Effect of Sodium Nitroprusside, a Nitric Oxide Donor, and Aminoguanidine, a Nitric Oxide Synthase Inhibitor, onIn VitroDevelopment of Buffalo (Bubalus bubalis) Embryos

Abstract

Nitric oxide (NO), a free radical molecule, is produced from L-arginine by the action of nitric oxide synthase (NOS), which exists in three isoforms: inducible (iNOS), endothelial (eNOS) and neuronal (nNOS, Clementi et al. 1999). There are several reports on the putative role NO pathway plays in oocyte maturation in mice (Sengoku et al. 2001) and cattle (Viana et al. 2007). However, the role of NO and iNOS in embryo development is not clear. This study was taken up to investigate the effects of dose-dependent application of sodium nitroprusside (SNP), a NO donor, or aminoguanidine (AG), an inhibitor of iNOS, to in vitro culture (IVC) medium on the development of buffalo (Bubalus bubalis) embryos.Oocytes were aspirated from slaughterhouse buffalo ovaries, and those with an unexpanded multilayered cumulus mass and homogeneous cytoplasmic granulation were subjected to in vitro maturation (IVM) by culture in 100 ll droplets (10-15 oocytes⁄droplet) of IVM medium (TCM-199 + 10% FBS + 5 lg⁄ml pFSH + 1 lg⁄ml estradiol)17b + 0.81 mM sodium pyruvate + 50 lg⁄ml gentamycin sulphate) for 24 h in a CO 2 incubator (5% CO 2 in air) at 38.5°C. In vitro fertilization was carried out (Chauhan et al. 1998) by incubating 2-4 million spermatozoa⁄ml for 18 h in a CO 2 incubator at 38.5°C. The presumed zygotes were then divided into different groups and were cultured in IVC medium consisting of modified Charles Rosenkrans2 medium with amino acids (mCR2aa, Kumar et al. 2007,) +0.8% BSA + 10% FBS containing SNP (0, 10 )8 , 10 )6 or 10 )4 M) or AG (0, 1, 10 or 50 mM) for up to 9 days postinsemination. The IVC medium was replaced with fresh medium containing respective concentrations of SNP or AG at 48 and 96 h post-insemination. The cleavage rate was recorded at 48 h post-insemination, whereas the embryonic development was examined at days 5, 7, 8 and 9 post-insemination. The data were analysed by one way analysis of variance followed by Fisher's LSD test using SYSTAT 7.0 (SPSS Inc., Chicago, IL, USA) after arcsin transformation of percentage values.At a concentration of 10 )4 M, SNP reduced (p < 0.01) the cleavage rate to less than half that of the controls and none of the cleaved embryos developed beyond the four-to eight-cell stage, whereas at the intermediate concentration of 10 )6 M, SNP decreased (p < 0.01) day 9 hatched blastocyst production rate (Table 1). However, at the lowest concentration of 10 )8 M, SNP increased (p < 0.05) the yield of day 7 blastocysts and day 8 hatched blastocysts.Aminoguanidine (50 mM) reduced (p < 0.01) the cleavage rate and none of the cleaved embryos developed beyond the four-to eight-cell stage (Table 2). In the presence of 10 mM AG, day 5 morulae production rates were reduced (p < 0.01) and none of the 42 cleaved embryos taken developed beyond the morula stage. Similarly, the percentage of cleaved embryos that developed to morulae on day 5, to blastocysts on day 8 or to blastocysts or hatched blastocysts on day 9 postinsemination was significantly lower (p < 0.01) with 1 mM AG compared to that of con...

Presence of Nitric Oxide Synthase Immunoreactivity and mRNA in Buffalo (Bubalus bubalis) Oocytes and Embryos

“…To our knowledge, this is the first report demonstrating the presence of immunoreactivity and mRNA for different NOS isoforms in buffalo oocytes and embryos at different stages of development. The results of the present study, in combination with those of our earlier report (Saugandhika et al. 2011) in which sodium nitroprusside, a NO donor and aminoguanidine, an inhibitor of iNOS were found to affect embryonic development in buffalo, indicate that NO is endogenously produced in buffalo oocytes and embryos and that it may play an important role in embryonic development in this species.…”

Reduction of nitric oxide level results in maturation promoting factor destabilization during spontaneous meiotic exit from diplotene arrest in rat cumulus oocytes complexes cultured in vitro

“…Few studies indicate that iNOS‐derived NO directly or indirectly increases cyclic nucleotides level and maintain meiotic arrest at diplotene stage (Saugandhika et al . ; Schwarz et al . ).…”

“…; Pandey & Chaube ) cattle (Saugandhika et al . ), canine (Ji‐Hoon et al . ), bovine (Bilodeau‐Goeseels ), porcine (Chmelikova et al .…”

Reduction of nitric oxide level leads to spontaneous resumption of meiosis in diplotene-arrested rat oocytes culturedin vitro

“…NOS activity and NO presence are necessary for the correct course of meiotic maturation in the oocyte. 7,13,18,[22][23][24][25] The presence of NOS inhibitors in culture medium inhibits the meiotic resumption in mouse, 23 goat, 26 and cattle oocytes in vitro. 21,25,27 NO is important for meiotic maturation of porcine oocytes.…”