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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.