Changes in protein phosphorylation during the maturation of mammalian oocytes in vitro

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Abstract
Cumulus‐enclosed sheep oocytes were cultured in gonadotrophin‐containing medium for up to 9 hr and were then incubated for 3 hr in the presence of [32P]phosphate. The incorporation of 32P into TCA‐insoluble material was measured, and oocyte proteins were separated by one‐ and two‐ dimensional gel electrophoresis. Incorporation of [32P]phosphate into protein increased after 3 hr of culture and again after 9 hr, the time of germinal vesicle breakdown (GVBD). Qualitative and quantitative changes in the phosphorylation of proteins occurred over the 12‐hr period studied. One of the most prominent changes was the appearance of a band of Mr 33,000, which was absent at 0–3 hr but appeared with increasing intensity with longer periods of culture. Two‐dimensional electrophoresis revealed that the bulk of material in this band was a neutral polypeptide. No significant incorporation of [32P]phosphate was found in ribosomal extracts of oocytes.