Estrogen Receptor Null Mice - Endocrine Reviews

June, 1999 ESTROGEN RECEPTOR NULL MICE 367
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