Browsing by Subject "Oocyte maturation"
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Item Examination of the role of different classes of progesterone receptors in oocyte maturation in zebrafish, Danio rerio(2007-12) Harris, Caleb Michael, 1979-; Thomas, P. (Peter)The overall goal of this thesis was to investigate the involvement of three potential progestin receptors in the oocyte maturation of a single model species, zebrafish. Potential mediators of oocyte maturation in lower vertebrates include the nuclear intracellular progesterone receptor (nPR) and two membrane bound progestin receptors, a seven-transmembrane progestin receptor (mPR) and a single transmembrane progestin receptor (PGRMC1). Inhibitory G-protein involvement was inspected as well as ligand activation, relative binding of various ligands and expression patterns of membrane receptors. Intracellular inhibitory G (Gi)-protein signal pathways were shown to be necessary for the steroid induction of oocyte maturation by injecting competent oocytes with pertussis toxin. Various native and synthetic steroid ligands were investigated for their ability to promote germinal vesicle breakdown (GVBD). Classical nPR antagonists were potent inducers of GVBD. Ligand effectiveness of various steroids in promoting GVBD in vitro was correlated to the steroids' relative binding affinity for recombinant zebrafish mPR[Greek small letter alpha] The expression of PGRMC1 was investigated and shown not to be correlated with any oocyte developmental stage. Gonadotropin in vitro incubation resulted in a modest but insignificant increase in PGRMC1 mRNA expression levels. Furthermore, abrogating PGRMC1 expression with antisense morpholino oligonucleotide injections did not significantly impair gonadotropin induction of oocyte maturational competence. This research supports the involvement of the seven transmembrane mPR receptor in the oocyte maturation of zebrafish. Though the involvement of nPR and PGRMC1 is not supported, some trends in data require further investigation as to potential involvement of PGRMC1 in oocyte function.Item Involvement of epidermal growth factor receptor (EGFR) signaling in estrogen inhibition of oocyte maturation mediated through G protein-coupled estrogen receptor 1 (GPER) in zebrafish (Danio rerio)(2010-05) Peyton, Candace Ann; Thomas, P. (Peter); Holt, Joan; Erdner, DeanaOocyte maturation (OM) in teleosts is under precise hormonal control by estrogens and progestins. We show here that estrogens activate an epidermal growth factor receptor (EGFR) signaling pathway through the G protein-coupled estrogen receptor (GPER) to maintain meiotic arrest of full-grown zebrafish (Danio rerio) oocytes in an in vitro germinal vesicle breakdown (GVBD) bioassay. A GPER- specific agonist decreased OM and a GPER-specific antagonist increased spontaneous OM, whereas specific nuclear estrogen receptor (ERα and ERβ) agonists did not affect OM, which suggests the inhibitory action of estrogens on OM are solely mediated through GPER. Furthermore, a peptide-bound estrogen, which cannot enter the oocyte, decreased GVBD, showing that these estrogen actions are mediated through a membrane receptor. Treatment of oocytes with actinomycin D, a transcription inhibitor, did not block the inhibitory effects of estrogens on OM, indicating that estrogens act via a nongenomic mechanism to maintain oocyte meiotic arrest. EGFR mRNA was detected in denuded zebrafish oocytes by reverse transcription polymerase chain reaction (RT-PCR). Therefore, the potential role of transactivation of EGFR in estrogen inhibition of OM was investigated. The matrix metalloproteinase inhibitor, ilomastat, which prevents the release of heparin-bound epidermal growth factor (HB-EGF), increased spontaneous OM. Moreover, specific EGFR1 (ErbB1) inhibitors and inhibitors of extracellular-related kinase 1 and 2 (ERK1/2) increased spontaneous OM. Previously, estrogens have been shown to increase 3’-5’-cyclic adenosine mono phosphate (cAMP) levels through GPER in zebrafish oocytes during meiotic arrest. Taken together these present results suggest that estrogens also act through GPER to maintain meiotic arrest through a second signaling pathway involving transactivation of EGFR and activation of ERK 1 and 2.