Analysis of apoptosis and expression of genes related to apoptosis in cultures of follicles derived from vitrified and non-vitrified ovaries

The aim of this study was to evaluate the incidence of apoptosis after in vitro culture of isolated follicles derived from vitrified and non-vitrified ovaries. Mouse ovaries were vitrified and their pre-antral follicles were mechanically isolated and cultured for 10 days. Growth and survival rates of the follicles were assessed during the culture period and the ultrastructure of the follicles was studied. The expression of p53, Bcl-2, Bax, Fas, FasL and survivin were analyzed by real-time RT-PCR in different follicular developmental stages. The percentages of apoptotic and necrotic cells were determined using a fluorescein-activated cell sorting (FACS) technique. There were no differences between the growth and survival rates of follicles in the vitrified and non-vitrified groups. All of the evaluated genes were expressed in the pre-antral, large pre-antral and antral follicles in both groups, except Fas mRNA, which was not expressed in the pre-antral follicles. The expression of p53, Bcl2, Bax and FasL mRNA was similar in vitrified and non-vitrified groups; however, Fas mRNAs were more strongly expressed in the antral follicles of the vitrified group than of the control group (P < 0.05). The expression of survivin 140 was lower in the antral follicles of the vitrified group than of the control group (P < 0.05). FACS analysis showed that the percentage of intact cells was lower in the vitrified group than in the non-vitrified group (P < 0.05). This study demonstrated no signs of apoptosis ultrastructurally in cultured follicles; however, vitrification was shown to affect the expression of some genes related to apoptosis.