Aritro Sen

Granulosa Cell-Specific Androgen Receptors Are Critical Regulators of Ovarian Development and Function

The physiological significance of androgens in female reproduction was unclear until female mice with global knockout of androgen receptor (AR) expression were found to have reduced fertility with abnormal ovarian function. However, because ARs are expressed in a myriad of reproductive tissues, including the hypothalamus, pituitary, and various ovarian cells, the role of tissue-specific ARs in regulating female fertility remained unknown. To examine the importance of ovarian ARs in female reproduction, we generated granulosa cell (GC)- and oocyte-specific AR-knockout (ARKO) mice by crossing AR-flox mice with MisRIIcre (GC-specific) or growth differentiation factor growth differentiation factor-9cre (oocyte-specific) mice. Relative to heterozygous and wild-type mice, GC-specific ARKO mice had premature ovarian failure and were subfertile, with longer estrous cycles and fewer ovulated oocytes. In addition, ovaries from GC-specific knockout mice contained more preantral and atretic follicles, with fewer antral follicles and corpus lutea. Finally, in vitro growth of follicles from GC-specific AR-null mice was slower than follicles from wild-type animals. In contrast to GC-specific AR-null mice, fertility, estrous cycles, and ovarian morphology of oocyte-specific ARKO mice were normal, although androgens no longer promoted oocyte maturation in these animals. Together, our data indicate that nearly all reproductive phenotypes observed in global ARKO mice can be explained by the lack of AR expression in GCs. These GC-specific ARs appear to promote preantral follicle growth and prevent follicular atresia; thus they are essential for normal follicular development and fertility.

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.

Paxillin regulates androgen- and epidermal growth factor- induced MAPK signaling and cell proliferation in prostate cancer cells

Although transcriptional effects of androgens have been extensively studied, mechanisms regulating transcription-independent (nongenomic) androgen actions are poorly understood. Previously, we have shown that paxillin, a multidomain adaptor protein, is a critical regulator of testosterone-induced MAPK-signaling during Xenopus oocyte maturation. Here we examine the nongenomic effects of dihydrotestosterone (DHT) in prostate cancer cells, focusing on how paxillin mediates Erk signaling and downstream physiologic actions. We show that in LnCAP cells DHT functions as a growth factor that indirectly activates the EGF-receptor (EGFR) via androgen receptor binding and matrix metalloproteinase-mediated release of EGFR ligands. Interestingly, siRNA-mediated knockdown of paxillin expression in androgen-dependent LnCAP cells as well as in androgen-independent PC3 cells abrogates DHT- and/or EGF-induced Erk signaling. Furthermore, EGFR-induced Erk activation requires Src-mediated phosphorylation of paxillin on tyrosines 31/118. In contrast, paxillin is not required for PKC-induced Erk signaling. However, Erk-mediated phosphorylation of paxillin on serines 83/126/130 is still needed for both EGFR and PKC-mediated cellular proliferation. Thus, paxillin serves as a specific upstream regulator of Erk in response to receptor-tyrosine kinase signaling but as a general regulator of downstream Erk actions regardless of agonist. Importantly, Erk-mediated serine phosphorylation of paxillin is also required for DHT-induced prostate-specific antigen mRNA expression in LnCAP cells as well as EGF-induced cyclin D1 mRNA expression in PC3 cells, suggesting that paxillin may regulate prostate cancer proliferation by serving as a liaison between extra-nuclear kinase signaling and intra-nuclear transcriptional signals. Thus, paxillin may prove to be a novel diagnostic or therapeutic target in prostate cancer.

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.

Paxillin mediates extranuclear and intranuclear signaling in prostate cancer proliferation

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.

Endocrine autoimmune diseases and female infertility

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.

JY-1, an oocyte-specific gene, regulates granulosa cell function and early embryonic development in cattle

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.

Developmental Sensitivity of the Bovine Corpus Luteum to Prostaglandin F2α (PGF2α) and Endothelin-1 (ET-1): Is ET-1 a Mediator of the Luteolytic Actions of PGF2α or a Tonic Inhibitor of Progesterone Secretion?

Abstract We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2α and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2α and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2α increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2α and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2α steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2α action. The lower efficacy of PGF2α in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2α to up-regulate ET-1.

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.

Evidence Supporting a Role for Cocaine- and Amphetamine-Regulated Transcript (CARTPT) in Control of Granulosa Cell Estradiol Production Associated with Dominant Follicle Selection in Cattle

We demonstrated previously a negative association of granulosa cell cocaine- and amphetamine-regulated transcript (CARTPT) expression with follicle health status and inhibitory effects of the mature CARTPT peptide (CART) on follicle-stimulating hormone (FSH) signal transduction in vitro, resulting in reduced bovine granulosa cell CYP19A1 mRNA and estradiol production. The objectives of this study were to investigate temporal regulation of granulosa cell CARTPT expression (granulosa cell mRNA and follicular fluid CART peptide concentrations) during follicular waves, CART regulation of androstenedione production (precursor for estradiol biosynthesis) by thecal tissue collected at specific stages of a follicular wave, FSH regulation of granulosa cell CARTPT mRNA expression, and the ability of CART to inhibit granulosa cell estradiol production and CYP19A1 mRNA expression when administered in vivo. CART concentrations in healthy, estrogen-active follicles (estradiol greater than progesterone in follicular fluid) decreased after dominant follicle selection, and CARTPT mRNA was lower in healthy, estrogen-active versus estrogen-inactive atretic follicles (progesterone greater than estradiol) collected at the predeviation and early dominance stages. CART treatment reduced luteinizing hormone-induced androstenedione production by thecal tissue collected at predeviation and early dominance stages but not at later stages of a follicular wave. The FSH or insulin-like growth factor 1 treatment in vitro reduced granulosa cell CARTPT mRNA in a dose-dependent fashion. Administration of CART in vivo into follicles at the early dominance stage reduced follicular fluid estradiol concentrations and granulosa cell CYP19A1 mRNA. Collectively, results support a potential stage-specific regulatory role for CART in negative regulation of estradiol production associated with selection of the dominant follicle.