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

Year 2019, , 112 - 116, 01.10.2019

Leyla Bener , Mehmet Kuran

https://doi.org/10.34248/bsengineering.587008

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.

Keywords

Oocyte, Maturation, Culture Temperature, 70 kDa Proteins, Cattle

References

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