αERKO Mice Research Articles

Estren Behaves as a Weak Estrogen Rather than a Nongenomic Selective Activator in the Mouse Uterus

A proposed membrane-mediated mechanism of rapid nongenomic response to estrogen has been the intense focus of recent research. Estren, a synthetic steroid, is reported to act selectively through a rapid membrane-mediated pathway, rather than through the classical nuclear estrogen receptor (ER)-mediated pathway, to maintain bone density in ovariectomized mice without uterotropic effects. To evaluate the mechanism and physiological effects of estren, we studied responses in adult ovariectomized mice. In a 3-d uterine bioassay, we found that 300 microg estren significantly increased uterine weight; in comparison, a more maximal response was seen with 1 mug estradiol (E2). The estren response was partly ERalpha independent, because ERalpha knockout (alphaERKO) uteri also exhibited a more moderate weight increase. Estren induced epithelial cell proliferation in wild-type, but not alphaERKO, mice, indicating ERalpha dependence of the epithelial growth response. Examination of estren-regulated uterine genes by microarray indicated that early (2 h) changes in gene expression are similar to the early responses to E2. These gene responses are ERalpha dependent, because they are not seen in alphaERKO mice. Later estren-induced changes in gene expression (24 h) are blunted compared with those seen 24 h after E2. In contrast to early genes, these later estren responses are independent of ERalpha, because the alphaERKO shows a similar response to estren at 24 h. We found that E2 or estren treatments lead to depletion of ERalpha in the uterine cytosol fraction and accumulation in the nuclear fraction within 30-60 min, consistent with the ability of estren to regulate genes through a nuclear ERalpha rather than a nongenomic mechanism. Interestingly, estren, but not E2, induces accumulation of androgen receptor (AR) in the nuclear fraction of both wild-type and alphaERKO samples, suggesting that AR might be involved in the later ERalpha-independent genomic responses to estren. In conclusion, our studies suggest that estren is weakly estrogenic in the mouse uterus and might induce nuclear ERalpha- and AR-mediated responses. Given its activity in our uterine model, the use of estren as a bone-selective clinical compound needs to be reconsidered.

The Proliferative Uterine Response of Nonclassical ERα (Estrogen Receptor α) Mice Reveals a Distinct Signaling Pathway

Examine the role of nonclassical ERα (non-estrogen response element dependent actions of ERα) expression in mediating the proliferative response of uterine stromal and epithelial cells to estrogen and tamoxifen. Experimental laboratory study. Morphometric analysis and immunohistochemistry. Statistical analysis was performed by ANOVA with appropriate post hoc tests. Previous reports detail the generation of a knock-in/knockout mouse (E207A/G208A, ERAA/-) which lacks classical ERα signaling by crossbreeding ERAA/WT to estrogen receptor-α knockout (αERKO, ER-/-) animals. The ERAA/- animals signal via the nonclassical ERα pathway. The nonclassical pathway involves a ligand bound ERα that interacts with transcription factors such as AP1 and Sp1. Analysis of the hemizygous mouse (ERAA/-) should indicate the contribution of nonclassical ERα signaling in vivo. Animals were ovariectomized at 10 weeks, rested for two weeks, followed by one of the following treatments: (i) control, (ii) 2 μg 17β-estradiol (E2), (iii) 50 μg 4-OH-tamoxifen (4-OHT). Mice were sacrificed at various times after hormone treatments. BrdU was given intraperitoneally 2 hours before sacrifice. The uterine epithelial proliferative response to 72 hours of E2 and 4-OHT was present in both ERAA/- and WT animals. The uterine luminal epithelial height increased with the restoration of estrogen signaling. The responsiveness of the ERAA/- uterus was confirmed by the presence of BrdU incorporation. The percentage of BrdU positive epithelial cells was significantly greater in the ERAA/- and WT uteri compared with the αERKO (p<0.001) animals. Only the ERAA/- uterus demonstrated nuclear localization of cyclin D2 in luminal and glandular epithelial cells following both E2 and 4-OHT treatments. The presence of ERAA/- did not facilitate an acute uterotrophic response six hours after a single subcutaneous injection of E2. The uterine wet weight of ERAA/- animals was larger than αERKO mice (22.7 vs. 11.3 mg, p<0.01) but did not differ from the ovariectomized ERAA/- control animals. Tabled 1 The presence of a single ERAA/- allele is sufficient to restore luminal epithelial proliferation. The proliferative response was confirmed by the presence of luminal and glandular epithelial BrdU incorporation. The nuclear localization of cyclin D2 provides a potential mechanism for the proliferative response in the ERAA/- animals. In addition, the finding of luminal epithelial proliferation in the absence of ERα suggests that classical ERαstromal signaling is not required for epithelial response to hormones. Nonclassical ERα signaling does not contribute to the acute uterotrophic response of water imbibition. The early events of edema and increased capillary permeability may require a membrane-bound ERα receptor. The in vivo demonstration of nonclassical ERα signaling may provide a means for evaluating the uterine epithelial response of new antiestrogens.

Effects of estrogens and the phytoestrogen genistein on adipogenesis and lipogenesis in males and females

Effects of estrogens and the phytoestrogen genistein on adipogenesis and lipogenesis in males and females

Differential distribution of estrogen receptor (ER)-α and ER-β in the midbrain raphe nuclei and periaqueductal gray in male mouse: Predominant role of ER-β in midbrain serotonergic systems

We examined the distribution of estrogen receptor (ER)-α and ER-β immunoreactive (ir) cells in the dorsal (DRN) and median/paramedian (MPRN) raphe nuclei in male mice. ER-α ir neurons were scattered across the three subdivisions (ventral, dorsal, and lateral) of the DRN and the MPRN. Robust ER-β ir cells were observed throughout the raphe nuclei, and were particularly abundant in the ventral and dorsal subdivisions of the DRN. Using dual-label immunocytochemistry for ER-α or ER-β with tryptophan hydroxylase (TPH), the rate-limiting enzyme for 5-hydroxytryptamine (5-HT) synthesis, over 90% of ER-β ir cells exhibited TPH-ir in all DRN subdivisions, whereas only 23% of ER-α ir cells contained TPH. Comparisons of ER-α knockout (αERKO) as well as ER-β knockout (βERKO) mice with their respective wild-type (WT) littermates revealed that gene disruption of either ER-α or ER-β did not affect the other ER subtype expression in the raphe nuclei. In situ hybridization histochemistry revealed that there was a small but statistically significant decrease in TPH mRNA expression in the ventral DRN subdivision in βERKO mice compared with βWT mice, whereas TPH mRNA levels were not affected in αERKO mice. These findings support a hypothesis that ER-β activation may contribute to the estrogenic regulation of neuroendocrine and behavioral functions, in part, by acting directly on 5-HT neurons in the raphe nuclei in male mice.

Characterization of the uterine phenotype of non-classical ERα (estrogen receptor α) signaling in vivo.

Determine the non-ERE (estrogen response element) dependent actions of ERα on growth, differentiation, and developmental gene expression in the female reproductive tract. Experimental laboratory study. Morphometric analysis, immunohistochemistry, and quantitative real-time PCR. Previous work from our laboratory demonstrated that the mutation of residues 207 and 208 of ERα (within the proximal P-box of the first zinc finger of the DNA binding domain) to alanines abolished classical ERα signaling via ERE. We generated a knock-in/knockout mouse (E207A/G208A, ERAA/-) lacking classical ERα signaling by crossbreeding ERAA/WTto estrogen receptor-α knockout (αERKO) animals. The ERAA/- animals signal via the non-classical ERα pathway. The non-classical pathway involves a ligand bound ERα that interacts with transcription factors such as AP1 and Sp1. Animals were sacrificed at 8 weeks of age. The presence of a single ERAA/- allele (n = 8) supported significant uterine growth compared with αERKO animals (n = 6). The uterine radius of ERAA/-animals was larger than age matched αERKO mice (321 μm ± 58 vs. 170 ± 62, p < .01). The width of the inner circular muscle (ICM) layer of ERAA/- mice was also increased (54 μm ± 34 vs. 38 ± 15, p < .05). The ERAA/-uterus also exhibited altered glandular development. The endometrial glands were more numerous but remained organized in clusters in the stroma. The mesenchymal differentiation characteristic of wild-type (WT) mice was restored with positive ICM coexpression for vimentin and α-actin in all ERAA/-animals. In contrast, the αERKO uterus lacked cytodifferentiation with no positive staining of the ICM for vimentin or α-actin. The expression of the Wnt genes (Wnt-7a, Wnt-5a, and Wnt-4a) and EGFR (epidermal growth factor receptor) did not differ between ERAA/-(n = 8) and αERKO (n = 7). However, the uterine expression of cyclin D2 (which lacks an ERE) increased 2.6-fold in ERAA/- compared to αERKO. The expression of PR (progesterone receptor isoform A and B) was down-regulated significantly in ERAA/- compared to αERKO (-2.5-fold, p < .05). Non-classical ERα signaling via the presence of a single ERAA/-allele is sufficient to restore many features of growth and differentiation of the murine uterus. In addition, glandulargenesis progresses beyond that observed in the αERKO uterus suggesting that non-classical signaling is involved in the epithelial-mesenchyme interactions required for complex gland formation. Developmental gene expression is not altered by the restoration of non-classical ERα signaling. The up-regulation of cyclin D2 and the loss of progesterone receptor expression provide a potential mechanism for the observed phenotype.

Critical in vivo roles for classical estrogen receptors in rapid estrogen actions on intracellular signaling in mouse brain.

Estrogen exerts classical genomic as well as rapid nongenomic actions on neurons. The mechanisms involved in rapid estrogen signaling are poorly defined, and the roles of the classical estrogen receptors (ERs alpha and beta) are unclear. We examined here the in vivo role of classical ERs in rapid estrogen actions by evaluating the estrogen-induced effects on two major signaling pathways within the brains of alphaER-, betaER-, and double alphabetaER-knockout (ERKO) ovariectomized female mice. Estrogen significantly (P < 0.05) increased the numbers of phospho-cAMP response element binding protein (phospho-CREB)-immunoreactive cells in specific brain regions of wild-type mice in a time-dependent manner beginning within 15 min. In brain areas that express predominantly ERbeta, this response was absent in betaERKO mice, whereas brain regions that express mostly ERalpha displayed no change in alphaERKO mice. In the medial preoptic nucleus (MPN), an area that expresses both ERs, the estrogen-induced phosphorylation of CREB was normal in both alphaERKO and betaERKO mice. However, estrogen had no effect on CREB phosphorylation in the MPN, or any other brain region, in double alphabetaERKO animals. Estrogen was also found to increase MAPK phosphorylation levels in a rapid (<15 min) manner within the MPN. In contrast to CREB signaling, this effect was lost in either alphaERKO or betaERKO mice. These data show that ERalpha and ERbeta play region- and pathway-specific roles in rapid estrogen actions throughout the brain. They further indicate an indispensable role for classical ERs in rapid estrogen actions in vivo and highlight the importance of ERs in coordinating both classical and rapid actions of estrogen.

Critical Role for Estrogen Receptor alpha in Negative Feedback Regulation of Gonadotropin-Releasing Hormone mRNA Expression in the Female Mouse

Estrogen exerts an important regulatory influence upon the functioning of the gonadotropin-releasing hormone (GnRH) neurons. Whether this is mediated by estrogen receptor α (ERα) or ERβ or both ERs is presently unclear. Using female mice with targeted disruptions of ERα and ERβ (αERKO and βERKO, respectively) we have investigated the in vivo role of the two ERs in the negative feedback influence of estrogen upon GnRH mRNA expression. Compared with intact wild-type mice, plasma luteinizing hormone (LH) levels were substantially (p < 0.01) higher in intact αERKO females and increased modestly (p < 0.05) in intact βERKO mice. Three weeks after ovariectomy, LH concentrations were elevated significantly in wild-type (p < 0.01) and βERKO (p < 0.05) mice but not changed in αERKO females. Quantitative analysis of GnRH mRNA expression using in situ hybridization revealed that cellular GnRH mRNA content was greater (p < 0.05) in intact αERKO mice compared with intact wild-type and βERKO mice. Following ovariectomy, GnRH mRNA expression was elevated in wild-type (p = 0.06) and βERKO (p < 0.05) females but not αERKO mice. These data demonstrate that both ERα and ERβ are involved in inhibiting LH levels at times of estrogen-negative feedback in vivo. However, only ERα appears to be critical for the estrogen-negative feedback suppression of GnRH mRNA expression in the female mouse.

Estrogen receptor β (ERβ) protein levels in neurons depend on estrogen receptor α (ERα) gene expression and on its ligand in a brain region-specific manner

Estrogen receptors (ERs) α and β are very similar estrogen-binding proteins, perhaps gene duplication products, which act as ligand-dependent transcription factors. While the estrogenic regulation of ERα has been well documented, little is known about how estrogen regulates ERβ and whether ERα plays a role in the expression and estrogenic regulation of ERβ. In the present study, we examined the effects of gonadectomy and estrogen replacement on ERβ immunoreactivity (ir) in wild-type (WT) and ERα knockout (αERKO) adult male mice in six brain regions, the medial preoptic area (MPOA), the bed nucleus of the stria terminalis (BNST), the paraventricular nucleus (PVN), the ventromedial nucleus of hypothalamus (VMH), the medial amygdala nucleus (MeAMY) and the dorsal raphe nucleus (DRN). Mice were divided into four different treatment groups: gonadally intact, gonadectomized (GDX), GDX+short-term treatment with estrogen (s.c. injection of estradiol benzoate (EB), 5 μg, at 48 h before perfusion) or GDX+long-term treatment with estrogen (implant of an EB pellet, 2.5 μg/day, for 10 days). In intact αERKO mice, the number of ERβ expressing cells was significantly decreased in the MPOA and increased in the BNST, compared to WT mice. Both in the MPOA and BNST, steroid hormone regulation of ERβ protein (an increase by GDX and a decline to intact levels by EB) was found only in WT, not in αERKO mice. In the VMH, GDX significantly increased the number of ERβ ir expressing cells in both genotypes. EB treatment tended to decrease the number of ERβ ir cells in WT mice, whereas EB treatment tended to increase ERβ ir cell counts in αERKO mice. No effects of GDX or EB treatment were found in the DRN and MeAMY regardless of genotype. These results suggest that gonadal steroid hormones may regulate ERβ protein in male mice and ERα may be involved in the expression and regulation of ERβ in a region-specific manner.

Estrogen action and male fertility: roles of the sodium/hydrogen exchanger-3 and fluid reabsorption in reproductive tract function.

Estrogen receptor alpha (ER alpha) is essential for male fertility. Its activity is responsible for maintaining epithelial cytoarchitecture in efferent ductules and the reabsorption of fluid for concentrating sperm in the head of the epididymis. These discoveries and others have helped to establish estrogen's bisexual role in reproductive importance. Reported here is the molecular mechanism to explain estrogen's role in fluid reabsorption in the male reproductive tract. It is shown that estrogen regulates expression of the Na(+)/H(+) exchanger-3 (NHE3) and the rate of (22)Na(+) transport, sensitive to an NHE3 inhibitor. Immunohistochemical staining for NHE3, carbonic anhydrase II (CAII), and aquaporin-I (AQP1) was decreased in ER alpha knockout (alpha ERKO) efferent ductules. Targeted gene-deficient mice were compared with alpha ERKO, and the NHE3 knockout and CAII-deficient mice showed alpha ERKO-like fluid accumulation, but only the NHE3 knockout and alpha ERKO mice were infertile. Northern blot analysis showed decreases in mRNA for NHE3 in alpha ERKO and antiestrogen-treated mice. The changes in AQP1 and CAII in alpha ERKO seemed to be secondary because of the disruption of apical cytoarchitecture. Ductal epithelial ultrastructure was abnormal only in alpha ERKO mice. Thus, in the male, estrogen regulates one of the most important epithelial ion transporters and maintains epithelial morphological differentiation in efferent ductules of the male, independent of its regulation of Na(+) transport. Finally, these data raise the possibility of targeting ER alpha in developing a contraceptive for the male.

Estrogen Receptor-α Knockout Mice Exhibit Resistance to the Developmental Effects of Neonatal Diethylstilbestrol Exposure on the Female Reproductive Tract

Data indicate that estrogen-dependent and -independent pathways are involved in the teratogenic/carcinogenic syndrome that follows developmental exposure to 17β-estradiol or diethylstilbestrol (DES), a synthetic estrogen. However, the exact role and extent to which each pathway contributes to the resulting pathology remain unknown. We employed the αERKO mouse, which lacks estrogen receptor-α (ERα), to discern the role of ERα and estrogen signaling in mediating the effects of neonatal DES exposure. The αERKO provides the potential to expose DES actions mediated by the second known ER, ERβ, and those that are ER-independent. Wild-type and αERKO females were treated with vehicle or DES (2 μg/pup/day for Days 1–5) and terminated after 5 days and 2, 4, 8, 12, and 20 months for biochemical and histomorphological analyses. Assays for uterine expression of the genes Hoxa10, Hoxa11, and Wnt7a shortly after treatment indicated significant decreases in DES-treated wild-type but no effect in the αERKO. In contrast, the DES effect on uterine expression of Wnt4 and Wnt5a was preserved in both genotypes, suggesting a developmental role for ERβ. Adult αERKO mice exhibited complete resistance to the chronic effects of neonatal DES exposure exhibited in treated wild-type animals, including atrophy, decreased weight, smooth muscle disorganization, and epithelial squamous metaplasia in the uterus; proliferative lesions of the oviduct; and persistent vaginal cornification. Therefore, the lack of DES effects on gene expression and tissue differentiation in the αERKO provides unequivocal evidence of an obligatory role for ERα in mediating the detrimental actions of neonatal DES exposure in the murine reproductive tract.

Studying endocrine disrupter action and toxicology in ER knock‐out mice

Estrogen receptors (ER) are thought to play a crucial role in development, reproduction, carcinogenesis and normal physiology. Using gene targeting techniques, we have produced lines of transgenic mice homozygous for the disrupted ERα gene (αERKO) and ERβ genes (βERKO). Comparable levels of ER‐β mRNA were found in tissues of αERKO mice suggesting that ER‐β expression is not dependent on ERα. αERKO mice were totally unresponsive to uterotropic assays with estradiol, Genistein, hydroxy TAM, DES or EGF treatment. Progesterone receptor (PR) mRNA was detected in αERKO mice, but not stimulated by estrogen in the uterus, mammary gland and ovary. Pituitary hormones are elevated in αERKO females but same as WT in βERKO mice indicating that ERα plays a major role in peptide hormone regulation. Genistien was equally effective in αERKO as WT mice in suppressing serum LH levels suggesting its action in this estrogen target response involved a non‐ER mediated mechanism compared to the uterine genistein activity. Analysis of the mammary glands of adult αERKO mice, showed a primitive ductal rudiment while the βERKO had the fully developed ductal tree seen in WT siblings. The influence of ERα activity on mammary tumorgenicity was evaluated by crossing αERKO with an oncogenic WNT‐1 transgenic mouse line having an increased incidence of mammary tumors. Mice with WT ER levels and WNT‐1 transgene show 98% tumor incidence at 27 weeks of age. αERKO/WNT‐1 mice showed a delayed onset (54 weeks). These results indicate that mammary tumors can develop from ER negative tissues, but with a delayed onset. A second cross with erB2 (Neu) transgenics shows that WT and hetero Neu mice have a tumor incidence at 54 wks of age. αERKO/Neu have an extremely low incidence past 90 wks. Compared to the WNT/ERKO the ERKO/Neu have a poorer tumor rate suggesting that ERα plays a more prominent role in this oncogenic tumor induction. Since the initial description of DES carcinogenesis in both humans and mice, a major question was what aspect of the DES action was ER mediated or non‐receptor mediated. Perinatal DES treatment to WT mice showed numerous phenotypic toxicological and carcinogenic effects including, persistent vaginal cornification, uterine hyperplasia, oviductal hypertrophy in females. In similarly treated males, DES induced seminal vesicle atrophy and prostatic hyperplasia. αERKO treated males and females showed none of the observed toxic effects indicating that ERα was involved in mediating these DES effects and not ERβ. Further characterization of the mice and comparison of the DES effects in βERKO mice and double ER gene KO mice will be needed to more fully the specific roles of the two different forms of ER in estrogen hormone toxicology and hormonal carcinogenesis.

The role of estrogen and estrogen receptor-α in male adipose tissue

Males and females both express estrogen receptor (ER) in white adipose tissue (WAT), and estrogens appear to play an important role in regulating WAT in females. However, the role of ER in male WAT was unclear. In this review, we describe our work, which used wild type (WT) and ERα-knockout (αERKO) male and female mice to determine the role of ERα in regulating WAT and brown adipose tissue (BAT). There were progressive increases in WAT with advancing age in αERKO compared with WT males; weights of various WAT depots in αERKO males were increased by more than 100% compared with WT controls during adulthood. Conversely, BAT weight was similar in αERKO and WT males at all ages. Adipocyte areas and numbers were also increased in WAT from αERKO compared with WT males. Compared with WT controls, αERKO females also had increases in WAT. The αERKO mice also had insulin resistance and impaired glucose tolerance, similar to humans lacking ERα or aromatase. The obesity in αERKO males appeared to involve decreased energy expenditure rather than hyperphagia. In summary, ERα absence causes adipocyte hyperplasia and hypertrophy in WAT, but not BAT, and is accompanied by insulin resistance and glucose intolerance in both males and females. These results are the first evidence that the estrogen/ERα signaling system is critical in female and male WAT deposition, and may have clinical implications.

Estrogen receptor-α is required by the supporting somatic cells for spermatogenesis

The gene for estrogen receptor-alpha (ERα) was disrupted in embryonic stem cells by homologous recombination and these cells were used to generate mice with a targeted mutation in the ERα gene (αERKO mice). It was found that males homozygous for the mutation are infertile, indicating that estrogen signaling through this nuclear hormone receptor is required for male reproductive function. Although spermatogenesis appears normal in juvenile and young adult αERKO mice, the sperm produced are unable to fertilize eggs in vitro. To determine whether ERα is required by somatic or germ cells in the male reproductive tract, we transplanted germ cells from homozygous mutant (ERα −/−) males to the testes of wild-type (ERα +/+) males depleted of germ cells by busulfan treatment. The recipients (‘surrogate fathers’) sired offspring heterozygous for the mutation (ERα +/−) and carrying the coat-color marker of the infertile donor males. This indicated that ERα −/− germ cells are able to produce sperm competent to fertilize when they are supported by ERα +/+ somatic cells. When ERα +/− offspring produced by germ cell transplantation were mated to produce ERα −/− males, these mice were found to have the same phenotype as originally reported for αERKO males. These studies showed that male germ cells do not require ERα for regulation of their own genes for development and function, and strongly imply that somatic cells of the male reproductive tract require ERα to support the production of sperm that are capable of fertilization.

Evidence That Epithelial and Mesenchymal Estrogen Receptor-α Mediates Effects of Estrogen on Prostatic Epithelium

In combination with androgens, estrogens can induce aberrant growth and malignancy of the prostate gland. Estrogen action is mediated through two receptor subtypes: estrogen receptors α (ERα) and β (ERβ). Wild-type (wt) and transgenic mice lacking a functional ERα (αERKO) or ERβ (βERKO) were treated with the synthetic estrogen diethylstilbestrol (DES). DES induced prostatic squamous metaplasia (SQM) in wt and βERKO but not in αERKO mice, indicating an essential role for ERα, but not ERβ, in the induction of SQM of prostatic epithelium. In order to determine the respective roles of epithelial and stromal ERα in this response, the following tissue recombinants were constructed with prostatic epithelia (E) and stroma (S) from wt and ERKO mice: wt-S+wt-E, αERKO-S+αERKO-E, wt-S+αERKO-E, and αERKO-S+wt-E. A metaplastic response to DES was observed in wt-S+wt-E tissue recombinants. This response to DES involved multilayering of basal epithelial cells, expression of cytokeratin 10, and up-regulation of the progesterone receptor. Tissue recombinants containing αERKO-E and/or -S (αERKO-S+αERKO-E, wt-S+αERKO-E, and αERKO-S+wt-E) failed to respond to DES. Therefore, full and uniform epithelial SQM requires ERα in the epithelium and stroma. These results provide a novel insight into the cell–cell interactions mediating estrogen action in the prostate via ERα.

Progesterone action and responses in the αERKO mouse

Ovarian steroids have important inter-related roles in many systems and processes required for mammalian reproduction. The female reproductive tract, ovaries, and mammary glands are all targets for both estrogen and progesterone. In addition, the actions of these hormones are intertwined in that, for example, progesterone attenuates the proliferative effect of estrogen in the uterus, whereas estrogen also induces the progesterone receptor (PR) mRNA and protein, thus enhancing progesterone actions. The generation of mice that lacks the progesterone receptor (PRKO) or the estrogen receptorα (αERKO) has provided numerous insights into the interacting roles of these hormones. The mammary glands of the PRKO mice develop with full epithelial ducts that lack side branching and lobular alveolar structures, whereas the αERKO mice develop only an epithelial rudiment. This indicates that estrogen is important for ductal morphogenesis, whereas progesterone is required for ductal branching and alveolar development. Both the αERKO and PRKO mice are also anovulatory, but exhibit different causal pathologies. The αERKO ovary seems to possess follicles up to the preantral stage and shows a polycystic phenotype as a result of chronic hyperstimulation by LH. The PRKO follicles seem to develop to an ovulatory stage, but are unable to rupture, indicating a role for progesterone in ovulation. The uteri of these two strains seem to develop normally; however, the function and hormone responses are abnormal in each. Because estrogen is known to induce PRs in the uterus, the progesterone responsiveness of the αERKO uterus was characterized. PR mRNA was detected but was not up-regulated by estrogen in the αERKO tissue. PRs are present in the αERKO tissue at 60% of the level in wild-type tissue and show a similar amount of A and B isoforms when measured by R5020 binding and detected by Western blotting. The PRs were able to mediate induction of two progesterone-responsive uterine genes: calcitonin and amphiregulin. The αERKO uterine tissue was also able to undergo a decidual reaction in response to hormonal and intraluminal treatments to mimic implantation; however, unlike normal wild-type uteri, this response was estrogen independent in the αERKO uterine tissue.