Decreasing the Relative Gene Expressions Associated With Apoptotic Factors in Granulosa Cells by Adding Estradiol

The objective of this study was to evaluate the apoptotic effects of increasing granulosa cell density and increasing estradiol on relative gene expression associated with apoptosis factors (Bcl-2, Bax and GADD45β) and cell cycle (CCDN-2). For this reason after collecting ovaries of cows from an abattoir, granulosa cells were separated from 4- to 8-mm follicles. Then, viable cells were counted and granulosa cell samples were cultured for 6 days. Treatments were included 1) 500,000 cell/1000 ml liquid culture (Control), 2) 500,000 cell/500 ml liquid culture (apoptotic group: increasing cell density cause apoptosis), 3) 500,000 cell/500 ml liquid culture and estradiol from day two of culture (apoptotic group+estradiol). Also, expression of genes associated with apoptosis factors and cell cycle, and estradiol of samples were evaluated. Results showed relative gene expression of Bax and GADD45β was greatest in apoptotic group compared to two other groups; but, with adding estradiol expression of these two genes decreased to control level. Bcl-2/Bax ratio decreased in apoptotic group compared with two other groups; while, adding estradiol significantly increased this ration, but, still it was lower than control group. Relative gene expression of CCDN-2 was lowest in apoptotic group compared to two other groups; but, no significant difference was observed between control and estradiol group. Overall results of the present study showed that estradiol can protect granulosa cells against apoptosis induced with increasing in cell density and probably it was done through progression of the cell cycle from G1 to S stage.