Estrogen Receptor ß Modulates Apoptosis Complexes and the Inflammasome to Drive the Pathogenesis of Endometriosis.

Alterations in estrogen-mediated cellular signaling play an essential role in the pathogenesis of endometriosis. In addition to higher estrogen receptor (ER) ß levels, enhanced ERß activity was detected in endometriotic tissues, and the inhibition of enhanced ERß activity by an ERß-selective antagonist suppressed mouse ectopic lesion growth. Notably, gain of ERß function stimulated the progression of endometriosis. As a mechanism to evade endogenous immune surveillance for cell survival, ERß interacts with cellular apoptotic machinery in the cytoplasm to inhibit TNF-α-induced apoptosis. ERß also interacts with components of the cytoplasmic inflammasome to increase interleukin-1ß and thus enhance its cellular adhesion and proliferation properties. Furthermore, this gain of ERß function enhances epithelial-mesenchymal transition signaling, thereby increasing the invasion activity of endometriotic tissues for establishment of ectopic lesions. Collectively, we reveal how endometrial tissue generated by retrograde menstruation can escape immune surveillance and develop into sustained ectopic lesions via gain of ERß function.

Aberrant uterine folding in mice disrupts implantation chamber formation and alignment of embryo-uterine axes.

The uterine luminal epithelium folds characteristically in mammals, including humans, horses and rodents. Improper uterine folding in horses results in pregnancy failure, but the precise function of folds remains unknown. Here, we uncover dynamic changes in the 3D uterine folding pattern during early pregnancy with the entire lumen forming pre-implantation transverse folds along the mesometrial-antimesometrial axis. Using a time course, we show that transverse folds are formed before embryo spacing, whereas implantation chambers form as the embryo begins attachment. Thus, folds and chambers are two distinct structures. Transverse folds resolve to form a flat implantation region, after which an embryo arrives at its center to attach and form the post-implantation chamber. Our data also suggest that the implantation chamber facilitates embryo rotation and its alignment along the uterine mesometrial-antimesometrial axis. Using WNT5A- and RBPJ-deficient mice that display aberrant folds, we show that embryos trapped in longitudinal folds display misalignment of the embryo-uterine axes, abnormal chamber formation and defective post-implantation morphogenesis. These mouse models with disrupted uterine folding provide an opportunity to understand uterine structure-based mechanisms that are crucial for implantation and pregnancy success. This article has an associated 'The people behind the papers' interview.

An oocyte-specific Cas9-expressing mouse for germline CRISPR/Cas9-mediated genome editing.

Cas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene-edited animals creates unique issues including ill-defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte-specific, Gdf9 promoter driven, Cas9 transgene (Gdf9-Cas9) targeted as a single copy into the Hprt1 locus. The X-linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9-Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9-Cas9 does not mediate genome editing, confirming that Gdf9-Cas9 is not expressed in embryos. Finally, we demonstrate that off-target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9-Cas9 transgene is a viable alternative to exogenous Cas9.

Chromatin architectural factor CTCF is essential for progesterone-dependent uterine maturation.

Receptors for estrogen and progesterone frequently interact, via Cohesin/CTCF loop extrusion, at enhancers distal from regulated genes. Loss-of-function CTCF mutation in >20% of human endometrial tumors indicates its importance in uterine homeostasis. To better understand how CTCF-mediated enhancer-gene interactions impact endometrial development and function, the Ctcf gene was selectively deleted in female reproductive tissues of mice. Prepubertal Ctcfd/d uterine tissue exhibited a marked reduction in the number of uterine glands compared to those without Ctcf deletion (Ctcff/f mice). Post-pubertal Ctcfd/d uteri were hypoplastic with significant reduction in both the amount of the endometrial stroma and number of glands. Transcriptional profiling revealed increased expression of stem cell molecules Lif, EOMES, and Lgr5, and enhanced inflammation pathways following Ctcf deletion. Analysis of the response of the uterus to steroid hormone stimulation showed that CTCF deletion affects a subset of progesterone-responsive genes. This finding indicates (1) Progesterone-mediated signaling remains functional following Ctcf deletion and (2) certain progesterone-regulated genes are sensitive to Ctcf deletion, suggesting they depend on gene-enhancer interactions that require CTCF. The progesterone-responsive genes altered by CTCF ablation included Ihh, Fst, and Errfi1. CTCF-dependent progesterone-responsive uterine genes enhance critical processes including anti-tumorigenesis, which is relevant to the known effectiveness of progesterone in inhibiting progression of early-stage endometrial tumors. Overall, our findings reveal that uterine Ctcf plays a key role in progesterone-dependent expression of uterine genes underlying optimal post-pubertal uterine development.

Steroid receptor coactivator-2 drives epithelial reprogramming that enables murine embryo implantation.

Although we have shown that steroid receptor coactivator-2 (SRC-2), a member of the p160/SRC family of transcriptional coregulators, is essential for decidualization of both human and murine endometrial stromal cells, SRC-2's role in the earlier stages of the implantation process have not been adequately addressed. Using a conditional SRC-2 knockout mouse (SRC-2d/d ) in timed natural pregnancy studies, we show that endometrial SRC-2 is required for embryo attachment and adherence to the luminal epithelium. Implantation failure is associated with the persistent expression of Mucin 1 and E-cadherin on the apical surface and basolateral adherens junctions of the SRC-2d/d luminal epithelium, respectively. These findings indicate that the SRC-2d/d luminal epithelium fails to exhibit a plasma membrane transformation (PMT) state known to be required for the development of uterine receptivity. Transcriptomics demonstrated that the expression of genes involved in steroid hormone control of uterine receptivity were significantly disrupted in the SRC-2d/d endometrium as well as genes that control epithelial tight junctional biology and the emergence of the epithelial mesenchymal transition state, with the latter sharing similar biological properties with PMT. Collectively, these findings uncover a new role for endometrial SRC-2 in the induction of the luminal epithelial PMT state, which is a prerequisite for the development of uterine receptivity and early pregnancy establishment.

Deficiency of PARP-1 and PARP-2 in the mouse uterus results in decidualization failure and pregnancy loss.

Miscarriage is a common complication of pregnancy for which there are few clinical interventions. Deficiency in endometrial stromal cell decidualization is considered a major contributing factor to pregnancy loss; however, our understanding of the underlying mechanisms of decidual deficiency are incomplete. ADP ribosylation by PARP-1 and PARP-2 has been linked to physiological processes essential to successful pregnancy outcomes. Here, we report that the catalytic inhibition or genetic ablation of PARP-1 and PARP-2 in the uterus lead to pregnancy loss in mice. Notably, the absence of PARP-1 and PARP-2 resulted in increased p53 signaling and an increased population of senescent decidual cells. Molecular and histological analysis revealed that embryo attachment and the removal of the luminal epithelium are not altered in uterine Parp1, Parp2 knockout mice, but subsequent decidualization failure results in pregnancy loss. These findings provide evidence for a previously unknown function of PARP-1 and PARP-2 in mediating decidualization for successful pregnancy establishment.

Progesterone receptor isoform B regulates the Oxtr-Plcl2-Trpc3 pathway to suppress uterine contractility.

Uterine contractile dysfunction leads to pregnancy complications such as preterm birth and labor dystocia. In humans, it is hypothesized that progesterone receptor isoform PGR-B promotes a relaxed state of the myometrium, and PGR-A facilitates uterine contraction. This hypothesis was tested in vivo using transgenic mouse models that overexpress PGR-A or PGR-B in smooth muscle cells. Elevated PGR-B abundance results in a marked increase in gestational length compared to control mice (21.1 versus 19.1 d respectively, P < 0.05). In both ex vivo and in vivo experiments, PGR-B overexpression leads to prolonged labor, a significant decrease in uterine contractility, and a high incidence of labor dystocia. Conversely, PGR-A overexpression leads to an increase in uterine contractility without a change in gestational length. Uterine RNA sequencing at midpregnancy identified 1,174 isoform-specific downstream targets and 424 genes that are commonly regulated by both PGR isoforms. Gene signature analyses further reveal PGR-B for muscle relaxation and PGR-A being proinflammatory. Elevated PGR-B abundance reduces Oxtr and Trpc3 and increases Plcl2 expression, which manifests a genetic profile of compromised oxytocin signaling. Functionally, both endogenous PLCL2 and its paralog PLCL1 can attenuate uterine muscle cell contraction in a CRISPRa-based assay system. These findings provide in vivo support that PGR isoform levels determine distinct transcriptomic landscapes and pathways in myometrial function and labor, which may help further the understanding of abnormal uterine function in the clinical setting.

Establishment of Murine Pregnancy Requires the Promyelocytic Leukemia Zinc Finger Transcription Factor.

Using an established human primary cell culture model, we previously demonstrated that the promyelocytic leukemia zinc finger (PLZF) transcription factor is a direct target of the progesterone receptor (PGR) and is essential for progestin-dependent decidualization of human endometrial stromal cells (HESCs). These in vitro findings were supported by immunohistochemical analysis of human endometrial tissue biopsies, which showed that the strongest immunoreactivity for endometrial PLZF is detected during the progesterone (P4)-dominant secretory phase of the menstrual cycle. While these human studies provided critical clinical support for the important role of PLZF in P4-dependent HESC decidualization, functional validation in vivo was not possible due to the absence of suitable animal models. To address this deficiency, we recently generated a conditional knockout mouse model in which PLZF is ablated in PGR-positive cells of the mouse (Plzf d/d). The Plzf d/d female was phenotypically analyzed using immunoblotting, real-time PCR, and immunohistochemistry. Reproductive function was tested using the timed natural pregnancy model as well as the artificial decidual response assay. Even though ovarian activity is not affected, female Plzf d/d mice exhibit an infertility phenotype due to an inability of the embryo to implant into the Plzf d/d endometrium. Initial cellular and molecular phenotyping investigations reveal that the Plzf d/d endometrium is unable to develop a transient receptive state, which is reflected at the molecular level by a blunted response to P4 exposure with a concomitant unopposed response to 17-ß estradiol. In addition to a defect in P4-dependent receptivity, the Plzf d/d endometrium fails to undergo decidualization in response to an artificial decidual stimulus, providing the in vivo validation for our earlier HESC culture findings. Collectively, our new Plzf d/d mouse model underscores the physiological importance of the PLZF transcription factor not only in endometrial stromal cell decidualization but also uterine receptivity, two uterine cellular processes that are indispensable for the establishment of pregnancy.

iCre recombinase expressed in the anti-Müllerian hormone receptor 2 gene causes global genetic modification in the mouse†.

Genetically engineered mice are widely used to study the impact of altered gene expression in vivo. Within the reproductive tract, the Amhr2-IRES-Cre(Bhr) mouse model is used to ablate genes in ovarian granulosa and uterine stromal cells. There are reports of Amhr2-IRES-Cre(Bhr) inducing recombination in non-target tissues. We hypothesized the inefficiency or off-target Cre action in Amhr2-IRES-Cre(Bhr) mice is due to lack of recombination in every cell that expresses Amhr2. To investigate, we created a new targeted knock-in mouse model, Amhr2-iCre(Fjd), by inserting a codon-optimized improved Cre (iCre) into exon 1 of the Amhr2 gene. Amhr2-iCre(Fjd)/+ males were mated with females that contain a lox-stop-lox cassette in the Sun1 gene so when DNA recombination occurs, SUN1-sfGFP fusion protein is expressed in a peri-nuclear pattern. In adult Amhr2-iCre(Fjd)/+ Sun1LsL/+ mice, Amhr2-iCre(Fjd)-mediated genetic recombination was apparent in uterine epithelial, stromal, and myometrial cells, while Amhr2-IRES-Cre(Bhr)/+ Sun1LsL/+ females demonstrated inter-mouse variability of Amhr2-IRES-Cre(Bhr) activity in uterine cells. Fluorescence was observed in Amhr2-iCre(Fjd)-positive mice at post-natal Day 1, indicating global genetic recombination, while fluorescence of individual Amhr2-IRES-Cre(Bhr)-positive pups varied. To determine the developmental stage that genetic recombination first occurs, Sun1LsL/LsL females were super-ovulated and mated with Amhr2-IRES-Cre(Bhr)/+ or Amhr2(iCre/+)Fjd males, then putative zygotes were collected and cultured. In the four-cell embryo, Amhr2-iCre(Fjd) and Amhr2-IRES-Cre(Bhr) activities were apparent in 100% and 25-100% of cells, respectively. In conclusion, Amhr2-IRES-Cre(Bhr) or Amhr2-iCre(Fjd) driven by the Amhr2 promoter is active in the early embryo and can lead to global genetic modification, rendering this transgenic mouse model ineffective.

Targeting progesterone signaling prevents metastatic ovarian cancer.

Effective cancer prevention requires the discovery and intervention of a factor critical to cancer development. Here we show that ovarian progesterone is a crucial endogenous factor inducing the development of primary tumors progressing to metastatic ovarian cancer in a mouse model of high-grade serous carcinoma (HGSC), the most common and deadliest ovarian cancer type. Blocking progesterone signaling by the pharmacologic inhibitor mifepristone or by genetic deletion of the progesterone receptor (PR) effectively suppressed HGSC development and its peritoneal metastases. Strikingly, mifepristone treatment profoundly improved mouse survival (∼18 human years). Hence, targeting progesterone/PR signaling could offer an effective chemopreventive strategy, particularly in high-risk populations of women carrying a deleterious mutation in the BRCA gene.

Inserting Cre recombinase into the Prolactin 8a2 gene for decidua-specific recombination in mice.

An estimated 75% of unsuccessful pregnancies are due to implantation failure. Investigating the causes of implantation failure is difficult as decidualization and embryo implantation is a dynamic process. Here, we describe a new decidua-specific iCre recombinase mouse strain. Utilizing CRISPR/Cas9-based genome editing, a mouse strain was developed that expresses iCre recombinase under the control of the endogenous prolactin family 8, subfamily a, member 2 (Prl8a2) promoter. iCre recombinase activity was examined by crossing with mTmG/+ or Sun1-GFP reporter alleles. iCre activity initiated reporter expression at gestational day 5.5 in the primary decidual zone and continued into mid-gestation (gestational day 9.5), with expression highly concentrated in the anti-mesometrial region. No reporter expression was observed in the ovary, oviduct, pituitary, or skeletal muscle, supporting the tissue specificity of the Prl8a2iCre in the primary decidual zone. This novel iCre line will be a valuable tool for in vivo genetic manipulation and lineage tracing to investigate functions of genetic networks and cellular dynamics associated with decidualization and infertility.

Spatial transcriptomic profiles of mouse uterine microenvironments at pregnancy day 7.5†.

Uterine dysfunctions lead to fertility disorders and pregnancy complications. Normal uterine functions at pregnancy depend on crosstalk among multiple cell types in uterine microenvironments. Here, we performed the spatial transcriptomics and single-cell RNA-seq assays to determine local gene expression profiles at the embryo implantation site of the mouse uterus on pregnancy day 7.5 (D7.5). The spatial transcriptomic annotation identified 11 domains of distinct gene signatures, including a mesometrial myometrium, an anti-mesometrial myometrium, a mesometrial decidua enriched with natural killer cells, a vascular sinus zone for maternal vessel remodeling, a fetal-maternal interface, a primary decidual zone, a transition decidual zone, a secondary decidual zone, undifferentiated stroma, uterine glands, and the embryo. The scRNA-Seq identified 12 types of cells in the D7.5 uterus including three types of stromal fibroblasts with differentiated and undifferentiated markers, one cluster of epithelium including luminal and glandular epithelium, mesothelium, endothelia, pericytes, myelomonocytic cell, natural killer cells, and lymphocyte B. These single-cell RNA signatures were then utilized to deconvolute the cell-type compositions of each individual uterine microenvironment. Functional annotation assays on spatial transcriptomic data revealed uterine microenvironments with distinguished metabolic preferences, immune responses, and various cellular behaviors that are regulated by region-specific endocrine and paracrine signals. Global interactome among regions is also projected based on the spatial transcriptomic data. This study provides high-resolution transcriptome profiles with locality information at the embryo implantation site to facilitate further investigations on molecular mechanisms for normal pregnancy progression.

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.

Cell-type specific analysis of physiological action of estrogen in mouse oviducts.

One of the endogenous estrogens, 17ß-estradiol (E2 ) is a female steroid hormone secreted from the ovary. It is well established that E2 causes biochemical and histological changes in the uterus. However, it is not completely understood how E2 regulates the oviductal environment in vivo. In this study, we assessed the effect of E2 on each oviductal cell type, using an ovariectomized-hormone-replacement mouse model, single-cell RNA-sequencing (scRNA-seq), in situ hybridization, and cell-type-specific deletion in mice. We found that each cell type in the oviduct responded to E2 distinctively, especially ciliated and secretory epithelial cells. The treatment of exogenous E2 did not drastically alter the transcriptomic profile from that of endogenous E2 produced during estrus. Moreover, we have identified and validated genes of interest in our datasets that may be used as cell- and region-specific markers in the oviduct. Insulin-like growth factor 1 (Igf1) was characterized as an E2 -target gene in the mouse oviduct and was also expressed in human fallopian tubes. Deletion of Igf1 in progesterone receptor (Pgr)-expressing cells resulted in female subfertility, partially due to an embryo developmental defect and embryo retention within the oviduct. In summary, we have shown that oviductal cell types, including epithelial, stromal, and muscle cells, are differentially regulated by E2 and support gene expression changes, such as growth factors that are required for normal embryo development and transport in mouse models. Furthermore, we have identified cell-specific and region-specific gene markers for targeted studies and functional analysis in vivo.

Estrogen receptor α (ERα)-binding super-enhancers drive key mediators that control uterine estrogen responses in mice.

Estrogen receptor α (ERα) modulates gene expression by interacting with chromatin regions that are frequently distal from the promoters of estrogen-regulated genes. Active chromatin-enriched "super-enhancer" (SE) regions, mainly observed in in vitro culture systems, often control production of key cell type-determining transcription factors. Here, we defined super-enhancers that bind to ERα in vivo within hormone-responsive uterine tissue in mice. We found that SEs are already formed prior to estrogen exposure at the onset of puberty. The genes at SEs encoded critical developmental factors, including retinoic acid receptor α (RARA) and homeobox D (HOXD). Using high-throughput chromosome conformation capture (Hi-C) along with DNA sequence analysis, we demonstrate that most SEs are located at a chromatin loop end and that most uterine genes in loop ends associated with these SEs are regulated by estrogen. Although the SEs were formed before puberty, SE-associated genes acquired optimal ERα-dependent expression after reproductive maturity, indicating that pubertal processes that occur after SE assembly and ERα binding are needed for gene responses. Genes associated with these SEs affected key estrogen-mediated uterine functions, including transforming growth factor ß (TGFß) and LIF interleukin-6 family cytokine (LIF) signaling pathways. To the best of our knowledge, this is the first identification of SE interactions that underlie hormonal regulation of genes in uterine tissue and optimal development of estrogen responses in this tissue.

An Ancient Fecundability-Associated Polymorphism Creates a GATA2 Binding Site in a Distal Enhancer of HLA-F.

Variation in female reproductive traits, such as fertility, fecundity, and fecundability, are heritable in humans, but identifying and functionally characterizing genetic variants associated with these traits have been challenging. Here, we explore the functional significance and evolutionary history of a G/A polymorphism at SNP rs2523393, which is an eQTL for HLA-F and is significantly associated with fecundability (the probability of being pregnant within a single menstrual cycle). We replicated the association between the rs2523393 genotype and HLA-F expression by using GTEx data and demonstrate that HLA-F is upregulated in the endometrium during the window of implantation and by progesterone in decidual stromal cells. Next, we show that the rs2523393 A allele creates a GATA2 binding site in a progesterone-responsive distal enhancer that loops to the HLA-F promoter. Remarkably, we found that the A allele is derived in the human lineage and that the G/A polymorphism arose before the divergence of modern and archaic humans and segregates at intermediate to high frequencies across human populations. Remarkably, the derived A allele is has also been identified in a GWAS as a risk allele for multiple sclerosis. These data suggest that the polymorphism is maintained by antagonistic pleiotropy and a reproduction-health tradeoff in human evolution.

Dynamic transcriptome, accessible genome, and PGR cistrome profiles in the human myometrium.

The myometrium undergoes structural and functional remodeling during pregnancy. We hypothesize that myometrial genomic elements alter correspondingly in preparation for parturition. Human myometrial tissues from nonpregnant (NP) and term pregnant (TP) human subjects were examined by RNAseq, ATACseq, and PGR ChIPseq assays to profile transcriptome, assessible genome, and PGR occupancy. NP and TP specimens exhibit 2890 differentially expressed genes, reflecting an increase of metabolic, inflammatory, and PDGF signaling, among others, in adaptation to pregnancy. At the epigenome level, patterns of accessible genome change between NP and TP myometrium, leading to the altered enrichment of binding motifs for hormone and muscle regulators such as the progesterone receptor (PGR), Krüppel-like factors, and MEF2A transcription factors. PGR genome occupancy exhibits a significant difference between the two stages of the myometrium, concomitant with distinct transcriptomic profiles including genes such as ENO1, LHDA, and PLCL1 in the glycolytic and calcium signaling pathways. Over-representation of SRF, MYOD, and STAT binding motifs in PGR occupying sites further suggests interactions between PGR and major muscle regulators for myometrial gene expression. In conclusion, changes in accessible genome and PGR occupancy are part of the myometrial remodeling process and may serve as mechanisms to formulate the state-specific transcriptome profiles.

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, ß-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.

Shift from androgen to estrogen action causes abdominal muscle fibrosis, atrophy, and inguinal hernia in a transgenic male mouse model.

Inguinal hernia develops primarily in elderly men, and more than one in four men will undergo inguinal hernia repair during their lifetime. However, the underlying mechanisms behind hernia formation remain unknown. It is known that testosterone and estradiol can regulate skeletal muscle mass. We herein demonstrate that the conversion of testosterone to estradiol by the aromatase enzyme in lower abdominal muscle (LAM) tissue causes intense fibrosis, leading to muscle atrophy and inguinal hernia; an aromatase inhibitor entirely prevents this phenotype. LAM tissue is uniquely sensitive to estradiol because it expresses very high levels of estrogen receptor-α. Estradiol acts via estrogen receptor-α in LAM fibroblasts to activate pathways for proliferation and fibrosis that replaces atrophied myocytes, resulting in hernia formation. This is accompanied by decreased serum testosterone and decreased expression of the androgen receptor target genes in LAM tissue. These findings provide a mechanism for LAM tissue fibrosis and atrophy and suggest potential roles of future nonsurgical and preventive approaches in a subset of elderly men with a predisposition for hernia development.

A distal super enhancer mediates estrogen-dependent mouse uterine-specific gene transcription of Igf1 (insulin-like growth factor 1).

Insulin-like growth factor 1 (IGF1) is primarily synthesized in and secreted from the liver; however, estrogen (E2), through E2 receptor α (ERα), increases uterine Igf1 mRNA levels. Previous ChIP-seq analyses of the murine uterus have revealed a potential enhancer region distal from the Igf1 transcription start site (TSS) with multiple E2-dependent ERα-binding regions. Here, we show E2-dependent super enhancer-associated characteristics and suggest contact between the distal enhancer and the Igf1 TSS. We hypothesized that this distal super-enhancer region controls E2-responsive induction of uterine Igf1 transcripts. We deleted 430 bp, encompassing one of the ERα-binding sites, thereby disrupting interactions of the enhancer with gene-regulatory factors. As a result, E2-mediated induction of mouse uterine Igf1 mRNA is completely eliminated, whereas hepatic Igf1 expression remains unaffected. This highlights the central role of a distal enhancer in the assembly of the factors necessary for E2-dependent interaction with the Igf1 TSS and induction of uterus-specific Igf1 transcription. Of note, loss of the enhancer did not affect fertility or uterine growth responses. Deletion of uterine Igf1 in a PgrCre;Igf1f/f model decreased female fertility but did not impact the E2-induced uterine growth response. Moreover, E2-dependent activation of uterine IGF1 signaling was not impaired by disrupting the distal enhancer or by deleting the coding transcript. This indicated a role for systemic IGF1, suggested that other growth mediators drive uterine response to E2, and suggested that uterine-derived IGF1 is essential for reproductive success. Our findings elucidate the role of a super enhancer in Igf1 regulation and uterine growth.