261 SUPPLEMENTATION OF SHEEP OOCYTE MATURATION MEDIA WITH NITRIC OXIDE SYNTHASE INHIBITORS MODIFIES THE ZONA PELLUCIDA AND DECREASES SPERM ZONA BINDING

Abstract

Nitric oxide plays a critical role in several reproductive functions, including sperm and egg maturation. Nitric oxide has complex and sometimes antagonistic biological effects. At low and controlled levels, NO aids gamete maturation, but at high levels, it has detrimental effects and often causes cell death. Both the female tract and egg vestment contain endothelial NO synthase (eNOS) and inducible NOS (iNOS). The knockout mouse for specific NOS has been shown to impair egg maturation and ovulation. The aim of this work was to study the effect of NOS inhibitors on in vitro sheep oocyte maturation and function. We evaluated the cumulus cell expansion, sperm zona binding, and time required to fully digest the zona pellucida (ZP). The 3 different NOS inhibitors used were 1 mM l-NAME (N-ω-nitro-l-arginine methyl ester, an eNOS inhibitor), 1 mM l-NMMA (N^G-monomethyl-l-arginine acetate, a competitive inhibitor of all three isoforms of NOS), and 1 mM aminoguanidine (eNOS and iNOS inhibitors). We also used a NO donor, S-nitroso-glutathione (GSNO; 50 µM). Oocytes were matured in TCM without supplementation or supplemented with GSNO or NOS inhibitors for 24 h at 38°C, 5% CO₂. The effect of the inhibitors on the ZP was assessed by determining the time necessary to complete digestion of the ZP with pronase (0.1%). To evaluate the number of sperm attached to the ZP, oocytes matured as described above were denuded and co-incubated for 4 h (38°C, 5% CO₂) with epididymal sheep sperm (1 × 10⁶ spermatozoa mL^–1 in TALP medium). At the end of the incubation, sperm unbound to the ZP were eliminated by several washings. The oocytes were fixed and stained with Hoechst to stain the sperm nucleus, and the number of sperm bound per zona was measured under an epifluorescence microscope. The times required for complete ZP digestion and spermatozoa attachment were analysed by one-way ANOVA. Mean digestion times (seconds) and numbers of spermatozoa bound to the ZP in the control, GSNO, l-NAME, aminoguanidine, and l-NMMA groups were 234.6^a, 297.8^a, 243.2^a, 323.0^ab, 398.4^b, and 20.2^a, 12.0^ab, 11.1^b, 7.7^b, and 8.1^b, respectively (^a,bP < 0.001). Our results showed that 1) egg maturation, as measured by cumulus expansion, was not affected by the NOS inhibitors or GSNO, 2) the time necessary to completely digest the ZP increased in the presence of aminoguanidine and l-NMMA, and 3) all three NOS inhibitors decreased the number of spermatozoa that could be attached to the ZP. The GSNO showed no effect because these levels represent physiological concentration observed during normal egg maturation. In conclusion, these results are in accordance with the importance of NO for egg maturation, and this study shows for the first time the possible involvement of NO in maintaining the structure of the ZP, which is important for sperm–egg binding and further fertilization. Such an effect on the ZP proteins could be due to a change in protein S-nitrosylation, which is a post-translational modification attributable to NO.