Presence of nitric oxide synthase immunoreactivity and mRNA in buffalo (Bubalus bubalis) oocytes and embryos

This study examined the presence of immunoreactivity and mRNA for different nitric oxide synthase (NOS) isoforms in immature and in vitro matured oocytes and in embryos at two-, four- and eight-cell, and morula and blastocyst stages in buffalo. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to in vitro maturation in TCM-199 + 10% FBS + 5 μg/ml pFSH + 1 μg/ml estradiol-17β + 0.81 mm sodium pyruvate + 10% buffalo follicular fluid + 50 μg/ml gentamycin sulphate for 24 h in a CO(2) incubator (5% CO(2) in air) at 38.5°C. Following in vitro fertilization carried out by incubating them with 2-4 million spermatozoa/ml for 18 h, the presumed zygotes were cultured in mCR2aa medium containing 0.6% BSA and 10% FBS for up to 8 days post insemination. Immunofluorescence staining of NOS using antibodies that cross-reacted either with all the NOS isoforms i.e., universal (uNOS) or specifically with inducible (iNOS) or endothelial (eNOS) isoforms revealed that NOS was present in oocytes and embryos at all the stages examined. Examination of the semi-quantitative expression of NOS genes by RT-PCR revealed that the iNOS, eNOS and nNOS mRNA was present in the immature and mature oocytes and in all the embryonic stages examined. In conclusion, it was demonstrated in the present study that immunoreactivity and mRNA for different NOS isoforms was present in buffalo oocytes and pre-implantation stage embryos.