Cilostamide and follicular hemisections inhibit oocyte meiosis resumption and regulate gene expression and cAMP levels in bovine cumulus-oocyte complexes

Abstract

This study investigated (1) the effects of cilostamide and follicular hemisections on in vitro oocyte meiotic resumption during a pre-maturation period of 12h, (2) the levels of cAMP in bovine cumulus oocyte complexes (COCs) after pre-maturation, and (3) the expression of mRNA for CONNEXIN 43(CX43), HAS2, MATER, NPM2 and ZAR1 in denuded oocytes and cumulus cells from COCs that were subjected to both pre-maturation and maturation in vitro. To evaluate the effects of follicular hemisections and cilostamide on meiosis resumption, COCs were subjected to pre-IVM for 12h in TCM-199 alone or in presence of 10µM cilostamide, follicular hemisections and both cilostamide and follicular hemisections. After 6h, COCs were used to measure the levels of cAMP, while after 12h of pre-IVM, COCs were fixed to assess meiotic progression or stored to evaluate mRNA expression. At the end of this pre-IVM period, COCs were matured in vitro and meiotic progression was evaluated. The results showed that the presence of both follicular hemisections and cilostamide in pre-maturation medium increased significantly the percentages of oocytes at germinal vesicle stage (94%) when compared with COCs cultured in control medium alone or in presence of either follicular hemisections or cilostamide. After this pre-maturation period, 75% of oocytes reached metaphase II after 16h of maturation, emphasizing that cilostamide did not have a toxic effect on oocytes. Moreover, oocytes cultured in medium containing cilostamide and follicular hemisections had significantly higher levels of cAMP when compared to other treatments. The presence of both cilostamide and follicular hemisections also increased the levels of mRNA expression for NPM2 and maintained those of MATER, ZAR1, HAS2 and CX43 similar to control. In conclusion, cilostamide and follicular hemisections interact and promotes the maintenance of oocytes at germinal vesicle stage by increasing the levels cAMP in cultured COCs.