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Conventionally viewed as male hormone, androgens play a critical role in female fertility. Although androgen receptors (AR) are transcription factors, to date very few direct transcriptional targets of ARs have been identified in the ovary. Using mouse models, this study provides three critical insights about androgen-induced gene regulation in the ovary and its impact on female fertility. First, RNA-sequencing reveals a number of genes and biological processes that were previously not known to be directly regulated by androgens in the ovary. Second, androgens can also influence gene expression by decreasing the tri-methyl mark on lysine 27 of histone3 (H3K27me3), a gene silencing epigenetic mark. ChIP-seq analyses highlight that androgen-induced modulation of H3K27me3 mark within gene bodies, promoters or distal enhancers have a much broader impact on ovarian function than the direct genomic effects of androgens. Third, androgen-induced decrease of H3K27me3 is mediated through (a) inhibiting the expression and activity of Enhancer of Zeste Homologue 2 (EZH2), a histone methyltransferase that promotes tri-methylation of K27 and (b) by inducing the expression of a histone demethylase called Jumonji domain containing protein-3 (JMJD3/KDM6B), responsible for removing the H3K27me3 mark. Androgens through the PI3K/Akt pathway, in a transcription-independent fashion, increase hypoxia-inducible factor 1 alpha (HIF1α) protein levels, which in turn induce JMJD3 expression. Furthermore, proof of concept studies involving in vivo knockdown of Ar in the ovary and ovarian (granulosa) cell-specific Ar knockout mouse model show that ARs regulate the expression of key ovarian genes through modulation of H3K27me3.

Although androgen excess is considered detrimental to women’s health and fertility, global and ovarian granulosa cell-specific androgen-receptor (AR) knockout mouse models have been used to show that androgen actions through ARs are actually necessary for normal ovarian function and female fertility. Here we describe two AR-mediated pathways in granulosa cells that regulate ovarian follicular development and therefore female fertility. First, we show that androgens attenuate follicular atresia through nuclear and extranuclear signaling pathways by enhancing expression of the microRNA (miR) miR-125b , which in turn suppresses proapoptotic protein expression. Second, we demonstrate that, independent of transcription, androgens enhance follicle-stimulating hormone (FSH) receptor expression, which then augments FSH-mediated follicle growth and development. Interestingly, we find that the scaffold molecule paxillin regulates both processes, making it a critical regulator of AR actions in the ovary. Finally, we report that low doses of exogenous androgens enhance gonadotropin-induced ovulation in mice, further demonstrating the critical role that androgens play in follicular development and fertility. These data may explain reported positive effects of androgens on ovulation rates in women with diminished ovarian reserve. Furthermore, this study demonstrates mechanisms that might contribute to the unregulated follicle growth seen in diseases of excess androgens such as polycystic ovary syndrome.

Maternal perturbations or sub-optimal conditions during fetal development can predispose the offspring to diseases in adult life. Animal and human studies show that prenatal androgen excess may be an underlying cause of polycystic ovary syndrome (PCOS) later in life. In women, PCOS is a common fertility disorder with comorbid metabolic dysfunction. Here, using a sheep model of PCOS phenotype, we elucidate the epigenetic changes induced by prenatal (30–90 day) testosterone (T) treatment and its effect on gene expression in fetal day 90 (D90) and adult year 2 (Y2) ovaries. RNA-seq study shows 65 and 99 differentially regulated genes in prenatal T-treated fetal and adult ovaries, respectively. Interestingly, there were no differences in gene inducing histone marks H3K27ac, H3K9ac, and H3K4me3 or in gene silencing marks, H3K27me3 and H3K9me3 in the fetal D90 ovaries of control and excess T-exposed fetuses. In contrast, except for H3K4me3 and H3K27me3, all the other histone marks were upregulated in the prenatal T-treated adult Y2 ovary. Chromatin immunoprecipitation (ChIP) studies in adult Y2 ovaries established a direct relationship between the epigenetic modifications with the upregulated and downregulated genes obtained from RNA-seq. Results show increased gene inducing marks, H3K27ac and H3K9ac, on the promoter region of upregulated genes while gene silencing mark, H3K9me3, was also significantly increased on the downregulated genes. This study provides a mechanistic insight into prenatal T-induced developmental programming and its effect on ovarian gene expression that may contribute to reproductive dysfunction and development of PCOS in adult life. Summary sentence A direct correlation between changes in epigenetic marks and gene expression in adult ovaries from prenatal T-treated sheep establishes epigenetic changes as one of the underlying causes for differential expression of genes in PCOS ovary.

The vagina plays a critical role in supporting the pelvic organs and loss of support leads to pelvic organ prolapse. It is unknown what microstructural changes influence prolapse progression nor how decreased elastic fibers contributes to vaginal remodeling and smooth muscle contractility. The objective for this study was to evaluate the effect of fibulin-5 haploinsufficiency, and deficiency with progressive prolapse on the biaxial contractile and biomechanical function of the murine vagina. Vaginas from wildtype (n = 13), haploinsufficient (n = 13), and deficient mice with grade 1 (n = 9) and grade 2 or 3 (n = 9) prolapse were explanted for biaxial contractile and biomechanical testing. Multiaxial histology (n = 3/group) evaluated elastic and collagen fiber microstructure. Western blotting quantified protein expression (n = 6/group). A one-way ANOVA or Kruskal–Wallis test evaluated statistical significance. Pearson’s or Spearman’s test determined correlations with prolapse grade. Axial contractility decreased with fibulin-5 deficiency and POP (p < 0.001), negatively correlated with prolapse grade (ρ = − 0.80; p < 0.001), and positively correlated with muscularis elastin area fraction (ρ = − 0.78; p = 0.004). Circumferential (ρ = 0.71; p < 0.001) and axial (ρ = 0.69; p < 0.001) vaginal wall stresses positively correlated with prolapse grade. These findings demonstrated that fibulin-5 deficiency and prolapse progression decreased vaginal contractility and increased vaginal wall stress. Future work is needed to better understand the processes that contribute to prolapse progression in order to guide diagnostic, preventative, and treatment strategies.

In prostate cancer, the signals that drive cell proliferation change as tumors progress from castration-sensitive (androgen-dominant) to castration-resistant states. While the mechanisms underlying this change remain uncertain, characterization of common signaling components that regulate both stages of prostate cancer proliferation is important for developing effective treatment strategies. Here, we demonstrate that paxillin, a known cytoplasmic adaptor protein, regulates both androgen- and EGF-induced nuclear signaling. We show that androgen and EGF promoted MAPK-dependent phosphorylation of paxillin, resulting in nuclear translocation of paxillin. We found nuclear paxillin could then associate with androgen-stimulated androgen receptor (AR). This complex bound AR-sensitive promoters, retaining AR within the nucleus and regulating AR-mediated transcription. Nuclear paxillin also complexed with ERK and ELK1, mediating c-FOS and cyclin D1 expression; this was followed by proliferation. Thus, paxillin is a liaison between extranuclear MAPK signaling and nuclear transcription in response to androgens and growth factors, making it a potential regulator of both castration-sensitive and castration-resistant prostate cancer. Accordingly, paxillin was required for normal growth of human prostate cancer cell xenografts, and its expression was elevated in human prostate cancer tissue microarrays. Paxillin is therefore a potential biomarker for prostate cancer proliferation and a possible therapeutic target for prostate cancer treatment.

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F 2α (PGF 2α ) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF 2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF 2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF 2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF 2α , resulting in Gα q -specific coupling and Ca 2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [F max 7.67 ± 0.63 (IC 50 21.26 nM), AUC 7.30 ± 0.32 (IC 50 50.43 nM), and frequency of Ca 2+ oscillations 7.66 ± 0.41 (IC 50 22.15 nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48 h post PGF 2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF 2α treatment increased the expression of MYLK , CALD1 , and CNN1 as well as the pro-labour genes OXTR , IL6 , and IL11 , which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN , FBLN1 , and PDGFRA . Our findings suggest that in addition to the well-described acute contractile effect, PGF 2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour–like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug.

Oocyte-specific gene products play a key role in regulation of fertility in mammals. Here, we describe the discovery, molecular characterization, and function of JY-1, a bovine oocyte-expressed gene shown to regulate both function of ovarian granulosa cells and early embryogenesis in cattle and characteristics of JY-1 loci in other species. The JY-1 gene encodes for a secreted protein with multiple mRNA transcripts containing an identical ORF but differing lengths of 3' UTR. JY-1 mRNA and protein are oocyte-specific and detectable throughout folliculogenesis. Recombinant JY-1 protein regulates function of follicle-stimulating hormone-treated ovarian granulosa cells, resulting in enhanced progesterone synthesis accompanied by reduced cell numbers and estradiol production. JY-1 mRNA of maternal origin is also present in early bovine embryos, temporally regulated during the window from meiotic maturation through embryonic genome activation, and is required for blastocyst development. The JY-1 gene has three exons and is located on bovine chromosome 29. JY-1-like sequences are present on syntenic chromosomes of other vertebrate species, but lack exons 1 and 2, including the protein-coding region, suggestive of species specificity in evolution and function of this oocyte-specific gene.

Key Points Despite mounting evidence for close interconnectivity, the clinical association of autoimmunity with female infertility has remained a subject of considerable controversy Following implantation, the female immune system usually induces tolerance towards the embryo, whereas tolerance induction is incomplete in a hyperactive immune system, reducing fertility and increasing the risk of miscarriage Autoimmunity can adversely affect female fertility by prematurely diminishing ovarian reserve, adversely affecting fertilization and implantation, and by increasing the risk of miscarriage and of various pregnancy complications Similar to other autoimmune diseases, autoimmune endocrine diseases affect reproduction, including fertility and miscarriage risk, even at prodromal clinical stages, often months to years before diagnosis of the disease Androgen supplementation in hypoandrogenic conditions of low ovarian reserve (whatever the aetiology) established a treatment paradigm, expanding interventions from the gonadotropin-sensitive stage (last 2 weeks) into earlier follicle maturation stages Insufficient knowledge about the interplay between autoimmunity and reproduction suggests that reproductive immunology is an urgent target area for multi-specialty research by reproductive biologists, rheumatologists, immunologists and endocrinologists Mounting evidence suggests that immune-mediated processes and autoimmunity affect female reproductive success. This Review summarizes the effect of individual autoimmune endocrine diseases on female fertility, and points towards selected developments expected in the near future. An increasing body of evidence suggests that immune-mediated processes affect female reproductive success at multiple levels. Crosstalk between endocrine and immune systems regulates a large number of biological processes that affect target tissues, and this crosstalk involves gene expression, cytokine and/or lymphokine release and hormone action. In addition, endocrine–immune interactions have a major role in the implantation process of the fetal (paternally derived) semi-allograft, which requires a reprogramming process of the maternal immune system from rejection to temporary tolerance for the length of gestation. Usually, the female immune system is supportive of all of these processes and, therefore, facilitates reproductive success. Abnormalities of the female immune system, including autoimmunity, potentially interfere at multiple levels. The relevance of the immune system to female infertility is increasingly recognized by investigators, but clinically is often not adequately considered and is, therefore, underestimated. This Review summarizes the effect of individual autoimmune endocrine diseases on female fertility, and points towards selected developments expected in the near future.

Androgen actions through androgen receptor (AR) are critical for follicle growth and female fertility. While excess androgen level leads to polycystic ovary syndrome (PCOS), a certain amount of direct androgen action is critical for normal ovarian function. Thus, with respect to androgen actions in the ovary, balance is key. However, how androgens regulate ovarian physiology or its underlying mechanism is still poorly understood. Androgen actions are mediated through ‘nuclear/genomic’ or ‘extra-nuclear/non-genomic’ actions of AR. Recently we reported that androgens through both the extra-nuclear and nuclear pathways inhibit the expression and activity of Ezh2, (a histone methyl-transferase involved in gene repression mark, H3K27me3) to induce different genes that are imperative for ovarian function. Here, we further extended our study to elucidate other histone modifications induced by androgens and its downstream physiological effect in follicular development. In addition to Ezh2, we have found that androgens also regulated the expression of a histone demethylase called Jumonji domain containing protein 3 (JMJD3/KDM6B), that is responsible of removing H3K27me3 mark. Results showed that DHT in GCs increased hypoxia-inducible factor 1 alpha (HIF1α) protein levels through the PI3K/Akt pathway, which in turn induced JMJD3 expression. We found that DHT had not affect HIF1α mRNA but increased HIF1α protein levels. This effect of DHT on HIF1α protein level was blocked by flutamide (AR inhibitor), LY294002 (PI3K inhibitor) as well as cyclohexamide (protein translational inhibitor). Interestingly the 5’ prime end of JMJD3 have HIF1α response elements (HRE) and it is well established that several JMJDs are induced by HIF1α under both normal conditions and hypoxic stress. Our studies showed that DHT treatment induced JMJD3 expression and decreased H3K27me3 levels. Inhibition of AR, PI3K/AKT pathway and siRNA-mediated knockdown of HIF1α attenuated androgen-induced expression of JMJD3. Moreover, ChIP studies showed increased binding of HIF1α on the JMJD3 promoter region with DHT treatment. In our efforts to understand the downstream effects of androgen-induced JMJD3 expression, we found Dicer1 was upregulated upon DHT treatment to mouse granulosa cells. Given the critical role of Dicer in follicular development, ovarian physiology and female fertility, in general, along with the importance of androgens in ovarian physiology, we propose that regulation of Dicer is one of the underlying mechanisms of androgen actions in the ovary. Androgens in a transcription-independent manner, through the PI3K/Akt signaling pathway, enhance HIF1α protein synthesis under normoxic conditions which in turn regulate the expression of Dicer through JMJD3. Altogether, this study provides a mechanistic insight of androgen actions in ovarian function by epigenetic modulation.