298 CYCLIC AMP AND CYCLIC GMP CONTENTS IN PORCINE OOCYTE–CUMULUS COMPLEXES, DENUDED OOCYTES, AND CUMULUS CELLS DERIVED FROM SMALL AND MIDDLE FOLLICLES DURING IN VITRO MATURATION

Abstract

Drastic changes in intracellular cAMP and cGMP levels play a critical role in the regulation of meiotic resumption. The objective of this study was to compare cAMP and cGMP contents in cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell masses (CC) derived from small (SF) and middle follicles (MF) during in vitro maturation (IVM). The COC were aspirated from SF (1–3 mm in diameter) or MF (3–6 mm in diameter) of prepubertal gilt ovaries. The COC were cultured in modified porcine oocyte medium (mPOM) with eCG, hCG, and dibutyryl cAMP for 20 h and then in fresh mPOM without those supplements for 4 h in an atmosphere of 5% CO₂ in air at 39°C. At 0, 10, 20, and 24 h of IVM, COC, DO, and CC were collected. The DO were prepared by removal of cumulus cells and zona pellucida. The CC were prepared by puncturing ooplasm by using 18-gauge needle. Intracellular contents of cAMP and cGMP were determined by direct enzyme immunoassay kits. Statistical analyses of 3 to 7 replicated data were performed by ANOVA. There were no significant differences in contents of cAMP and cGMP between DO from SF and MF in all observation points (P > 0.05). Cyclic AMP contents in COC and CC derived from MF were higher than those from SF at 20 h of IVM (MF 33.0 ± 0.5 fmol/COC v. SF 28.4 ± 1.0 fmol/COC, MF 20.9 ± 0.9 fmol/CC v. SF 14.6 ± 0.8 fmol/CC; P < 0.05), whereas there were no significant differences between origins of those (SF v. MF, P > 0.05) at 0, 10, and 24 h of IVM. Furthermore, although cAMP content in CC from MF was not significantly different between 10 and 20 h of IVM (25.4 ± 1.7 and 20.9 ± 0.9 fmol/CC, respectively; P > 0.05), the content in CC from SF significantly decreased between 10 and 20 h (23.1 ± 1.2 , and 14.6 ± 0.8 fmol/CC, respectively; P < 0.05). At 0 and 10 h of IVM, cGMP contents in COC and CC from MF were significantly higher than those from SF (0 h: 81.8 ± 4.5 fmol/COC from MF v. 41.7 ± 10.6 fmol/COC from SF and 82.7 ± 7.5 fmol/CC from MF v. 10.7 ± 2.7 fmol/CC from SF; 10 h: 64.8 ± 8.4 fmol/COC from MF v. 24.8 ± 8.2 fmol/COC from SF, 49.3 ± 14.9 fmol/CC from MF v. 13.5 ± 4.8 fmol/CC from SF; P < 0.05). However, there were no significant differences in cGMP contents in COC and CC between the origins (MF v. SF) at 20 and 24 h of IVM (P > 0.05). From these results, we conclude that cAMP and cGMP contents in cumulus cells are significantly differences between the origins (MF v. SF) during IVM.