The long term objective of this research program is to identify specific molecules and pathways that govern mammalian oocyte development, and resolve their mechanism of action. There is strong evidence that elevated oocyte cyclic adenosine monophosphate (cAMP) levels are physiologically important for maintaining oocyte meiotic arrest.
The aims of this proposal focus on specific molecules that could regulate levels of cAMP and the responses to cAMP in mouse oocytes. Hypoxanthine is the principal low molecular weight substance found in porcine and murine ovarian follicular fluid that maintains meiotic arrest in murine oocytes. Accordingly, experiments in the first part of this proposal are designed to test the following hypotheses. (1) Granulosa cells produce hypoxanthine and other purines that could participate in the maintenance of meiotic arrest. (2) Hypoxanthine, or molecules derived from it, may function to maintain meiotic arrest by inhibiting oocyte phosphodiesterase. (3) Hypoxanthine, or its derivatives, is transferred from somatic cells directly to the oocyte. (4) Elevating cAMP levels in granulosa cells induces oocyte maturation in cultured oocyte-granulosa cell complexes even though the hypoxanthine concentration of the surrounding medium remains unchanged. (5) Oocytes that are first maintained in meiotic arrest with hypoxanthine in vitro, and then allowed to mature spontaneously after withdrawal of hypoxanthine, can be fertilized and undergo development to live young. Oocytes from strain I/LnJ mice express a very marked retardation of oocyte maturation kinetics, a trait that will be exploited in the second part of this proposal to identify specific molecules or pathways that regulate maturation. Strain I/LnJ mice are known to have a deficiency of muscle phosphorylase kinase, an enzyme that induces maturation of frog oocytes. Therefore, experiments are proposed to determine whether retarded maturation kinetics is the result of a deficiency in oocyte phosphorylase kinase or some other lesion that may, or may not, be related to the phosphorylase kinase deficiency. For example, it is proposed to assess whether retarded maturation kinetics is caused by a phosphodiesterase deficiency or other lesions that could prolong cAMP-dependent maintenance of meiotic arrest.
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