260. Synergistic effects of cAMP modulating agents in pre-IVM and in IVM on bovine cumulus and oocyte functions

Abstract

Cyclic AMP plays a crucial role in oocyte maturation. It has been shown that prolonging spontaneous maturation by modulating oocyte cAMP levels during in vitro maturation (IVM) improves developmental outcomes. This study sought to assess the effect of inclusion of cAMP modulators during a pre-IVM phase and during IVM on cumulus cell (CC) and oocyte function. Bovine cumulus-oocyte complexes (COCs) were precultured for 2 h in medium supplemented with/without cAMP modulators: 0.5 mM IBMX and 0.1 mM forskolin (FK). Cyclic AMP in control COCs dropped significantly from 15 ± 4 to 2 ± 1 fmol/COC within 30 min, whereas FK+IBMX during pre-IVM increased levels to 180 ± 19 fmol/COC, which had notable persistent effects on several COC functions throughout IVM. Cyclic AMP modulators delayed oocyte GV progression; after 2 h of pre-IVM, 67 ± 5% were at GVII stage compared (P < 0.0001) to 12 ± 3% in controls, 66 ± 5% of which had progressed to GVIII. This was associated with a significantly higher level of oocyte-CC gap junction mediated communication (1000 ± 148 v. 340 ± 73 units). We next assessed the interaction of cAMP modulators in pre-IVM and IVM (20uM cilostamide) phases. After 9 h of IVM, significantly more oocytes were arrested at GV stage when modulators were present in both pre-IVM and IVM phases, compared with in IVM alone (96 ± 1 v. 74 ± 2%, P < 0.05). Moreover, progression to MII was delayed by modulators in pre-IVM and IVM (24 ± 1, 75 ± 2, 92 ± 1% MII at 20, 24 and 28 h, respectively), compared with in IVM alone (78 ± 1, 95 ± 1, 98 ± 2% MII). After 24 h of IVM, intra-oocyte cAMP levels remained 15-fold higher (P < 0.05) with modulators in both phases compared with in IVM alone. These results show that the efficacy of cAMP modulators during IVM is substantially improved by management of COC cAMP levels during the pre-IVM phase. Such an approach has profound effects on CC and oocyte function, including increasing oocyte-CC communication and delaying meiotic resumption.