278 INFLUENCE OF NITRIC OXIDE AND PHOSPHODIESTERASE INHIBITORS ON CYCLIC NUCLEOTIDES AND MEIOSIS RESUMPTION IN BOVINE OOCYTES IN VITRO MATURED

Abstract

Nitric oxide (NO) is a local action mediator of essential hormones and neurotransmitters that regulate several physiological processes in reproduction. Nitric oxide activates soluble guanylate cyclase (sGC), and results in the production of cyclic guanosine monophosphate (cGMP). This nucleotide is involved in oocyte maturation and, consequently, in the success of fertilization. In addition to cGMP, another cyclic nucleotide, cAMP, is also related to maturation. The levels of these nucleotides are balanced by synthesis and degradation is made by phosphodiesterases (PDE). The aim of this study was to analyse the effect of NO and PDE inhibitors on cyclic nucleotides concentrations and meiosis resumption in bovine oocytes matured in vitro. In experiment 1, cumulus-oocyte complexes (COC) were cultured in maturation medium with a NO donor (0.1 µM S-nitroso-N-acetylpenicillamine; SNAP) associated or not with a PDE5 inhibitor (10 µM sildefanil; SILD) for 1 h; after this period, cGMP levels (40 COC/group) were measured in COC using commercial enzyme immunoassay kits (EIA). Immature (0 h) and control COC (untreated) were measured as well. In experiment 2, COC were cultured for 9 h in maturation medium with an NO donor (0.1 µM SNAP) associated or not with inhibitors the PDE5 (10 µM SILD), PDE3 (20 µM cilostamide; CIL), or PDE8 (50 µM dipyridamole; DIP) and assessed for meiosis resumption (%GVBD; ± 100 COC/treatment). In experiment 3, COC were cultured as in experiment II and cAMP levels (10 COC/group) were measured after 1 h in culture using commercial EIA kits. Immature COC were also measured. Statistical analyses were performed using the SAS system. Data were tested for normal distribution and were transformed (arcsine; GVBD). The percentages of GVBD were analysed by one-way ANOVA followed by Bonferroni post-hoc test (4 replicates). The cGMP and cAMP levels were analysed by two-way ANOVA followed by Bonferroni post-hoc testing (5 replicates). Differences with probabilities of P < 0.05 were considered significant. In experiment 1, cGMP levels decreased from 0 to 1 h in control and SNAP groups (P < 0.05). When SNAP was associated with SILD, cGMP levels were similar to immature oocytes (P > 0.05%). In experiment 2, all treatments delayed meiosis by decreasing GVBD rates (24.7 ± 2.8 to 56.9 ± 8.6%) when compared to controls (77.1 ± 1.8%, P < 0.05). However, SNAP + CIL had the lowest GVBD rates (24.7 ± 2.8%, P < 0.05). In experiment 3, cAMP levels declined from imature COC (93 ± 7 fmol/COC) to 1 h in all treatments (21 ± 6 to 10 ± 5 fmol/COC; P < 0.05). The results indicate that NO and PDE inhibitors during maturation were ineffective in preventing the decrease in cyclic nucleotides levels during the first hour of maturation, but were still effective in delaying meiosis resumption, and the effectiveness depends on the PDE isoform which is inhibited (SNAP + CIL was most effective).