Improved development of in vitro-derived bovine embryos by use of a nitric oxide scavenger in a cumulus-granulosa cell coculture system.

Abstract

This study was conducted to examine the hypothesis that nitric oxide (NO) affects prehatching development of bovine oocytes fertilized in vitro. In experiment 1, inseminated oocytes were cultured in a cumulus-granulosa cell (CG) coculture system to which 0.008 or 0.04 mM of sodium nitroprusside (SNP), a spontaneous NO releaser, was added at 18 or 60 hr postinsemination. Embryo development was greatly (P < 0.001) inhibited by the addition of SNP, regardless of time of addition or SNP concentration. In experiment 2, eight-cell embryos were cultured singly in a defined medium, to which 0.0016, 0.008, or 0.04 mM of SNP was added. Development to the blastocyst stage was greatly (P < 0.001) decreased after addition of SNP compared with no addition. Higher (P < 0.02) concentration of NO metabolites was found in developmentally arrested embryos than in developing embryos at 144 hr postinsemination (experiment 3). In experiment 4, blastocyst formation of oocytes cocultured with CGs was significantly (P < 0.02) increased after addition of hemoglobin (Hb, 1 microgram/ml), an NO scavenger. Prehatching development of oocytes was significantly (P < 0.05) increased after addition of Hb at different time intervals (18, 60, or 144 hr postinsemination) in experiment 5. Embryo development was not enhanced by Hb addition to the culture medium in the absence of CGs (experiment 6). Prehatching development of eight-cell embryos derived from a Hb-containing culture system was not promoted by the further addition of Hb after transfer of the embryos to a defined and CG-free single-embryo culture system (experiment 7). In conclusion, NO, which may be secreted from CGs, has an inhibitory role in prehatching development of bovine oocytes fertilized in vitro, and use of an NO scavenger, Hb, in a coculture system enhances blastocyst formation.