Determination of 70 kDa Proteins for Different Temperatures in Cattle Oocytes Matured in vitro

Abstract

This study was conducted to determine the effect of 36.5 (low) or 38.5 °C (conventional) culture temperatures during in vitro maturation (IVM) on 70 kDa proteins in cattle oocytes. In the study, cattle cumulus oocyte complexes (COC) were subjected to IVM in bicarbonate-buffered TCM–199 supplemented with 10% FCS for 22 hours with a humidified 5% CO2 in air at either 36.5 or 38.5 °C culture temperatures. Maturation of COCs was determined according to fully cumulus expansion at the end of IVM. The cumulus cells of matured COCs were removed by vortexing in TCM-199 with HEPES buffered TCM-199 containing 0.1% hyaluronidase enzyme. Following, denuded oocytes were washed in 9.6 % phosphate buffered saline (PBS) solution for protein isolation. Denuded oocytes (n = 100 for both groups) were centrifuged for 5 min at 300 rpm at 4 °C for PBS removed than the lysis buffer (100 μl) was added and shake 15 min on ice for 30 minutes. For protein isolation, the protein was centrifuged again at 13,000 rpm for 5 minutes and the protein samples were stored at -20 ° C. To increase the concentration of isolated protein samples, the samples were frozen at -80 °C for 24 hours and lyophilized at 0.140 hPa in a lyophilizer. Lyophilized protein samples were stored at -20 °C until SDS-PAGE analysis. The level of 70 kDa proteins in oocytes were determined by SDS-PAGE method. There were no significant differences between low (81.11%) or conventional (84.41%) incubation temperatures in IVM in terms of full cumulus expansion. The 70 kDa proteins band size in SDS-PAGE polyacrylamide gel image of cattle oocytes maturated at low culture temperature was weak compared to cattle oocytes maturated at conventional culture temperature. The results of this study may show that low incubation temperature during IVM decreases the amount of 70 kDa proteins in cattle oocytes.