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The present study was undertaken to investigate the influence of epidermal growth factor (EGF) and follicle-stimulating hormone (FSH) during in vitro maturation on cytoplasmic maturation of porcine oocytes as revealed by the success of fertilization and by the changes in the pattern of protein synthesis in oocytes and cumulus cells. For fertilization studies, oocyte-cumulus cell complexes (OCC) were cultured in media containing human recombinant EGF (1 ng/ml) or FSH (1.5 microg/ml) or both for 44 hr prior to fertilization with fresh sperm for 6-8 hr. The oocytes were then fixed, stained, and examined as whole mounts following an additional 14 hr of culture. Addition of EGF, FSH, and EGF + FSH significantly increased the proportion of oocytes reaching MII stage. The addition of EGF alone significantly decreased the percentage of polyspermic oocytes and increased the proportion of monospermic oocytes forming 2 normal pronuclei. FSH abolished these effects of EGF and significantly increased the percentage of polyspermic oocytes forming more than 2 pronuclei when added alone or with EGF. For protein analysis, OCC were cultured in media containing the above hormones for 6, 24, and 44 hr and exposed to 0.5 mCi/ml L-[35S]methionine during the last 3 hr of cultures. The oocytes and cumulus cells were separated prior to lysis in SDS sample buffer, and denatured polypeptides were separated by 1-dimensional SDS-PAGE. In the oocyte, addition of EGF and FSH alone stimulated the synthesis of 34, 45, and 97 kDa proteins after 6 hr of culture; however, the addition of EGF and FSH together was without any effect. After 24 hr, EGF alone inhibited the synthesis of these peptides, whereas FSH alone and with EGF maintained the stimulation of synthesis of 34 and 45 kDa proteins. Two additional peptides corresponding to 66 and 200 kDa appeared at this time as a result of exposure to FSH alone or with EGF. After 44 hr of culture, these 2 new peptides were observed in all groups and the stimulatory effect of FSH and FSH + EGF was still evident. An additional peptide of 26 kDa appeared at this time as a result of FSH and EGF + FSH treatments. In the cumulus cells, EGF and FSH each alone induced the synthesis of a new peptide of 26 kDa after 6 hr of culture. FSH when added alone or with EGF induced the synthesis of an additional peptide of 29 kDa, the synthesis of which remained unchanged at 24 and 44 hr. After 24 hr, FSH alone and in combination with EGF induced the synthesis of an additional 38 kDa peptide and its synthesis was still maintained at 44 hr. EGF alone had no effect on protein synthesis in cumulus cells at 24 and 44 hr. These studies indicate that EGF may have a physiological role in the regulation of cytoplasmic maturation of porcine oocytes. |
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