Effect of activin A and insulin-like growth factor-I on in vitro development of preantral follicles isolated from cryopreserved ovarian tissues in the mouse.

摘要

Cryopreservation of ovarian tissue has been reported to delay the development of preantral follicles by temporary suppression of granulosa cell proliferation during in vitro culture. This delay might be overcome by treatment with activin A and/or IGF-I, known to stimulate granulosa cell proliferation. However, the effects of these growth factors, on delayed follicle development induced by ovarian tissue cryopreservation, have not been evaluated. Therefore, we studied the effects of activin A and/or IGF-I on granulosa cell proliferation and follicle development in preantral follicles isolated from mouse cryopreserved ovarian tissues. The preantral follicles isolated from fresh ovarian tissues were cultured with control medium (CM) for 10 days. The preantral follicles isolated from cryopreserved ovarian tissues were cultured with CM and with CM+activin A (100 ng/ml), IGF-I (50 ng/ml) or activin A+IGF-I added for 10, 12 and 14 days. The follicles were stimulated with hCG at the end of culture. The granulosa cell proliferation was evaluated by measuring the PCNA expression and the follicle development assessed by comparing the follicle diameter and oocyte maturation. The expressed level of PCNA was significantly decreased in the cryopreserved preantral follicles cultured with CM, compared to the fresh group (p<0.05), but increased to the level of the fresh group by the addition of activin A, IGF-I or activin A and IGF-I. The maximum follicle diameter and oocyte maturation rate were obtained in the fresh group after 10 days of culture, while the diameter and oocyte maturation rate of cryopreserved preantral follicles reached similar levels after 14 days. Under conditions of CM with added activin A or activin A+IGF-I, both the diameter and oocyte maturation rate of the cryopreserved preantral follicles improved to the levels of the fresh group after 12 days. However, the stimulatory effect was not different in comparisons between activin A and activin A+IGF-I. In addition, adding activin A significantly increased the survival rate of cryopreserved preantral follicles compared to IGF-I (p<0.05). On the other hand, the diameter of the cryopreserved preantral follicles did not increase to the level of the fresh group by addition of IGF-I, although the oocyte maturation rate was improved to the level of fresh group after 12 days of culture. These findings indicate that activin A has a greater stimulatory effect on the growth of preantral follicles than does IGF-I. In conclusion, the addition of activin A to culture media stimulated granulosa cell proliferation to overcome the delay in culture of the development of preantral follicles isolated from cryopreserved mouse ovaries.