Progesterone Receptor Cre Research Articles

YAP1 and WWTR1 are required for murine pregnancy initiation.

Endometrial stromal cell decidualization is required for pregnancy success. Although this process is integral to fertility, many of the intricate molecular mechanisms contributing to decidualization remain undefined. One pathway that has been implicated in endometrial stromal cell decidualization in humans in vitro, is the Hippo signaling pathway. Two previously conducted studies showed that the effectors of the Hippo signaling pathway, YAP1 and WWTR1, are required for decidualization of primary endometrial stromal cells in vitro. To investigate the in vivo role of YAP1 and WWTR1 in decidualization and pregnancy initiation, we generated Progesterone receptor Cre mediated mutation of a combination of Yap1 and Wwtr1 alleles. Female Yap1 and Wwtr1 triple allele mutants exhibited subfertility, a compromised decidualization response, decreased endometrial receptivity, delayed embryonic development, and a unique transcriptional profile at 7.5 days post coitus. Bulk mRNA sequencing revealed aberrant maternal remodeling evidenced by significant alterations in extracellular matrix encoding genes at 7.5 days post-coitus in mutant dams and enrichment for terms associated with fertility-compromising diseases like pre-eclampsia and endometriosis. In addition, differentially expressed genes overlapped directionally with Estrogen receptor and Epidermal growth factor receptor regulated genes as identified by microarray. Our results indicate that Yap1 and Wwtr1 are necessary for successful mammalian pregnancy initiation.

DCAF2 is essential for the development of uterine epithelia and mouse fertility.

The successful outcome of a pregnancy depends on the proper functioning uterine epithelium. DNA damage binding protein 1 and cullin 4-associated factor 2 (DCAF2), a conserved substrate receptor for the cullin 4-RING E3 ubiquitin ligase (CRL4) complex, is essential for maintaining genome stability by facilitating ubiquitin-mediated degradation of substrates. To better understand the physiological role of DCAF2 in female reproduction, we conducted a study using mice with conditional knockout (cKO) of DCAF2 in the uterus using the progesterone receptor Cre (Pgr Cre/+) mouse model. Our results showed the cKO mice were completely infertile, despite having ovarian function. The cKO mice exhibited severely thin uteri, demonstrating notable defects in both the uterine epithelium and a lack of glands. In addition, there were impaired proliferation and differentiation of epithelial cells in the cKO mice, ultimately resulting in failed implantation. Moreover, through deciphering the uterine transcriptome of cKO mice, we revealed crucial differentially expressed genes associated with steroid signaling. Further experiments have demonstrated cKO mice exhibit elevated uterine PGR signaling and reduced estrogen receptor signaling, although the levels of progesterone and estrogen remained unaltered. These alterations may contribute to defects in epithelium. Overall, our findings highlight a previously unrecognized but indispensable role for DCAF2 in the development of uterine luminal and glandular epithelium by orchestrating PGR and estrogen receptor responses. Its deficiency in the uterus leads to mouse infertility.

Uterine-specific SIRT1 deficiency confers premature uterine aging and impairs invasion and spacing of blastocyst, and stromal cell decidualization, in mice.

A distinct age-related alteration in the uterine environment has recently been identified as a prevalent cause of the reproductive decline in older female mice. However, the molecular mechanisms that underlie age-associated uterine adaptability to pregnancy are not known. Sirtuin 1 (SIRT1), a multifunctional NAD+-dependent deacetylase that regulates cell viability, senescence and inflammation during aging, is reduced in aged decidua. Thus, we hypothesize that SIRT1 plays a critical role in uterine adaptability to pregnancy and that uterine-specific ablation of Sirt1 gene accelerates premature uterine aging. Female mice with uterine ablation of Sirt1 gene using progesterone receptor Cre (PgrCre) exhibit subfertility and signs of premature uterine aging. These Sirt1-deficient mothers showed decreases in litter size from their 1st pregnancy and became sterile (25.1 ± 2.5 weeks of age) after giving birth to the third litter. We report that uterine-specific Sirt1 deficiency impairs invasion and spacing of blastocysts, and stromal cell decidualization, leading to abnormal placentation. We found that these problems traced back to the very early stages of hormonal priming of the uterus. During the window of receptivity, Sirt1 deficiency compromises uterine epithelial-stromal crosstalk, whereby estrogen, progesterone and Indian hedgehog signaling pathways are dysregulated, hampering stromal cell priming for decidualization. Uterine transcriptomic analyses also link these causes to perturbations of histone proteins and epigenetic modifiers, as well as adrenomedullin signaling, hyaluronic acid metabolism, and cell senescence. Strikingly, our results also identified genes with significant overlaps with the transcriptome of uteri from aged mice and transcriptomes related to master regulators of decidualization (e.g. Foxo1, Wnt4, Sox17, Bmp2, Egfr and Nr2f2). Our results also implicate accelerated deposition of aging-related fibrillar Type I and III collagens in Sirt1-deficient uteri. Collectively, SIRT1 is an important age-related regulator of invasion and spacing of blastocysts, as well as decidualization of stromal cells.

Rictor/mTORC2 is involved in endometrial receptivity by regulating epithelial remodeling.

Successful embryo implantation requires well-functioning endometrial luminal epithelial cells to establish uterine receptivity. Inadequate uterine receptivity is responsible for approximately two thirds of implantation failures in humans. However, the regulatory mechanism governing this functional process remains largely unexplored. A previous study revealed that the expression of Rictor, the main member of mTORC2, in mouse epithelial cells is increased on the fourth day of gestation (D4). Here, we provide the first report of the involvement of Rictor in the regulation of endometrial receptivity. Rictor was conditionally ablated in the mouse endometrium using a progesterone receptor cre (PRcre ) mouse model. Loss of Rictor altered polarity remodeling and the Na+ channel protein of endometrial cells by mediating Rac-1/PAK1(pPAK1)/ERM(pERM) and Sgk1/pSgk1 signaling, respectively, ultimately resulting in impaired fertility. In the endometrium of women with infertility, the expression of Rictor was changed, along with the morphological transformation and Na+ channel protein of epithelial cells. Our findings demonstrate that Rictor is crucial for the establishment of uterine receptivity in both mice and humans. The present study may help improve the molecular regulatory network of endometrial receptivity and provide new diagnostic and treatment strategies for infertility.

Loss of basigin expression in uterine cells leads to subfertility in female mice†.

Basigin (BSG) is a transmembrane glycoprotein involved in cell proliferation, angiogenesis, and tissue remodeling. BSG has been shown to be essential for male and female reproduction although little is known about its role in normal uterine function. To study the potential function of BSG in the female reproductive tract, we generated mice with conditional knockout of Bsg in uterine cells using progesterone receptor-Cre and hypothesized that BSG is required for normal pregnancy in mice. Fertility study data showed that the conditional knockout mice had significantly reduced fertility compared to controls. Ovarian function of the conditional knockout mice appeared normal with no difference in the number of superovulated oocytes collected or in serum progesterone levels between the conditional knockout and the control mice. Uterine tissues collected at various times of gestation showed increased abnormalities in implantation, decidualization, placentation, and parturition in the conditional knockout mice. Uterine cross sections on Day 5 of pregnancy showed implantation failure and abnormal uterine epithelial differentiation in a large proportion of the conditional knockout mice. There was a compromised decidual response to artificial decidualization stimuli and decreased mRNA and protein levels for decidualization genes in the uteri of the conditional knockout mice. We also observed altered protein expression of monocarboxylate transporter 1 (MCT1), as well as impaired angiogenesis in the conditional knockout uteri compared to the controls. These results support that BSG is required for successful pregnancy through its functions in implantation and decidualization.

Vaginal Squamous Cell Carcinoma Develops in Mice with Conditional Arid1a Loss and Gain of Oncogenic Kras Driven by Progesterone Receptor Cre

Oncogenic KRAS mutations are a common finding in endometrial cancers. Recent sequencing studies indicate that loss-of-function mutations in the ARID1A gene are enriched in gynecologic malignant tumors. However, neither of these genetic insults alone are sufficient to develop gynecologic cancer. To determine the role of the combined effects of deletion of Arid1a and oncogenic Kras, Arid1aflox/flox mice were crossed with KrasLox-Stop-Lox-G12D/+ mice using progesterone receptor Cre (PgrCre/+). Histologic analysis and immunohistochemistry of survival studies were used to characterize the mutant mouse phenotype. Hormone dependence was evaluated by ovarian hormone depletion and estradiol replacement. Arid1aflox/flox; KrasLox-Stop-Lox-G12D/+; PgrCre/+ mice were euthanized early because of invasive vaginal squamous cell carcinoma. Younger mice had precancerous intraepithelial lesions. Immunohistochemistry supported the pathological diagnosis with abnormal expression and localization of cytokeratin 5, tumor protein P63, cyclin-dependent kinase inhibitor 2A, and Ki-67, the marker of proliferation. Ovarian hormone deletion in Arid1aflox/flox; KrasLox-Stop-Lox-G12D/+; PgrCre/+ mice resulted in atrophic vaginal epithelium without evidence of vaginal tumors. Estradiol replacement in ovarian hormone-depleted Arid1aflox/flox; KrasLox-Stop-Lox-G12D/+; PgrCre/+ mice resulted in lesions that resembled the squamous cell carcinoma in intact mice. Therefore, this mouse can be used to study the transition from benign precursor lesions into invasive vaginal human papillomavirus-independent squamous cell carcinoma, offering insights into progression and pathogenesis of this rare disease.

Oviductal Retention of Embryos in Female Mice Lacking Estrogen Receptor α in the Isthmus and the Uterus.

Estrogen receptor α (ESR1; encoded by Esr1) is a crucial nuclear transcription factor for female reproduction and is expressed throughout the female reproductive tract. To assess the function of ESR1 in reproductive tissues without confounding effects from a potential developmental defect arising from global deletion of ESR1, we generated a mouse model in which Esr1 was specifically ablated during postnatal development. To accomplish this, a progesterone receptor Cre line (PgrCre) was bred with Esr1f/f mice to create conditional knockout of Esr1 in reproductive tissues (called PgrCreEsr1KO mice) beginning around 6 days after birth. In the PgrCreEsr1KO oviduct, ESR1 was most efficiently ablated in the isthmic region. We found that at 3.5 days post coitus (dpc), embryos were retrieved from the uterus in control littermates while all embryos were retained in the PgrCreEsr1KO oviduct. Additionally, serum progesterone (P4) levels were significantly lower in PgrCreEsr1KO compared to controls at 3.5 dpc. This finding suggests that expression of ESR1 in the isthmus and normal P4 levels allow for successful embryo transport from the oviduct to the uterus. Therefore, alterations in oviductal isthmus ESR1 signaling and circulating P4 levels could be related to female infertility conditions such as tubal pregnancy.

Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit Is Required for Uterine Epithelial Integrity

Normal proliferation and differentiation of uterine epithelial cells are critical for uterine development and function. Enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a core component of polycomb repressive complexes 2, possesses histone methyltransferase activity that catalyzes the trimethylation of lysine 27 of histone H3. EZH2 has been involved in epithelial-mesenchymal transition, a key event in development and carcinogenesis. However, its role in uterine epithelial cell function remains unknown. To determine the role of uterine EZH2, Ezh2 was conditionally deleted using progesterone receptor Cre recombinase, which is expressed in both epithelial and mesenchymal compartments of the uterus. Loss of EZH2 promoted stratification of uterine epithelium, an uncommon and detrimental event in the uterus. The abnormal epithelium expressed basal cell markers, including tumor protein 63, cytokeratin 5 (KRT5), KRT6A, and KRT14. These results suggest that EZH2 serves as a guardian of uterine epithelial integrity, partially via inhibiting the differentiation of basal-like cells and preventing epithelial stratification. The observed epithelial abnormality was accompanied by fertility defects, altered uterine growth and function, and the development of endometrial hyperplasia. Thus, the Ezh2 conditional knockout mouse model may be useful to explore mechanisms that regulate endometrial homeostasis and uterine function.

Activin-like kinase 5 (ALK5) inactivation in the mouse uterus results in metastatic endometrial carcinoma

The endometrial lining of the uterine cavity is a highly dynamic tissue that is under the continuous control of the ovarian steroid hormones, estrogen and progesterone. Endometrial adenocarcinoma arises from the uncontrolled growth of the endometrial glands, which is typically associated with unopposed estrogen action and frequently occurs in older postmenopausal women. The incidence of endometrial cancer among younger women has been rising due to increasing rates of obesity, a major risk factor for the disease. The transforming growth factor β (TGFβ) family is a highly conserved group of proteins with roles in cellular differentiation, proliferation, and cancer. Inactivating mutations in the genes encoding the TGFβ cell surface receptors (TGFBR1/ALK5 and TGFBR2) have been detected in various human cancers, indicating that a functional TGFβ signaling pathway is required for evading tumorigenesis. In this study, we present a mouse model with conditional inactivation of activin receptor-like kinase 5 (ALK5) in the mouse uterus using progesterone receptor cre ("Alk5 cKO") that develops endometrial adenocarcinoma with metastasis to the lungs. The cancer and metastatic lung nodules are estrogen dependent and retain estrogen receptor α (ERα) reactivity, but have decreased levels of progesterone receptor (PR) protein. The endometrial tumors develop only in Alk5 cKO mice that are mated to fertile males, indicating that TGFβ-mediated postpartum endometrial repair is critical for endometrial function. Overall, these studies indicate that TGFβ signaling through TGFBR1/ALK5 in the endometrium is required for endometrial homeostasis, tumor suppression, and postpartum endometrial regeneration.

FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation.

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, β-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.

Forkhead box a2 (FOXA2) is essential for uterine function and fertility

Establishment of pregnancy is a critical event, and failure of embryo implantation and stromal decidualization in the uterus contribute to significant numbers of pregnancy losses in women. Glands of the uterus are essential for establishment of pregnancy in mice and likely in humans. Forkhead box a2 (FOXA2) is a transcription factor expressed specifically in the glands of the uterus and is a critical regulator of postnatal uterine gland differentiation in mice. In this study, we conditionally deleted FOXA2 in the adult mouse uterus using the lactotransferrin Cre (Ltf-Cre) model and in the neonatal mouse uterus using the progesterone receptor Cre (Pgr-Cre) model. The uteri of adult FOXA2-deleted mice were morphologically normal and contained glands, whereas the uteri of neonatal FOXA2-deleted mice were completely aglandular. Notably, adult FOXA2-deleted mice are completely infertile because of defects in blastocyst implantation and stromal cell decidualization. Leukemia inhibitory factor (LIF), a critical implantation factor of uterine gland origin, was not expressed during early pregnancy in adult FOXA2-deleted mice. Intriguingly, i.p. injections of LIF initiated blastocyst implantation in the uteri of both gland-containing and glandless adult FOXA2-deleted mice. Although pregnancy was rescued by LIF and was maintained to term in uterine gland-containing adult FOXA2-deleted mice, pregnancy failed by day 10 in neonatal FOXA2-deleted mice lacking uterine glands. These studies reveal a previously unrecognized role for FOXA2 in regulation of adult uterine function and fertility and provide original evidence that uterine glands and, by inference, their secretions play important roles in blastocyst implantation and stromal cell decidualization.

Abstract LB-136: The role of ER chaperone GRP94 in endometrial cancer progression

Abstract Endometrial cancer is the most prevalent gynecologic cancer in the United States and accounts for nearly 50,000 incident cases annually. One of the most common mutations detected in the more prevalent Type 1 endometrial cancer is the loss of the tumor suppressor gene, Pten (phosphatase and tensin homolog). Pten mutations are involved in a wide variety of human cancers, including >60% of endometrial cancer. The glucose-regulated protein 94 (GRP94), also known as glycoprotein 96 (gp96), is a major chaperone protein in the ER. The client proteins of GPR94 include important regulators of cell growth and cell adhesion such as IGF-1, Toll-like receptors and integrins. GRP94 is known to play major roles in immune modulation. GRP94 also possesses anti-apoptotic properties. GRP94 elevation has been reported in a variety of human cancers and has been implicated in cancer development. Previously, GRP94 knockout in multiple myeloma reduced cell proliferation and survival, through inhibiting Wnt-LRP-survivin pathway. GRP94 knockout in macrophages reduced inflammatory colon tumorigenesis. On the other hand, GRP94 knockout in hepatocytes resulted in liver progenitor cell proliferation and acceleration of liver cancer, associating with repopulation of GRP94-positive hepatocytes. Therefore, the role of GRP94 in cancer appears to be context-dependent due to its multifaceted functions. Currently, little is known about GRP94 expression and function in endometrial cancer. We examined GRP94 expression in human endometrial cancer tissues and robust GRP94 expression was observed. In human endometrial cancer cell line, GRP94 knockdown by siRNA led to decreased proliferation and cell migration. To examine the requirement of GRP94 in the initiation and development of endometrial cancer, we utilized the Progesterone Receptor-Cre to specifically delete genes in the mouse endometrium. Before studying GRP94 in endometrial cancer, we first determined the effect of GRP94 depletion in normal uteri. Surprisingly, GRP94 single knockout uteri showed squamous cell metaplasia, confirmed by expression of K14 and p63, and reduction in size. Next, we created the biallelic mutant mice with concurrent deletion of Pten and Grp94 in the endometrium. We monitored the progress of endometrial cancer development, with mice bearing Pten knockout alone serving as the positive controls. As expected, Pten single knockout mice develop endometrial cancer by 4 week and initiate invasion into myometrium by 8 week. Analysis of the mice uteri indicated that GRP94 deficiency delayed the onset of endometrial cancer, associating with altered cell adhesion properties and pro-oncogenic signaling pathways. This study expands our understanding of GRP94 function in the progression of solid tumors and provides the first evidence that GRP94 could be a target for combating endometrial cancer. Citation Format: Jieli Shen, Yvonne G. Lin, Louis Dubeau, Amy S. Lee. The role of ER chaperone GRP94 in endometrial cancer progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-136. doi:10.1158/1538-7445.AM2015-LB-136

Role of Foxl2 in uterine maturation and function.

Foxl2 codes for a forkhead/HNF3 transcription factor essential for follicular maturation and maintenance of ovarian identity. FOXL2 mutations are associated with Blepharophimosis, Ptosis and Epicanthus inversus Syndrome (BPES) characterized by eyelid malformations (types I and II) and premature ovarian insufficiency (type I). We show that Foxl2 is not only expressed by the ovary, but also by other components of the mouse female reproductive tract, including the uterus, the cervix and the oviduct. In the uterus, Foxl2 expression is first observed in the neonatal mesenchyme and, during uterine maturation, persists in the stroma and in the deep inner myometrial layer (IML). In the adult, Foxl2 is expressed in the differentiated stromal layer, but no longer in the myometrium. Conditional deletion of Foxl2 in the postnatal (PN) uterus using Progesterone Receptor-cre (Pgr(cre/+)) mice results in infertility. During PN uterine maturation Pgr(cre/+); Foxl2(flox/flox) mice present a severely reduced thickness of the stroma layer and an hypertrophic, disorganized IML. In adult Pgr(cre/+); Foxl2(flox/flox) mice a supplementary muscular layer is present at the stroma/myometrium border and vascular smooth muscle cells fail to form a coherent layer around uterine arteries. Wnt signalling pathways play a central role in uterine maturation; in Pgr(cre/+); Foxl2(flox/flox) mice, Wnt genes are deregulated suggesting that Foxl2 acts through these signals. In humans, thickening of the IML (also called "junctional zone") is associated with reduced fertility, endometriosis and adenomyosis. Our data suggest that Foxl2 has a crucial role in PN uterine maturation and could help to understand sub-fertility predisposition in women.

Ovarian LGR5 is critical for successful pregnancy

Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) is expressed in many organs, including female reproductive organs, and is a stem cell marker in the stomach and intestinal epithelium, hair follicles, and ovarian surface epithelium. Despite ongoing studies, the definitive physiological functions of Lgr5 remain unclear. We utilized mice with conditional deletion of Lgr5 (Lgr5(d/d)) in the female reproductive organs by progesterone receptor-Cre (Pgr(Cre)) to determine Lgr5's functions during pregnancy. Only 30% of plugged Lgr5(d/d) females delivered live pups, and their litter sizes were lower. We found that pregnancy failure in Lgr5(d/d) females was due to insufficient ovarian progesterone (P4) secretion that compromised decidualization, terminating pregnancy. The drop in P4 levels was reflected in elevated levels of P4-metabolizing enzyme 20α-hydroxysteroid dehydrogenase in corpora lutea (CL) inactivated of Lgr5. Of interest, P4 supplementation rescued decidualization failure and supported pregnancy to full term in Lgr5(d/d) females. These results provide strong evidence that Lgr5 is critical to normal CL function, unveiling a new role of LGR5 in the ovary.

Dysregulation of Uterine Signaling Pathways in Progesterone Receptor-Cre Knockout of Dicer

Epithelial-stromal interactions in the uterus are required for normal uterine functions such as pregnancy, and multiple signaling pathways are essential for this process. Although Dicer and microRNA (miRNA) have been implicated in several reproductive processes, the specific roles of Dicer and miRNA in uterine development are not known. To address the roles of miRNA in the regulation of key uterine pathways, we generated a conditional knockout of Dicer in the postnatal uterine epithelium and stroma using progesterone receptor-Cre. These Dicer conditional knockout females are sterile with small uteri, which demonstrate significant defects, including absence of glandular epithelium and enhanced stromal apoptosis, beginning at approximately postnatal d 15, with coincident expression of Cre and deletion of Dicer. Specific miRNA (miR-181c, -200b, -101, let-7d) were down-regulated and corresponding predicted proapoptotic target genes (Bcl2l11, Aldh1a3) were up-regulated, reflecting the apoptotic phenomenon. Although these mice had normal serum hormone levels, critical uterine signaling pathways, including progesterone-responsive genes, Indian hedgehog signaling, and the Wnt/β-catenin canonical pathway, were dysregulated at the mRNA level. Importantly, uterine stromal cell proliferation in response to progesterone was absent, whereas uterine epithelial cell proliferation in response to estradiol was maintained in adult uteri. These data implicate Dicer and appropriate miRNA expression as essential players in the regulation of multiple uterine signaling pathways required for uterine development and appropriate function.

Cdh1 Is Essential for Uterine Gland Development and Adult Uterine Function.

Epithelial differentiation and growth are key processes in mammalian development, which are central to the formation of many organs. Adherens junctions play a role in the establishment of cell polarity and maintenance of the epithelial phenotype, and are essential for epithelial development including cell differentiation and proliferation. CDH1 (E-cadherin) belongs to the cadherin superfamily of cell adhesion molecules, and forms structures known as adherens junctions that mediate intercellular adhesion through dynamic interactions with CTNNB1 (beta-catenin). The objective of this study is to determine a biological role of Cdh1 on postnatal uterine morphogenesis and adult uterine function. Because global disruption of Cdh1 in mice causes early embryo death, we generated the mice with Cdh1 conditionally deleted in the uterus using the progesterone receptor Cre knockin (Pgr-Cre) model. First, histological analysis was conducted in the uteri of Pgr+/+Cdh1f/f (control) and PgrCre/+Cdh1f/f (mutant) mice. Examination of the 4-5-week old uteri of control and mutant mice showed ablation of uterine glands and abnormal organization of the luminal epithelium (LE) in Cdh1 mutant mice. We did not see any significant differences of uterine size and weight at 5-week old. Further, critical regulators for uterine development (CTNNB1, Wnt4, Wnt5a, Wnt7a, Hoxa10 and Hoxa11), and PGR and ESR1 were not significantly altered by loss of Cdh1 in the uterus, but the expression of Foxa2, a marker of glandular epithelium, was reduced in Cdh1 mutant mice. We also examined whether CDH1 expression was regulated by endocrine disruptors: diethylstilbestrol (DES) or progesterone (P4). Although each model has several different phenotypes, both can lead to multiple patterning defects in the female reproductive tract including loss of endometrial glands and gene expression (Wnt and Hox). The exposure of neonatal mice to either DES or P4 did not affect CDH1 expression in the epithelium even though both hormones disrupted uterine gland development. The uteri of the 6-month old Cdh1 mutant mice showed an increase in uterine diameter. Examination of the uterine histology revealed a larger uterine lumen, thinner stroma, and thicker myometrium in Cdh1 mutant mice. Interestingly, immunohistochemical analysis of cyclin D1 and Ki67 indicated no cell proliferation in the LE, but an increase of stromal cell proliferation in Cdh1 mutant mice. To determine whether Cdh1 has a role in pregnancy, Female control and mutant mice were mated to wild-type male mice for 6 months. We have confirmed the presence of vaginal plugs in both control and Cdh1 mutant mice. During the time of observation, the control mice exhibited normal fecundity, whereas the mutant mice were found to be infertile. Further, we determined effects of ovarian steroid hormone on CDH1 expression in the adult uterus. The ovariectomized mice were given an injection of sesame oil (vehicle control, estradiol-17beta (E), P4, or E+P, subcutaneously. However, CDH1 was not altered by ovarian steroids in the adult mouse uterus. These results suggest that Cdh1 is essential for uterine gland development and adult uterine function. The defective mechanisms of gland development or adult uterine function in Cdh1 mutant mice may be different from those seen with disruption by endocrine disruptors in neonate or with regulation by steroid hormones in adult. Supported by NIH HD058222. (platform)

Demonstrating the Critical Role of Uterine Erbb Signaling in Fertility.

The establishment of the appropriate embryo-uterine interactions required for successful implantation and pregnancy require the coordinate actions of growth factors and their receptors. The spatiotemporal expression of Erbb ligands and receptors in the periimplantation uterus and embryo suggests that they play a role in reproduction. Numerous Erbb ligands have overlapping expression patterns in the uterus during pregnancy, and gene ablations of individual Erbb ligands do not show a major uterine phenotype due to compensation by other family members. We have identified EGFR (ERBB1) as being induced during the implantation period and hypothesize that EGFR signaling regulates the ability of the endometrium to support embryo implantation. This hypothesis is being tested in vivo using mouse models and in vitro using primary human endometrial stromal (HES) cell culture and a novel high-throughput microscopy assay. In order to circumvent the embryonic lethality of Egfr-/- mice, conditional ablation of Egfr was achieved by crossing an Egfr floxed (Egfr f/f) mouse with a progesterone receptor cre (PRcre) mouse, resulting in Egfr deletion in all PR positive cells (PRcre/+ Egfr f/f; Egfr d/d). Egfr d/d mice are severely subfertile and exhibit reduced implantation and abnormal embryos. Mutant mice demonstrate an ablation of artificial decidualization including defects in morphology, proliferation, apoptosis, differentiation and gene expression. EGFR is induced during in vitro HES cell decidualization and that the use of EGFR knockdown impedes decidualization. Furthermore, these results have been confirmed using a novel, automated image-based microscopy assay. These results strongly indicate that Erbb signaling plays a critical role in female reproduction. Continuation of ongoing studies determining the mechanism of EGFR action and the role of Erbb2 will provide valuable insight and allow further elucidation of complex pathways and the orchestrated molecular interactions vital to reproductive health. This research was supported by NIH Grant RO1CA077530 (to J.P.L.), by NIH R01HD057873 (to JW.J), and by the Eunice Kennedy Shriver NICHD/NIH through cooperative agreement U54 HD07495 as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research. (platform)

ACTIVATION OF PPAR GAMMA FUNCTION BY ARACHIDONIC ACID METABOLITES CONTROLS OVULATION BY INDUCING THE EXPRESSION OF PRO-INFLAMMATORY AND VASOACTIVE MOLECULES IN THE OVARY

Ovulation is a key female reproductive event, which involves the release of a fertilizable oocyte from an ovarian follicle. This process is initiated when follicular tissue is stimulated by the pituitary gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH). A surge of LH induces a cascade of gene expression in the FSH-primed ovary that culminates in the rupture of the follicle. In order to explore the pathways that control ovulation, we subjected mice to a superovulation protocol and performed global gene expression profiling at 5 h and 11 h after administration of human chorionic gonadotropin (hCG) following 48 h of equine chorionic gonadotropin (eCG) treatment. We found that the gene encoding the peroxisome proliferator-activated receptor gamma (PPARr) is one of several genes that are induced in the mouse ovary at 5 h post hCG. The expression of PPARr is induced primarily in the granulosa cells of the preovulatory follicles. To address the functional role of this gene during ovulation, we created a conditional knockout mouse model by crossing mice harboring “floxed” PPARr gene with progesterone receptor Cre knockin (PR-Cre) mice. This resulted in the generation of females, in which the PPARr gene undergoes Cre-mediated excision in the mural granulosa cells of the preovulatory follicles. The conditional PPARr knockout (KO) mice exhibited impaired ovulation in response to gonadotropins. Histological analysis of the ovaries of conditional PPARr- KO mice revealed defects in follicular rupture. Using these mutant mice, we identified endothelin 2 (ET-2), a potent vasoactive molecule, and interleukin-6, a proinflammatory molecule, as primary targets of regulation by PPARr in the preovulatory follicles. Recent studies indicated that antagonist-mediated blockade of ET-2 action in the preovulatory phase prevented follicular rupture, supporting the concept that it is a critical mediator of PPARr function during ovulation. Since PPARr is activated by certain metabolites of arachidonic acid in other tissues, we examined the effects of indomethacin, an inhibitor of arachidonic acid metabolism and a suppressor of ovulation, on the expression of PPARr-target genes. Treatment with this drug markedly reduced the expression of ET-2 and IL-6, confirming that PPARr signaling is indeed activated by arachidonic acid metabolites during the ovulatory period. Collectively, our studies indicated that PPARr controls ovulation by generating important biological mediators of vaso-regulatory and inflammatory pathways, which are likely to play critical roles during follicular rupture. (Supported by NIH grant.) (platform)

Steroid receptor coactivator 2 is essential for progesterone-dependent uterine function and mammary morphogenesis: Insights from the mouse—implications for the human

While the indispensability of the progesterone receptor (PR) in female reproduction and mammary morphogenesis is acknowledged, the coregulators preferentially recruited by PR to mediate its in vivo effects have yet to be fully delineated. To further parse the roles of steroid receptor coactivator (SRC)/p160 family members in P-dependent physiological processes, genetic approaches were employed to generate a mouse model (PR Cre/+SRC-2 flox/flox) in which SRC-2 function was ablated specifically in cell-types that express the PR. Fertility evaluation revealed that while ovulation occurred normally in the PR Cre/+SRC-2 flox/flox mouse, uterine function was markedly affected. Absence of SRC-2 in PR positive uterine cells contributed to an early block in embryo implantation, a phenotype not shared by knockouts for SRC-1 or -3. Although the PR Cre/+SRC-2 flox/flox uterus could mount a partial decidual response, removal of SRC-1 in the PR Cre/+SRC-2 flox/flox uterus resulted in a complete block in decidualization, confirming that uterine SRC-2 and -1 are both required for P-initiated transcriptional programs which lead to full decidualization. In the case of the mammary gland, whole-mount and histological analyses revealed the absence of significant branching morphogenesis in the hormone-treated PR Cre/+SRC-2 flox/flox mammary gland, reinforcing an important role for mammary SRC-2 in cellular proliferative events that require PR. Based on the above and the observation that SRC-2 is expressed in many of the uterine and mammary cell-lineages in the human as observed in the mouse, we suggest that further investigations are warranted to gain additional insights into SRC-2's involvement in normal (and possibly abnormal) uterine and mammary cellular responses to progestins.

Cre-mediated recombination in cell lineages that express the progesterone receptor

Using gene-targeting methods, a progesterone receptor Cre knockin (PR-Cre) mouse was generated in which Cre recombinase was inserted into exon 1 of the PR gene. The insertion positions the Cre gene downstream (and under the specific control) of the endogenous PR promoter. As for heterozygotes for the progesterone receptor knockout (PRKO) mutation, mice heterozygous for the Cre knockin insertion are phenotypically indistinguishable from wildtype. Crossing the PR-Cre with the ROSA26R reporter revealed that Cre excision activity is restricted to cells that express PR in progesterone-responsive tissues such as the uterus, ovary, oviduct, pituitary gland, and mammary gland. Initial characterization of the PR-Cre mouse underscores the utility of this model to precisely ablate floxed target genes specifically in cell lineages that express the PR. In the wider context of female reproductive tissue ontology, this model will be indispensable in tracing the developmental fate of cell lineages that descend from PR positive progenitors.