and suggests an active role for 

these receptors in neurotransmitter release (reviewed in Ref. 

338). Recently, Razandi et al. (363) reported the detection of 

membrane ER� and ER� receptors in Chinese hamster ovary 

(CHO) cells transfected with an expression vector of the 

respective receptor cDNA, indicating that the membrane and 

nuclear forms of each ER originate from the same transcript 

and exhibit similar affinities for estradiol. These studies further 

demonstrated that the membrane-bound ERs were G 

protein linked and able to elicit a variety of signal transduction 

events, including the induction of cell proliferation (363). 

In contrast, Gu et al. (364) recently employed the �ERKO 

mouse to illustrate that the documented rapid action of estradiol 

on kainate-induced currents in the hippocampus occurs 

in the absence of a functional ER� gene, nor does ICI- 

182,780 have an inhibitory effect, suggesting that ER� is not 

involved as well. Therefore, the putative membrane receptor 

involved in mediating the neuronal effect of estradiol in the 

hippocampus described by Gu et al. appears to be distinct 

from the intracellular nuclear form of the ER as well as that 

described by Razandi et al. (363). Regardless, the ultimate 

function of the nongenomic signaling pathways of the gonadal 

steroids in the proper organization and function of the 

mammalian brain remains unclear. Therefore, the ERKO mu- 

tant mice provide an excellent model to not only study the 

role of the nuclear receptors, but also further the investigations 

of steroid hormone actions that may be nuclear receptor 

independent. 

A comprehensive review of the neuroendocrine system is 

beyond the scope of this discussion and has been reviewed 

in detail elsewhere (337, 347). However, as was expected, 

distinct phenotypes in the neuroendocrine system have become 

evident in mice after disruption of the ER� gene. The 

ultimate consequences of the lack of ER� action in the neuroendocrine 

system are manifested in the ovary of the 

�ERKO female and as severe deficits in sexual and field 

behavior in both sexes of the �ERKO mice. Due to the relatively 

short time in which the �ERKO model has been 

available for study, no detailed characterizations of possible 

phenotypes in the hypothalamic-pituitary axis of this model 

have been carried out. Therefore, this section of the review 

will concentrate on what is currently known about the 

�ERKO, but will attempt to shed light on the possible distinct 

roles of both ERs based on the limited observations of the 

�ERKO. 

A. Hypothalamic-pituitary axis 

The hypothalamus may be thought of as the interface 

between the central nervous system and the endocrine system, 

i.e., the pituitary. The anatomical location of the hypothalamus, 

forming the base of the brain and residing just 

above the pituitary, is conducive to a function of translating 

neuronal signals from the brain into humoral factors that 

stimulate the appropriate actions in the anterior pituitary 

(365). The two components are connected by the hypothalamo-hypophyseal 

portal system, within which blood 

flows predominantly from the hypothalamus to the anterior 

pituitary, carrying the appropriate hormonal factors (365). 

These hormones act as releasing or inhibiting factors to control 

the secretory activity of the pituitary. In contrast, the 

posterior pituitary is connected directly to the hypothalamus 

via neurons passing through the pituitary stalk and functions 

as a storage organ for the hypothalamic hormones, oxytocin 

and vasopressin. 

The anterior pituitary is composed of at least five distinct 

cell types, all derived from a common primordium, which 

have been categorized by the particular peptide hormone 

they produce and secrete. These cell types are as follows, 

with the secretory hormone in parentheses: gonadotrophs 

(FSH and LH), corticotrophs (ACTH), thyrotrophs (TSH), 

somatotrophs (GH), and lactotrophs (PRL). Early studies 

employing steroid autoradiography demonstrated estrogen 

binding to varied degrees throughout the different cells of 

the anterior pituitary, although discrepancies among species 

are evident (reviewed in Ref. 339). These studies have been 

followed by those using in situ hybridization and immunohistochemistry, 

indicating that the majority of estradiol binding 

in the anterior pituitary is due to the expression of ER� 

(366, 367). In most species described, gonadotrophs and lactotrophs 

exhibit the greatest level of ER� followed by lower 

and varied levels of localization to the other cell types (339). 

Furthermore, Shupnik et al. (29, 368) have described in the rat 

pituitary a number of ER� mRNA isoforms characterized by


the removal of single or multiple exons as well as 5�-sequences 

that exhibit little homology to the full-length wildtype 

ER�. Although a distinct function for these ER� isoforms 

has not yet been determined, their transcription as well 

as that of the full-length wild-type ER� gene appears to be 

regulated by ovarian steroids (368, 369). Further complexity 

has been introduced by several recent descriptions of the 

presence of ER� mRNA in the anterior pituitary of the human 

(100), monkey (96), and rat (70, 98, 99). Wilson et al. (98) 

report that ER� mRNA levels were easily detectable in the 

pituitary of the 15-day-old rat, exceeding the levels of ER� 

mRNA. These levels decreased in the adult, whereupon a 

distinct sex difference became evident in which the anterior 

pituitary of the female expressed much higher levels of ER� 

mRNA (98). Still, previous studies employing radiolabeled 

ligand do not corroborate this difference in the rat anterior 

pituitary between the sexes at the level of estradiol binding 

(370, 371). Furthermore, there are contrasting reports concerning 

the cellular localization of the ER� transcripts in the 

rat anterior pituitary. Mitchner et al. (99) reported varied 

levels of ER� mRNA throughout the different cell types of 

the anterior pituitary. In contrast, Wilson et al. (98) describe 

the detection of both ER� and ER� mRNAs in the gonadotrophs, 

whereas the lactotrophs appear to possess ER� only. 

Nonetheless, as previously mentioned, we find low to undetectable 

levels of ER� mRNA in the pituitary of the adult 

mouse, including that of the �ERKO (93). However, in light 

of the above studies described in the prepubertal rat, which 

indicate a possible developmental role for the ER�, similar 

assays in the mouse are warranted. 

Although the rate of gonadotropin secretion from the anterior 

pituitary is directly modulated by the hypothalamus, 

the level of circulating sex steroids is the most important 

physiological determinant of serum gonadotropin levels in 

animals and humans (reviewed in Ref. 251). The positive and 

negative regulatory loops of the hypothalamic-pituitary-gonadal 

axis have been reviewed in detail and will be summarized 

briefly here (251, 362, 372). In short, the hypothalamus 

stimulates the synthesis and subsequent secretion of 

the gonadotropins into the circulatory system, which then act 

to induce gametogenesis and steroidogenesis in the gonads. 

The functional gonadotropins, FSH and LH, are composed of 

dimers of the common �-glycoprotein subunit (�GSU), with 

a distinct �-subunit that confers specificity to the hormone, 

i.e., active FSH consists of a FSH-� (FSH�)/�GSU dimer, 

whereas LH consists of a LH-� (LH�)/�GSU dimer (373). 

Hypothalamic stimulation of the anterior pituitary is via the 

release of GnRH, a deca-peptide that functions to positively 

regulate the synthesis and secretion of the gonadotropins 

from the anterior pituitary (365). A reciprocal hypothalamic 

factor that may act to inhibit pituitary secretion of gonadotropins 

has not yet been found. Hypothalamic secretion of 

GnRH is not tonic but rather in the form of pulses, driven by 

a poorly understood oscillating pulse generator (374, 375). 

The pulsatile stimulation of the anterior pituitary by GnRH 

thereby results in pulsatile gonadotropin release, which has 

been shown to be necessary for gonadal function and reproductive 

success (374, 375). Gonadotropin stimulation of the 

gonads subsequently results in gametogenesis and the synthesis 

of gonadal steroid and peptide hormones, which then 

feed back to the hypothalamus and pituitary to regulate FSH 

and LH secretion (251). However, differences in the system 

of feedback loops are apparent between the sexes. While 

there exists a tonic system to maintain a constant level of 

gonadotropins in both sexes, the female has been endowed 

with the ability to produce a surge of gonadotropin secretion 

to produce transient levels of hormone that are several fold 

higher than the tonic level (365). This massive gonadotropin 

surge provides for the reproductive cycle in the female and 

is critical to ovulation. 

1. Female-negative gonadotropin regulation. There is ample experimental 

evidence in several species that estradiol can suppress 

the secretion of gonadotropins from the anterior pituitary 

(reviewed in Refs. 251, 372, 373, 376, and 377). 

Ovariectomy of the female rodent is known to result in significant 

elevations in serum FSH and LH that can be returned 

to intact levels with physiological treatments of estradiol 

(376, 378, 379). The effects of ovariectomy are mirrored in the 

gonadotrophs of the anterior pituitary, which exhibit equally 

elevated mRNA levels for the gonadotropin subunit genes 

(373, 376). Studies in the female rat indicate that by 21 days 

post ovariectomy, LH� mRNA levels rise to as high as 20fold, 

whereas the increases in FSH� and �GSU mRNA levels 

plateau at 4- to 5-fold (373). Daily treatments with estradiol 

will return the gonadotropin subunit mRNAs to the pregonadectomy 

levels within 7 days in the rat (373, 376). 

The ability of estradiol to maintain a tonic level of gonadotropins 

via regulation of both the expression of the gonadtropin 

subunit genes and the ultimate secretion of the 

peptide hormones is well accepted. However, the precise site 

at which the steroid exerts this effect has been difficult to 

ascertain. This is partly due to the fact that the ER has been 

detected in both the hypothalamic regions controlling pituitary 

function as well as the anterior pituitary itself, as previously 

discussed. Although there is evidence to support a 

direct effect of estradiol on both components of the neuroendocrine 

axis, it is believed that the hypothalamus may be the 

primary site of action in the negative feedback actions (reviewed 

in Refs. 251, 372, 373, and 376). In the laboratory 

animal, it is believed that castration principally results in an 

increased frequency of GnRH pulses and therefore increased 

tonic levels of gonadotropins, both of which can be restored 

to normal with exogenous estradiol replacement (373, 380– 

383). 

Numerous studies have produced data to indicate that 

estrogen regulation of hypothalamic GnRH secretion may be 

the predominant pathway by which transcription of the gonadotropin 

subunit genes and gonadotropin secretion is 

maintained (373, 376). The most prominent include the characterization 

of transgenic mice that possess, within the anterior 

pituitary, a measurable reporter gene under the regulation 

of a gonadotropin subunit gene promoter, including 

the promotor of the human �GSU gene (384), the rat �GSU 

gene (385), and the bovine LH� gene (386). As expected, 

ovariectomy of the transgenic females resulted in increased 

transcription and activity of the transgene reporter gene. Keri 

et al. (386) illustrated that estradiol was able to reduce the 

postovariectomy rise in transcription of the reporter gene 

despite the lack of detectable DNA binding of the ER� to the


gonadotropin promoter sequences of the construct. Furthermore, 

the postovariectomy rise in promoter activity could be 

prevented by administration of a GnRH antagonist to the 

transgenic animal, thereby inhibiting GnRH action at the 

gonadotrope. Therefore, a loss of estradiol action via ovariectomy 

appeared to result in increased GnRH release from 

the hypothalamus, which in turn stimulated increased transcription 

of the reporter constructs. However, the possibility 

of estradiol actions at the level of the gonadotrope that may 

directly effect gene transcription or alter GnRH responsiveness 

can not be ruled out. 

Regardless of the precise site at which estrogens negatively 

regulate gonadotropin expression and release, genetic disruption 

of the ER� gene was expected to have effects in the 

female hypothalamic-pituitary axis that mimic ovariectomy. 

Northern blot analysis of RNA from pituitaries of intact 

wild-type and �ERKO females indicates this to be true. 

Scully et al. (282) demonstrated that in the �ERKO female, the 

levels of the �GSU transcript are elevated 4-fold, whereas 

FSH� and LH� mRNAs are as high as 7-fold compared with 

wild-type. Ovariectomy in wild-type female littermates produced 

elevated gonadotropin subunit mRNAs that approximated 

the levels observed in the intact �ERKO, indicating 

that the effects of an acute loss of estrogen action are similar 

to those produced by a hereditary loss of ER� (282). Therefore, 

despite the fact that the hypothalamic-pituitary axis of 

the �ERKO female is chronically exposed to elevated levels 

of estradiol, the significantly increased level of all three gonadotropin 

subunit transcripts resemble those of an ovariectomized 

female. These data provide strong support for a 

critical role of ER�, rather than ER�, in the negative regulation 

of transcription of the gonadotropin subunit genes. 

However, at this time, studies of the �ERKO have not provided 

data to further elucidate the precise mechanism or site 

at which a loss in ER� action has resulted in this effect. 

Although the transcript levels of both the LH�, FSH�, and 

�GSU are significantly elevated in the �ERKO pituitary, this 

effect does not extend to the serum levels of the gonadotropins. 

Whereas serum LH is elevated 4- to 7-fold in the adult 

�ERKO female, levels of FSH appear to be within the normal 

range (Table 2) (252). A similar effect is reported in the PRKO 

female mice, although the serum LH levels in this mutant 

female are not nearly as elevated as those in the �ERKO 

female (387). In addition, ovariectomy in the PRKO female 

results in a further elevation of serum LH (387), most likely 

due to the loss of serum estrogens. This effect is not observed 

in the �ERKO female (252), indicating that estradiol is the 

predominant steroid hormone maintaining tonic levels of LH 

in the female. 

As shown in Table 2, the serum gonadotropin levels in the 

�ERKO female indicate that only LH is significantly elevated, 

whereas FSH is within the wild-type range. This is in 

contrast to the levels of FSH� mRNA in the anterior pituitary 

of the �ERKO, which are elevated 7-fold and equal to those 

exhibited by an ovariectomized wild-type female. Furthermore, 

assays of pituitary homogenates from intact �ERKO 

females for FSH protein indicate levels within the wild-type 

range, suggesting that the divergence between gene expression 

and serum levels for FSH does not appear to be due to 

a decreased secretory rate of the hormone but rather at the 

level of translation. This is in contrast to the ArKO female 

mouse, in which serum levels of both gonadotropins are 

reportedly elevated 3-fold compared with the wild-type 

(257). 

There are a number of possible explanations for this observation 

in the �ERKO female. Whereas estradiol treatment 

of ovariectomized rats has been reported to completely block 

the expected increases in LH� mRNA and LH secretion, it 

appears to be only partially effective in reducing transcription 

of the FSH� gene and secretion of FSH (376). It is now 

known that FSH� gene expression and FSH secretion is selectively 

regulated in a positive or negative nature by the 

peptides activin and inhibin, respectively (reviewed in Ref. 

388). This is illustrated in the knockout mouse model of the 

activin receptor type II gene, which exhibits suppressed FSH 

levels in the adult of both sexes, supporting a role for activin 

in the positive regulation of FSH secretion (248). Although it 

is believed that the reciprocal effects of the activin/inhibin 

peptides are mediated at the level of the anterior pituitary, 

the precise mechanisms of action remain unclear. Inhibin has 

been shown to alter the levels of GnRH receptor (389) and 

decrease FSH� mRNA levels (390–392), as well as inhibit 

translation of FSH� mRNA (391, 393). Activin appears to 

utilize similar mechanisms to exert opposite effects on FSH� 

mRNA transcription and translation and ultimate secretion 

of the hormone (394, 395). Therefore, the normal levels of 

FSH in the pituitary and serum of female �ERKO mice may 

indicate that disruption of the ER� gene has no effect on the 

pattern of activin and inhibin secretion. This is supported by 

the apparent negative regulation of FSH� at the translational 

level in the �ERKO female, suggesting the presence of an 

active inhibin-signaling pathway. Further support is provided 

by studies indicating that ovariectomy of the �ERKO 

female, and therefore a loss of ovarian inhibin secretion, 

results in elevated levels of serum FSH similar to those seen 

in ovariectomized wild types (252). Estradiol replacement 

was partially effective in reducing the serum FSH in the 

ovariectomized wild-type but completely ineffective in the 

ovariectomized �ERKO (252). These studies provide for at 

least two conclusions: 1) the partial effectiveness of estradiol 

in reducing serum FSH levels in the wild-type was likely via 

functional ER� in the hypothalamus that may complement 

the actions of inhibin in the intact female; and 2) ovarian 

factors other than estradiol, most likely inhibin, are maintaining 

normal serum FSH levels in the �ERKO female, possibly 

by mechanisms that override the loss of ER� action. It 

is also possible that both activin and inhibin synthesis and 

secretion may be altered in the �ERKO female, but do not 

result in a net difference in serum FSH levels. Future investigations 

to determine the serum levels of the activin/inhibin 

subunit peptides and their functional dimers in the �ERKO 

female are warranted. 

Another possible explanation for the selective increase in 

serum LH in the �ERKO female may be that a lack of ER� 

action has resulted in aberrations in the GnRH pulse frequency 

and amplitude that are more conducive to LH secretion 

from the anterior pituitary. Normal levels of �GSU 

transcripts can be maintained with constant GnRH stimulation 

of the anterior pituitary (376). However, normal expression 

of both gonadotropin �-subunit genes and gonadotro-


pin secretion requires pulsatile stimulation from the 

hypothalamus, and each responds differently depending on 

the amplitude and frequency of GnRH stimulation (373, 376). 

Varied expression of the GnRH receptor on the cell surface 

of the gonadotrophs also varies with the level of hypothalamic 

stimulation (396). This mechanism, by which the level 

GnRH receptors can be differentially regulated and thereby 

modify the gonadotropin responsiveness to GnRH, has been 

proposed to be a key element in the differential regulation of 

the two different gonadotropins by the same releasing hormone 

(397). Studies in the rat have revealed that more rapid 

GnRH pulses (15–60 min) favor the secretion of LH, whereas 

slower pulses (120 min) allow for secretion of FSH (251, 376). 

Positive regulation of LH synthesis and secretion is also more 

sensitive to the amplitude of GnRH stimulation (251, 376). 

Therefore, a loss of ER� action during development and 

maturation of the hypothalamic-pituitary axis may have resulted 

in a pattern of GnRH secretion that favors the translation 

and secretion of LH rather than FSH. The influence that 

ER� may have, including a possible compensatory role in the 

�ERKO hypothalamus, remains to be evaluated. 

2. Female-positive gonadotropin regulation. In addition to maintaining 

tonic levels of serum gonadotropins, estradiol also 

plays a central role in the preovulatory gonadotropin surge 

mode in the female (reviewed in Refs. 263, 264, and 377). 

Differentiation of the neuroendocrine system results in the 

development of mechanisms necessary to produce a dramatic 

preovulatory rise in serum gonadotropins in response 

to the positive feedback of ovarian steroids. In the rodent, 

developmental differentiation of this pathway in the neuroendocrine 

system is unique to the female (365). The surge 

in serum LH and FSH is the hallmark of the female cycle and 

is critical to ovulation as well as the synchronized induction 

of appropriate sexual behavior and, therefore, is vital to the 

female ovarian cycle and fertility. 

Numerous studies have demonstrated that estradiol is 

required for the preovulatory gonadotropin surge, and that 

the timing and dose of estradiol exposure may be the most 

critical parameters (reviewed in Ref. 377). As with the negative 

regulatory effects of estradiol, the precise site of action 

for the sex steroids during the preovulatory gonadotropin 

surge also remains unclear. However, it is believed to be the 

result of the combined effects of external and internal cues 

transduced from the brain and the positive feedback of gonadal 

hormones that provide for a synchronized pattern of 

GnRH secretion upon an anterior pituitary that has been 

rendered transiently hypersensitive to the releasing hormone 

(reviewed in Ref. 377). In the monkey, destruction of the 

neurons involved in GnRH production can be overcome with 

pulsatile administration of exogenous GnRH, whereupon a 

gonadotropin surge can be produced with exogenous estradiol, 

indicating the pituitary as the predominant factor in the 

surge (398). However, this is not possible in the rodent, 

apparently due to a greater level of interdependency among 

the components of the neuroendocrine axis (365). 

Therefore, although the rises in ovarian estrogen secretion 

are critical to the generation of a gonadotropin surge, the 

pathways involved are poorly understood. A number of 

mechanisms involving direct actions of estrogen in the brain 

have been proposed and recently reviewed (264). However, 

it is unlikely that the actions of estrogen are via direct interaction 

with GnRH neurons since these processes appear to 

be devoid of ER (264). Therefore, the actions of estradiol 

appear to result in indirect stimulation of the hypothalamic 

neurons that synthesize and release GnRH. Possible mechanisms 

include steroid interaction with receptor-positive 

monoaminergic and opoid neurons that may mediate the 

ultimate effects to GnRH neurons, possibly via modifications 

in the levels of catacholamines, glutamate, �-aminobutyric 

acid, neuropeptide Y, �-endorphins, and galanin (reviewed 

in Refs. 263 and 264). At the level of the anterior pituitary, the 

preovulatory increases in estradiol may act in concert with 

GnRH to enhance gonadotrope sensitivity to the forthcoming 

rise in releasing hormone by increasing the levels of GnRH 

receptor (263, 377). Furthermore, the 5�-flanking region of the 

rat LH� gene has been shown to possess an imperfect estrogen-responsive 

element that is able to bind ER� and confer 

estrogen responsiveness to a chimeric promoter-reporter 

gene construct in vitro (399). Therefore, the estradiol-ER� 

complex may also act to directly increase LH� mRNA levels 

before the LH surge (251). 

Attempts to elicit an LH surge in the ovariectomized 

model with acute estradiol treatments have been reported to 

be only partially effective, indicating that ovarian factors 

other than estradiol are also required for a full physiological 

response (reviewed in Ref. 263). It is now known that the 

actions of progesterone and the PR are also a necessary 

component in the induction of the gonadotropin surge (reviewed 

in Ref. 263). The role of progesterone and PR in 

facilitating the preovulatory surge may be to induce a rapid 

release of GnRH from the hypothalamus, as well as possibly 

mediate a decrease in ER levels in the anterior pituitary, 

thereby possibly counteracting the inhibitory effects of estradiol 

(263). Although the precise mechanism of action may 

be unclear, the complete lack of a preovulatory surge in intact 

PRKO female mice provides strong support for the requirement 

of this steroid receptor (387). 

A cooperative role between the estrogen- and progestronesignaling 

pathways in the induction of the preovulatory 

surge may include the ability of estradiol to stimulate increased 

PR levels in both the hypothalamus and anterior 

pituitary, thereby increasing the sensitivity of these tissues to 

progesterone (400, 401). Shughrue et al. have shown that 

estradiol-induced increases in PR expression in the preoptic 

nucleus are possible in the �ERKO female (Fig. 8) and suggest 

that this may be a compensatory action of ER� (400). 

These same studies demonstrated that the preoptic nucleus 

of the hypothalamus in intact �ERKO females possessed a 

significantly greater level of PR mRNA when compared with 

wild-types, perhaps due to chronic stimulation of ER� by the 

elevated serum estradiol (400). Evidence to support this hypothesis 

is the apparent decrease in the level of PR transcripts 

observed after ovariectomy in the �ERKO, which can be 

returned to intact levels 6 h after a single treatment with 

estradiol (Fig. 8) (400). 

The preoptic area of the hypothalamus, more specifically, 

the anteroventral periventricular nucleus of the preoptic region 

(AVPV), is thought to play a critical role in transducing 

the gonadotropin surge via interactions with the GnRH neu-


FIG. 8.In situ hybridization for progesterone receptor (PR) mRNA in female WT and �ERKO hypothalamus. A, PR mRNA was detected in the 

medial preoptic nucleus of wild-type (a) and �ERKO females (b) 5 days afer ovariectomy. Also shown is the increased detection of PR mRNA 

in ovariectomized wild-type (c) and �ERKO (d) females 6 h after treatment with 5 �g of estradiol. Asterisks indicate the third ventricle. B, 

Quantitative analysis of the hybridization signal shown in panel A. Note the dramatic increase in PR hybridization signal when ovariectomized 

(OVX) wild-type mice were treated with estradiol (E 2). Similarly, the hybridization signal seen in intact �ERKO females is attenuated by 

ovariectomy, but augmented to intact levels when ovariectomized females were treated with estradiol. Statistical significance is indicated as 

follows: **, P � 0.01, ***, P � 0.001. [Reproduced with permission from P. J. Shughrue et al.: Proc Natl Acad Sci USA 94:11008–11012, 1997 

(400). © National Academy of Sciences, USA] 

rons. Unlike most sexually dimorphic nuclei, the AVPV is 

actually larger and composed of a greater number of dopaminergic 

neurons in the female compared with the male 

(402). In male rodents, this region is rendered inoperative by 

the actions of testosterone during differentiation (365), an 

effect that can be reproduced in females with neonatal testosterone 

or estradiol exposure (403, 404). Therefore, masculinization 

of this portion of the brain involves the destruction 

of a large portion of these neurons and is believed to be 

due to local aromatization of testosterone to estradiol and 

subsequent activation of ER-mediated pathways (405). In 

support of this hypothesis, Simerly et al. (405) reported that 

the AVPV region of �ERKO males possess a population of 

dopaminergic neurons more characteristic of a wild-type 

female, confirming a critical role of ER� in this differentiation 

process. Furthermore, the numbers of dopaminergic neurons 

in the female �ERKO are only slightly reduced when compared 

with wild-type, indicating a morphologically normal 

AVPV region (405). Therefore, with the �ERKO female exhibiting 

an apparent preservation of estrogen-induced increases 

in hypothalamic PR and a wild-type-like female phenotype 

in the AVPV region, it is conceivable that the 

hypothalamic mechanisms required for induction of the preovulatory 

surge may be intact. 

3. Males: gonadotropin regulation. Because of the more prominent 

role of testosterone in the male, certain issues specific 

to the male hypothalamic-pituitary axis are worthy of discussion. 

Of course, lower aromatase activity in the testis 

results in circulating levels of estradiol in the male that do not 

approach those observed in the intact cycling female. Therefore, 

it would be expected that distinct mechanisms of steroid 

feedback and regulation of gonadotropin synthesis and secretion 

from the hypothalamic-pituitary axis have evolved in 

males, presumably one likely to be more dependent on tes- 

tosterone. This difference is thought to occur at the level of 

the hypothalamus since the anterior pituitary generally exhibits 

no sexual differentiation (365) and possesses receptors 

for all sex steroids (339). 

A critical role of testosterone and AR-mediated actions in 

the negative regulation of gonadotropin secretion in the male 

is illustrated by the elevated serum LH levels in Tfm mice 

(301) and in humans with androgen insensitivity syndromes 

(38). As in the female, transcription of the gonadotropin 

subunit genes is significantly elevated after castration in the 

male, although peak levels are reached much earlier (�7 

days) (373). Furthermore, FSH� mRNA levels appear to return 

to precastration levels by 28 days, whereas the levels of 

LH� and �GSU transcripts remain elevated (373). Estradiol 

is equally effective as testosterone in reducing serum LH 

levels that result after castration in the male (reviewed in Ref. 

373). These data, along with the documented presence of 

P450 arom activity (reviewed in Refs. 406 and 407) and wide 

distribution of ER in the hypothalamic-pituitary axis support 

a role for locally synthesized estradiol and ER action in male 

gonadotropin synthesis and/or secretion (88, 339). Furthermore, 

adult male ArKO mice exhibit elevated levels of serum 

LH despite possessing significantly high circulating testosterone 

(257). Therefore, the roles of estradiol and testosterone 

often appear overlapping as well as distinct, making obvious 

the complexity of the steroid-feedback mechanisms that exist 

in the male. 

Any specific role the ER� may play in the regulation of 

gonadotropin synthesis and secretion in the male would be 

expected to become apparent in the �ERKO. Adult �ERKO 

males exhibit levels of hypothalamic GnRH, pituitary FSH� 

mRNA, and serum FSH that are within the normal range 

when compared with wild-type littermates (Table 2) (317). 

The normal levels of FSH� mRNA in the pituitary of �ERKO


males are in stark contrast to the significantly elevated levels 

found in the female �ERKO mice (282). This contrast between 

the sexes may reflect differences in the inhibin/activin levels 

or may represent a definitive sexual differentiation in the 

transcriptional regulation of the FSH� gene in the mouse, 

indicating that androgens are the primary acting steroids in 

the male. However, although not as extreme as those found 

in the �ERKO female, pituitary LH� mRNA and serum LH 

levels are increased 2-fold in the adult �ERKO male (Table 

2) (317). 

In a series of experiments, Lindzey et al. (317) demonstrated 

that castration results in the expected elevated levels 

of serum LH in the wild-type, and a further increase in the 

already elevated LH levels in �ERKO males. The rise in 

serum LH that occurs upon castration even in the �ERKO 

male suggests that either estradiol-ER� or androgen-mediated 

mechanisms are maintaining the lower LH levels in the 

intact animal. Once again however, the mouse pituitary (including 

the �ERKO) appears to possess very little if any ER� 

mRNA (93), although ER� is expressed normally in the hypothalamic 

regions in the �ERKO (93, 352). Estradiol treatment 

of castrated animals over a period of 3 weeks reduced 

the serum LH levels to normal in the wild-type males, 

whereas no effect was observed in the �ERKO, indicating a 

requirement for ER� in this process (317). Although treatments 

of similar castrated males with testosterone was completely 

effective in producing an inhibitory effect on LH 

release in the wild-types, it was only partially effective in the 

�ERKO (317). The authors therefore concluded that the ability 

of testosterone to fully restore normal levels of LH in the 

sera of castrate wild-type males but only partially in the 

�ERKO males suggests that local aromatization of testosterone 

to estradiol and subsequent activation of ER�-mediated 

pathways act to enhance the negative feedback effects of 

androgens in the male hypothalamic-pituitary axis (317). 

However, the inability of testosterone to completely suppress 

the serum LH in the �ERKO male may be related to the 

dosage used in these studies. Strong evidence of AR-dependent 

regulation of LH secretion in the male �ERKO is found 

in preliminary experiments in which treatment with an antiandrogen 

(flutamide) increased serum LH by 3- to 10-fold 

in wild-type and �ERKO males, respectively. This suggests 

that �ERKO males have come to rely entirely on AR-mediated 

actions to regulate LH secretion, whereas the ER� continues 

to play a role in the wild-type. 

In these same studies, Lindzey et al. (317) illustrated that 

prolonged treatment with DHT, the more potent and nonaromatizable 

androgen, resulted in no reduction in the castrate 

levels of serum LH in wild-type but was partially effective 

in the �ERKO male. However, the DHT was effective 

in restoring hypothalamic GnRH content levels to normal in 

castrate males of both genotypes (317). Therefore, the enhanced 

effect of DHT in negatively affecting the hypothalamic-pituitary 

regulation of serum LH, including the inhibition 

of hypothalamic GnRH release, remains a puzzling 

phenomenom unique to the �ERKO male. It is possible that 

a lack of ER� action during development resulted in a “reorganization” 

of the hypothalamic-pituitary axis in the 

�ERKO male, and thereby somehow allowed for an increased 

sensitivity to androgens (317). Further studies in the 

�ERKO as well as the �ERKO males may help elucidate these 

unexpected results. 

4. PRL regulation. PRL possesses more biological actions than 

all of the other anterior pituitary hormones combined. A 

recent review by Bole-Feysot et al. (279) thoroughly covered 

the current knowledge of the diverse actions of PRL, including 

its functions as a hormone, growth factor, neurotransmitter, 

and immunoregulator. A reflection of the multiple 

functions of PRL is the equally broad distribution of PRLbinding 

sites throughout the many physiological systems in 

vertebrates (279). The well known effects of PRL in reproduction 

include a critical role in the differentiation and function 

of the lactating mammary gland, as a luteotrophic hormone 

in the function of the corpus luteum and thereby as a 

promotor of blastocyst implantation, and an overall enhancement 

of the physiological functions in the tissues of the male 

reproductive tract (279). Nonreproductive roles of PRL include 

an involvement in osmoregulation; promotion of 

growth, development, and differentiation in several tissues; 

enhancement of metabolic activities in the brain, liver, pancreas, 

and adrenals; and various actions in immunoregulation 

(279). 

It has long been known that estradiol is a critical hormone 

in the regulation of PRL synthesis and secretion from the 

lactotrophs in the anterior pituitary. Estradiol has also been 

shown to stimulate lactotroph cell growth (reviewed in Ref. 

281) and has been implicated as a possible factor in the 

promotion of PRL-secreting tumors in humans (reviewed in 

Ref. 339). Furthermore, the lactotrophs of several species 

have been shown to possess significant levels of ER�, 

strongly suggesting that the actions of estradiol are receptormediated 

(reviewed in Ref. 339). As discussed above, recent 

descriptions have indicated the presence of ER� in the lactotrophs 

of the rat anterior pituitary, although contrasting 

reports exist, possibly due to strain variations. Whereas Wilson 

et al. (98) describe the presence of only ER� in lactotrophs, 

Mitchner et al. (99) report variable levels of ER� 

mRNA throughout the various cell types of the rat anterior 

pituitary. Shupnik et al. (100) have also reported the detection 

of ER� transcripts in human PRL-secreting tumors. Once 

again, we find only low to undetectable levels of ER� mRNA 

in the pituitary of the adult mouse, including the �ERKO 

(93). 

The upstream regulatory sequences of the rat PRL gene 

have been found to possess an estrogen-responsive element 

that binds ER� and functions synergistically with the pituitary-specific 

factor, Pit-1, to promote expression (281, 408, 

409). The required function of the ER� in the positive regulation 

of the PRL gene is nicely illustrated in the �ERKO 

mouse. The �ERKO females exhibit a 20-fold decrease in PRL 

mRNA levels in the anterior pituitary, whereas the �ERKO 

males exhibit a 10-fold decrease when each is compared with 

sex-matched wild-type controls (282). Although not as drastic, 

this reduction in the expression of the PRL gene is mirrored 

in the serum levels of the hormone in the �ERKO 

female, which possess an approximate 5-fold reduction in 

serum PRL (Table 2). Therefore, given the plethora of roles 

in which PRL is involved, it is likely that several of the 

phenotypes observed in the �ERKO mice may be due to a


direct loss of or simply enhanced by the concurrent decrease 

in PRL signaling. 

Interestingly, the extremely low levels of PRL mRNA in 

the anterior pituitary of the �ERKO female are even significantly 

less than that observed 14 days after ovariectomy in 

the wild-type (282). Therefore, the loss of ER� during development 

and differentiation of the lactotrophs in the anterior 

pituitary has resulted in a phenotype that is more 

severe than that induced by postpubertal ovariectomy, possibly 

due to a decrease in lactotroph cell number. It has been 

proposed that the lactotroph and somatotroph cell types of 

the adult anterior pituitary may be derived from a common 

cell that expresses both the genes for GH and PRL during 

development (reviewed in Ref. 410). The factors that may be 

involved in the terminal differentiation of this stem cell into 

a distinct cell type secreting only one of the respective hormones 

remain elusive. Because the appearance of the ER and 

the ontogeny of PRL expression appear to coincide in the 

developing pituitary, estrogen action has been proposed as 

a possible factor (410–412). However, a defect in the cell 

lineage of the lactotrophs that may be expected due to a loss 

of ER� action was not apparent in the �ERKO, as immunostaining 

for both PRL and GH localized expression of the 

genes to distinct cell types (282). 

Furthermore, estrogen has also been shown to stimulate 

proliferation of the lactotrophs and PRL-secreting cell lines 

(reviewed in Ref. 339). Therefore, since the marked difference 

in PRL mRNA levels observed between the �ERKO female 

and the ovariectomized wild-type is not apparently due to a 

defect in the differentiation of the lactotrophs, it may possibly 

be due to a decreased number of lactotrophs in the anterior 

pituitary of the �ERKO. Scully et al. (282) provided evidence 

against this hypothesis, by once again employing immunohistochemical 

staining to illustrate only a modest decrease in 

lactotroph cells in the anterior pituitary of the �ERKO mice. 

Therefore, ER� action does not appear to be required for 

either differentiation or proliferation of the lactotrophs in the 

mouse anterior pituitary. However, a recent report by Chun 

et al. (413) has illustrated a distinct contrast in the level of 

occupied ER required to elicit proliferation and that required 

for PRL synthesis in PR1 cells, a PRL-secreting cell line. 

Whereas approximately 50% of the cellular pool of ER was 

required to be complexed with estradiol for half-maximal 

stimulation of the PRL gene, only 0.1% was required to 

induce cellular proliferation (413). These results suggest that 

the mechanisms required for estrogen-induced lactotroph 

proliferation are hypersensitive in this cell line compared 

with the mechanisms involved in regulation of the PRL gene 

(413). Therefore, it is possible that the small amount of the 

active ER� splicing variant known to be present in the 

�ERKO (see Section II.C.) has allowed for sufficient estrogen 

signaling and lactotroph proliferation during develpment, 

resulting in the apparent lack of a somewhat expected phenotype 

of decreased lactotroph cell number in the pituitary 

of �ERKO mice. 

B. Behavior 

There are obvious effects of the gonadal steroids on sexual 

behavior in vertebrates; however, a more defined knowledge 

of these actions has become evident from a series of classical 

experimental schemes. These laboratory studies often relied 

on perinatal castration and/or developmental exposure to 

exogenous steroids followed by studies of the activational 

abilities of the different steroids during adulthood. The majority 

of such investigations have been carried out in the rat, 

but similar results have been described in other species (345, 

414). Breifly, studies on sexual behavior in the rat have 

shown that 1) castration on the day of birth results in a 

feminized adult male that exhibits a female pattern of behavioral 

responses when treated with estradiol and progesterone, 

and 2) neonatal testosterone or estradiol treatment of 

a female results in a masculinized adult that exhibits a malelike 

pattern of behaviors and is refractory to estradiol and 

progesterone (131, 337). The culmination of the data collected 

from such experimental schemes has led to the conclusion 

that testosterone secreted from the perinatal testes during a 

critical developmental window results in permanent changes 

in the hypothalamic nuclei of the brain that mediate male 

sexual behavior. However, the data indicating that developmental 

exposure to estradiol results in an adult phenotype 

that is similar to that elicited by testosterone suggest that 

many of the masculinizing effects of perinatal testosterone 

may be via local aromatization of the hormone to estradiol 

and subsequent activation of the ER signaling pathway (reviewed 

in Refs. 406 and 407). In addition, estradiol is also 

necessary for normal development of the female brain, although 

in lower amounts (131). Therefore, sex steroid-mediated 

sexual differentiation of the various regions of the 

brain that are critical to behavior relies not only on the nature 

of the steroid ligand, but also on the dose and timing of 

exposure (348). 

Before the availability of the �ERKO mouse, McCarthy et 

al. (415) employed an elaborate technique of infusing anti- 

ER� oligodeoxynucleotides into the neonatal rat hypothalamus 

to elucidate a direct role for ER� in the sexual differentiation 

of the female brain. This experimental scheme was 

based on the hypothesis that the presence of specific ER� 

antisense oligodeoxynucleotides in the hypothalamus would 

interfere with proper expression of the ER� gene during a 

critical period of differentiation (415). The experimental 

groups included neonatal rats treated with testosterone plus 

or minus the infusion of the ER� antisense oligodeoxynucleotides. 

As adults, those females infused with the ER� antisense 

oligodeoxynucleotides exhibited more female sexual 

behavior compared with those treated with androgen alone. 

The investigators thereby concluded that the reduced ER� 

expression protected the infused female rats from the masculinizing 

effects of testosterone exposure (415), providing 

strong evidence that local aromatization and subsequent estradiol 

activation of the ER� pathway plays a primary role 

in the masculinization of the rat brain. However, the experimental 

scheme of McCarthy et al. does not allow for a direct 

comparison with the �ERKO female, due to the caveats discussed 

(see Section II.C.1). It is important to recognize that the 

�ERKO are deficient in ER� throughout development, 

whereas McCarthy’s scheme produced a lack of ER� action 

that was only transient and most likely not as complete. 

The use of technologies to target individual genes has 

created numerous models available for studies in the behav-


ioral sciences. A recent review by Nelson and Young (128) 

summarized and compared the behavioral or lack of behavioral 

phenotypes in a select 50 murine knockout models. Due 

to the lack of any grossly apparent behavioral phenotypes in 

the �ERKO mice, only those studies concerning the �ERKO 

will be discussed here. 

1. �ERKO female. The dependence of female sexual behavior 

on the synchronized fluctuations in estradiol and progesterone 

that occur during the ovarian cycle have been described 

in detail (reviewed in Ref. 416). In the rodent, circulating 

estrogens continue to rise as ovulation approaches, eventually 

leading to the gonadotropin surge that not only triggers 

the release of the oocyte from the ovary but also a marked 

increase in serum progesterone. This dramatic rise in circulating 

progesterone is required for an optimal display of the 

lordosis posture, a measurable response required for successful 

copulation (416). The gonadotropin surge from the 

hypothalamic-pituitary axis is due to the postive-feedback 

actions of estradiol. The development of this pathway in the 

rodent is unique to the female as a result of sexual differentiation 

of the neurons in the anteroventral periventricular 

nucleus of the preoptic area that serve to regulate hypothalamic 

function (264, 365, 405). As discussed above, feminization 

of the brain involves the actions of estradiol during 

fetal and neonatal development that may rely heavily on the 

dose and time of exposure. Therefore, knockout models for 

ER� and ER�, as well as the PR, have and will continue to 

serve as invaluable resources for dissecting the role of each 

receptor-signaling system in female sexual behavior. 

In general, evaluation of the aberrant sexual behaviors of 

the �ERKO female must consider not only the absence of ER� 

signaling, but also the elevated levels of serum testosterone 

that exist in the adult female (Table 2). Despite the presence 

of the hormones presumably required for sexual behavior, 

intact adult �ERKO females exhibit behavior that resembles 

that of a male in terms of parental, aggressive, and sexual 

activities (417, 418). When placed in the presence of a stud 

male, �ERKO females display a complete lack of sexual receptivity, 

measured as prelordotic behavior and a lordosis 

posture (418). In fact, intact �ERKO females were often 

treated as intruders and attacked by the stud male (417). 

However, similar studies using ovariectomized females indicate 

that this behavior of the stud male was most likely 

elicited by the significantly elevated levels of circulating testosterone 

in the �ERKO female (418, 426). These same studies, 

employing ovariectomized females coupled with steroid 

replacement of varied combinations, illustrated a complete 

resistance to estradiol (418, 419) and a minimal effect of 

progesterone in inducing a lordosis response in the �ERKO 

female (418). 

Although the above studies have indicated a prominent 

role for ER� in sexual behavior in the mouse, the precise 

pathways disrupted by a lack of ER� remain unclear. Not 

surprisingly, the PRKO female mice also exhibit a lack of 

normal sexual behavior and are unable to produce a lordosis 

posture even when treated with doses of either estradiol 

and/or progesterone (44). However, this same study reported 

an inability of estradiol alone to induce lordosis but 

rather a requirement for both estradiol and progesterone in 

wild-type females (44). This is in contrast to the capacity of 

estradiol to solely induce lordosis in the wild-type controls 

employed by Rissman et al. (419) and may be a reflection of 

the differences in background strain and/or experimental 

design employed in the two studies. 

The �ERKO and PRKO models nicely illustrate a requirement 

for both estradiol and progesterone action for a full 

lordotic response. As early as 1939, estrogen exposure before 

progesterone treatment was found to be required for a full 

display of sexual behavior in the rat (420). As in the uterus, 

the expression and induction of PR in certain regions of the 

brain is under estrogen regulation. Studies have indicated 

that estradiol elicits detectable increases in PR in the hypothalamus, 

strongly suggesting that this estrogen-action is 

required for ultimate progesterone-induced sexual behaviors 

(346, 421, 422). Therefore, disruption of the ER� gene may be 

expected to cause abnormally low levels of PR expression in 

those areas of the brain that mediate female sexual behavior 

and therefore may explain the lack of such behavior in the 

�ERKO mouse. However, two separate reports have described 

the ability of estradiol to induce increases in PR 

transcripts in the forebrain of the �ERKO female mouse, 

including regions of the preoptic area (400) (Fig. 8) arcuate 

nucleus, caudal ventromedial hypothalamus, and posterodorsal 

medial hypothalamus (423). However, the extent 

of estrogen-induced increases in hypothalamic PR in the 

�ERKO female is slightly attenuated compared with that 

observed in similarly treated wild-type mice (400, 423). It is 

possible that this observed estrogen action in the �ERKO 

female is either mediated by a splicing variant of the disrupted 

ER� gene or by ER�. Regardless, the level of estrogeninduced 

PR in the �ERKO female does not appear to allow 

for a response to progesterone that is sufficient to elicit sexual 

behavior. These data support the conclusion that normal 

expression of female sex behavior requires the sequential 

activation of the ER�- and PR-signaling pathways. 

Significant deficits in parental behavior and a greater tendency 

toward infanticide is also observed in �ERKO females 

compared with wild-type littermates (418). These phenotypes 

do not dramatically differ between intact and ovariectomized 

�ERKO females; however, levels of infanticide 

were reduced in tests carried out after a prolonged postgonadectomy 

period (65 days) (418). The �ERKO female 

exhibits aggressive behaviors that are significantly elevated 

compared with the wild-type female littermates, and in stark 

contrast to the dramatically reduced aggressive behavior 

displayed by the �ERKO male (to be discussed below) (418). 

Remarkably, acute treatment of ovariectomized females with 

estradiol resulted in the expected reduction in aggressive 

behavior in both the wild-type and �ERKO females (418). 

Previous studies have shown that whereas both testosterone 

and estradiol can elicit aggressive behaviors in the castrated 

male mouse, only testosterone is effective in the ovariectomized 

female mouse (424, 425). These studies, in combination 

with the findings in the �ERKO, indicate that differentiation 

as well as activation of aggressive behaviors in the 

female mouse are testosterone dependent. However, the preserved 

ability of estradiol to reduce aggression in the ovariectomized 

�ERKO female is puzzling and may indicate an 

ER�-mediated pathway.


The elevated levels of infanticide and aggressive behavior 

exhibited by the �ERKO females may be contributed to by 

the elevated levels of testosterone secreted by the acyclic 

ovary (Table 2). As discussed earlier, experimental evidence 

suggests that disruption of the ER� gene has resulted in a 

hypothalamic-pituitary axis with an enhanced capacity to 

respond to androgens in the �ERKO male. In support of this 

possibility, Ogawa et al. (418, 426) reported their preliminary 

finding of increased androgen receptor levels in the brain of 

the �ERKO female as early as 12 days of age. 

2. �ERKO male. Given the apparent role of ER�-mediated 

estrogen actions in the masculinization of the brain, it was 

expected that the �ERKO males would exhibit a female-like 

behavioral phenotype. Surprisingly, however, Ogawa et al. 

(318) observed that a lack of hypothalamic ER� during development 

has little effect on the sexual behavior of the intact 

�ERKO male in terms of mounting and sexual attraction 

toward wild-type females. In contrast, Wersinger et al. (427) 

report that tests of male sexual behavior carried out in a 

neutral arena, as opposed to the male’s home cage as done 

in the study of Ogawa et al., demonstrates that the number 

of mounting attempts exhibited by the �ERKO males is reduced. 

Interestingly, the studies of Ogawa et al. illustrated 

that �ERKO males, however, exhibit an almost complete lack 

of intromission and ejaculation, even though the number and 

frequency of mounts were similar to those of wild-type males 

(318). Furthermore, treatment of castrated males with estradiol 

or the nonaromatizable androgen, DHT, resulted in no 

differences in sexual behavior compared with the findings in 

the intact �ERKO males (428). These results differ from those 

described by Ono et al. (429) and Olsen (131) in the androgeninsensitive 

Tfm mouse, which exhibits no male-like sexual 

behavior including a lack of mounting as well as intromission 

and ejaculation. However, the �ERKO and Tfm males are 

similar in terms of exhibiting complete insensitivity to the 

effects of both estradiol and testosterone as behavioral activators 

during adulthood. 

The �ERKO male behavioral phenotype described above 

is obviously a contributing factor to the infertility that results 

after disruption of the ER� gene. The culmination of the 

studies indicate that a discrete component of sexual behavior 

in the male mouse, i.e., consummatory activity, is dependent 

on the actions of ER�, whereas testosterone or possibly ER�mediated 

estradiol actions may regulate motivational aspects 

(428). Although reports of categorical studies on sexual 

behavior are not available, both the �ERKO (47) and ArKO 

(257) male mice appear to be fertile and able to sire multiple 

litters, suggesting a minor role for ER� in sexual behavior. 

The possibility of compensatory mechanisms mediated by 

ER� in the �ERKO cannot be ruled out. It is interesting, 

however, that the ArKO males, presumably lacking physiological 

levels of estradiol throughout life, show no obvious 

deficits in sexual behavior that result in infertility (257). It 

might be expected, given the apparent need of ER�-mediated 

estrogen actions illustrated by the �ERKO, that the ArKO 

male would display a similar phenotype. Perhaps, exposure 

to maternal steroid hormones during gestation in the ArKO 

mouse has allowed for the proper “organization” of the 

neuronal circuitry regulating sexual behavior. More detailed 

studies may elucidate subtle behavioral phenotypes that exist 

in both the �ERKO and ArKO models and will help 

further define the precise role that estradiol and testosterone 

play in the regulation of sexual behavior. 

A dichotomy similar to that observed for the elements of 

male sexual behavior in the �ERKO is observed when behavioral 

assays for aggression and parental instincts are considered. 

Intact �ERKO males demonstrate a relatively normal 

pattern of parental behavior as measured by levels of 

infanticide when placed in the presence of newborn pups 

(428). However, despite the fact that �ERKO males possess 

serum testosterone levels that exceed the norm by as much 

as 2-fold and show no reduction in the levels of AR (430) or 

ER� (93, 352) in the brain, they consistently exhibit a significant 

deficit in all male aggression indices tested (428, 430). 

Therefore, as in the case of certain components of sexual 

behavior, ER�-mediated actions appear critical to the development 

and/or activation of aggressive behaviors, whereas 

parental instincts appear to be independent of ER� action 

(428). 

VII. Phenotypes in Peripheral Tissues 

The ER and the estrogen-signaling pathway have been 

described in several peripheral organ systems (reviewed in 

Ref. 431). A discussion of the phenotypes that may occur in 

each of these after disruption of either of the ER genes is 

beyond the scope of this review. Therefore, we have chosen 

to focus on three areas, all of which have received great 

attention as sites of estrogen action that are critical to human 

health. These are the bone, cardiovascular system, and adipogenesis. 

Furthermore, studies toward specifically defining 

a role that ER may play in mediating the actions of 

estrogen in each of these systems have begun to employ the 

�ERKO, and eventually the �ERKO. 

A. Skeletal system 

The link between the onset of osteoporosis and the decreasing 

estrogen levels associated with menopause has been 

realized since the report of Albright et al. in 1941 (432). Postmenopausal 

estrogen replacement therapy is currently the 

most commonly prescribed drug treatment in the United 

States (433, 434). In most patients, the increased risks associated 

with long-term estrogen replacement therapy, such as 

breast and endometrial cancer, are strongly overshadowed 

by the well established reduction in the risk of osteoporosis 

and bone fracture (433). Bone is a dynamic tissue that is 

constantly being resorbed to serve as a mineral source for the 

body and remodeled to replace this reservoir as well as 

maintain skeletal strength. Osteoporosis is a defined pathology 

characterized by a loss in bone mass and strength and 

is believed to be due to a disruption in the equilibrium 

between bone resorption and formation (435). Current evidence 

supports the hypothesis that excess bone resorption 

occurs in the postmenopausal years, acting to strip the bone 

of mass and further remove the foundation upon which new 

bone may be formed (435). Several therapies are known to 

reduce the postmenopausal increases in bone resorption, 

including the intake of calcium and vitamin D, calcitonin,


bisphosphates, and estrogens (435). The obvious beneficial 

effects of estrogen replacement therapy have evoked an intense 

research effort for bone-specific estrogen agonists that 

lack the potentially harmful side effects in the breast and 

reproductive tissues. Ironically, the currently available “selective 

ER modulators” or SERMs, as these drugs have come 

to be termed, have been selected from the pool of nonsteroidal 

estrogen antagonists (reviewed in Refs. 8, 434, and 

436). 

For several years it was believed that the effects of estrogens 

on bone physiology were indirect, inferred from the 

inability to detect ER within bone and bone cell cultures. 

However, in 1988, Komm et al. (437) and Eriksen et al. (438) 

simultaneously reported the detection of high-affinity, competable 

estradiol binding, ER� mRNA, and the induction of 

estrogen-responsive genes in cultured rat and human osteoblast-like 

cells, the bone-forming cell. We have reported similar 

findings, including the inhibition of estradiol transactivational 

activity with antiestrogens, in two separate 

osteoblast-like cell lines from the rat (439). Oursler et al. (440) 

have since demonstrated ER� and estrogenic activity in cultured 

avian osteoclasts, the bone-resorbing cell type, including 

estrogen induction of the genes for c-fos and c-jun. Using 

in situ RT-PCR analysis, Hoyland et al. (441) demonstrated 

the presence of ER� mRNA in both osteoblasts and osteoclasts 

in bone grafts from human females. Immunocytochemical 

methods have also been used to demonstrate the 

presence of ER� in multiple bone cell lines (442). Recently, 

Bodine et al. (443) reported significant increases in the levels 

of ER� transcripts during dexamethasone-induced differentiation 

of rat osteoblasts in vitro. Therefore, there is adequate 

experimental evidence to support the presence of a direct 

ER�-mediated estrogen-signaling pathway in bone. 

The discovery of the ER� introduced renewed vigor in the 

search for SERMs, allowing for the greater possibility of 

finding a receptor-selective agonist. The distinct expression 

pattern of the two ERs among various tissues has further 

enhanced the possibility of finding tissue-specific SERMs. 

Several recent studies have reported the detection of ER� in 

bone cells. Onoe et al. (444) employed RT-PCR to demonstrate 

the presence of both ER� and ER� mRNA in immortalized 

as well as primary osteoblast cell cultures from the rat. 

Similar to reports of ER�, both Onoe et al. (444) and Arts et 

al. (95) report significant increases in ER� mRNA levels during 

in vitro dexamethasone-induced differentiation of osteoblasts 

derived from the rat and human, respectively. Therefore, 

the generation of mice lacking ER� or ER� will once 

again prove invaluable in delineating the roles of the two 

receptors in bone physiology. 

The majority of animal studies concerning the role of estrogens 

in bone morphology and metabolism have been carried 

out in the rat (reviewed in Ref. 445). Ovariectomy in the 

rodent results in increased bone turnover similar to that seen 

in postmenopausal women; however, the mechanisms of 

action may differ between the species (445). The effects of 

ovariectomy in the rat include decreases in bone mineral 

density, cancellous bone area, and bone strength, whereas 

increases are observed in radial and longitudinal growth, 

osteoblast and osteoclast activity, and overall rates of bone 

turnover (445). Estrogen replacement, including those com- 

pounds with mixed agonist/antagonist activity, has been 

shown to reverse several of the effects induced by ovariectomy 

(445). However, the extent and direction of the changes 

induced by ovariectomy, as well as the protection provided 

by estrogen replacement, vary depending on the bone parameter, 

sex, and type of bone being evaluated, e.g., femur, 

tibia, calvaria, or vertebrae (445). Interestingly, a study of the 

androgen-resistant Tfm rat describes a bone phenotype similar 

to a wild-type female, i.e., shorter and thinner femurs, 

indicating that androgen action may also be critical to longitudinal 

and radial bone growth in the male rat (446). However, 

endogenous gonadal estrogens were able to maintain 

a normal cancellous bone mass in the Tfm rat (446). It is 

noteworthy that the first description of a human case of 

estrogen insensitivity due to a spontaneous mutation of the 

ER� gene exhibits severe osteoporosis as well as significant 

increases in longitudinal growth of bones (see Section VIII.C) 

(116). 

Unfortunately, few studies of the effects of steroids on 

bone physiology have been carried out in the mouse. Analysis 

of femoral bone length in �ERKO mice indicates a significant 

decrease in length and diameter in females and a 

slight decrease in males, when compared with age- and sexmatched 

wild-type controls (447). However, measurements 

of bone density and mineral content indicate the opposite 

effect, i.e., �ERKO males exhibited significant decreases 

throughout the femur (448), whereas the �ERKO females 

demonstrate just slight and localized decreases (447). In 

agreement with the ovariectomized rat model, �ERKO female 

mice exhibit increased bone resorption-remodeling 

rates (448). However, the decreased femur length observed 

in the �ERKO is in contrast to that reported in the ovariectomized 

rat and the ER�-deficient human male. Interestingly, 

a series of studies by Migliaccio et al. (449, 450) illustrated 

that prenatal and neonatal exposure to the synthetic 

estrogen, DES, also results in significantly shorter femur 

lengths as well as increased cortical bone thickness and increased 

trabecular bone at the epiphysis of the femur during 

adulthood in female mice. Therefore, it appears that in the 

mouse, aberrant estrogen exposure during development or 

a hereditary loss of ER� action leads to decreased longitudinal 

bone growth, contrasting experimental schemes resulting 

in a similar phenotype. 

These data indicate that pathways other than ER� may 

mediate the negative regulatory effects of estradiol on bone 

growth, suggesting a possible role for ER�. Longitudinal 

bone growth is a poorly understood process that depends on 

chondrocyte activity, including proliferation, hypertrophy, 

and the secretion of extracellular matrix at the growth plate 

(445). Estrogen is thought to slow this process by reducing 

the recruitment, proliferation, and synthetic activity of chondrocytes, 

thereby resulting in a maturation of the epiphyseal 

plate and inhibition of further longitudinal growth (445). The 

detection of both ER� (451) and ER� (452) in human epiphyseal 

chondrocytes has recently been described. Therefore, it 

is possible that ER�, in the context of significantly elevated 

levels of estradiol, results in an inhibition of long bone 

growth in the �ERKO mouse. It is also possible that the 

significantly elevated levels of serum androgens in the 

�ERKO female may be playing an influential role. This may


also be true in the �ERKO male, which exhibit slightly 

shorter femur lengths in the context of only a 2-fold increase 

in serum androgens. The possibility that a loss of ER� during 

bone development results in abnormal genomic imprinting 

and increased ER� and/or AR levels as a compensatory 

mechanism must also be considered. 

B. Cardiovascular system 

Since the early part of this century, a remarkable genderrelated 

contrast in the risk of cardiovascular disease has been 

known. Although women generally possess a greater incidence 

of the multiple risk factors associated with cardiovascular 

disease when compared with men, e.g., obesity, diabetes, 

elevated blood pressure, and plasma cholesterol, 

epidemiological studies continue to indicate their relative 

risk of developing this disease is significantly lower (453, 

454). It is now believed that the protective factor against 

cardiovascular disease in females is their inherently increased 

exposure to estrogens (reviewed in Refs. 433 and 

454). The protective effects of estrogens have been documented 

in a number of epidemiological studies documenting 

the reduced rate of cardiovascular disease in postmenopausal 

women receiving estrogen replacement therapy (433, 

454). This correlation between the administration of estradiol 

and a reduced risk of vascular disease has been reproduced 

in laboratory animal studies involving several different species 

(454). 

The possible mechanisms by which estrogens reduce vascular 

disease remain unclear but are likely to include positive 

modifications of a number of physiological parameters that 

are believed to impinge upon the development of cardiovascular 

pathlogy (reviewed in Ref. 454). Most notable of 

these is the ability of estrogen replacement therapy to significantly 

lower total cholesterol, with a profound effect on 

levels of the more damaging low-density lipoproteins (LDLs) 

(454). Hypercholesterolemia is strongly associated with the 

development of cardiovascular disease. The ability of estrogen 

therapy to reduce total cholesterol levels is believed to 

account for a large portion of the cardiovascular protective 

effects of the hormone (433, 454). Lundeen et al. (455) reported 

that the significant decreases in serum cholesterol are 

specific to estrogen action in the ovariectomized rat, whereas 

other gonadal steroids tested had little effect. Furthermore, 

cotreatment with the antagonist, ICI-182,780, was shown to 

inhibit the cholesterol-lowering effects of estrogens, strongly 

indicating that this is a receptor-mediated effect (455). One 

possible site at which the actions of estradiol may converge 

with the pathways of cholesterol metabolism is via the upregulation 

of the apo E protein and the apolipoprotein (apo) 

B/E LDL receptor within and on the cell surface of hepatocytes, 

respectively (454). During hydrolysis of triglycerides 

in the circulation, the apo E protein is integrated into the apo 

B-LDL complexes and thereby functions as a ligand for the 

hepatocyte apo B/E LDL receptor (454). When the apo Econtaining 

LDL complex is bound by the apo B/E LDL receptor 

on the cell surface of hepatocytes, the complex is 

internalized, thereby providing for a means of clearing LDL 

from the blood (454). The importance of the apo E protein in 

maintaining serum cholesterol levels and providing protec- 

tion against vascular disease is evident from studies in transgenic 

mice that either overexpress the protein or possess a 

targeted disruption of the apo E gene. Transgenic mice in 

which an apo E gene is overexpressed are significantly protected 

from atherosclerosis (456), whereas the apo E knockout 

is highly susceptible to the vascular disease (457). 

Therefore, much of the cholesterol-lowering ability of estradiol 

is thought to be due to increased clearance of LDL 

from the circulation via the mechanism described above 

(454). Regulation of the apo E gene by estradiol has been 

demonstrated in the rat (458, 459). Furthermore, Srivastava 

et al. (460) recently reported that hepatic levels of apo E 

mRNA are similar in wild-type and �ERKO male littermates, 

although a slight decrease in serum levels of the protein was 

observed in the �ERKO. However, 6 days of estradiol exposure 

(via implantation of a 3 �g E 2/g body weight/day 

pellet) elicited an almost 2-fold increase in serum apo E levels 

in the wild-type compared with a 1.2-fold increase in the 

�ERKO (460). Therefore, these data provide evidence that 

ER�, acting at the level of translation, is required to mediate 

the estradiol up-regulation of apo E expression in mice (460). 

In addition to the favorable effects of estrogens on the lipid 

profile, it is now believed the steroid may also play a beneficial 

function directly at the level of the vasculature. The 

proposed mechanisms of estrogen action on the vasculature 

include modulations of vascular adhesion molecules, chemoattractants, 

vasodilators (e.g., nitric oxide), vasoconstrictors 

(e.g., endothelin-1), as well as possibly acting as an antioxidant 

(reviewed in Refs. 454 and 461). Several of the 

effects of estrogens, as well as other steroid hormones, on 

blood vessel physiology have been proposed to be nongenomic 

and independent of the classical nuclear activational 

pathway of steroid receptors (reviewed in Ref. 355). 

However, ER has been detected in both the endothelial and 

smooth muscle cells of the vasculature in several species, 

suggesting a role for receptor-mediated actions as well (reviewed 

in Refs. 431, 454, and 462). Furthermore, a recent 

study reported that the level of ER in atherosclerotic plaques 

from human coronary arteries was reduced compared with 

levels found in nonlesioned sections (463). Studies have indicated 

the presence of ER� in the vasculature as well. Analysis 

by ribonuclease protection assay for ER� and ER� 

mRNA in the mouse indicate only detectable levels of ER� 

transcripts in the aorta (93). However, Iafrati et al. (464) report 

the detection of ER� transcripts by RT-PCR in mouse aorta 

and blood vessels, including those of the �ERKO. The apparent 

contrast in ER� detection between these two reports 

in the �ERKO vasculature is most likely due to differences 

in the sensitivity of the techniques used, since ER� mRNA 

was detected only by the more sensitive RT-PCR method. 

This is likely a reflection of the low levels of this receptor 

compared with ER�. In the rat aorta, Petersen et al. (70) 

described the detection of full-length and variant ER� 

mRNA by RT-PCR. Recent reports also describe the detection 

of ER� transcripts by RT-PCR in aortic smooth muscle cells 

and coronary artery (465), aorta, and cardiac muscle (96) 

from monkey. An intriguing report of the tissue distribution 

for ER� and ER� mRNA in human vasculature was that of 

Tschugguel et al., which described the presence of ER� 

mRNA only in cultures from larger blood vessels, i.e., aorta


and pulmonary artery, whereas ER� transcripts were predominant 

in cultures from the endothelium of smaller vessels, 

such as those from the uterus (466). Therefore, with the 

apparent variations that exist in the distribution of the two 

forms of ER in the vasculature, depending on the species and 

possibly even the vessel of study, the ERKO mice provide a 

unique tool to discern the direct role that ER� and/or ER� 

may play on vascular biology. 

One of the initial studies of the vasculature in the ERKO 

mice was that by Rubanyi et al. (467) in which the level of the 

vasodilator, nitric oxide, was characterized in the aortic rings 

of the �ERKO male. This study demonstrated that the male 

C57BL/6J mice, the background strain of the �ERKO, possess 

a significantly greater amount of ER in the aorta than 

female littermates, when quantified by radiolabeled E 2 binding 

(467). Furthermore, the basal levels of endothelial-derived 

nitric oxide were also found to be significantly higher 

in the male tissue compared with female, suggesting that the 

level of ER, rather than the amount of ligand, may be the most 

critical parameter in modulating endothelial nitric oxide production 

(467). In agreement with this hypothesis was the 

observation of significantly reduced basal nitric oxide levels 

in aortic tissue from male �ERKO mice (467). However, the 

levels of stimulated vasodilatation achieved after treatment 

with acetylcholine to induce endogenous nitric oxide release 

or nitroglycerin, a nitric oxide donor, did not differ between 

the sexes or ER� genotypes (467). Therefore, it appeared that 

only the ability to synthesize basal nitric oxide levels was 

altered by ER� gene disruption, whereas the capability of the 

vasculature to respond to the vasodilator effects of nitric 

oxide were not altered (467). Interestingly, the susceptibility 

to hypercholesterolemia-induced atherosclerosis in this 

strain of mice is in direct correlation with the level of ER 

detected, i.e., male C57BL/6J mice appear less likely to develop 

the vascular disease than female counterparts (468). 

Therefore, it may be speculated that the �ERKO mice possess 

an inherent susceptibility to vascular disease as well. These 

data also indicate that a contributing factor to the protective 

effects of estrogen action on the vasculature may be caused 

by a convergence of the estrogen and nitric oxide systems 

and may, in fact, be due to ligand-independent ER� actions. 

In contrast to the above description of a measurable vascular 

defect in the �ERKO mice, Mendelsohn et al. (464) 

demonstrated no apparent difference in the response between 

wild-type and �ERKO female mice in a carotid vascular 

injury model. This model involves the monitoring of 

endothelial and smooth muscle cell growth and proliferation 

that is spontaneously elicited in carotid arteries after artificial 

endothelial denudation (469). This response has been shown 

to be inhibited by estradiol, suggesting a mechanism by 

which estrogens may protect the vessel from atherosclerosis 

(462). Removal of estrogen action via ovariectomy in wildtype 

and �ERKO mice resulted in similar levels of endothelial 

and smooth muscle cell proliferation 2 weeks after carotid 

injury, as measured both morphometrically as well as with 

BrdU incorporation (464). However, daily estradiol treatments 

commencing 1 week before injury and continued for 

the 2 weeks after were able to inhibit the cellular proliferation 

to similar levels in both the wild-type and �ERKO animals 

(464). Therefore, the “protective” effect of estradiol illus- 

trated by this model system appears to be independent of 

ER� action and may possibly be mediated by ER�. Similar 

studies in the �ERKO mice are currently underway and will 

prove interesting in this regard. 

Estrogens may also exert direct effects at the level of the 

heart and have been proposed to play an important role in 

cardiac hypertrophy and remodeling after myocardial infarction 

(462, 470). In a recent report, Grohé et al. (471) described 

the presence of both ER� and ER� as well as the 

P450 arom enzyme in cardiac myocytes cultured from neonatal 

rats. This same study demonstrated that androgen-induced 

up-regulation of ER� was evident in cardiac myocytes derived 

from female rats, whereas ER� levels were unaffected 

and remained stable in the cells from both sexes (471). Furthermore, 

estrogens may modulate cardiac contractility via 

regulation of Ca 2� channel activity. Previous studies have 

indicated that estrogen may reduce Ca 2� channel activity in 

several tissues; however, demonstrative studies in the heart 

usually involved superphysiological doses and therefore remain 

an issue of controversy (355, 472). However, Johnson et 

al. (472) demonstrated an increased number of L-type Ca 2� 

channels in �ERKO male heart, presumably due to a hereditary 

loss of ER�. Whole hearts from �ERKO males exhibited 

an approximate 46% increase in the number of L-type Ca 2� 

channels and a corresponding delay in cardiac repolarization 

when compared with those from wild-type (472). Therefore, 

estradiol ER�-mediated actions appear to modulate cardiac 

contractility via regulation of the number of calcium channels, 

possibly providing another function to complement the 

protective effects of estrogens in the cardiovascular system. 

C. Adipogenesis 

A role for gonadal steroid action in adipogenesis and adipocyte 

function has been realized for some time (473). This is 

evidenced by the well known gender differences that exist in the 

distribution of white adipose tissue in humans. Whereas men 

tend to accumulate fat stores in the thoracic and abdominal 

regions, accumulations in women are found in the upper portions 

of the arms and legs. Furthermore, obese woman that also 

exhibit androgen excess demonstrate a male pattern of fat distribution, 

termed android adiposity (474). Supporting evidence 

for a functional relationship between the gonadal and adipogenesis 

systems is also provided by the direct correlation that 

exists between nutrition, body mass, and fat content with the 

onset of menarche in young females (475). In addition, white 

adipose tissue serves not only as an energy reservoir, but is also 

a site of steroid storage and metabolism that can supplement 

gonadal and adrenal synthesis and thereby influence the overall 

endocrine milieu (292, 473). 

The roles of several members of the nuclear hormone 

receptor superfamily in adipogenesis have been intensely 

researched, especially the glucocorticoid receptor, peroxisome 

proliferator-activated receptor, retinoic acid receptor, 

and the thyroid receptor (reviewed in Ref. 473). There is little 

research data concerning any direct role that ER may play in 

adipocyte function and distribution, but it is well known that 

ovariectomy results in increased fat stores and body weight 

in females of some strains of rodents (197, 476, 477). This 

effect can be prevented with estrogen replacement as well as


reproduced with prolonged antiestrogen treatments in intact 

females (197, 476, 477). Interestingly, a similar increased 

body weight is observed in �ERKO females of the C57/BL 

background. Gross observations upon necropsy of adult 

�ERKO females indicate an obvious increase in the amount 

of white adipose fat in the pads of the mammary gland and 

those lining the lateral-ventral portions of the body cavity. As 

early as 4 months of age, �ERKO females exhibit an increased 

body weight compared with female wild-type littermates, at 

28.7 g (�0.91) vs. 23.3 g (�0.43), respectively. This difference 

in body weight between wild-type and �ERKO females increases 

at 8 months of age, at which time the average weight 

of �ERKO females exceeds that of age-matched wild-types 

by almost 35% (t test; P � 0.01). However, by 12 months of 

age, increases in the body weight of wild-type females appear 

to decrease the gap between the two genotypes. 

The fact that the increased fat stores in the �ERKO female 

are similar to those observed in the classical ovariectomized 

model indicate that a loss of ER�-mediated estrogen action 

may alter metabolism and adipocyte physiology. Fisher et al. 

(257) reported a similar phenotype in the ArKO female mice, 

in which the weight of the gonadal and mammary fat pads 

were increased by 50–80%. In both �ERKO and ArKO females, 

serum testosterone levels are substantially elevated 

but do not appear to have a lessening effect on fat pad weight 

(257). Furthermore, there are no reports of increased body 

weight in the androgen-resistant Tfm mice (478), indicating 

that androgen action may play a lesser role than estrogen in 

fat storage. It is unknown at this time whether a phenotype 

similar to the �ERKO and ArKO mice may also exist in the 

�ERKO mice. However, sexually mature �ERKO mice of 

both sexes exhibit no significant differences in body weight 

and appear to possess a relatively normal distribution of 

white adipose tissue in the peritoneum at the ages examined. 

Although the above evidence strongly supports a role for 

ER�-mediated estrogen action in adipocyte physiology, the 

mechanisms involved remain unclear. Adipose tissue has been 

shown to possess ER� (479, 480, 486–488) and the enzymes 

necessary for estradiol synthesis (292, 480). Recent studies have 

also indicated that the gonadal sex steroids can alter the activity 

of lipoprotein lipase, a critical enzyme in adipocyte growth and 

fat storage (474). Interestingly, Lubahn et al. (481) reported that 

�ERKO female mice fed a diet enriched with the phytoestrogen, 

genistein, exhibited a reduced body weight compared with 

�ERKO females fed the control diet. This may indicate a non- 

ER�-mediated yet estrogenic action of genistein, a naturally 

occurring isoflavone shown to possess hormone-like actions in 

mammalian cells that are devoid of ER (482). Therefore, the 

ER�-independent actions of genistein, such as the inhibition of 

protein tyrosine kinases and influence on growth factor action, 

complicate the interpretation of the above study in terms of 

defining a role for ER� in fat stores. 

VIII. Comparison with Human Disease and Models of 

Deficient Estrogen Action 

A. Ovarian carcinogenesis 

Ovarian cancer is the leading cause of death from gynecological 

cancers and accounts for 5% of all cancer deaths in 

Western countries (483). However, the etiology of ovarian 

carcinoma is complicated by paradoxes similar to those concerning 

breast cancer, i.e., although risk is significantly reduced 

with each pregnancy, the use of oral steroid contraceptives 

also appears to reduce the risk (483). Approximately 

80–90% of human ovarian cancers are derived from the ovarian 

surface epithelium (484), a portion of the ovary known to 

be rich in ER� expression. Although the causal factors remain 

unclear, evidence indicates that incessant ovulation, resulting 

in constant rupture and estrogen-mediated repair of the 

surface epithelium, increases the probability of spontaneous 

genetic abnormalities that may lead to tumorigenesis (483, 

484). The exact role that estrogen and the ER may play in the 

induction and promotion of ovarian carcinoma remains unclear 

(reviewed in Refs. 483–485). A number of immortalized 

cell lines have been generated and characterized from human 

ovarian tumors and exhibit varied levels and responses to 

estrogen agonists and antagonists (reviewed in Ref. 484). 

Brandenberger et al. (94) recently reported the detection of 

ER� and ER� mRNA in the human ovary, ovarian tumors, 

and ovarian tumor cell lines. Their findings include the description 

of ER� mRNA predominantly in the granulosa cells 

of normal ovaries and a marked reduction in the levels of 

ER� transcripts in ovarian carcinomas (94). In contrast, a cell 

line derived from a human ovarian surface epithelium and 

several human ovarian carcinomas were reported to express 

high levels of ER� mRNA (94). As mentioned previously, 

ovarian tumors are most often derived from the outer surface 

epithelium, whereas granulosa cell-derived carcinoma in humans 

is rare. Interestingly, we observe an approximately 40% 

incidence of granulosa/thecal cell tumors of the ovary in 

�ERKO females between the ages of 15–20 months. No such 

ovarian tumors have been observed in the wild-type or heterozygous 

littermates of the �ERKO. A similar incidence and 

type of spontaneous tumor is reported in transgenic mice 

possessing significantly elevated levels of LH caused by 

overexpression of the LH� subunit gene (254, 255). Therefore, 

hypergonadotropin stimulation appears to play a significant 

role in the etiology of this type of ovarian tumor in 

the mouse. The similarities in the incidence and type of 

tumor observed between the �ERKO and bLH�-CTP mouse 

indicate that ER� probably plays a minor role. Preliminary 

analysis in tumor samples for the �ERKO females indicates 

normal to elevated levels of ER� expression. Therefore, elevated 

gonadotropins and estradiol may result in chronic 

induction of the granulosa cells to proliferate and is the likely 

stimulus for the ovarian tumors observed in both transgenic 

models. Future investigations to determine the incidence of 

tumors in the �ERKO ovary will prove useful in further 

elucidating the etiology of this neoplasia. 

B. Chronic anovulation 

Targeted gene disruption of the ER genes has resulted in 

partial and complete anovulation in the �ERKO and �ERKO 

females, respectively. In the clinical setting, chronic anovulation 

is often categorized by the presence or lack of estrogen 

synthesis (204). Chronic anovulation in the absence of estrogen 

is diagnosed in women who experience little to no 

menstrual bleeding after progesterone withdrawal and is


most often associated with hypogonadotropism resulting 

from impaired function of the hypothalamic GnRH neurons 

or disorders of the pituitary (204). Chronic anovulation in the 

presence of estrogen synthesis can be caused by several different 

functional abnormalities, such as Cushing’s syndrome, 

hyper/hypothyroidism, adrenal hyperplasia, and 

ovarian tumors (204). However, the majority of cases of 

anovulation in the presence of estrogen are associated with 

polycystic ovarian syndrome (PCOS), a heterogeneous series 

of clinical and endocrine abnormalities thought to be due to 

inappropriate steroid feedback regulation of the hypothalamic 

pituitary axis (reviewed in 204, 486, 487). Although the 

ovaries of both the �ERKO and �ERKO females synthesize 

estradiol, the inherent insensitivity of certain target tissues to 

the hormone may render effects similar to those of insufficient 

estrogen synthesis and therefore may offer some analogy 

to the human syndrome. The primary defect resulting in 

inefficient ovulation in the �ERKO appears to be due to 

defects directly within the ovarian tissue, more precisely, an 

inability to appropriately respond to estradiol in the granulosa 

cells of maturing follicles. However, until further studies 

are carried out, an impaired ability to produce an adequate 

gonadotropin surge must also be considered as a 

possible contributory factor to the decreased ovulatory rates 

observed in the �ERKO. Still, the reduced number of ovulations 

observed in the �ERKO even after stimulation with 

exogenous gonadotropin suggests the presence of a significant 

defect within the ovary. 

Although an analogy of the �ERKO ovarian phenotype 

with a human pathology may be obscure at this point, the 

ovarian phenotype of the �ERKO is strikingly similar to that 

of PCOS patients. The most common pathological symptoms 

of PCOS include anovulation, hirsutism, and the presence of 

bilateral enlarged, white, smooth, and sclerotic ovaries with 

a thickened outer capsule (204, 486, 487). Internal characterization 

of the ovaries of a PCOS patient most often indicates 

the presence of follicles at various stages of atresia, a strikingly 

hypertrophied thecal/stromal compartment, and the 

observation of rare or absent corpora lutea (204). The follicles 

presented usually possess a reduced number of granulosa 

cells and little aromatase activity (204). Biochemical findings 

are most often characterized by elevated serum androgen 

and LH levels, whereas progesterone and FSH levels remain 

low to normal (204, 486, 487). However, there appear to be 

wide variations in the extent and occurrence of each of these 

symptoms, illustrated by the diagnosis of PCOS in women 

with apparently normal LH levels and no menstrual abnormalities, 

indicating there is likely more than one etiological 

factor involved in this syndrome (204, 487). 

The onset of PCOS most often coincides with menarche in 

young females, suggesting the existence of endocrine abnormalities 

even before puberty (204, 486). The first clinical 

indications are dysfunctional and/or unpredictable uterine 

bleeding or oligo/amenorrhea (204, 486). This is often accompanied 

by a high incidence of hirsutism (male-pattern 

hair growth) in 70–90% of PCOS females, determined to be 

due to increased serum androgens (204, 486). However, the 

causal agents that lead to increased circulating steroids and 

the initiation of PCOS remain unclear, although a strong 

familial component is believed to be involved (204, 488). 

Franks et al. (489) have proposed that the primary cause of 

PCOS is ovarian in nature and that endocrine abnormalities 

are secondary. Others have provided evidence to indicate 

that inappropriate steroid feedback at the hypothalamic-pituitary 

axis, resulting in increased frequency and amplitude 

of LH pulses, results in hyperstimulation of an otherwise 

normal ovary (204, 486). The initiation of the abnormal steroid 

signaling to the hypothalamic-pituitary axis may stem 

from extragonadal conversion of elevated circulating androgens 

to estrone and estradiol (204, 486, 487). The source of the 

initial increases in androgen that may trigger PCOS remain 

unclear and may be ovarian or adrenal in nature (487). Regardless, 

extraglandular aromatase activity in the adipose 

tissue is believed to result in the initial acyclic increases in 

estrogens that lead to the ovarian syndrome (204, 486). Obesity 

is reported in 40% of PCOS patients and is thought to be 

a contributory factor in the adipose tissue-derived estrogen 

production (204, 486). The abnormally high levels of estradiol 

then feed back to the hypothalamic-pituitary axis in a positive 

fashion, resulting in chronically elevated LH levels (204, 

486). Increased secretion of LH leads to hypertrophy of the 

ovarian thecum, a hallmark of the PCOS ovary, and further 

increases in androgen production (204, 486). Therefore, once 

initiated, it is thought that ovarian-derived androgens, followed 

by extraglandular conversion to estrogens, act to selfperpetuate 

the syndrome. Treatment of PCOS patients with 

a GnRH antagonist has proven to reduce circulating androgen 

and subsequent estrogen levels, indicating the ovary as 

the primary source of the androgen (204, 486). Furthermore, 

women with PCOS can be induced to ovulate with administration 

of FSH, suggesting that the primary defect is extragonadal 

in nature (204). 

The similarities of the �ERKO ovarian phenotype and 

those associated with PCOS are worth noting. However, the 

initial stimulus of abnormal feedback to the hypothalamicpituitary 

axis that leads to the phenotype may be different in 

the human and �ERKO mouse. As described above in the 

human female, it is believed that extragonadal synthesis of 

estrogens results in acyclic positive regulation of LH secretion 

by the hypothalamic-pituitary axis. In contrast, the 

�ERKO ovary is the principal source of both androgen and 

estradiol, and acyclic increases in serum LH are primarily 

due to a lack of negative feedback at the hypothalamicpituitary 

axis. However, preliminary studies in the �ERKO 

female have indicated that the signaling pathways for positive 

regulation of the LH secretion may be intact and therefore 

may also contribute to the increased gonadotropin levels 

observed. Therefore, although the means by which the elevated 

levels of LH are achieved in human PCOS and the 

�ERKO female mouse may differ, some features of the ovarian 

phenotypes are similar. Certain morphological abnormalities 

in the ovaries are comparable, i.e., polycystic follicles 

with reduced numbers of granulosa cells, hypertrophied thecal 

and stromal tissue, and the absence of corpora lutea, 

although the �ERKO ovary does not exhibit the thickened 

outer capsule seen in PCOS patients. It is possible that encapsulation 

of the polycystic ovary in the human is an ER�mediated 

event and therefore not possible in the �ERKO. 

However, the LH-overexpressing bLH�-CTP mice, which 

possess a functional ER� gene as well as several character-


istics associated with PCOS, also do not demonstrate this 

phenotype (254, 255). Several of the endocrine parameters 

associated with human PCOS are also observed in the 

�ERKO, especially the presence of elevated serum androgens 

and LH. Although the observation of hirsutism caused by 

elevated serum androgens is not possible in mice, analogous 

biological manifestations of elevated androgen levels are 

exhibited in the �ERKO female, including masculinized preputial 

(clitoral) glands and a thickened dermis. Furthermore, 

obesity is reported in a large proportion of PCOS patients and 

is thought to be a contributory factor in the extragonadal 

conversion of androgens to estrogens. As described above 

(Section VII.C), adult �ERKO females significantly outweigh 

their age-matched wild-type littermates because of excessive 

white adipose tissue. Interestingly, insulin resistance and 

hyperinsulinemia are often reported in PCOS patients and 

thought to contribute to further androgen synthesis in the 

ovarian thecum (204, 487). Taylor and Lubahn (490) reported 

an abnormal glucose tolerance test in �ERKO females that 

could be corrected with ovariectomy, suggesting PCOS-like 

insulin resistance. Therefore, the similarities of the endocrine 

abnormalities and ovarian phenotypes in the �ERKO and 

bLH�-CTP mice with those of human PCOS patients provide 

support that the cause may be extragonadal in nature, and 

possibly due to elevated LH in the presence of normal FSH. 

Although the initial cause of the abnormally high LH levels 

in �ERKO females may differ from that postulated in the 

PCOS patient, the resulting effects on the ovary are similar 

and may allow the �ERKO to be a useful experimental model 

in future investigations of this human disease. 

C. ER� and aromatase deficiency 

The first and only reported case of estrogen insensitivity 

in a human is that of Smith et al. (116) in which a male (46, 

XY) was determined to be homozygous for a single-point 

mutation in exon 2 of the ER� gene that resulted in a premature 

stop codon. Similar reports of human estrogen deficiency 

were also lacking in the clinical literature until recently. 

However, five cases of aromatase deficiency and 

therefore a lack of estrogen synthesis have been reported in 

both human males (119, 120) and females (117, 118, 120, 491). 

Therefore, the existence of humans that exhibit insufficient 

estrogen action due to either a lack of functional receptor or 

hormone, along with the successful generation of the ER null 

mice, suggests that estrogen is not essential to embryonic and 

fetal development in mammals. However, like the ER null 

mice, a distinct syndrome of phenotypes is apparent in both 

sexes of humans lacking in estradiol or ER. 

All three reports of females homozygous for inactivating 

mutations in the P450 arom gene describe pseudohermaphroditism, 

i.e., 46XX genotype, the presence of internal female 

reproductive structures but ambiguous external genitalia 

(117, 120, 491). This phenotype is due to the lack of aromatase 

activity in the fetus and placental unit during gestation, 

leading to the accumulation of androgens, which in turn elicit 

masculinizing effects on the fetus (117, 120, 491). A similar 

phenotype of ambiguous external genitalia was described in 

an infant in which placental aromatase deficiency was determined, 

although enzyme activity in the fetus was not 

evaluated at the time of the report (118). In the two cases of 

a complete lack of aromatase, the mother also exhibited virilization 

during pregnancy (120, 491), whereas a traceable 

level of placental aromatase activity appeared to inhibit this 

symptom in the case of Conte et al. (117). In all three cases, 

development of the internal structures of the female reproductive 

tract did not appear to be altered by a lack of estrogen 

action (117, 120, 491), although a compensatory role fulfilled 

by maternal estrogens cannot be ruled out. Serum levels of 

androgen, androgen precursors, FSH, and LH were elevated 

in the P450 arom-deficient patients as early as infancy (117, 

491) and continued to be elevated as the females approached 

puberty (117, 120). At 12–14 yr of age, secondary development 

of the breasts, a pubertal growth spurt, and menarche 

were all absent, but virilization of the external genitalia was 

reported (117, 120). In all three cases, hyperstimulation of the 

ovary and the development of multifollicular cysts was illustrated, 

even as early as 2 yr of age (117, 120, 491). The 

ovarian pathology exhibited was similar to that observed in 

the �ERKO females and was compatible with a diagnosis of 

PCOS (120). Therefore, intraovarian estrogen action does not 

appear to play a role in the development of the multifollicular 

ovarian cysts in humans. Estrogen and progesterone replacement 

therapy alleviated the above phenotypes, resulting in 

normal gonadotropin and androgen levels, regression of the 

ovarian cysts, and in the two pubertal patients reported, the 

onset of breast development, a growth spurt, and menarche 

(117, 120, 491). 

The clinical syndromes exhibited by the two reported human 

male cases of aromatase deficiency (119, 120) and the 

single known human case of an inactivating mutation of the 

ER� gene (116) are strikingly similar. All three patients exhibited 

a normal onset of puberty and no gender-identity 

disorders, as well as normal external genitalia and prostate 

size (116, 119, 120). The patient lacking functional ER� exhibited 

a testicular volume and sperm density within the 

norm for men of his age (116). A normal testicular volume 

was also reported in the P450 arom-deficient patient of Morishima 

et al. (120). However, Carani et al. (119) reported small 

testis and severe oligozoospermia and infertility in the other 

male lacking aromatase, although the presence of similar 

findings in a brother with a normal P450 arom gene suggested 

other possible familial factors for this finding. Serum levels 

of androgens, FSH, and LH were all consistently elevated in 

the P450 arom-deficient males (119, 120), as well as estrone and 

estradiol in the ER�-deficient male (116). Estrogen replacement 

therapy resulted in the return of androgen and gonadotropin 

levels to the normal range in the P450 arom-deficient 

males. However, similar estrogen therapy induced no such 

changes in the ER�-deficient male (116). In fact, estrogen 

replacement therapy in the ER�-deficient male achieved circulating 

levels of the steroid that exceeded the norm by 

nearly 5-fold, yet no alleviation of the above pathologies or 

side effects were observed, such as impotence and gynecomastia 

(116). Alterations in the cardiovascular system have 

been reported recently in the ER�-deficient male, including 

dysfunctions in vasodilation (492) and premature coronary 

artery disease (493). 

The most overt phenotypes in all patients lacking sufficient 

estrogen action involved the skeleton. Females homozygous


for mutations of the P450 arom gene displayed a delay in bone 

age, a significant decrease in bone mineral density of the 

lumbar spine, and absence of the pubertal growth spurt (117, 

491). Both the ER�-deficient male and the two males lacking 

aromatase were evaluated as adults and exhibited strikingly 

similar skeletal phenotypes. All three were characterized by 

tall stature (�95th percentile) with continued slow linear 

growth, a low upper/lower body segment ratio (�0.88, vs. 

0.96, average for men), unfused epiphyses, bone age of 14–15 

yr, and decreased bone density (116, 119, 120). Epiphyseal 

closure and increases in bone mineral density were observed 

after estrogen replacement therapy in the P450 arom-deficient 

patients (119, 120). However, as with the phenotypes described 

above in the ER�-deficient male, exogenous estrogen 

treatments resulted in no changes in bone physiology (116). 

Therefore, although it was once believed that estrogens were 

the major sex steroid influencing bone physiology in females 

and androgen fulfilled similar roles in the male, the phenotypes 

described above strongly indicate that estrogen action 

is critical to the pubertal growth spurt, bone mineralization, 

and epiphyseal maturation in both males and females. 

IX. Summary 

All scientific investigations begin with distinct objectives: 

first is the hypothesis upon which studies are undertaken to 

disprove, and second is the overall aim of obtaining further 

information, from which future and more precise hypotheses 

may be drawn. Studies focusing on the generation and use 

of gene-targeted animal models also apply these goals and 

may be loosely categorized into sequential phases that become 

apparent as the use of the model progresses. Initial 

studies of knockout models often focus on the plausibility of 

the model based on prior knowledge and whether the generation 

of an animal lacking the particular gene will prove 

lethal or not. Upon the successful generation of a knockout, 

confirmatory studies are undertaken to corroborate previously 

established hypotheses of the function of the disrupted 

gene product. As these studies continue, observations of 

unpredicted phenotypes or, more likely, the lack of a phenotype 

that was expected based on models put forth from 

past investigations are noted. Often the surprising phenotype 

is due to the loss of a gene product that is downstream 

from the functions of the disrupted gene, whereas the lack of 

an expected phenotype may be due to compensatory roles 

filled by alternate mechanisms. As the descriptive studies of 

the knockout continue, use of the model is often shifted to the 

role as a unique research reagent, to be used in studies that 

1) were not previously possible in a wild-type model; 2) 

aimed at finding related proteins or pathways whose existence 

or functions were previously masked; or 3) the subsequent 

effects of the gene disruption on related physiological 

and biochemical systems. 

The �ERKO mice continue to satisfy the confirmatory role 

of a knockout quite well. As summarized in Table 4, the 

phenotypes observed in the �ERKO due to estrogen insensitivity 

have definitively illustrated several roles that were 

previously believed to be dependent on functional ER�, including 

1) the proliferative and differentiative actions critical 

to the function of the adult female reproductive tract and 

mammary gland; 2) as an obligatory component in growth 

factor signaling in the uterus and mammary gland; 3) as the 

principal steroid involved in negative regulation of gonadotropin 

gene transcription and LH levels in the hypothalamic-pituitary 

axis; 4) as a positive regulator of PR expression 

in several tissues; 5) in the positive regulation of PRL 

synthesis and secretion from the pituitary; 6) as a promotional 

factor in oncogene-induced mammary neoplasia; and 

7) as a crucial component in the differentiation and activation 

of several behaviors in both the female and male. 

The list of unpredictable phenotypes in the �ERKO must 

begin with the observation that generation of an animal 

lacking a functional ER� gene was successful and produced 

animals of both sexes that exhibit a life span comparable to 

wild-type. The successful generation of �ERKO mice suggests 

that this receptor is also not essential to survival and 

was most likely not a compensatory factor in the survival of 

the �ERKO. In support of this is our recent successful generation 

of double knockout, or ��ERKO mice of both sexes. 

The precise defects in certain components of male reproduction, 

including the production of abnormal sperm and the 

loss of intromission and ejaculatory responses that were observed 

in the �ERKO, were quite surprising. In turn, certain 

estrogen pathways in the �ERKO female appear intact or 

unaffected, such as the ability of the uterus to successfully 

exhibit a progesterone-induced decidualization response, 

and the possible maintenance of an LH surge system in the 

hypothalamus. Furthermore, it is apparent that several of the 

�ERKO phenotypes may be aggravated by the downstream 

attenuation of progesterone and PRL action or even enhanced 

by increased androgen sensitivity, including those 

observed in the mammary gland, gonads, bone, and behavior. 

In addition, disruption of the ER� gene may have unmasked 

estrogen-signaling systems that were not easily detectable 

in the wild-type, such as 1) the apparent estrogen 

actions of the 4-OH-E 2 in the �ERKO uterus that are independent 

of ER� and ER�; 2) the ability of estrogen to induce 

increased levels of hypothalamic PR in the �ERKO female; 3) 

estradiol-induced potentiation of select neurons of the hippocampus; 

and 4) the protective effects of estrogen in the 

carotid vascular injury model. Finally, the concurrent descriptions 

of human mutations resulting in a lack of estrogen 

action due to a loss in ligand or functional receptor have 

illustrated the utility of the ER null mice as a model system 

to further understand the pathologies that result. 

Therefore, the �ERKO mice have illustrated the several 

ways in which data collected from a knockout model can 

quickly contribute to the current knowledge concerning the 

function of a particular gene. However, it is worth noting the 

distinct differences in thought that occurred during the generation 

of the �ERKO and �ERKO models. At the time the 

work was initiated to generate the �ERKO mice, much was 

known about the many roles of estrogen and the ER; therefore, 

several educated predictions of the possible phenotypes 

were possible and have since been confirmed, rejected, or 

reevaluated. However, the conception of the �ERKO mice 

occurred only 2 yr after the discovery of the ER�. Because 

little was known about the function and role that the ER� 

might play, it was difficult to make sound predictions. There-


TABLE 4. Summary of reported phenotypes in the �ERKO and �ERKO mice 

ERKO model Description of phenotype Ref. 

Lethality of mutation 

ER� No 46, 116 

ER� 

Fertility 

No 47 

�ERKO Both sexes are infertile 46 

�ERKO 

General 

Female is subfertile (reduced litter size); male is fertile 47 

�ERKO Exhibit normal expression of the ER� gene 93, 121, 352 

Female reproductive tract 

�ERKO Tract undergoes normal pre- and neonatal development but is insensitive to estradiol, DES, and hydroxy 46, 123, 153 

tamoxifen during adulthood 

Presence of non-ER� or -ER� receptor-mediated estrogenic pathway for 4-OH-estradiol and methoxychlor 173, 174 

Loss of mitogenic actions of EGF 170 

Responsive to mitogenic actions of androgens 154 

Responsive to progesterone and able to undergo artificially induced decidualization 183 

Ovaries undergo normal pre- and neonatal development, but are anovulatory during adulthood, exhibit 

multiple hemorrhagic cysts, and no corpora lutea 

46, 142 

30–40% incidence of ovarian tumors by 18 months of age Herein 

�ERKO Tract undergoes normal pre- and neonatal development and appears sensitive to ovarian estrogen cycling 47 

during adulthood 

Ovaries undergo normal pre- and neonatal development, but do not exhibit normal frequency of spontaneous 

ovulations during adulthood, exhibit a severely attenuated response to superovulation treatment with 

reduced numbers of oocytes and multiple trapped preovulatory follicles 

Mammary gland 

�ERKO Undergoes normal prenatal development but is insensitive to estrogen-induced development during puberty 

and adulthood 

269 

Responsive to exogenous progesterone and prolactin In preparation 

Susceptible to proto-oncogene (Wnt-1) induced ductal hyperplasia and lobuloalveolar adenocarcinoma but 

tumors exhibit a delayed growth rate compared to those in wild-type 

297 

�ERKO Undergoes normal prenatal and pubertal development, virgin gland is grossly indistinguishable from that of 47 

age-matched wild-type 

Undergoes normal differentiation and lactation during pregnancy and motherhood 

Male reproductive tract 

47 

�ERKO Tract undergoes normal pre- and neonatal development 315, 316 

Age-related phenotype of attenuated fluid resorption in efferent ducts leads to dilation of Rete testis, 315, 327 

atrophy of the seminiferous epithelium, and decreasing sperm counts 

Disrupted sperm function illustrated by an inability to fertilize 315 

Age-related decreases in testis weight Herein 

Age-related increases in seminal vesicle weight Herein 

�ERKO Undergoes normal pre- and neonatal development with no apparent defects in spermatogenesis that impede 47 

fertility 

Neuroendocrine system: females 

�ERKO Anterior pituitary possesses all of the expected cell types but exhibits elevated transcripts for the 

gonadotropin subunits (�-gonadotropin subunit, LH-�, FSH-�) 

282 

Elevated serum levels of estradiol, testosterone, and LH, but normal serum levels of progesterone and FSH 123, 252 

Normal lactotroph differentiation and number in the anterior pituitary, but exhibits significant deficits in 

transcription of the PRL gene and serum PRL levels 

282, Herein 

Medial preoptic region of the hypothalamus exhibits elevated levels of PR transcripts which reduce with 400, 423 

ovariectomy and return with estradiol treatment 

Rapid actions of estradiol on hippocampal neurons are preserved 364 

�ERKO Normal serum levels of estradiol Herein 

Neuroendocrine system: males 

�ERKO Anterior pituitary possesses all the expected cell types but exhibit elevated LH-� transcripts 282, 317 

Elevated serum levels of estradiol, testosterone, and LH, but normal serum levels of progesterone and FSH 317, Herein 

Behavior: females 

�ERKO Exhibit a lack of estradiol and progesterone-induced sexual behavior, increased aggression, and infanticide 417, 418, 419 

�ERKO Exhibit no defects in sexual behavior that impede fertility 47 

Behavior: males 

�ERKO Exhibit normal mounting and attraction toward wild-type females but a complete lack of intromission and 318, 427, 428 

ejaculation; display reduced aggression 

�ERKO Exhibit no defects in sexual behavior that impede fertility 47 

Cardiovascular 

�ERKO Exhibit reduced estradiol-induced angiogenesis and reduced basal levels of vascular nitric oxide 467 

Exhibit wild-type response to estradiol in the carotid artery injury model 464 

Exhibit increased expression of L-type Ca 2� channels 472 

Exhibit reduced response to estrogen-induced increases in serum apolipoprotein E 460 

Other 

�ERKO Both sexes exhibit growth arrest of longitudinal bones 447, 448 

Impaired glucose tolerance 490 

No apparent defect on B lymphopoiesis 507 

47


fore, the �ERKO mice have played a significant role in confirming 

many of the roles thought to be fulfilled by estrogen, 

whereas the �ERKO mice have and will continue to provide 

primary insight into the functions of the ER�. The generation 

of both models, as well as a forthcoming description of the 

��ERKO, will prove invaluable in elucidating the precise 

roles fulfilled by each ER, as well as any possible cooperative 

roles the two receptors might play within the same tissue or 

even within the same cell. 

Acknowledgments 

The authors are grateful to several individuals for their dedication, 

effort, and insight that has made the work described above possible: first 

and foremost, our original collaborators in the generation of the ERKO 

mice, Dr. Oliver Smithies for his efforts leading to the generation of both 

ERKO models; Dr. Dennis Lubahn for the �ERKO; and Drs. John Krege, 

Jeff Hodges, and Jan-Åke Gustafsson and laboratory for the �ERKO. The 

authors are exceptionally indebted to Sylvia Curtis, Todd Washburn, Dr. 

Jonathan Lindzey, Dr. Wayne Bocchinfuso, Mariana Yates, Linwood 

Koonce, James Clarke, Page Myers, Dr. Motohiko Taki, and Dr. Sean 

Kimbro for their efforts and dedication. We would also like to thank our 

several collaborators, Drs. Ralph Cooper, Richard DiAugustine, E. Mitch 

Eddy, Thomas Golding, Paul Kincaide, Diane Klotz, Michael Mendolsohn, 

Robert Moss, Jeffrey Moyer, Sonoko Ogawa, Donald Pfaff, Gabor 

Rubanyi, David Schomberg, Allen Silverstone, and Harold Varmus. We 

would also like to acknowledge Drs. Paul Shughrue and Istvan Merchenthaler 

for the contribution of Fig. 8. 

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