Isolation, identification and characterization of secretory proteins of IVMFC embryos and blood circulation of estrus and early pregnant goat

Abstract

The aim of the present study was to isolate, identify and characterize the secretory proteins of IVM oocytes and IVMFC embryos to evaluate its immunogenecity and identify of such proteins if any, in blood circulation of estrus and early pregnant goats. Oocytes were matured in TCM-199 with 1μg/ml, estradiol -17β; 0.5 μg/ml, FSH: 100 IU/ml , LH and 10% FCS on granulosa cell monolayer. After 18 hr of maturation, oocytes were further cultured in maturation medium containing 3 mg/ml polyvinyl alcohol (PV A) without serum and BSA for 12 hr and medium was collected. The IVF embryos of 4-8 cell stage were cultured in medium containing PVA without serum and BSA. Embryo culture medium was collected after 24 hr of culture and was pooled. The proteins were analyzed on SDS-PAGE (12.5%). Four secretory proteins of oocytes with approximately molecular weight of 45, 55, 65 and 95 kDa and three secretory proteins of embryos 45, 55 and 65 kDa were obtained on SDS-PAGE in silver staining. The protein profile of midluteal, estrus and early pregnant goat serum was similar and no variation was observed among the proteins on SDS-PAGE. Two secretory proteins of 55 and 65 kDa of both IVM oocytes and IVMFC embryos were observed on Western analysis. None of such proteins was observed in midluteal, estrus and early pregnant goat serum on western blotting. It can be concluded that IVM oocytes and IVMFC embryos secrete proteins in medium and two of them can develop antibody.The proteins secreted from embryos till morula stage was similar to that of oocytes. None of these oocyte/embryo released proteins were observed in blood circulation of estrus and early pregnant goats.