Hen Prizant

Androgens regulate ovarian follicular development by increasing follicle stimulating hormone receptor and microRNA-125b expression.

Significance Androgens are primarily considered detrimental to women’s health. However, androgen-receptor KO mouse models have been used to establish that androgens are actually necessary for normal ovarian function and female fertility. Despite these observations, how androgens regulate female fertility is not known. Here we show that androgens promote follicular development via two mechanisms: (i) prevention of follicular atresia by inducing the expression of an antiapoptotic microRNA (miR), miR-125b; and (ii) promotion of follicle growth by increasing follicle-stimulating hormone receptor levels in a transcription-independent fashion. These data considerably change our understanding of androgen effects in female reproduction, and help explain the ovarian physiology seen in patients with too little or too much androgen. 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.

Androgen actions in the ovary: balance is key

For many decades, elevated androgens in women have been associated with poor reproductive health. However, recent studies have shown that androgens play a crucial role in womens fertility. The following review provides an overall perspective about how androgens and androgen receptor-mediated actions regulate normal follicular development, as well as discuss emerging concepts, latest perceptions, and controversies regarding androgen actions and signaling in the ovary.

Understanding extranuclear (nongenomic) androgen signaling: What a frog oocyte can tell us about human biology

Steroids are key factors in a myriad of mammalian biological systems, including the brain, kidney, heart, bones, and gonads. While alternative potential steroid receptors have been described, the majority of biologically relevant steroid responses appear to be mediated by classical steroid receptors that are located in all parts of the cell, from the plasma membrane to the nucleus. Interestingly, these classical steroid receptors modulate different signals depending upon their location. For example, receptors in the plasma membrane interact with membrane signaling molecules, including G proteins and kinases. In contrast, receptors in the nucleus interact with nuclear signaling molecules, including transcriptional co-regulators. These extranuclear and intranuclear signals function together in an integrated fashion to regulate important biological functions. While most studies on extranuclear steroid signaling have focused on estrogens, recent work has demonstrated that nongenomic androgen signaling is equally important and that these two steroids modulate similar signaling pathways. In fact, by taking advantage of a simple model system whereby a physiologically relevant androgen-mediated process is regulated completely independent of transcription (Xenopus laevis oocyte maturation), many novel and conserved concepts in nongenomic steroid signaling have been uncovered and characterized.

GPCR/EGFR Cross Talk Is Conserved in Gonadal and Adrenal Steroidogenesis but Is Uniquely Regulated by Matrix Metalloproteinases 2 and 9 in the Ovary

Previous work has demonstrated that cross talk between G protein-coupled LH receptors and epidermal growth factor receptors (EGFR) is essential for LH-induced steroid production in ovarian follicles and testicular Leydig cells. Here we demonstrate that G protein-coupled receptor (GPCR)/EGFR cross talk is also required for ACTH-induced steroidogenesis in Y1 adrenal cells. Moreover, we confirm that the signaling pathway from GPCR to Erk activation is conserved in all three steroidogenic tissues. ACTH or LH induces Gα(s), resulting in elevated cAMP and protein kinase A activation. cAMP/protein kinase A then triggers EGFR trans-activation, which promotes Erk signaling and subsequent steroidogenesis. Interestingly, although EGFR trans-activation is conserved in all three tissues, the specific mechanisms regulating this receptor cross talk differ. ACTH and LH trigger matrix metalloproteinase (MMP)-mediated release of EGFR ligands in adrenal and gonadal cells, respectively. However, this extracellular, ligand-dependent EGFR transactivation is required only for LH-induced steroidogenesis in ovarian follicles, reflecting the unique requirement of cell-cell cross talk for ovarian steroid production. Furthermore, MMP2 and MMP9 appear to regulate LH-induced steroidogenesis in mouse ovarian follicles, because a specific MMP2/9 inhibitor as well as the MMP2/9 inhibitor doxycycline suppress LH-induced follicular steroid production in vitro. Notably, although EGFR or MMP inhibition minimally affects estrous cycling in female mice, they attenuate ovarian steroidogenesis in response to LHR overstimulation in vivo. These results may have implications with regard to EGFR inhibitor use in various cancers as well as in polycystic ovarian syndrome, where excess LH-driven ovarian androgen production might be controlled by MMP2/9 inhibition.

Uterine-Specific Loss of Tsc2 Leads to Myometrial Tumors in Both the Uterus and Lungs

Lymphangioleiomyomatosis (LAM) is a rare disease characterized by proliferation of abnormal smooth-muscle cells in the lungs, leading to functional loss and sometimes lung transplantation. Although the origin of LAM cells is unknown, several features of LAM provide clues. First, LAM cells contain inactivating mutations in genes encoding Tsc1 or Tsc2, proteins that limit mTORC1 activity. Second, LAM tumors recur after lung transplantation, suggesting a metastatic pathogenesis. Third, LAM is found almost exclusively in women. Finally, LAM shares features with uterine leiomyomas, benign tumors of myometrial cells. From these observations, we proposed that LAM cells might originate from uterine leiomyomas containing Tsc mutations. To test our hypothesis, and to develop mouse models for leiomyoma and LAM, we targeted Tsc2 deletion primarily in uterine cells. In fact, nearly 100% of uteri from uterine-specific Tsc2 knockout mice developed myometrial proliferation and uterine leiomyomas by 12 and 24 weeks, respectively. Myometrial proliferation and mTORC1/S6 activity were abrogated by the mTORC1 inhibitor rapamycin or by elimination of sex steroid production through ovariectomy or aromatase inhibition. In ovariectomized Tsc2 null mice, mTORC1/S6 activity and myometrial growth were restored by estrogen but not progesterone. Thus, even without Tsc2, estrogen appears to be required for myometrial mTORC1/S6 signaling and proliferation. Finally, we found Tsc2 null myometrial tumors in lungs of older Tsc2 uterine-specific knockout females, suggesting that lung LAM-like myometrial lesions may indeed originate from the uterus. This mouse model may improve our understanding of LAM and leiomyomas and might lead to novel therapeutic strategies for both diseases.

Leptin-Induced CART (Cocaine- and Amphetamine- Regulated Transcript) Is a Novel Intraovarian Mediator of Obesity-Related Infertility in Females

Obesity is considered detrimental to womens reproductive health. Although most of the attention has been focused on the effects of obesity on hypothalamic function, studies suggest a multifactorial impact. In fact, obesity is associated with reduced fecundity even in women with regular cycles, indicating that there may be local ovarian effects modulating fertility. Here we describe a novel mechanism for leptin actions directly in the ovary that may account for some of the negative effects of obesity on ovarian function. We find that normal cycling, obese, hyperleptinemic mice fed with a high-fat diet are subfertile and ovulate fewer oocytes compared with animals fed with a normal diet. Importantly, we show that leptin induces expression of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in the granulosa cells (GCs) of ovarian follicles both in vitro and in vivo. CART then negatively affects intracellular cAMP levels, MAPK signaling, and aromatase mRNA expression, which leads to lower estradiol synthesis in GCs and altered ovarian folliculogenesis. Finally, in human samples from patients undergoing in vitro fertilization, we show a significant positive correlation between patient body mass index, CART mRNA expression in GCs, and CART peptide levels in follicular fluid. These observations suggest that, under obese conditions, CART acts as a local mediator of leptin in the ovary to cause ovarian dysfunction and reduced fertility.

Estrogen maintains myometrial tumors in a lymphangioleiomyomatosis model

Lymphangioleiomyomatosis (LAM) is a rare disease in women. Patients with LAM develop metastatic smooth-muscle cell adenomas within the lungs, resulting in reduced pulmonary function. LAM cells contain mutations in tuberous sclerosis genes (TSC1 or TSC2), leading to up-regulation of mTORC1 activity and elevated proliferation. The origin of LAM cells remains unknown; however, inactivation of Tsc2 gene in the mouse uterus resulted in myometrial tumors exhibiting LAM features, and approximately 50% of animals developed metastatic myometrial lung tumors. This suggests that LAM tumors might originate from the uterine myometrium, possibly explaining the overwhelming prevalence of LAM in female. Here, we demonstrate that mouse Tsc2-null myometrial tumors exhibit nearly all the features of LAM, including mTORC1/S6K activation, as well as expression of melanocytic markers and matrix metalloproteinases (MMPs). Estrogen ablation reduces S6K signaling and results in Tsc2-null myometrial tumor regression. Thus, even without TSC2, estradiol is required to maintain tumors and mTORC1/S6K signaling. Additionally, we find that MMP-2 and -9, as well as neutrophil elastase (NE), are overexpressed in Tsc2-null myometrial tumors in an estrogen-dependent fashion. In vivo fluorescent imaging using MMP- or NE-sensitive optical biomarkers confirms that protease activity is specific to myometrial tumors. Similar to LAM cells, uterine Tsc2-null myometrial cells also overexpress melanocytic markers in an estrogen-dependent fashion. Finally, we identify glycoprotein NMB (GPNMB) as a melanocytic marker up-regulated in Tsc2-null mouse uteri and human LAM samples. Our data highlight the potential importance of estradiol in LAM cells, suggesting that anti-estrogen therapy may be a treatment modality. Furthermore, proteases and GPNMB might be useful LAM biomarkers.

Minireview: Lymphangioleiomyomatosis (LAM): The “Other” Steroid-Sensitive Cancer

Lymphangioleiomyomatosis (LAM) is a devastating rare lung disease affecting primarily childbearing age women in which tumors consisting of abnormal smooth-muscle-like cells grow within the lungs and progressively lead to loss of pulmonary function. LAM cells metastasize to the lungs, predominantly through the lymphatics; however, the source of the LAM cell is still unknown. LAM cells contain inactivating mutations in genes encoding tuberous sclerosis 1 or 2, proteins that normally limit cell growth through suppression of mammalian target of rapamycin complex 1. As of today, sirolimus (an mammalian target of rapamycin complex 1 inhibitor) is the only treatment, available for LAM patients that is approved by the Food and Drug Administration; however, this drug and others in its class provide stabilization but not remission of LAM. One of the biggest problems in treating LAM is that both the origin of the LAM cells and the mechanism of the sexual dimorphism in LAM are still not understood. LAM cells express estrogen and progesterone receptors, and lung function declines during periods of high circulating estrogen levels. Moreover, numerous basic research studies find that estrogen is a key driving force in LAM cell proliferation, migration, and metastasis. In this review, we highlight recent insights regarding the role of steroid hormones in LAM and discuss possible explanations for the profound female sexual dimorphism of LAM.

Androgens Regulate Ovarian Gene Expression Through Modulation of Ezh2 Expression and Activity

&NA; A substantial amount of evidence suggests that androgen signaling through classical androgen receptors is critical for both normal and pathologic ovarian physiology. Specifically, we and others have shown that, in mouse granulosa cells, androgen actions through both extranuclear and nuclear androgen receptor signaling are critical for normal follicle development and ovulation. Here, we show that androgens through the PI3K/Akt pathway rapidly (within minutes) phosphorylate and inhibit activity of the Polycomb group protein enhancer of zeste homolog 2 (Ezh2). Over the course of 24 to 48 hours, androgens then induce expression of the microRNA miR‐101, which targets Ezh2 messenger RNA (mRNA), leading to a nearly complete loss of Ezh2 protein expression. This long‐term androgen‐induced loss of Ezh2 actions ultimately results in sustained reduction of the H3K27me3‐repressive mark in the promoter region of the Runt‐related transcription factor‐1 (Runx1) gene, a luteinizing hormone (LH)‐induced transcription factor essential for ovulation, leading to increased Runx1 mRNA expression. Accordingly, blocking androgen‐induced inhibition of Ezh2 in vivo adversely affects LH‐induced Runx1 mRNA expression and subsequent ovulation. Importantly, although estrogen treatment of granulosa cells similarly causes rapid activation of the PI3K/Akt pathway and short‐term phosphorylation of Ezh2, it does not induce miR‐101 expression and thereby does not reduce overall Ezh2 expression, demonstrating the androgen specificity of long‐term Ezh2 suppression. Thus, this study provides insight regarding how androgen‐induced extranuclear kinase signaling and intranuclear transcription through Ezh2 modifications may influence the expression pattern of genes, ultimately affecting various downstream physiological processes.