Estrogen Receptor Null Mice - Endocrine Reviews

368 COUSE AND KORACH Vol. 20, No. 3
�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-