Steroid-induced maturation of Xenopus oocytes has long served as a model for studying meiosis. Progesterone has been considered the relevant steroid controlling maturation, perhaps through interactions with classical progesterone receptors. In this study, we provide evidence that androgens, rather than progesterone, are the physiologic mediators of Xenopus oocyte maturation. Androgens were equal or more potent activators of maturation in vitro relative to progesterone and were significantly more abundant in the serum and ovaries of ␤-human chorionic growth hormone-stimulated frogs. Androgen action appeared to be mediated by classical androgen receptors (ARs) expressed in oocytes, as androgen-induced maturation and signaling was specifically attenuated by AR antagonists. Interestingly, we found that progesterone was rapidly converted to the androgen androstenedione in isolated oocytes by the enzyme CYP17, suggesting that androgens may be promoting maturation even under conditions typical for ''progesterone-mediated'' maturation assays. Androgens are thought to play an important role in ovarian development as well as pathology, and signaling through the AR may prove to be a major regulatory mechanism mediating these processes.T he phenomenon of progesterone-induced maturation of Xenopus oocytes has served as an in vitro experimental model for studying meiosis and cell cycle regulation for over 30 years (1-3), with recent work implicating the classical nuclear͞cytoplasmic progesterone receptor (PR) as the mediator of these processes (4, 5). Although progesterone is a potent promoter of Xenopus oocyte maturation in vitro, little is known about its role in mediating maturation in vivo. In other animals, progesterone does not appear to be the primary physiologic mediator of oocyte maturation. For example, oocyte maturation in fish is mediated by the progesterone metabolite 17␣, 20␤-dihydroxyprogesterone (6). In mice, ovaries from females lacking the PR gene still contain well-developed follicles with mature oocytes (7), suggesting that progesterone and its receptor are not necessary for oocyte maturation and that other factors are therefore important. Finally, mifepristone (RU486), a potent inhibitor of both mammalian and Xenopus PR-mediated transcription, does not block progesterone-mediated maturation (5), implying that, even in vitro, signaling via the PR may not be the only mechanism behind progesterone-induced maturation.In an effort to clarify the physiologic importance of progesterone in Xenopus oocyte maturation, we measured the serum and ovarian steroid content of female frogs injected with ␤-human chronic growth hormone (␤-hCG). We show that progesterone is barely detectable in the serum and ovaries of these animals but that androgen concentrations are Ͼ10 times higher than that of progesterone. Further, we demonstrate that oocytes are equally or more sensitive to signaling by androstenedione (AD) and testosterone than progesterone. We identify classical Xenopus androgen receptors (ARs) in oocytes and show t...

Progesterone-induced maturation of Xenopus oocytes is a well known example of nongenomic signaling by steroids; however, little is known about the early signaling events involved in this process. Previous work has suggested that G proteins and G protein-coupled receptors may be involved in progesterone-mediated oocyte maturation as well as in other nongenomic steroid-induced signaling events. To investigate the role of G proteins in nongenomic signaling by progesterone, the effects of modulating G␣ and G␤␥ levels in Xenopus oocytes on progesterone-induced signaling and maturation were examined. Our results demonstrate that G␤␥ subunits, rather than G␣, are the principal mediators of progesterone action in this system. We show that overexpression of G␤␥ inhibits both progesterone-induced maturation and activation of the MAPK pathway, whereas sequestration of endogenous G␤␥ subunits enhances progesterone-mediated signaling and maturation. These data are consistent with a model whereby endogenous free Xenopus G␤␥ subunits constitutively inhibit oocyte maturation. Progesterone may induce maturation by antagonizing this inhibition and therefore allowing cell cycle progression to occur. These studies offer new insight into the early signaling events mediated by progesterone and may be useful in characterizing and identifying the membrane progesterone receptor in oocytes.Steroid hormones are traditionally known to mediate their signaling and subsequent biological activities via nuclear receptors (1). Interestingly, many steroid-induced signaling events appear to be triggered independently from the classic nuclear receptor pathways. In fact, these processes likely involve steroid signaling via membrane receptors (2). Examples of rapid, nongenomic signaling by steroids are myriad, including aldosterone-induced increases in intracellular calcium in vascular smooth muscle cells (3-8), estrogen-mediated induction of nitric-oxide synthase in vascular endothelial cells (9 -12), vitamin D-induced increases in intracellular calcium in osteosarcoma cells (13)(14)(15), and progesterone-mediated maturation of amphibian and fish oocytes (16 -18).The phenomenon of progesterone-induced maturation of Xenopus oocytes serves as a useful experimental model for studying nongenomic steroid signaling (16, 19 -21). The maturation of an oocyte refers to the meiotic stage at which an oocyte rests. "Immature" oocytes are arrested in prophase of meiosis I. Before ovulation, oocytes are induced to re-enter the cell cycle, finally resting in metaphase II. These "mature" oocytes are then competent for ovulation and subsequent fertilization, after which the final stages of meiosis are completed (16).Evidence suggests that progesterone-induced maturation of Xenopus oocytes is mediated by cell surface rather than nuclear receptors. First, maturation is unaffected by the transcriptional inhibitor actinomycin D (21). Second, progesterone covalently attached to either polymers or bovine serum albumin and therefore unable to diffuse through the oocyte mem...

Steroids can induce both transcription-dependent (genomic) and independent (nongenomic) signaling. Here, several classical androgen receptor ligands were tested for their ability to modulate genomic and nongenomic responses, focusing on the role of the oocyte-expressed Xenopus classical androgen receptor (XeAR) in mediating these processes. Cellular fractionation and immunohistochemistry revealed that the XeAR was located throughout oocytes, including within the plasma membrane. RNA interference and oocyte maturation studies suggested that androgen-induced maturation was mediated in part by the XeAR in a transcription-independent fashion, perhaps by altering G protein-mediated signaling. While inducing minimal transcription in oocytes, all AR ligands promoted significant XeAR-mediated transcription in CV1 cells. In contrast, only testosterone and androstenedione potently induced oocyte maturation, whereas dihydrotestosterone and R1881 actually inhibited testosterone and human chorionic gonadotropin-induced maturation and signaling. These results suggest that the nature of a steroid-induced signal (genomic vs. nongenomic) may depend on the type of target cell, the receptor location within cells, as well as the ligand itself. The identification of molecules capable of selectively altering genomic vs. nongenomic signaling may be useful in delineating the roles of these pathways in mediating androgen responses and might lead to the development of novel compounds that specifically modulate these signals in vivo.

Classical steroid receptors mediate many transcription-independent (nongenomic) steroid responses in vitro, including activation of Src and G proteins. Estrogen-triggered activation of Src can be regulated by the modulator of nongenomic actions of the estrogen receptor (MNAR), which binds to estrogen receptors and Src to create a signaling complex. In contrast, the mechanisms regulating steroid-induced G protein activation are not known, nor are the physiologic responses mediated by MNAR. These studies demonstrate that MNAR regulates the biologically relevant process of meiosis in Xenopus laevis oocytes. MNAR was located throughout oocytes, and reduction of its expression by RNA interference markedly enhanced testosterone-triggered maturation and activation of MAPK. Additionally, Xenopus MNAR augmented androgen receptor (AR)-mediated transcription in CV1 cells through activation of Src. MNAR and AR coimmunoprecipitated as a complex involving the LXXLL-rich segment of MNAR and the ligand binding domain of AR. MNAR and Gbeta also precipitated together, with the same region of MNAR being important for this interaction. Finally, reduction of MNAR expression decreased Gbetagamma-mediated signaling in oocytes. MNAR therefore appears to participate in maintaining meiotic arrest, perhaps by directly enhancing Gbetagamma-mediated inhibition of meiosis. Androgen binding to AR might then release this inhibition, allowing maturation to occur. Thus, MNAR may augment multiple nongenomic signals, depending upon the context and cell type in which it is expressed.

No abstract