Estrogen Receptor Null Mice - Endocrine Reviews
Estrogen Receptor Null Mice - Endocrine Reviews
Estrogen Receptor Null Mice - Endocrine Reviews
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
368 COUSE AND KORACH Vol. 20, No. 3<br />
�ERKO female and ovarian function in the �ERKO female.<br />
The consequences of ER gene disruption on the individual<br />
components of the female reproductive tract is the topic of<br />
this portion of the review.<br />
Before we continue, we believe it is necessary to briefly<br />
reiterate those studies carried out to verify successful targeting<br />
of the ER� gene in the �ERKO. This discussion is<br />
appropriate for this portion of the review because the majority<br />
of these experiments were performed on uterine tissue.<br />
To determine the effectiveness of the gene targeting, Western<br />
blots of adult �ERKO uterine nuclear and cytosolic extracts<br />
were probed with the H222 antibodies, a rat monoclonal<br />
antibody specific to the ligand-binding domain of the human<br />
ER� (10). Our studies, as well as those of others, have demonstrated<br />
that this antibody possesses high cross-reactivity to<br />
the mouse ER� (139–141). These assays detected no wildtype<br />
ER� or any other immunoreactive fragments unique to<br />
the �ERKO uterus. Similar results were obtained when blots<br />
were probed with the rabbit antiserum ER-21, directed toward<br />
the 21 amino-terminal residues of the rat ER� (141).<br />
However, binding assays using 3 H-E 2 on �ERKO uterine<br />
extracts indicated the presence of high-affinity binding of the<br />
hormone at levels approximately 3–9% of the wild type (123).<br />
In agreement with these data, sucrose gradient analysis with<br />
3 H-E2 on low-salt cytosol extracts from �ERKO uteri indicated<br />
a binding factor with an 8S sedimentation value, similar<br />
to that of the wild-type ER� (123). The discovery of the<br />
ER�, reported approximately 3 yr after the generation of the<br />
�ERKO, prompted a renewed assessment of this �ERKO<br />
estrogen-binding data in several publications. Unfortunately,<br />
in a number of these reports, the original datum<br />
discussed above is not evaluated in full, and the authors<br />
elude to ER� as the likely binding source in the �ERKO uteri.<br />
Certainly at the time of the initial characterization, concern<br />
over the residual level of binding in the �ERKO uteri was<br />
often mixed with the wonder of possibly discovering an<br />
unknown ER. However, during these studies we also demonstrated<br />
that when the H222 antibodies were included in<br />
the sucrose gradient assays, the estradiol binding peak in the<br />
�ERKO uterine extract was shifted accordingly (123). The<br />
H222 antibodies have been shown by us, as well as by other<br />
laboratories, to be ER� specific and unable to recognize ER�<br />
by Western blot analysis or immunohistochemistry (142). As<br />
described earlier in this review, our RT-PCR analysis on<br />
mRNA from �ERKO uteri demonstrated the presence of a<br />
splicing variant of the disrupted ER� gene that could encode<br />
a mutant ER� possessing both the ability to bind estradiol as<br />
well the H222 epitope (123). Furthermore, we have recently<br />
shown that ER� mRNA is undetectable in the uteri of adult<br />
wild-type as well as �ERKO mice when assayed by ribonuclease<br />
protection assay (93). Therefore, we believe that relatively<br />
conclusive data have been generated to indicate that<br />
the estradiol-binding factor present in �ERKO uteri is most<br />
likely not ER�.<br />
A. Uterus<br />
1. Uterine phenotype and estrogen insensitivity. The ER has been<br />
detected by steroid autoradiography and immunohistochemical<br />
methods in the ductal structures of the rodent fe-<br />
male reproductive tract during several stages of development,<br />
including the late fetal and neonatal stages through<br />
puberty and adulthood (reviewed in Refs. 112 and 143).<br />
Several reports describe the initial appearance of ER immunoreactivity<br />
in the developing uterus as early as fetal day 15<br />
(112, 143). ER immunoreactivity was first detectable in mesenchymal<br />
cells, whereas induction in the epithelial cells occurs<br />
during the late fetal stages and increases significantly<br />
during the neonatal period (112, 143). The fully developed<br />
uterus is composed of many heterogeneous cell types comprising<br />
three major anatomical compartments, the outer<br />
myometrium, endometrial stroma, and luminal/glandular<br />
epithelium. In the immature CD-1 mouse, ER� immunoreactivity<br />
is easily detectable in the stroma on day 1 and continues<br />
to rise to a maximal level on day 10, whereas the<br />
appearance of epithelial ER� is delayed and reaches a peak<br />
around day 16 (144). Other reports indicate variations in the<br />
exact timing of the appearance of ER� among different<br />
strains and species, most likely reflecting temporal deviations<br />
in development (112, 143).<br />
The presence of an intact estrogen-signaling system appears<br />
to coincide with the appearance of ER�. In several<br />
species, estrogen treatment of fetal and neonatal females<br />
results in the stimulation of increased uterine levels of nucleic<br />
acid (136, 145), protein synthesis (146), ornithine decarboxylase<br />
(147), progesterone receptor (148), and cellular<br />
proliferation (145, 149, 150). However, a full biological response<br />
to estradiol in terms of maximum increases in uterine<br />
weight is not possible in the neonatal uterus, and can be<br />
observed only after the animal approaches weaning age<br />
(146). Furthermore, significant differences in the uterine response<br />
to estradiol between the neonate and sexually mature<br />
rodent are known (151). For example, estrogen stimulates<br />
cellular proliferation in all tissues of the immature uterus,<br />
whereas this response becomes limited to the epithelial compartment<br />
during adulthood (151, 152). Therefore, sexual maturation<br />
of the uterus is not simply marked by the presence<br />
of ER�, but rather the acquisition of the capacity to undergo<br />
the correct synchronized phases of proliferation and differentiation<br />
elicited by the ovary-derived sex steroids.<br />
As shown in Fig. 3, the uteri of both adult �ERKO and<br />
�ERKO females possess all three definitive uterine compartments,<br />
the myometrium, endometrial stroma, and epithelium.<br />
However, in the �ERKO, each is hypoplastic and results<br />
in whole uterine weights that are approximately half<br />
that recorded for wild-type littermates (46). In contrast, the<br />
uteri of adult �ERKO females appear normal and able to<br />
undergo the cyclic changes associated with the ovarian steroid<br />
hormones (47). Therefore, perinatal development of the<br />
female reproductive tract in the mouse appears to be independent<br />
of ER� and ER� actions. However, estrogen responsiveness<br />
and subsequent sexual maturity in the uterus has<br />
been ablated by disruption of the ER� gene. The �ERKO<br />
endometrial stoma is characterized by a less organized structure<br />
and hypotrophy, with a sparse distribution of uterine<br />
glands compared with that of the wild type (153). Luminal<br />
and glandular epithelial cells in the �ERKO uterus most often<br />
appear healthy, but are consistently cuboidal and lack the<br />
normal “estrogenized” morphology of a tall columnar shape<br />
and basally located nucleus (Fig. 3). This phenotype is in-