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.

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.

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.

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.

Ovary-predominant wnt4 expression during gonadal differentiation is not conserved in the rainbow trout (Oncorhynchus mykiss).

The Wnt/ß-catenin pathway is crucial for ovarian differentiation in mammals, and WNT4 is an important protein that regulates this process. While the role of Wnt4 in gonadal differentiation is relatively well characterized in mammals, little is known regarding its role in teleost fish. Therefore, we investigated the potential activity of wnt4 in gonadal differentiation in rainbow trout (Oncorhynchus mykiss), focusing on the teleost and salmonid gene duplications. Phylogenetic and synteny analyses demonstrated that teleost fish possess two wnt4 genes, wnt4a and wnt4b, as a consequence of the teleost-specific whole-genome duplication (3R). In rainbow trout, we also identified an additional wnt4 gene, which is a wnt4a paralog that likely resulted from the salmonid-specific whole-genome duplication (4R). These two Wnt4a proteins (Wnt4a1 and Wnt4a2) share a high identity (>80%) with other vertebrate Wnt4 proteins, whereas Wnt4b is clearly more divergent (60% identity). During embryogenesis and adulthood, the wnt4a1/2 transcripts were expressed in various tissues, including the ovaries and testes. In contrast, wnt4b expression was restricted to the nervous system, suggesting a sub- or a neo-functionalization of this divergent paralog. During early gonadal differentiation in both males and females, the wnt4a1/2 transcripts were detected in the somatic cells surrounding the germ cells, with a slight sexual dimorphism in favor of males. These results demonstrate that, unlike mammals, rainbow trout do not display an ovary-predominant wnt4 expression profile during early gonadal differentiation.

The duplicated rainbow trout (Oncorhynchus mykiss) T-box transcription factors 1, tbx1a and tbx1b, are up-regulated during testicular development.

Tbx1 is a member of the T-box transcription factor gene family involved in embryogenesis and organogenesis. Recently, within a pan-genomic screen using rainbow trout (Oncorhynchus mykiss) cDNA microarrays, we identified a tbx1 homolog with testicular over-expression during sex differentiation. Here, we characterized two very similar rainbow trout tbx1 paralogs, tbx1a and tbx1b. In adult tissues, tbx1a expression is restricted to the gonads, with high expression in the testis, while tbx1b is more widely expressed in gonads, gills, brains, muscle, and skin. During gonadal differentiation, both genes are differentially expressed in favor of testis formation shortly after hatching. These genes are expressed in somatic cells surrounding germ cells of the differentiating testis, while no or only weak expression was observed in the differentiating ovary. tbx1a and tbx1b were also both down-regulated in the differentiating testis during feminization with estrogens and up-regulated in the differentiating ovary during masculinization with an aromatase inhibitor. These results suggest that tbx1a and tbx1b are probably involved in the regulation of testicular differentiation in rainbow trout. Since Tbx1 is known to interact with the retinoic acid (RA) signaling pathway, we also examined the effect of RA on the rainbow trout tbx1 expression pattern. Expression of tbx1a and tbx1b was down-regulated in RA-treated male gonads, suggesting that tbx1 interacts with the RA signaling pathway and thus could be involved in the control of rainbow trout gonadal differentiation.