THE ROLE OF NITRIC-OXIDE IN OOCYTE MEIOTIC MATURATION AND OVULATION -MEIOTIC ABNORMALITIES OF ENDOTHELIAL NITRIC-OXIDE SYNTHASE KNOCK-OUT MOUSE OOCYTES

Evidence supports the involvement of nitric oxide (NO) in ovulation, s teroidogenesis, and atresia-related apoptosis. This study was designed to investigate the role of endothelial nitric oxide synthase (eNOS)-d erived NO in ovulation, oocyte meiotic maturation, and ovarian steroid ogenesis using wild-type (WT) mice and mice in which the gene for eNOS had been deleted (eNOS knock-out). We observed that mature eNOS knock -out females have significantly fewer pups born in each litter and a h igher mortality rate of pups than those born to heterozygote or WT fem ales (P < 0.05). To determine the influence of eNOS deficiency on ovar ian function, immature WT and eNOS knockout mice were superovulated by injecting PMSG (5 IU) followed by an injection of hCG (5 IU, ip) 48 h later. To determine whether murine oocytes expressed eNOS before (0 a nd 8 h post-hCG) and after ovulation (16 h post-hCG), we performed imm unofluorescent staining. Positive specific staining for eNOS was obser ved on the surface of ovarian and ovulated oocytes obtained from WT mi ce, but not on oocytes from eNOS knock-out mice. To determine the role of eNOS-derived NO in ovulation, ovulated oocytes were counted 16 h p ost-hCG. eNOS knock-out females showed a significant reduction (by 63% ; P < 0.0001) in ovulatory efficiency compared with WT females. The re duction in the ovulation rate in eNOS-deficient mice compared with tha t in WT mice was also associated with a higher concentration of estrad iol (P < 0.01) without significant changes in the plasma progesterone level. eNOS deficiency impaired not only ovulation, but also oocyte me iotic maturation. Ovulated oocytes were classified as being in one of the following stages of meiosis: metaphase I, metaphase II, or showing atypical (degenerative) morphology. We observed that fewer oocytes fr om eNOS knock-out mice had entered metaphase II of meiosis, and a grea ter percentage remained in metaphase I or were atypical (P < 0.002) re lative to those in WT mice. Furthermore, many oocytes that showed eith er a delay in meiotic maturation or abnormal morphology were undergoin g cell death. Our results support a role for NO in the ovulatory proce ss. The ovarian defects observed in the eNOS knock-out mice suggest th at; eNOS-derived NO is a modulator of oocyte meiotic maturation.