350 EFFECT OF INDIRECT MAINTENANCE OF INTRACELLULAR cAMP IN CUMULUS–OOCYTE COMPLEXES USING 3-ISOBUTYL-1-METHYLXANTHINE ON GAP JUNCTIONAL COMMUNICATIONS AND OOCYTE MEIOTIC MATURATION

Abstract

Oocyte and cumulus cells communicate through an extensive network of gap junctions (GJs), which permit the transfer of small molecules such as cAMP. Gonadotropin strongly enhances the intracellular cAMP concentration in cumulus cells, and induces oocyte meiotic resumption. Enhanced cAMP also triggers a reduction of GJ communications (GJCs) in cumulus-oocyte complexes (COCs), accompanied by cumulus expansion. Intracellular cAMP is modulated by both adenylate cyclase (AC) for synthesizing and phosphodiesterase (PDE) for degrading. Addition of AC to gonadotropin-free medium induces meiotic resumption of bovine oocytes without cumulus expansion, suggesting that maintenance of cAMP at a certain level in COCs may be crucial for either prolonged maintenance of GJCs or the timing of oocyte meiotic resumption. In the present study, we investigated the intracellular cAMP concentrations in porcine COCs or oocytes, and GJCs during in vitro oocyte maturation culture using PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Porcine COCs obtained from prepubertal gilts were cultured for 20 h (1st culture) using M199 containing 10% FCS (basic medium, BM group) with FSH (FSH group) or IBMX (IBMX group). Following this, the COCs were transferred into the basic medium containing FSH and LH, and cultured for another 24 h (2nd culture). At 6, 12, and 20 h of the 1st culture, intracellular cAMP in COCs or oocytes was measured. To determine GJCs in each COC, Lucifer Yellow fluorescent dye was microinjected into cumulus-enclosed oocytes at 6 or 12 h of the 1st culture, and the ability of dye transfer, which is related to the GJCs, from the oocyte to the surrounding cumulus cells was observed. At the end of the 1st culture, 30.8 ± 6.0% of the oocytes in the FSH group underwent germinal vesicle breakdown (GVBD), whereas only a few oocytes in the BM group (8.6 ± 2.4%) and the IBMX group (5.8 ± 3.0%) achieved GVBD (P < 0.05). In contrast, ratios of metaphase-II (M-II) stage oocytes at the end of the 2nd culture did not differ between the FSH group (75.7 ± 3.9%) and the IBMX group (68.2 ± 6.8%), although a few oocytes in BM group (10.1 ± 3.7%) reached the M-II stage (P < 0.05). Concentrations of cAMP in COCs and oocytes increased drastically in the FSH group compared to those of the BM and IBMX groups (P < 0.05). In addition, the concentration of cAMP in IBMX group oocytes was also higher than that in the BM group, with a significant difference detected at 20 h (P < 0.05). The GJCs in the FSH group were gradually closed, depending on the length of time in culture (54.9 ± 3.7% of COCs closed their GCJs at 12 h of the 1st culture). In contrast, in the IBMX group, only 23.0 ± 3.7% of COCs closed their GJCs at 12 h of the 1st culture, which was significantly different from that of the other two groups (P < 0.05). These results suggest that treatment with IBMX during the first half of IVM culture can induce subsequent meiotic resumption of porcine oocytes, and that a moderate increase of cAMP concentration in COCs or oocytes prolongs GJCs during the treatment.