Cilostamide and forskolin maintain gap junction function of incubated dog follicles

Disruption of the communication between the oocyte and granulosa cells is one of the major causes of poor development of in vitro grown ovarian follicles and oocytes. The present study investigated the effect of two cAMP modulators, cilostamide and forskolin, on in vitro growth of isolated dog secondary follicles and enclosed oocytes, communication between the gamete and surrounding granulosa cells, expression of GJA1 and GDF9, as well as cAMP level. Secondary follicles were incubated with cilostamide or forskolin alone or a combination of 20 μM cilostamide +1 μM forskolin, and the diameter of the incubated follicles and enclosed oocytes assessed every 72 h. Gap junction activity, GJA1 and GDF9 expression and cAMP level were assessed on Days 6 and 12 and transzonal projection (TZP) density was evaluated on Day 12. Neither cilostamide nor forskolin alone enhanced in vitro growth of dog follicles and the enclosed oocytes (P > 0.05). However, these two cAMP modulators dose dependently sustained gap junction activity and stimulated cAMP production compared with the non-supplemented control. Cilostamide at the high dosage (20 μM) also upregulated GJA1 expression. The combination of cilostamide and forskolin supported oocyte growth during the first 9 days and upregulated GJA1 and GDF9 expression at Day 12 of in vitro culture. This combination treatment also sustained gap junction activity, cAMP production, and increased TZP function (calcein intensity: TZP density ratio). The findings indicated that a combination of cilostamide and forskolin supported growth and survival of oocytes enclosed within cultured follicles by sustaining cAMP production and gap junction activity.