Oocytes of hamsters and other mammals removed from follicles and matured an fertilized in vitro undergo meiotic maturation, activation and sperm penetration. However, unlike in vivo matured oocytes, decondenstion of sperm chromatin and formation of the male pronucleus commonly fails to occur. This and other impairments encountered during early embryonic development has led to the idea that preovulatory oocyte maturation is comprised of both meiotic (nuclear) maturation and what is loosely defined as cytoplasmic maturation. The objectives of this proposal are to determine the hormonal and cellular mechanisms regulating cytoplasmic maturation. To study this problem, a model using follicular oocytes of golden hamsters has been developed which links in vitro meiotic maturation to subsequent in vitro fertilization with a visual assay (presence or absence of male pronuclear formation) for cytoplasmic maturation. Preliminary experiments show this model exhibits high frequencies of meiotic maturation, sperm penetration and failure of pronuclear formation. Using this model, 9 specific experiments are designed to determine the role of: 1) gonadotropins in inducing cytoplasmic maturation, 2) cells of the cumulus oophorus in mediating gonadotropic induction of cytoplasmic maturation, 3) steroids in the induction of cytoplasmic maturation, and 4) mRNA and protein synthesis in cytoplasmic maturation. The steroids to be studied in mediating possible effects of LH or FSH on cytoplasmic maturation are progesterone, estradial-17Beta and testosterone. Their role will be studied by inhibition with aminoglutethimide phosphate and cyanoketone and by specific replacement. The involvement of mRNA synthesis in cytoplasmic maturation will be determined by blocking RNA synthesis with Alpha-amanatin and actinomycin D. The necessity for protein synthesis will be assessed by inhibiting synthesis with puromycin and cyclohexamide.
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