Progesterone receptor mediates ovulatory transcription through RUNX transcription factor interactions and chromatin remodelling.

Progesterone receptor (PGR) plays diverse roles in reproductive tissues and thus coordinates mammalian fertility. In the ovary, rapid acute induction of PGR is the key determinant of ovulation through transcriptional control of a unique set of genes that culminates in follicle rupture. However, the molecular mechanisms for this specialized PGR function in ovulation is poorly understood. We have assembled a detailed genomic profile of PGR action through combined ATAC-seq, RNA-seq and ChIP-seq analysis in wildtype and isoform-specific PGR null mice. We demonstrate that stimulating ovulation rapidly reprograms chromatin accessibility in two-thirds of sites, correlating with altered gene expression. An ovary-specific PGR action involving interaction with RUNX transcription factors was observed with 70% of PGR-bound regions also bound by RUNX1. These transcriptional complexes direct PGR binding to proximal promoter regions. Additionally, direct PGR binding to the canonical NR3C motif enable chromatin accessibility. Together these PGR actions mediate induction of essential ovulatory genes. Our findings highlight a novel PGR transcriptional mechanism specific to ovulation, providing new targets for infertility treatments or new contraceptives that block ovulation.

Loss of Runx1 Induces Granulosa Cell Defects and Development of Ovarian Tumors in the Mouse.

Genetic alterations of the RUNX1 gene are associated with a variety of malignancies, including female-related cancers. The role of RUNX1 as either a tumor suppressor gene or an oncogene is tissue-dependent and varies based on the cancer type. Both the amplification and deletion of the RUNX1 gene have been associated with ovarian cancer in humans. In this study, we investigated the effects of Runx1 loss on ovarian pathogenesis in mice. A conditional loss of Runx1 in the somatic cells of the ovary led to an increased prevalence of ovarian tumors in aged mice. By the age of 15 months, 27% of Runx1 knockout (KO) females developed ovarian tumors that presented characteristics of granulosa cell tumors. While ovaries from young adult mice did not display tumors, they all contained abnormal follicle-like lesions. The granulosa cells composing these follicle-like lesions were quiescent, displayed defects in differentiation and were organized in a rosette-like pattern. The RNA-sequencing analysis further revealed differentially expressed genes in Runx1 KO ovaries, including genes involved in metaplasia, ovarian cancer, epithelial cell development, tight junctions, cell-cell adhesion, and the Wnt/beta-catenin pathway. Together, this study showed that Runx1 is required for normal granulosa cell differentiation and prevention of ovarian tumor development in mice.

Becoming female: Ovarian differentiation from an evolutionary perspective.

Differentiation of the bipotential gonadal primordium into ovaries and testes is a common process among vertebrate species. While vertebrate ovaries eventually share the same functions of producing oocytes and estrogens, ovarian differentiation relies on different morphogenetic, cellular, and molecular cues depending on species. The aim of this review is to highlight the conserved and divergent features of ovarian differentiation through an evolutionary perspective. From teleosts to mammals, each clade or species has a different story to tell. For this purpose, this review focuses on three specific aspects of ovarian differentiation: ovarian morphogenesis, the evolution of the role of estrogens on ovarian differentiation and the molecular pathways involved in granulosa cell determination and maintenance.

Somatic cell fate maintenance in mouse fetal testes via autocrine/paracrine action of AMH and activin B.

Fate determination and maintenance of fetal testes in most mammals occur cell autonomously as a result of the action of key transcription factors in Sertoli cells. However, the cases of freemartin, where an XX twin develops testis structures under the influence of an XY twin, imply that hormonal factor(s) from the XY embryo contribute to sex reversal of the XX twin. Here we show that in mouse XY embryos, Sertoli cell-derived anti-Mullerian hormone (AMH) and activin B together maintain Sertoli cell identity. Sertoli cells in the gonadal poles of XY embryos lacking both AMH and activin B transdifferentiate into their female counterpart granulosa cells, leading to ovotestis formation. The ovotestes remain to adulthood and produce both sperm and oocytes, although there are few of the former and the latter fail to mature. Finally, the ability of XY mice to masculinize ovaries is lost in the absence of these two factors. These results provide insight into fate maintenance of fetal testes through the action of putative freemartin factors.

Constitutive expression of Steroidogenic factor-1 (NR5A1) disrupts ovarian functions, fertility, and metabolic homeostasis in female mice.

Steroid hormones regulate various aspects of physiology, from reproductive functions to metabolic homeostasis. Steroidogenic factor-1 (NR5A1) plays a central role in the development of steroidogenic tissues and their ability to produce steroid hormones. Inactivation of Nr5a1 in the mouse results in a complete gonadal and adrenal agenesis, absence of gonadotropes in the pituitary and impaired development of ventromedial hypothalamus, which controls glucose and energy metabolism. In this study, we set out to examine the consequences of NR5A1 overexpression (NR5A1+) in the NR5A1-positive cell populations in female mice. Ovaries of NR5A1+ females presented defects such as multi-oocyte follicles and an accumulation of corpora lutea. These females were hyperandrogenic, had irregular estrous cycles with persistent metestrus and became prematurely infertile. Furthermore, the decline in fertility coincided with weight gain, increased adiposity, hypertriglyceridemia, hyperinsulinemia, and impaired glucose tolerance, indicating defects in metabolic functions. In summary, excess NR5A1 expression causes hyperandrogenism, disruption of ovarian functions, premature infertility, and disorders of metabolic homeostasis. This NR5A1 overexpression mouse provides a novel model for studying not only the molecular actions of NR5A1, but also the crosstalk between endocrine, reproductive, and metabolic systems.

Increased FOXL2 expression alters uterine structures and functions†.

The transcription factor forkhead box L2 (FOXL2) regulates sex differentiation and reproductive function. Elevated levels of this transcription factor have been observed in the diseases of the uterus, such as endometriosis. However, the impact of elevated FOXL2 expression on uterine physiology remains unknown. In order to determine the consequences of altered FOXL2 in the female reproductive axis, we generated mice with over-expression of FOXL2 (FOXL2OE) by crossing Foxl2LsL/+ with the Progesterone receptor Pgrcre model. FOXL2OE uterus showed severe morphological abnormality including abnormal epithelial stratification, blunted adenogenesis, increased endometrial fibrosis, and disrupted myometrial morphology. In contrast, increasing FOXL2 levels specifically in uterine epithelium by crossing the Foxl2LsL/+ with the lactoferrin Ltficre mice resulted in the eFOXL2OE mice with uterine epithelial stratification but without defects in endometrial fibrosis and adenogenesis, demonstrating a role of the endometrial stroma in the uterine abnormalities of the FOXL2OE mice. Transcriptomic analysis of 12 weeks old Pgrcre and FOXL2OE uterus at diestrus stage showed multiple signaling pathways related with cellular matrix, wnt/ß-catenin, and altered cell cycle. Furthermore, we found FOXL2OE mice were sterile. The infertility was caused in part by a disruption of the hypophyseal ovarian axis resulting in an anovulatory phenotype. The FOXL2OE mice failed to show decidual responses during artificial decidualization in ovariectomized mice demonstrating the uterine contribution to the infertility phenotype. These data support that aberrantly increased FOXL2 expressions in the female reproductive tract can disrupt ovarian and uterine functions.

Aberrant and constitutive expression of FOXL2 impairs ovarian development and functions in mice.

Development and functions of the ovary rely on appropriate signaling and communication between various ovarian cell types. FOXL2, a transcription factor that plays a key role at different stages of ovarian development, is associated with primary ovarian insufficiency and ovarian cancer as a result of its loss-of-function or mutations. In this study, we investigated the impact of aberrant, constitutive expression of FOXL2 in somatic cells of the ovary. Overexpression of FOXL2 that started during fetal life resulted in defects in nest breakdown and consequent formation of polyovular follicles. Granulosa cell differentiation was impaired and recruitment and differentiation of steroidogenic theca cells was compromised. As a consequence, adult ovaries overexpressing FOXL2 exhibited defects in compartmentalization of granulosa and theca cells, significant decreased steroidogenesis and lack of ovulation. These findings demonstrate that fine-tuned expression of FOXL2 is required for proper folliculogenesis and fertility.

In utero exposure to arsenite contributes to metabolic and reproductive dysfunction in male offspring of CD-1 mice.

In utero exposure to arsenite (iAs) is known to increase disease risks later in life. We investigated the effect of in utero exposure to iAs in the drinking water on metabolic and reproductive parameters in male mouse offspring at postnatal and adult stages. Pregnant CD-1 mice were exposed to iAs (as sodium arsenite) in the drinking water at 0 (control), 10 ppb (EPA standard for drinking water), and 42.5 ppm (tumor-inducing dose in mice) from embryonic day (E) 10-18. At birth, pups were fostered to unexposed females. Male offspring exposed to 10 ppb in utero exhibited increase in body weight at birth when compared to controls. Male offspring exposed to 42.5 ppm in utero showed a tendency for increased body weight and a smaller anogenital distance. The body weight in iAs-exposed pups continued to increase significantly compared to control at 3 weeks and 11 weeks of age. At 5 months of age, iAs-exposed males exhibited greater body fat content and glucose intolerance. Male offspring exposed to 10 ppb in utero had higher circulating levels of leptin compared to control. In addition, males exposed to 42.5 ppm in utero exhibited decreased total number of pups born compared to controls and lower average number of litters sired over a six-month period. These results indicate that in utero exposure to iAs at either human relevant concentration or tumor-inducing concentration is a potential cause of developmental origin of metabolic and reproductive dysfunction in adult male mice.

Canonical Wnt/ß-catenin activity and differential epigenetic marks direct sexually dimorphic regulation of Irx3 and Irx5 in developing mouse gonads.

Members of the Iroquois B (IrxB) homeodomain cluster genes, specifically Irx3 and Irx5, are crucial for heart, limb and bone development. Recently, we reported their importance for oocyte and follicle survival within the developing ovary. Irx3 and Irx5 expression begins after sex determination in the ovary but remains absent in the fetal testis. Mutually antagonistic molecular signals ensure ovary versus testis differentiation with canonical Wnt/ß-catenin signals paramount for promoting the ovary pathway. Notably, few direct downstream targets have been identified. We report that Wnt/ß-catenin signaling directly stimulates Irx3 and Irx5 transcription in the developing ovary. Using in silico analysis of ATAC- and ChIP-Seq databases in conjunction with mouse gonad explant transfection assays, we identified TCF/LEF-binding sequences within two distal enhancers of the IrxB locus that promote ß-catenin-responsive ovary expression. Meanwhile, Irx3 and Irx5 transcription is suppressed within the developing testis by the presence of H3K27me3 on these same sites. Thus, we resolved sexually dimorphic regulation of Irx3 and Irx5 via epigenetic and ß-catenin transcriptional control where their ovarian presence promotes oocyte and follicle survival vital for future ovarian health.

RUNX1 maintains the identity of the fetal ovary through an interplay with FOXL2.

Sex determination of the gonads begins with fate specification of gonadal supporting cells into either ovarian pre-granulosa cells or testicular Sertoli cells. This fate specification hinges on a balance of transcriptional control. Here we report that expression of the transcription factor RUNX1 is enriched in the fetal ovary in rainbow trout, turtle, mouse, goat, and human. In the mouse, RUNX1 marks the supporting cell lineage and becomes pre-granulosa cell-specific as the gonads differentiate. RUNX1 plays complementary/redundant roles with FOXL2 to maintain fetal granulosa cell identity and combined loss of RUNX1 and FOXL2 results in masculinization of fetal ovaries. At the chromatin level, RUNX1 occupancy overlaps partially with FOXL2 occupancy in the fetal ovary, suggesting that RUNX1 and FOXL2 target common sets of genes. These findings identify RUNX1, with an ovary-biased expression pattern conserved across species, as a regulator in securing the identity of ovarian-supporting cells and the ovary.

Enhanced Recovery After Surgery to Change Process Measures and Reduce Opioid Use After Cesarean Delivery: A Quality Improvement Initiative.

OBJECTIVE: To evaluate implementation of an enhanced recovery after surgery (ERAS) program for patients undergoing elective cesarean delivery by comparing opioid exposure, multimodal analgesia use, and other process and outcome measures before and after implementation. METHODS: An ERAS program was implemented among patients undergoing elective cesarean delivery in a large integrated health care delivery system. We conducted a pre-post study of ERAS implementation to compare changes in process and outcome measures during the 12 months before and 12 months after implementation. RESULTS: The study included 4,689 patients who underwent an elective cesarean delivery in the 12 months before (pilot sites: March 1, 2015-February 29, 2016, all other sites: October 1, 2015-September 30, 2016), and 4,624 patients in the 12 months after (pilot sites: April 1, 2016-March 31, 2017, all other sites: November 1, 2016-October 31, 2017) ERAS program implementation. After ERAS implementation mean inpatient opioid exposure (average daily morphine equivalents) decreased from 10.7 equivalents (95% CI 10.2-11.3) to 5.4 equivalents (95% CI 4.8-5.9) controlling for age, race-ethnicity, prepregnancy body mass index, patient reported pain score, and medical center. The use of multimodal analgesia (ie, acetaminophen and neuraxial anesthesia) increased from 9.7% to 88.8%, the adjusted risk ratio (RR) for meeting multimodal analgesic goals was 9.13 (RR comparing post-ERAS with pre-ERAS; 95% CI 8.35-10.0) and the proportion of time patients reported acceptable pain scores increased from 82.1% to 86.4% (P<.001). Outpatient opioids dispensed at hospital discharge decreased from 85.9% to 82.2% post-ERAS (P<.001) and the average number of dispensed pills decreased from 38 to 26 (P<.001). The hours to first postsurgical ambulation decreased by 2.7 hours (95% CI -3.1 to -2.4) and the hours to first postsurgical solid intake decreased by 11.1 hours (95% CI -11.5 to -10.7). There were no significant changes in hospital length of stay, surgical site infections, hospital readmissions, or breastfeeding rates. CONCLUSIONS: Implementation of an ERAS program in patients undergoing elective cesarean delivery was associated with a reduction in opioid inpatient and outpatient exposure and with changes in surgical process measures of care without worsened surgical outcomes.

The unusual rainbow trout sex determination gene hijacked the canonical vertebrate gonadal differentiation pathway.

Evolutionary novelties require rewiring of transcriptional networks and/or the evolution of new gene functions. Sex determination (SD), one of the most plastic evolutionary processes, requires such novelties. Studies on the evolution of vertebrate SD revealed that new master SD genes are generally recruited from genes involved in the downstream SD regulatory genetic network. Only a single exception to this rule is currently known in vertebrates: the intriguing case of the salmonid master SD gene (sdY), which arose from duplication of an immune-related gene. This exception immediately posed the question of how a gene outside from the classical sex differentiation cascade could acquire its function as a male SD gene. Here we show that SdY became integrated in the classical vertebrate sex differentiation cascade by interacting with the Forkhead box domain of the female-determining transcription factor, Foxl2. In the presence of Foxl2, SdY is translocated to the nucleus where the SdY:Foxl2 complex prevents activation of the aromatase (cyp19a1a) promoter in cooperation with Nr5a1 (Sf1). Hence, by blocking a positive loop of regulation needed for the synthesis of estrogens in the early differentiating gonad, SdY disrupts a preset female differentiation pathway, consequently allowing testicular differentiation to proceed. These results also suggest that the evolution of unusual vertebrate master sex determination genes recruited from outside the classical pathway like sdY is strongly constrained by their ability to interact with the canonical gonadal differentiation pathway.

Genome-wide identification of FOXL2 binding and characterization of FOXL2 feminizing action in the fetal gonads.

The identity of the gonads is determined by which fate, ovarian granulosa cell or testicular Sertoli cell, the bipotential somatic cell precursors choose to follow. In most vertebrates, the conserved transcription factor FOXL2 contributes to the fate of granulosa cells. To understand FOXL2 functions during gonad differentiation, we performed genome-wide analysis of FOXL2 chromatin occupancy in fetal ovaries and established a genetic mouse model that forces Foxl2 expression in the fetal testis. When FOXL2 was ectopically expressed in the somatic cell precursors in the fetal testis, FOXL2 was sufficient to repress Sertoli cell differentiation, ultimately resulting in partial testis-to-ovary sex-reversal. Combining genome-wide analysis of FOXL2 binding in the fetal ovary with transcriptomic analyses of our Foxl2 gain-of-function and previously published Foxl2 loss-of-function models, we identified potential pathways responsible for the feminizing action of FOXL2. Finally, comparison of FOXL2 genome-wide occupancy in the fetal ovary with testis-determining factor SOX9 genome-wide occupancy in the fetal testis revealed extensive overlaps, implying that antagonistic signals between FOXL2 and SOX9 occur at the chromatin level.

Effects of in Utero Exposure to Arsenic during the Second Half of Gestation on Reproductive End Points and Metabolic Parameters in Female CD-1 Mice.

BACKGROUND: Mice exposed to high levels of arsenic in utero have increased susceptibility to tumors such as hepatic and pulmonary carcinomas when they reach adulthood. However, the effects of in utero arsenic exposure on general physiological functions such as reproduction and metabolism remain unclear. OBJECTIVES: We evaluated the effects of in utero exposure to inorganic arsenic at the U.S. Environmental Protection Agency (EPA) drinking water standard (10 ppb) and at tumor-inducing levels (42.5 ppm) on reproductive end points and metabolic parameters when the exposed females reached adulthood. METHODS: Pregnant CD-1 mice were exposed to sodium arsenite [none (control), 10 ppb, or 42.5 ppm] in drinking water from gestational day 10 to birth, the window of organ formation. At birth, exposed offspring were fostered to unexposed dams. We examined reproductive end points (age at vaginal opening, reproductive hormone levels, estrous cyclicity, and fertility) and metabolic parameters (body weight changes, hormone levels, body fat content, and glucose tolerance) in the exposed females when they reached adulthood. RESULTS: Arsenic-exposed females (10 ppb and 42.5 ppm) exhibited early onset of vaginal opening. Fertility was not affected when females were exposed to the 10-ppb dose. However, the number of litters per female was decreased in females exposed to 42.5 ppm of arsenic in utero. In both 10-ppb and 42.5-ppm groups, arsenic-exposed females had significantly greater body weight gain, body fat content, and glucose intolerance. CONCLUSION: Our findings revealed unexpected effects of in utero exposure to arsenic: exposure to both a human-relevant low dose and a tumor-inducing level led to early onset of vaginal opening and to obesity in female CD-1 mice.

Gonadal Identity in the Absence of Pro-Testis Factor SOX9 and Pro-Ovary Factor Beta-Catenin in Mice.

Sex-reversal cases in humans and genetic models in mice have revealed that the fate of the bipotential gonad hinges upon the balance between pro-testis SOX9 and pro-ovary beta-catenin pathways. Our central query was: if SOX9 and beta-catenin define the gonad's identity, then what do the gonads become when both factors are absent? To answer this question, we developed mouse models that lack either Sox9, beta-catenin, or both in the somatic cells of the fetal gonads and examined the morphological outcomes and transcriptome profiles. In the absence of Sox9 and beta-catenin, both XX and XY gonads progressively lean toward the testis fate, indicating that expression of certain pro-testis genes requires the repression of the beta-catenin pathway, rather than a direct activation by SOX9. We also observed that XY double knockout gonads were more masculinized than their XX counterpart. To identify the genes responsible for the initial events of masculinization and to determine how the genetic context (XX vs. XY) affects this process, we compared the transcriptomes of Sox9/beta-catenin mutant gonads and found that early molecular changes underlying the XY-specific masculinization involve the expression of Sry and 21 SRY direct target genes, such as Sox8 and Cyp26b1. These results imply that when both Sox9 and beta-catenin are absent, Sry is capable of activating other pro-testis genes and drive testis differentiation. Our findings not only provide insight into the mechanism of sex determination, but also identify candidate genes that are potentially involved in disorders of sex development.

Building an ovary: insights into establishment of somatic cell lineages in the mouse.

The molecular pathways that drive the differentiation of somatic cell populations in the testis and ovary have been the subjects of intensive research over the past decade. It is now clear that ovarian differentiation is a coordinate event driven by secreted factors including R-spondin1, WNT4, and follistatin and transcriptional regulators such as ß-catenin and FOXL2. These factors direct bipotential somatic cell lineages toward an ovarian fate and simultaneously suppress the emergence of testis-determining processes. This review summarizes the molecular pathways responsible for establishment of the ovary and discusses the current hypotheses on the origin(s) of somatic cell lineages and how these somatic cells acquire the characteristics necessary for their function during ovarian development and maintenance.

Heritable targeted inactivation of the rainbow trout (Oncorhynchus mykiss) master sex-determining gene using zinc-finger nucleases.

Gene targeting is a powerful tool for analyzing gene function. Recently, new technology for gene targeting using engineered zinc-finger nucleases (ZFNs) has been described in fish species. However, it has not yet been widely used for cold water and slow developing species, such as Salmonidae. Here, we present the results of successful ZFN-mediated disruption of the sex-determining gene sdY (sexually dimorphic on the Y chromosome) in rainbow trout (Oncorhynchus mykiss). Three pairs of ZFN mRNA targeted to different regions of the sdY gene were injected into fertilized rainbow trout eggs. Sperm from 1-year-old male founders (parental generation one or P1) carrying a ZFN-induced mutation in their germline were then used to produce F1 non-mosaic animals. In these F1 populations, we characterized 14 different mutations in the sdY gene, including one mutation leading to the deletion of leucine 43 (L43) and 13 mutations at other target sites that had different effects on the SdY protein, i.e., amino acid insertions, deletions, and frameshift mutations producing premature stop codons in the mRNA. The gonadal phenotype analysis of the F1-mutated animals revealed that the single L43 amino acid deletion did not lead to a male-to-female sex reversal, but all other mutations induced a clear ovarian phenotype. These results show that targeted gene disruption using ZFN is efficient in rainbow trout but depends on the ZFN design. We also characterized new sdY mutations resulting in male-to-female sex reversal, and we conclude that L43 seems dispensable for SdY function.

Divergent expression regulation of gonad development genes in medaka shows incomplete conservation of the downstream regulatory network of vertebrate sex determination.

Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of "master sex-determining genes" at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY, a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought.

The sexually dimorphic on the Y-chromosome gene (sdY) is a conserved male-specific Y-chromosome sequence in many salmonids.

All salmonid species investigated to date have been characterized with a male heterogametic sex-determination system. However, as these species do not share any Y-chromosome conserved synteny, there remains a debate on whether they share a common master sex-determining gene. In this study, we investigated the extent of conservation and evolution of the rainbow trout (Oncorhynchus mykiss) master sex-determining gene, sdY (sexually dimorphic on the Y-chromosome), in 15 different species of salmonids. We found that the sdY sequence is highly conserved in all salmonids and that sdY is a male-specific Y-chromosome gene in the majority of these species. These findings demonstrate that most salmonids share a conserved sex-determining locus and also strongly suggest that sdY may be this conserved master sex-determining gene. However, in two whitefish species (subfamily Coregoninae), sdY was found both in males and females, suggesting that alternative sex-determination systems may have also evolved in this family. Based on the wide conservation of sdY as a male-specific Y-chromosome gene, efficient and easy molecular sexing techniques can now be developed that will be of great interest for studying these economically and environmentally important species.