Effect of Retinol in the Vitrification Medium on Viability of Vitrified Ovine Preantral Follicles and Expression of Key Developmental and Apoptosis Related Genes.

BACKGROUND:Vitrification increases the production of reactive oxygen species (ROS) and the antioxidants in the vitrification solution may be beneficial by reducing excessive ROS production.OBJECTIVE:To evaluate the effect of retinol supplementation in vitrification solution on viability, apoptosis and development-related gene expression in vitrified sheep preantral follicles.MATERIALS AND METHODS:Preantral follicles were isolated and randomly assigned into one of five groups: Group1, control fresh preantral follicles; Group 2, vitrification treatment; Group 3, vitrification + 2 μM retinol; Group 4, vitrification + 5 μM retinol; Group 5, vitrification + 10 μM retinol. Preantral follicles were placed in vitrification solutions and then plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue exclusion method and for gene expression.RESULTS:Vitrification with 5 μM retinol positively affected viability in comparison with vitrification without retinol (P < 0.05). There was no significant difference in viability among the Group 1, Group 2, Group 3 and Group 5. Expression of apoptotic genes BAX and Casp 3 were higher in the vitrified group, and vitrification with 5 μM retinol (Group 4) is comparable to the control fresh. Expressions of other apoptosis-related genes (i.e., BCL2L1, BAD and BAK) showed significant difference between the control fresh group and the vitrification group with 5 μM retinol. Expression of Annexin5 was also significantly different among various groups. The expression of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in the Group vitrified with 5 μM retinol.CONCLUSION:The supplementation of 5 μM retinol in vitrification solution was beneficial for the vitrification of ovine preantral follicles.