Epithelial progesterone receptor exhibits pleiotropic roles in uterine development and function

The ovarian steroid progesterone, acting through the progesterone receptor (PR), coordinates endometrial epithelial-stromal cell communication, which is critical for its development and function. PR expression in these cellular compartments is under tight temporal and endocrine control. Although ex vivo studies demonstrated the importance of stromal PR expression, they failed to show a role for epithelial PR in uterine function. Here, the in vivo role of PR in the uterine epithelium is defined using floxed PR (PRf/f) mice crossed to Wnt7a-Cre mice. Progesterone was unable to stimulate the expression of its epithelial target genes, including Ihh, in the Wnt7a-Cre+PRf/− mice. Analysis was conducted on Ihh to determine whether PR directly regulates epithelial gene transcription. ChIP-on-chip analysis identified PR binding sites in the 5′-flanking region of Ihh. Cotransfection of the proximal Ihh promoter with PR demonstrated that PR directly regulates Ihh transcription. Female Wnt7a-Cre+PRf/− mice are infertile due to defects in embryo attachment, stromal cell decidualization, and the inability to cease estrogen-induced epithelial cell proliferation. Finally, progesterone was unable to inhibit neonatal endometrial glandular development in Wnt7a-Cre+PRf/− mice. Thus, epithelial PR is necessary for the regulation of progesterone epithelial target gene expression, as well as uterine function and development.—Franco, H. L., Rubel, C. A., Large, M. J., Wetendorf, M., Fernandez-Valdivia, R., Jeong, J.-W., Spencer, T. E., Behringer, R. R., Lydon, J. P., DeMayo, F. J. Epithelial progesterone receptor exhibits pleiotropic roles in uterine development and function.

embryo implantation, proliferation, adenogenesis The ovarian steroid hormones, estrogen (E2) and progesterone (P4), mediate uterine epithelial-stromal crosstalk to promote embryo implantation (1). An imbalance in steroid hormone signaling or a breakdown in epithelial-stromal communication leads to infertility. P4 has been shown to regulate a number of genes in both the uterine epithelium and stroma that mediate this crosstalk to regulate embryo implantation (2–5). Its actions are mediated through its receptor, the progesterone receptor (PR), which exists as two predominant isoforms, PR-A and PR-B (6). Female mice with ablation of PR [PR knockout (PRKO)] are infertile due to defects in multiple reproductive tissues. In the uterus of these mice, the endometrial stromal cells show defects in stromal cell proliferation and differentiation, and the epithelial cells fail to respond to P4 in the inhibition of E2-induced epithelial proliferation (7, 8). In addition to regulating normal uterine function, inappropriate exposure of the developing neonatal uterus to P4 in both sheep and mice inhibits uterine gland formation, indicating that for normal uterine function and development, PR levels must be controlled (9–11). During pregnancy, PR is transiently expressed in the luminal epithelium just prior to embryo implantation (12, 13). At the time of embryo implantation, PR expression in the luminal epithelium is rapidly down-regulated, and its expression in the uterine stroma increases and persists throughout decidualization. The dynamic changes in the expression pattern of the PR have resulted in much speculation as to the compartment-specific functions of the PR. Previous experiments using tissue recombination in a xenograft model have addressed the compartment-specific roles of the PR. These studies determined that PR expressed in the stromal compartment is the major regulator of the expression of the P4 target gene Indian hedgehog (Ihh) and the ability of P4 to inhibit E2-induced epithelial cell proliferation (14, 15). Because the ability of P4 to inhibit endometrial epithelial proliferation and Ihh has been shown to be critical in the preparation of the uterus for embryo implantation, these studies would indicate that stroma PR is critical for the regulation of uterine function. However, these studies failed to describe a role for epithelial PR in uterine biology. To address the epithelial-specific roles of PR in the endometrium in vivo during normal development and pregnancy, the PR (8) was conditionally ablated specifically in the uterine epithelium using the Wnt7a-Cre mouse model (16). In contrast to the tissue recombination studies, here, it is demonstrated that on an acute treatment of P4, epithelial PR regulates the expression of epithelial P4 target genes, including Ihh, through interactions at the Ihh promoter. Epithelial PR also acts to inhibit E2-induced epithelial proliferation, and loss of epithelial PR rendered female mice infertile due to embryo implantation defects. Finally, epithelial PR acts through IHH to regulate the inhibition of endometrial gland formation after neonatal P4 administration. Therefore, epithelial PR is essential for uterine development and function as a key regulator of uterine epithelial-stromal crosstalk.

Animals and hormone treatments Mice were maintained in the designated animal care facility at Baylor College of Medicine, according to the institutional guidelines for the care and use of laboratory animals. Wnt7a-Cre mice were generated on the B6SJLF2 background, and floxed PR (PRf/f) mice were generated on the B6/129 mixed background (8, 16). Fertility was assessed by mating 8-wk-old female mice with wild-type male mice for 6 mo and determining the number and size of litters delivered. Embryo implantation was assessed by mating 8-wk-old female mice with wild-type male mice. The presence of the postcoital vaginal plug was designated d 0.5. The mice were sacrificed on d 4.5, and the uteri were fixed in 4% paraformaldehyde (PFA; v/v) overnight for serial sectioning and hematoxylin and eosin (H&E) staining. Analysis of uterine gene expression during pseudopregnancy was accomplished by mating 8-wk-old female mice with vasectomized male mice. The mice were sacrificed on the designated day, and the uteri were either fixed in 4% PFA (v/v) overnight or flash-frozen and stored at −80°C. The artificial induction of decidualization has been previously described (17). Briefly, 6-wk-old female mice were ovariectomized and treated with 3 daily injections of 100 ng E2/mouse. After 2 d rest, mice were then treated with daily injections of 1 mg P4 and 6.7 ng E2/mouse for 3 d, s.c. At 6 h after the last injection, one uterine horn was traumatized by the injection of 50 μl of sesame oil. Mice were given daily injections of 1 mg P4 and 6.7 ng E2 per mouse, s.c. following the trauma. Mice were sacrificed at 5 d after the trauma by cervical dislocation while anesthetized with 2,2,2-tribromoethyl alcohol (Avertin; Sigma-Aldrich, St. Louis, MO, USA). At the time of dissection, uterine tissues were placed in 4% PFA (v/v) or flash-frozen and stored at −80°C. The acute P4 response was assessed in adults by ovariectomizing mice at 6 wk of age. After 2 wk of rest, mice were administered vehicle (sesame oil) or 1 mg P4, s.c. The acute P4 response in neonates was assessed by injecting postnatal d 5 mice with vehicle (sesame oil) or 40 μg/kg P4, s.c. Mice were sacrificed 6 h later. At the time of dissection, uterine tissues were flash-frozen and stored at −80°C. For the 3-d hormone treatments, mice were ovariectomized at 6 wk of age. After 2 wk of rest, mice were administered vehicle (sesame oil), 100 ng E2, or 100 ng E2 plus 1 mg P4, s.c., daily. At 6 h after the injection, mice were sacrificed, and uterine tissues were placed in 4% PFA (v/v) or flash-frozen and stored at −80°C. The protocol to assess epithelial proliferation was previously described (18). Briefly, mice were ovariectomized at 6 wk of age. Following 2 wk of rest, mice received 2 daily injections of 100 ng E2, s.c. After 2 d rest, the mice were divided into 3 groups and received either vehicle (sesame oil) daily for 4 d; vehicle for 3 d followed by 50 ng E2, s.c.; or 1 mg P4 daily for 3 d followed by 50 ng E2 plus 1 mg P4, s.c. After 11 h, 0.6 mg of BrdU was injected i.p. (GE Healthcare, Piscataway, NJ, USA). The mice were sacrificed 13 h later, and the uterine tissues were placed in 4% PFA (v/v). P4 inhibition of gland formation was achieved by injecting mice with vehicle (sesame oil) or 50 μg/kg P4, s.c., from postnatal d 2 through 9. The mice were sacrificed on postnatal d 10, and the uteri were fixed in 4% PFA (v/v). Immunohistochemistry Uteri were fixed overnight in 4% PFA (v/v), followed by thorough washing in 70% ethanol, and tissues were processed, embedded in paraffin, and sectioned. Uterine sections from paraffin-embedded tissue were cut at 5 μm and mounted on silane-coated slides, deparaffinized, and rehydrated in a graded alcohol series. Antigen retrieval was achieved using Vector Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA, USA). Sections were preincubated with 10% normal goat serum in PBS (pH 7.5) according to manufacturer's instructions (Vector Laboratories) and then incubated with anti-CCND1 (1:500, RB-9041; Neomarkers, Fremont, CA, USA), anti-minichromosome maintenance 3 (MCM3; 1:500, sc-9850; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-histone H3 (1:2000, 06-570; Upstate, Billerica, MA, USA), or anti-PR (1:200, A0098; Dako, Carpinteria, CA, USA) in 10% normal serum in PBS (pH 7.5; Vector Laboratories). On the following day, sections were washed in PBS and incubated with biotinylated secondary antibody (5 μl/ml; Vector Laboratories) for 1 h at room temperature. BrdU incorporation was assessed after blocking with the NB blocking buffer (Renaissance TSA Fluorescence Systems Tyramide Signal Amplification; PerkinElmer Life Sciences, Waltham, MA, USA) and incubated with biotinylated anti-BrdU (1:15 dilution, BrdU In-Situ Detection Kit II; BD Biosciences, San Diego, CA, USA) overnight at room temperature. The sections were then incubated with Vectastain Elite ABC R.T.U. reagent (Vector Laboratories), according to manufacturer's instructions. Immunoreactivity was detected using the DAB substrate kit (Vector Laboratories) and was visualized as brown staining. Alkaline phosphatase activity assay Uteri were fixed in 4% PFA (v/v) overnight, followed by sucrose gradients in PBS (15 and 30%), and then embedded in optimal cutting temperature (OCT) compound. Tissue sections were cut at 16 μm. Slides were postfixed in 0.2% gluteraldehyde, washed in PBS, and incubated with a 100 mM Tris buffer (pH 9.5) containing chromogenic substrates for alkaline phosphatase (168.5 μl of 100 mg/ml nitro blue tetrazolium salt in dimethylformamide and 175 μl of 50 mg/ml 5-bromo-4-chloro-3-indoyl phosphate/toluidinium salt in dimethylformamide added to 50 ml of the Tris buffer; Roche, Boulder, CO, USA). The development of a purple color indicates alkaline phosphatase activity. Tissues were counterstained with nuclear fast red. Quantitative real-time PCR (qPCR) analysis Total RNA was isolated from frozen uterine tissue using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA (1 μg) was reverse transcribed into cDNA with M-MLV (Invitrogen) in a 20-μl volume. Expression levels of mRNA were measured by qPCR TaqMan analysis using the ABI Prism 7700 Sequence Detector System (Applied Biosystems Inc., Foster City, CA, USA), according to the manufacturer's instructions. Real-time probes and primers were purchased from Applied Biosystems: amphiregulin (Areg; Mm00437583_m1), Ihh (Mm00439613_m1), interleukin 13 receptor α 2 (Il13ra2; Mm00515166_m1), leukemia inhibitory factor (Lif; Mm00434761_m1). Standard curves were generated by serial dilution of a preparation of total cDNA isolated from whole-mouse uterus. All real-time PCR was done using independent RNA sets. All mRNA quantities were normalized against 18S RNA using ABI rRNA control reagents. Chromatin immunoprecipitation (ChIP) assays Mouse uteri were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by adding lysis buffer, followed by disruption with a handheld mechanical homogenizer, followed by 30 strokes with a Dounce homogenizer. Lysates were sonicated, and the DNA was sheared to an average length of 300–500 bp. Genomic DNA (input) was prepared by treating aliquots of chromatin with RNase, proteinase K, and heat for decrosslinking, followed by ethanol precipitation. Pellets were resuspended, and the resulting DNA was quantified on a NanoDrop spectrophotometer (NanoDrop, Wilmington, DE, USA). An aliquot of chromatin (30 μg) was precleared with protein A agarose beads (Invitrogen). ChIP was performed using an antibody against PR (Santa Cruz Biotechnology). After incubation at 4°C overnight, protein A agarose beads were used to isolate the immune complexes. Complexes were washed, eluted from the beads with SDS buffer, and treated with RNase and proteinase K. Cross-links were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. PR binding sites were identified using ChIP-on-chip. ChIP and input DNAs were amplified by whole-genome amplification (WGA) using the GenomePlex WGA Kit (Sigma-Aldrich). The resulting amplified DNAs were purified, quantified, fragmented, and labeled using the DNA Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA), and then hybridized to Affymetrix Mouse Tiling 2.0R Array A (containing mouse chromosomes 1, 9, and 19) at 45°C overnight. Arrays were washed and scanned, and the resulting CEL files were analyzed using Affymetrix TAS software. For ChIP-qPCR, primers were designed to amplify regions of PR binding identified through ChIP-on-chip. qPCR reactions were carried out in triplicate on specific genomic regions (Untr6: F-5′-TCAGGCATGAACCACCATAC, R-5′-AACATCCACACGTCCAGTGA; Ihh+566: F-5′-GCGATCTTGCCTTCGTAGC, R-5′-CTCGTGCCTCTTGCCTACA; Ihh+19Kb: F-5′-AAAGAAAGCAACTGAATGTGG, R-5′-ATGTGCAGGGATGAGTCCTT) using SYBR Green Supermix (Bio-Rad, Hercules, CA, USA). The resulting signals were normalized to input DNA. Transfection and luciferase assay Human endometrial carcinoma (HEC-1A) epithelial cells were transfected using Qiagen SuperFect reagent (Qiagen, Valencia, CA, USA), according to manufacturer's instructions, with 200 ng of 1 kb of the mouse Ihh promoter fused to luciferase along with 50 ng of human PR-A, human PR-B, or pcDNA empty vector control. The cells were treated with 10−8 M R5020 (Sigma-Aldrich) or vehicle control (ethanol) in McCoy's 5A medium (Invitrogen) for 24 h at 37°C. The cells were harvested and lysed using the Promega passive lysis buffer (Promega, Madison, WI, USA). Luciferase activity was measured using the Promega luciferase assay system in a Centro LB 960 luminometer (Berthold Technologies, Oak Ridge, TN, USA). Protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad). The luciferase activity was normalized to total protein concentration. Statistical analysis Statistical analyses were performed using the Student's t test or 1-way ANOVA followed by Tukey's post hoc multiple-range test with the Instat package from GraphPad (San Diego, CA, USA).

Generation of epithelial-specific ablation of the PR in the uterus To ablate PR specifically in the uterine epithelium, PRf/f mice (8) were crossed to the Wnt7a-Cre mouse model (16). Because of Cre recombinase expression in the testes (data not shown), a null allele is generated, resulting in mice of the genotypes PRf/− and Wnt7a-Cre+PRf/−. In order to validate epithelial specific ablation of PR, immunohistochemical analysis of PR was conducted on uteri from d 2.5 and 3.5 pseudopregnant mice. These time points were chosen because PR is highly and transiently expressed in the luminal epithelium prior to embryo implantation (13). On d 2.5, the PRf/− uteri show robust expression of PR in the luminal epithelium with reduced, although evident, expression in the uterine stroma, whereas the Wnt7a-Cre+PRf/− mice display PR expression only in the stroma (Fig. 1). On d 3.5, PR expression is reduced in the luminal epithelium of the PRf/− mice and not observed in the Wnt7a-Cre+PRf/− epithelium but is retained in the stroma of both the PRf/− and Wnt7a-Cre+PRf/− uteri. Therefore, PR was ablated specifically in the uterine epithelium by Wnt7a-Cre. Role of epithelial PR in the regulation of P4-regulated gene expression To determine whether epithelial PR regulates known P4 target genes, female PRf/−, Wnt7a-Cre+PRf/−, and PRKO mice were ovariectomized and treated with vehicle or P4 for 6 h. qPCR analysis showed a robust induction of the P4 target genes Ihh, Gata binding protein 2 (Gata2), Areg, cytochrome p450 26a1 (Cyp26a1), and Il13ra2 in the P4-treated PRf/− uteri, whereas there was no induction in the P4-treated PRKO uteri (Fig. 2A–C and data not shown). Interestingly, there was no induction of Ihh, Gata2, Areg, and Cyp26a1 in the P4-treated Wnt7a-Cre+PRf/− uteri; however, Il13ra2 was induced. As Ihh, Gata2, Areg, and Cyp26a1 localize to the epithelium, and Il13ra2 is found in the stroma (4, 19–21), these results suggest that epithelial PR regulates epithelial, but not stromal, target genes. IHH has been previously shown to be a critical regulator of uterine function and to be regulated by stromal, not epithelial, PR (15, 22) In order to determine the mechanism by which PR regulates Ihh, ChIP was conducted with antibodies for PR on whole uterine lysates from P4-treated, ovariectomized mice, and the resulting DNA was hybridized to an Affymetrix tiling array containing mouse chromosomes 1, 9, and 19. Ihh is located on mouse chromosome 1. Two potential PR binding sites were identified near the Ihh locus: one in the proximal promoter (−566 bp) and one 19 kb upstream from the transcriptional start site (Fig. 2D). In vivo ChIP-qPCR analyses validated these binding sites and demonstrated that this binding was induced by P4 (Fig. 2E). The functionality of the proximal site was assessed through transient transfection analysis of PR with 1 kb of the mouse Ihh promoter in human endometrial epithelial HEC-1A cells. Activation of the Ihh promoter by both PR isoforms was observed on treatment with the P4 analog R5020 (Fig. 2F). Therefore, PR directly regulates Ihh expression through an interaction with its promoter. Effect of epithelial PR ablation on P4 inhibition of estrogen actions In the uterus, P4 has inhibitory effects on E2 actions (23, 24). PRKO mice demonstrated the critical role of PR in the P4 inhibition of E2 action (7, 8, 25), and subsequent tissue recombination experiments pointed to stromal PR as being critical for this action (25). Here, the contribution of epithelial PR to the P4 inhibition of E2 actions was examined using an in vivo mouse model. PRf/−, Wnt7a-Cre+PRf/−, and PRKO mice were ovariectomized and administered vehicle, E2, or E2 plus P4. After 3 d of treatment, uterine weights were examined. In all three genotypes, E2 treatment induced uterine weight gain compared to the vehicle-treated uteri (Supplemental Fig. S1). After E2 plus P4 treatment, the uterine weight in the PRf/− mice was reduced significantly compared to that in the E2-treated mice. The PRKO uteri did not significantly differ in weight in the E2 plus P4 treatment compared to the E2 treatment. In the E2 plus P4-treated Wnt7a-Cre+PRf/− mice, uterine weight was also reduced compared to the E2-treatment group. Therefore, epithelial PR does not mediate P4 inhibition of E2-induced uterine weight gain. Next, it was determined whether epithelial PR mediates the P4 inhibition of E2-induced epithelial proliferation in the uterus. Female PRf/− and Wnt7a-Cre+PRf/− mice were ovariectomized and subjected to a hormone priming approach to examine proliferation, as described previously (18). E2 was able to promote epithelial proliferation similarly in both genotypes, as revealed by BrdU incorporation (Fig. 3A, B). In contrast, P4 was only able to inhibit this induction in the PRf/− mice, not in the Wnt7a-Cre+PRf/− mice. However, stromal proliferation was induced normally in the Wnt7a-Cre+PRf/− mice (Fig. 3A, C). Therefore, epithelial PR mediates P4 inhibition of E2-induced epithelial cell proliferation, but not stromal cell proliferation, which differs from the results of the tissue recombination studies. Previously, the PR was shown to inhibit epithelial proliferation through its regulation of cyclin D1 and members of the MCM family of proteins (26, 27). Therefore, the expression of cyclin D1 and MCM3 in the Wnt7a-Cre+PRf/− was determined. Comparable patterns of cyclin D1 and MCM3 expression were observed in the vehicle and E2-treated Wnt7a-Cre+PRf/− and control mice, with their expression localized to the epithelium (Supplemental Fig. S2 and Fig. 3D). However, while cyclin D1 and MCM3 expression were down-regulated in the uterine epithelium and up-regulated in the stroma of E2 plus P4 PRf/− mice, the expression of these proliferation markers was still apparent in the uterine epithelium of the Wnt7a-Cre+PRf/− mice, with a normal increase in the uterine stroma (Supplemental Fig. S2 and Fig. 3D). The role of epithelial PR in inhibiting epithelial proliferation during natural pregnancy was next investigated. Proliferation was assessed during pregnancy by phosphohistone H3 (pH3) immunohistochemical analysis on d 2.5 and 3.5 of pseudopregnancy. In wild-type mice, epithelial proliferation was decreased, and stromal proliferation was increased from d 2.5 to 3.5 of pregnancy (Fig. 4). This decrease in epithelial proliferation was not observed in the Wnt7a-Cre+PRf/− mice, whereas stromal proliferation was normally induced. Therefore, these results demonstrate that epithelial PR mediates P4 inhibition of E2-induced epithelial proliferation during pregnancy. Effect of epithelial PR on female fertility The proliferation data implied that epithelial PR might play a critical role during pregnancy. Therefore, its potential role in female fertility was assessed by conducting mating trials of female PRf/− and Wnt7a-Cre+PRf/− mice with wild-type male mice for 6 mo (n=7). The PRf/− mice displayed normal fertility, delivering a total of 314 pups with an average litter size of 8.49 ± 0.47 pups. Conversely, the Wnt7a-Cre+PRf/− mice were infertile and never delivered any pups. Therefore, epithelial PR has a critical role in female fertility. To determine the cause of the infertility in the Wnt7a-Cre+PRf/− mice, embryo attachment during natural pregnancy and the ability of the uterus to undergo an artificial induction of stromal cell decidualization were assayed. Embryo attachment was observed in the uteri of d 4.5 PRf/− mice, but not in Wnt7a-Cre+PRf/− mice (Fig. 5A). At 5 d after receiving the decidual stimulus, PRf/− mice exhibited a robust decidual response as seen grossly, by the ratio of the horn weights, and by alkaline phosphatase activity of the decidual horn compared to the control horn (Fig. 5B–D). However, the Wnt7a-Cre+PRf/− mice failed to display any response. Thus, PR in the epithelium is critical for embryo attachment and the ability of the stromal cells to undergo a normal decidual response. Mechanism by which epithelial PR regulates pregnancy Because epithelial PR has been shown to be critical for the P4 induction of Ihh in the ovariectomized uterus, and ablation of both epithelial PR and Ihh shows a similar phenotype with respect to uterine function, it was hypothesized that Ihh induction during early pregnancy would be inhibited in the uteri of Wnt7a-Cre+PRf/− mice. At the peak of Ihh induction on d 2.5 (4, 21, 22), Ihh expression was virtually absent in the uteri of PRKO mice, whereas its expression was ∼50% reduced (although not significant) in the Wnt7a-Cre+PRf/− mice compared to PRf/− mice (Fig. 6A). Therefore, unlike the observation that epithelial PR is the sole regulator of Ihh in the ovariectomized uterus treated acutely with P4, epithelial PR is not the only regulator of Ihh during early pregnancy. To determine what other pathways known to regulate pregnancy were altered in the Wnt7a-Cre+PRf/− mice, the expression of Lif, another key regulator of embryo implantation, was assayed (28). Lif expression on d 3.5 of pseudopregnancy was significantly reduced in the uteri of Wnt7a-Cre+PRf/− mice (Fig. 6B). Therefore, the embryo-implantation defect observed in the Wnt7a-Cre+PRf/− mice may be due to a reduction in Lif expression. Role of epithelial PR and Ihh in adenogenesis Previously, it was shown that neonatal treatment with progestins inhibited gland formation in sheep and mice (9–11). In mice, gland formation initiates on postnatal d 5 with invaginations of the luminal epithelium, which results in the formation of uterine glands by postnatal d 15 (29). During gland formation, PR is expressed solely in the uterine epithelium (30), and treatment of neonatal mice with P4 inhibits endometrial gland development (11). Therefore, it was determined whether epithelial PR mediates P4 inhibition of endometrial gland development. Neonate mice received vehicle or P4 from postnatal d 2 to 9, and the uterus was examined on postnatal d 10. In the vehicle-treated PRf/− uteri, uterine glands were apparent, and the luminal epithelium exhibited invaginations into the stroma that signifies tubulogenesis (Fig. 7A). However, in the P4-treated PRf/− uteri, no uterine glands were observed, and the luminal epithelium failed to display invaginations. Of particular note, uterine glands were evident in both the vehicle and P4-treated Wnt7a-Cre+PRf/− uteri. Therefore, epithelial PR mediates P4 inhibition of endometrial gland development during the neonatal period. Previously, constitutive activation of the hedgehog signaling pathway in the uterus was shown to result in a gland formation defect (31). As Ihh is a P4-regulated gene in the neonatal mouse uterus (15), it was hypothesized that P4 inhibition of uterine gland formation may occur through its induction of the hedgehog signaling pathway. In the neonate PRf/− mice, Ihh was robustly induced by 6 h of P4 treatment, but not in the neonate Wnt7a-Cre+PRf/− mice (Fig. 7B). Thus, epithelial PR regulates Ihh expression in the neonate uterus. Next, it was determined whether Ihh is necessary for the P4 inhibition of uterine gland formation using a previously described PRcre/+Ihhf/f mouse model (22). Vehicle or P4 was administered to neonatal mice as described previously. The Ihhf/f mice displayed a similar effect of P4 on gland inhibition as the PRf/− uteri (data not shown). In contrast, uterine glands were observed in both the vehicle and P4-treated PRcre/+Ihhf/f mice on postnatal d 10 (Fig. 7A). Therefore, P4 inhibition of endometrial gland formation in the uterus occurs via epithelial PR activation of the Ihh signaling pathway.

The PR is critical for the establishment and maintenance of pregnancy (7, 8). Because of the dynamic changes in the expression pattern of PR during pregnancy and the role of epithelial-stromal crosstalk in uterine biology, much interest has been generated in understanding the compartment-specific roles of the PR in uterine biology. To address the in vivo role of epithelial PR, PR (8) was conditionally ablated specifically in the uterine epithelium using the Wnt7a-Cre mouse model (16). While the PR is also expressed in the granulosa cells of the ovary and the anterior pituitary gland (32), the Wnt7a-Cre mouse model is not active in those regions (data not shown), indicating that the observed phenotypes are, in fact, due to ablation of PR in the uterine epithelium. Epithelial PR was found to regulate the induction of Ihh, Areg, Gata2, and Cyp26a1 in response to acute treatment of ovariectomized mice with P4. Epithelial PR was also shown to be a critical regulator of Ihh during neonatal development. This observation differs from the tissue recombination experiments, which indicated the necessity and sufficiency of stromal PR in regulating Ihh expression (15). However, epithelial PR was not the sole regulator of Ihh during the preimplantation period. Collectively, these studies suggest that Ihh expression is regulated by both epithelial and stromal PR, depending on the physiological and endocrine status of the uterus. To determine whether PR directly regulates Ihh transcription, a molecular analysis of its regulation was conducted. From in vivo ChIP-on-chip analyses, two potential binding sites for PR in the Ihh promoter were identified and validated. The functionality of the proximal binding site was examined in the human endometrial epithelial cell line HEC-1A. In transient transfection analysis, both PR-A and PR-B were able to activate the Ihh promoter in a P4-dependent manner. The PR can interact with DNA directly through PR response elements (PREs) or indirectly by interacting with other transcription factors (33). Examination of the proximal Ihh promoter found PRE half sites and an extensive GC-rich region. Previously, PR was shown to interact with the GC-rich binding transcription factors, AP1, Sp1, and Bteb1, to regulate target gene expression (34–37). Examination of the distal binding site revealed PRE half sites, as well as GATA, AP1, and Sp1 binding sites. Future studies are needed to determine the mechanism by which PR regulates its target gene expression at specific target genes, as well as on a global scale through in vivo ChIP-Seq analyses that will determine all the PR binding sites in the mouse uterus in vivo. P4 also acts to inhibit uterine E2 actions that need to be precisely regulated in order to modulate uterine receptivity (23, 24, 38). Normal P4 inhibition of E2-induced uterine weight gain on ablation of epithelial PR was observed, indicating that it is dispensable for this phenomenon. Examination of the inhibition of epithelial proliferation demonstrated that epithelial PR is required for the inhibition of epithelial proliferation, but not for the induction of stromal proliferation. While sustained epithelial proliferation has not been demonstrated to impede embryo implantation, a precise window of implantation is defined by the duration of the estrogenic signaling, suggesting that the maintenance of the estrogenic response, including epithelial proliferation, may, in fact, affect embryo implantation (38). Mechanistically, P4 is thought to inhibit epithelial proliferation through its regulation of cyclin D1 and down-regulation of the MCM proteins in the epithelium. Here, it is demonstrated that epithelial PR inhibits epithelial proliferation through its regulation of cyclin D1 and the MCM proteins. The mechanism by which this is achieved is suggested to be through the PI3K/Akt signaling pathway (27). Therefore, future studies are needed to determine whether it is through this pathway or others that epithelial PR regulates epithelial proliferation. Again, these results differ from the findings from the tissue recombination studies, which indicated that stromal PR mediates P4 inhibition of epithelial proliferation (25). For both Ihh induction and P4 inhibition of E2-induced epithelial proliferation, the in vivo results differ from those obtained using tissue recombination. The reason for the differences between the in vivo analysis and the tissue recombination experiments may be due to differences in absolute levels of PR or altered coregulator expression in the compartments after transplantation to the kidney capsule. Regardless, these studies stress the importance of using in vivo mouse models for the study of uterine biology in order to accurately represent uterine events. To take advantage of this in vivo mouse model, the effect of epithelial PR ablation on female fertility was assessed. It was determined that epithelial PR is essential for female fertility as a key regulator of embryo attachment and subsequent decidualization. Epithelial PR is also critical for both processes. As PR expression is intact in the uterine stroma, these data suggest that factors from the luminal and glandular epithelium are necessary for stromal cell decidualization. Thus, an epithelial PR-regulated gene is needed for the decidualization of the stromal cells. As conditional ablation of Ihh demonstrated its critical role in embryo attachment and subsequent decidualization, and it is regulated by epithelial PR (22, 39), it was hypothesized that it is through this pathway that epithelial PR regulates embryo implantation. However, Ihh expression was not completely lost after epithelial PR ablation, as observed in the PRKO mice, demonstrating that epithelial PR is not the sole regulator of Ihh expression during pregnancy and implicating stromal PR in its regulation during pregnancy. In addition, these data suggest that, while the Ihh signaling pathway is critical for embryo implantation, it may not be the sole pathway through which epithelial PR regulates embryo implantation. Future studies aimed at determining the epithelial PR transcriptome will identify these players; however, it is important that this assay be done under physiological conditions in order to identify the functionally relevant genes. Interestingly, epithelial PR was shown to alter Lif expression during pregnancy. Lif−/− mice display a defect in embryo implantation, demonstrating its critical role in this process (28). However, the mechanism by which Lif is regulated by epithelial PR is unclear. Ablation of epithelial PR does not affect E2 induction of Lif or other estrogenic genes, such as lactoferrin, indicating that estrogen responsiveness is intact (data not shown). Also, Lif is not regulated by an acute treatment of mice with P4 (19, 40). Thus, further investigations are needed to identify the mechanism by which epithelial PR regulates Lif expression. Even though the regulation of Ihh by epithelial PR depends on the physiological state of the uterus, a novel role for this pathway in the regulation of uterine gland formation has been identified. The proper development of uterine glands is vital for uterine function, as they secrete molecules, such as LIF, that are essential for embryo implantation (28, 41–43). In mice, uterine gland formation begins with the invagination of the luminal epithelium on postnatal d 5, leading to gland formation by postnatal d 15 (29). Using both sheep and mice, Bartel et al. (9) and Stewart et al. (11) demonstrated that the neonatal administration of progestin inhibited gland formation. This inhibition is mediated through epithelial PR and its regulation of the Ihh signaling pathway. These results are consistent with previous observations of an inhibition of gland formation on constitutive activation of the hedgehog signaling pathway in the uterus (31). Thus, a novel mechanism of uterine gland formation involving epithelial PR and Ihh signaling has been identified that can be used to further study this important developmental process. The generation of this novel mouse model represents the first step in understanding the in vivo compartment-specific roles of the PR in uterine function. Using this mouse model, we demonstrated that epithelial PR exerts a critical role in uterine function through its regulation of epithelial P4 target genes and inhibition of E2-induced epithelial proliferation. In addition, it mediates P4 inhibition of uterine gland formation through the hedgehog signaling pathway. The studies presented highlight the advantage of using in vivo mouse models to study uterine biology and provide the means for additional studies into the mechanism of P4 action in uterine development and function. Through these future studies, new knowledge will be gained to aid not only in the treatment of female infertility, but also in the treatment of uterine disorders, such as endometriosis and endometrial cancer, that are associated with altered P4 signaling. Supplementary Material Acknowledgments The authors thank Sungnam Cho, Jie Yang, Genpathway, Inc., and Grant Orvis for technical assistance and Janet DeMayo for manuscript preparation. This work was supported by U.S. National Institutes of Health (NIH) grants R01HD042311 (to F.J.D.), R01HD057873 (to J.W.J.), R01CA77530 (to J.P.L), and HD30284 (to R.R.B.), and Reproductive Biology Training grant 5T32HD07165 and a scholarship from Baylor Research Advocates for Student Scientists, supported, in part, by the Winterman Foundation and the Houston Livestock Show and Rodeo Scholarship Fund (to H.L.F.). This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information. - Areg - amphiregulin - Cyp26a1 - cytochrome p450 26a1 - E2 - estrogen - Gata2 - Gata binding protein 2 - H&E - hematoxylin and eosin - Ihh - Indian hedgehog - Il13ra2 - interleukin 13 receptor α 2 - Lif - leukemia inhibitory factor - MCM - minichromosome maintenance - MCM3 - minichromosome maintenance3 - P4 - progesterone - PFA - paraformaldehyde - pH3 - phosphohistone 3 - PR - progesterone receptor - PR-A - progesterone receptor A - PR-B - progesterone receptor B - PRE - progesterone receptor response element - PRf/f - floxed PR - PRKO - progesterone receptor knockout.

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