Estrogen Receptor Null Mice - Endocrine Reviews
Estrogen Receptor Null Mice - Endocrine Reviews
Estrogen Receptor Null Mice - Endocrine Reviews
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June, 1999 ESTROGEN RECEPTOR NULL MICE 367<br />
disrupting insert is accurately removed via the conventional<br />
donor and acceptor splice sites, preserving the normal reading<br />
frame of the gene (124). Studies in three different human<br />
genes have demonstrated that point mutations resulting in a<br />
premature stop codon can lead to complete excision of the<br />
exon possessing the mutation (125, 126). Furthermore, Reed<br />
and Maniatis (127) used artificial deletions and insertions<br />
within exons of genes that normally display alternative splicing<br />
to demonstrate that the proximity of the acceptor and<br />
donor splicing sequences to one another plays a role in splicing<br />
mechanisms. It is possible that insertion of sequences as<br />
large as those used in the ERKO mice may disrupt the spatial<br />
requirements necessary for proper mRNA splicing. Therefore,<br />
the above studies, along with our experiences, are relevant<br />
to the practice of targeting genes by insertion of a large<br />
disrupting sequence possessing internal stop codons.<br />
1. Interpretation of phenotypes in receptor null mice. The use of<br />
methodologies to target and disrupt individual genes has<br />
created numerous models available for study (reviewed in<br />
Refs. 109, 110, and 128). Furthermore, this impact has been<br />
felt in several facets of the biological sciences. Although it<br />
may be initially thought that a particular gene plays no role<br />
in the physiology of certain animal systems, such as reproduction<br />
or behavior, disruption of the gene and the subsequent<br />
phenotypes often prove otherwise. Therefore, transgenic<br />
and knockout technologies have spawned a number of<br />
collaborative efforts between investigators of varied disciplines<br />
that may have never occurred.<br />
An issue that has become apparent from the numerous<br />
gene disruption studies and interdisciplinary collaborative<br />
efforts is a collection of caveats to be considered when evaluating<br />
phenotypical data from a knockout model. These have<br />
arisen mostly from the behavioral sciences (reviewed in Refs.<br />
129 and 130), but have expanding application to all areas of<br />
study provided by transgenic animals. The first of these<br />
caveats is one that may be most relevant to the steroid receptor<br />
mutant models, i.e., when studying the target tissue of<br />
an adult receptor-knockout mouse, one must realize the tissue<br />
passed through all the stages of development and “organization”<br />
in the absence of the respective receptor. Therefore,<br />
this tissue, and in essence the whole animal, cannot be<br />
assumed to be truly identical to the wild type in all aspects<br />
except for the absence of the targeted gene product. Any<br />
genetic redundancies or compensatory mechanisms that<br />
took place during development cannot be readily detected or<br />
accounted for during most experiments. Therefore, the lack<br />
of a phenotype does not necessarily discount the function of<br />
the disrupted gene in the physiology being studied. Additionally,<br />
it is difficult to distinguish between an organizational<br />
vs. activational basis for an observed deficit in a particular<br />
physiology when studying the adult mutant. For<br />
example, observed resistance to a hormone due to alterations<br />
downstream of the function of the disrupted gene may be<br />
apparent during adulthood, but may have been imprinted<br />
during development.<br />
Other caveats of interpreting data from receptor null mice<br />
are founded in the methods used to generate and maintain<br />
a line of knockout mice. The standard protocol for generating<br />
a knockout mouse involves the incorporation of embryonic<br />
stem cells of a 129 strain of mice that carry the desired<br />
mutation into the blastocyst of a C57BL/6 strain. The resulting<br />
chimeric animals are then often back-crossed to the<br />
C57BL/6 strain once again until mice homozygous for the<br />
disruption are acquired. Therefore, early generations of<br />
knockout mice are composed of a somewhat chimeric genome,<br />
especially in the chromosomal regions closest to the<br />
targeted locus. This is of special importance in behavioral<br />
studies in light of the known variations in the sexual behavior<br />
of different strains of mice (131). However, most relevant to<br />
the ERKO, significant variations in estrogen responsiveness<br />
of the female reproductive tract among the different strains<br />
of mice are also known to exist (132, 133). A recent report by<br />
Roper et al. (134) has further defined the genetic basis for the<br />
variations that exist in the effects of estradiol on classical<br />
uterine parameters in mice. These limitations can be overcome<br />
to some degree by increasing experimental sample<br />
sizes and including parental strains as a control group in all<br />
experiments (135). In addition, the various models of hormone<br />
and steroid receptor deficiency that are now available<br />
no longer make necessary the complete interpretation of data<br />
from any one model. Therefore the data from these models,<br />
when interpreted as a whole, should prove invaluable to<br />
elucidating the roles of the different sex steroid receptors in<br />
both development and adult physiology.<br />
III. Reproductive Tract Phenotypes of the Female<br />
The most well characterized estrogen target tissues are<br />
those of the mammalian female reproductive tract, comprised<br />
of the ovaries, oviducts, uterus, cervix, and vagina.<br />
Reproductive capabilities in the female are dependent on the<br />
sequential processes of differentiation during the embryonic<br />
and prenatal periods and the maturation of these tissues<br />
during puberty. Differentiation of the fetal gonads to ovaries<br />
results from a lack of the Y chromosome, and therefore the<br />
testis-determining genes, and the presence of putative, yet<br />
unidentified, autosomal ovary-determining genes (111). The<br />
ductal organs of the tract subsequently result from differentiation<br />
of the fetal ambisexual precursor, the Müllerian<br />
ducts, due to the lack of testicular hormones. Early studies<br />
indicated that the female reproductive tract is the default<br />
phenotypical sex and will differentiate and develop normally<br />
in the absence of ovaries and adrenal glands (107, 136). Therefore,<br />
it appears that estrogens are not required for differentiation<br />
and initial development of the female reproductive<br />
tract, whereas testosterone is critical to differentiation of the<br />
male genitalia. This conclusion has been reconfirmed by the<br />
male-to-female sex reversal observed in male mice homozygous<br />
for a targeted disruption of the gene encoding SF-1, a<br />
transcription factor that regulates the steroid hydroxylases in<br />
steroidogenic cells (137, 138). Despite an inability to synthesize<br />
steroids and a complete lack of gonads, all SF-1 knockout<br />
mice develop female internal genitalia, regardless of genetic<br />
sex (137). It is therefore not surprising that the �ERKO and<br />
�ERKO female mice exhibit a properly differentiated female<br />
reproductive tract possessing the constituent structures (46,<br />
47). However, estrogen insensitivity has severely disrupted<br />
sexual maturation of the whole reproductive tract in the