This is a new proposal aimed at studying the changes which occur in the zonae pellucidae of mammalian oocytes as follicles develop. The zonae pellucidae is the extracellular glycoprotein matrix formed during oocyte maturation and follicular development. It later plays a fundamental role in the fertilization process as well as aids in the protection of the early embryo. The rabbit and pig zonae have proven to be excellent models for study of proteins which constitute the zonae since tens of thousands of zonae can now be rapidly isolated using methods developed by the Principal Investigator. Using immunochemical and automated two-dimensional gel electrophoresis methods to analyze zonae proteins, it is now possible to clarify the origin of specific zonae proteins as well as to study their synthesis and secretion in more detail. The overall objectives of this proposal are to: (1) Evaluate changes in rabbit zona pellucide proteins as ovarian follicles develop and relate these to hormonal profiles during ovarian development. a. Examine the appearance and changes in cellular localization of specific zona proteins during follicular development by immunocytochemistry using antisera to specific zona components. b. Use radioimmunioassay methods to quantitate changes in levels of zona proteins during development along with changes in calmodulin and tubulin. c. Evaluate levels of blood hormones including estrogen, progesterone, leutinizing hormone and follicle stimulating hormone. (2) Utilize immature rabbits to determine the regulation of zona protein accumulation in vivo. (3) Use female rabbits which have been actively immunized with zonae proteins as a model to study oocyte maturation and zona protein synthesis and secretion in vivo. (4) Utilize organ and cell culture methods to determine the cellular origin of the major rabbit zonae proteins and to study the regulation of their synthesis and secretion.
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