Browsing by Subject "Oocytes"
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Item Control of Oocyte Reawakening by Kit(2016-08-01) Saatcioglu, Hatice Duygu; Amatruda, James F.; Castrillon, Diego H.; Hamra, F. Kent; Brekken, Rolf A.In mammals, oocyte reawakening is a fundamental biological process controlling follicle maturation, female fertility, the onset of the menopause, and thus, overall aging. We demonstrate through complementary genetic experiments that Kit is the upstream receptor regulating reawakening within oocytes. Although many factors have been proposed as candidates, the data have remained contradictory, and definitive genetic evidence in support of any factor has been lacking. We engineered two novel Kit alleles in mice, one a dominant gain-of-function point mutation active in the germline, the other a floxed allele for conditional inactivation. These alleles permitted us to conduct elegant genetic experiments whereby Kit was activated or inactivated in primordial oocytes. The results were complementary and striking. Oocyte-specific Kit activation resulted in a syndrome of female sterility due to global and premature reawakening leading to ovarian failure. In contrast, Kit inactivation also led to female sterility, albeit via a contrasting and opposite phenotype: a complete failure of primordial follicles to reawaken. Additional studies demonstrated that Foxo3 was the mediator of both phenotypes, linking our findings to prior discoveries. These complementary genetic experiments thus definitively incriminate Kit as the upstream receptor regulating reawakening. We believe our study will be of interest to general scientific and lay audiences as well as geneticists, given the importance of oocyte maintenance in the menopause, overall female aging, and reproduction.Item EGFR Signaling Is Necessary for Gonadotropin-Induced Steroidogenesis and Oocyte Maturation(2006-12-19) Jamnongjit, Michelle Thuc-Quyen; Hammes, Stephen R.The mid-cycle luteinizing hormone (LH) surge triggers ovarian processes that are critical for normal female fertility, including steroidogenesis, oocyte maturation, and subsequent ovulation. Studies were designed to examine the LH-induced signaling pathways that regulate ovarian steroidogenesis, as well as the subsequent role of steroids and their receptors on oocyte and follicle development. Initial work focused on transcriptionindependent, or nongenomic, steroid-induced maturation of oocytes. We demonstrated for the first time that steroids trigger oocyte maturation in several different mouse models, activating G protein signaling, as well as MAPK and CDK1/cdc2 intracellular signaling cascades. Pharmacologic studies using receptor antagonists as well as transgenic mouse models suggested that steroid-induced maturation is mediated through classical steroid receptors. Subsequent studies focused on LH-induced signals that regulate steroidogenesis in the mouse ovary. We found that EGF triggered steroid production in three separate models of follicle culture and surprisingly, EGF receptor signaling was absolutely necessary for LHinduced steroidogenesis in an intact follicle. In addition, studies performed in testicular Leydig cells and adrenal cells revealed that EGF receptor signaling is required for normal steroid production, making this a universal signaling pathway in steroid physiology. In summary, our data suggest a model whereby stimulation of LH receptors in theca and mural granulosa cells activates matrix metalloproteinases (MMP) to cleave membrane-bound EGF moieties. The soluble EGF molecules then stimulate EGFRs on granulosa cells (and possibly theca cells), leading to activation of StAR and increased steroidogenesis. The steroids in turn act upon the oocyte to promote maturation, allowing for ovulation to occur. Notably, MMPs appear to be important only for ovarian steroid production, offering us a novel target for specifically regulating steroidogenesis in the ovary. Accordingly, in cycling and superovulated mice, we were able to show that treatment with the broad-spectrum MMP inhibitor Galardin in vivo, decreased ovarian steroid production without affecting adrenal steroidogenesis. Given these findings we propose that MMP inhibitors may be useful to specifically down-regulate excess ovarian steroid production in diseases such as polycystic ovarian syndrome (PCOS), the leading cause of infertility in women of reproductive age.Item G Protein and Androgen Signaling in Ovarian Function(2009-09-04) Deng, James Yang; Hammes, Stephen R.Oocytes are held in meiotic arrest in prophase I until ovulation when gonadotropins trigger a subpopulation of oocytes to resume meiosis in a process termed "maturation." Meiotic arrest is maintained through a mechanism whereby constitutive cAMP production exceeds phosphodiesterase-mediated degradation, leading to elevated intracellular cAMP. Studies have implicated a constitutively activated Gas-coupled receptor, G protein-coupled receptor 3 (GPR3), as one of the molecules responsible for maintaining meiotic arrest in mouse oocytes. Here, we characterized the signaling and functional properties of GPR3 by using the more amenable model system of Xenopus laevis oocytes. We cloned the Xenopus laevis isoform of GPR3 (XGPR3) from oocytes and showed that overexpressed XGPR3 elevated intra-oocyte cAMP, in large part due to Gbg signaling. Overexpressed XGPR3 suppressed steroid-triggered kinase activation and maturation of isolated oocytes, as well as gonadotropin-induced maturation of follicle-enclosed oocytes. In contrast, depletion of XGPR3 using antisense oligodeoxynucleotides enhanced steroid- and gonadotropin-mediated oocyte maturation. Interestingly, collagenase treatment of Xenopus oocytes cleaved and inactivated cell surface XGPR3, which enhanced steroid-triggered oocyte maturation and activation of MAPK. In addition, hCG treatment of follicle-enclosed oocytes triggered matrix metalloproteinase-mediated cleavage of XGPR3 at the oocyte cell surface. Collectively, these results suggest that GPR3 moderates the oocyte response to maturation-promoting signals, and that gonadotropin-mediated activation of metalloproteinases may sensitize oocytes for maturation by inactivating constitutive GPR3 signaling.Item Gonadotropin-induced Steroidogenesis and Downstream Signals Leading to Oocyte Maturation(2009-01-09) Evaul, Kristen Elizabeth; Hammes, Stephen R.The Hammes laboratory is interested in understanding the process of steroid-mediated oocyte maturation. This includes examining both steroid production and steroid signaling. In these studies, gonadotropin-induced steroid production was examined in the gonads using mouse models, as well as steroid-induced oocyte maturation in frog models. cAMP signaling is known to be important for steroid production, but further downstream pathways were not well characterized. These studies illuminate other downstream signaling pathways triggered by luteinizing hormone (LH) that regulate steroid production in the testes using Leydig cells, which are the primary steroidogenic cells in the testes. A novel downstream pathway was found involving epidermal growth factor receptor (EGFR) transactivation, downstream mitogen-activated protein kinase (MAPK) and steroidogenic acute regulatory protein (StAR) activation that was essential for short, but not long-term LH-induced steroidogenesis in MLTC-1 and primary mouse leydig cells. Despite this discrepancy in vitro, EGFR signaling was required in vivo for testicular testosterone production. To study the effects of steroids on oocyte maturation, the Xenopus laevis frog model was used. It has been shown that G-beta gamma, as well as other signals, keep the oocyte in meiotic arrest. Steroids block this constitutive signal, leading to oocyte maturation. To directly measure rapid changes in G-beta gamma signaling in oocytes, G-beta gamma sensitive-inward rectifying potassium channel currents (GIRKS) were exogenously expressed in Xenopus oocytes. Adding testosterone, the physiologic mediator of oocyte maturation in Xenopus, decreased the G-beta gamma mediated signal. This happened rapidly supporting the well known idea that maturation is a transcription-independent process. It was also seen that the classical androgen receptor (AR) was being used for this process. When the AR was knocked down, testosterone could only decrease GIRK signal at higher concentrations. This showed that testosterone is working, at least partially, through the AR. These studies may help elucidate novel targets for polycystic ovary syndrome (PCOS), which is characterized by excess androgen due to improper steroid production.Item Paxillin is a Novel Regulator of Xenopus Oocyte Maturation(2010-05-14) Young, Melissa Rasar; Hammes, Stephen R.Oocyte maturation is triggered by steroids in a transcription-independent fashion that involves an unusual positive feedback loop whereby MOS (a germ cell specific Raf) activates MEK1, which in turn activates ERK2. ERK2 then acts back on MOS to enhance its expression resulting in amplification of the kinase signaling cascade. To date, little is known regarding other factors that regulate this powerful feedback kinase cascade. Here we present the scaffolding molecule, Paxillin, as a newly recognized essential regulator of meiosis in Xenopus leavis oocytes. Reduction of Paxillin expression using RNA interference and antisense oligonucleotides completely abrogates steroid-triggered meiotic resumption. Detailed signaling studies reveal that Paxillin is acting early in the kinase cascade, as it is required for accumulation of MOS protein and complete activation of downstream kinase signaling in response to steroids. Surprisingly, full Paxillin activity also requires serine phosphorylation by a kinase downstream of MOS and MEK1, possibly ERK2. Together, these data suggest that Paxillin is an important regulator of the positive feedback effects of MEK/ERK signaling on MOS protein expression. The ability of Paxillin to function as a MAPK scaffold was analyzed, revealing Paxillin can interact with MOS in mammalian cells. Furthermore, the ability of Paxillin to regulate activity of proteins important for translation, specifically polyadenylation binding proteins, is briefly explored. In all, these experiments reveal a novel and critical function for Paxillin in meiosis, and support the notion that Paxillin may be general modulator of MAPK signaling and/or mRNA translation by polyadenylation binding proteins.