Localization of nitric oxide synthase activity in unfertilized oocytes and fertilized embryos during preimplantation development in mice

Changes in the activities of nitric oxide synthase (NOS) during embryonic development, and the distribution of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) isoforms were examined in unfertilized mouse oocytes at the second meiotic metaphase (MII) stage and in fertilized mouse embryos during preimplantation development. In addition, the effects of NOS inhibitors on mouse preimplantation development in vitro were investigated. The activities of NOS in MII oocytes and fertilized embryos during the preimplantation period were determined by NADPH-diaphorase staining. Although NOS activity was detected in unfertilized MII oocytes, the intensity of staining was much weaker than that of fertilized embryos at the one-cell stage. There was a decrease in NOS activity in embryos from the four-cell to the eight-cell stage; however, NOS activity increased again in embryos at the morula stage, particularly in the inner cell population. In the expanded blastocysts, staining was confined to the inner cell mass. Immuno-cytochemical staining showed that eNOS and iNOS were expressed in the cytoplasm of oocytes and embryos during the preimplantation period, and eNOS was also distributed in the nuclei of the embryos. When one-cell embryos were treated with 1 mmol N-omega-nitro-L-arginine methyl ester (L-NAME) l(-1), their development in vitro was arrested at the two-cell stage. This inhibition of development was overcome by the addition of 1 mmol L-arginine l(-1) to the medium. These observations indicate that nitric oxide plays an important role as a diffusible regulator of cell proliferation and differentiation, especially at the developmental transition from the two-cell to the four-cell stage during preimplantation development of mice.