Requirement of nitric oxide for murine oocyte maturation, embryo development, and trophoblast outgrowth in vitro

We investigated the extent to which NO participates in the developmental competence (oocyte maturation, fertilization and embryo development to blastocyst) using an in vitro culture system adding sodium nitroprusside (SNP), NO donor, and NOS inhibitor (N-omega-nitro-L-arginine methyl ester, L-NAME). We also assessed the effects of NO/NOS system on blastocyst implantation using an in vitro trophoblast outgrowth assay. The treatment of low concentrations of SNP (10(-7) M) significantly stimulated meiotic maturation to metaphase II stages in cumulus enclosed oocytes. In contrast, 10(-3) and 10(-5) M L-NAME demonstrated a significant suppression in resumption of meiosis. This inhibition was reversed by the addition of SNP. No development beyond the four-cell stage was observed by the addition of high concentration of SNP (10(-3) M). Inhibition of embryo development, especially the conversion of morulae to blastocysts, was also observed in the treatment of lower doses of SNP (10(-5) and 10(-7) M). Similarly, inhibition of NO by NOS inhibitor resulted in the dose-dependent inhibition of embryo development and hatching rates, but the concomitant addition of SNP with L-NAME reversed the inhibitory effect by each SNP or L-NAME treatment. Furthermore, low concentration of SNP (10(-7) M) but not high concentration of SNP (10(-3) M) significantly stimulated trophoblast outgrowth, whereas the addition of L-NAME suppressed the spreading of blastocysts in a dose-dependent manner. These results suggest that NO may have crucial roles in oocyte maturation and embryogenesis including the process of implantation. The observed differences in required amount of NO and the sensitivity to cytotoxicity of NO in each developmental stage embryos may also suggest that NO/NOS system is tightly regulated in developmental stage specific manner.