Effect of inhibition of synthesis of inducible nitric oxide synthase-derived nitric oxide by aminoguanidine on the in vitro maturation of oocyte–cumulus complexes of cattle

Abstract

The aim of the present study was to investigate the effects of inhibition of the enzyme inducible nitric oxide synthase (iNOS) by aminoguanidine (AG) on the in vitro maturation of oocyte–cumulus cell complex(es) (COC) of cattle. COC were cultured with different concentrations of AG (0, 1, 10, and 100 mM) for 24 h. In Experiment 1, the extent of cumulus complex expansion, nuclear maturation status and plasma membrane integrity of oocytes and cumulus cells from each treatment were assessed. Nitrate/nitrite (NO 3 −/NO 2 −) concentrations were determined in culture medium by the Griess method. Addition of different concentrations of AG to maturation medium promoted a dose–response inhibitory effect on cumulus expansion ( P < 0.05). Addition of 1 and 10 mM AG to IVM medium did not affect plasma membrane integrity of oocytes or nuclear maturation rates ( P > 0.05), but it did reduce plasma membrane integrity in cumulus cells. One hundred millimolar inhibited pre-metaphase I (pre-MI) to metaphase II (MII) transition, promoted plasma membrane damage in oocytes ( P < 0.05), and increased NO 3 −/NO 2 − concentration when compared to controls ( P < 0.05). To evaluate if this effect was reversible, 10 −5 M sodium nitroprusside (SNP, NO donor) was added, only in the treatment with 100 mM AG that inhibited the nuclear maturation. However, association of 10 −5 M SNP to 100 mM AG did not reverse the effects of AG, but increased NO 3 −/NO 2 −concentration ( P < 0.05). In Experiment 2, the effect of different AG concentrations on cytoplasmic maturation in vitro was assessed based on cortical granule migration, and embryonic development. There was a dose effect on cortical granule migration rate, in which 1 mM AG (83.9 ± 6.2%) did not differ from control oocytes (83.6 ± 8.2%; P > 0.05), but 10 mM partially inhibited migration (3.8 ± 6.4%) and 100 mM totally inhibited migration ( P < 0.05). SNP (10 −5 M) did not revert this inhibitory effect on cortical granules migration in oocytes treated with 100 mM AG. Only those concentrations that did not inhibit IVM were used to assess cleavage and blastocyst development. Addition of 10 mM AG to IVM medium reduced (73.0 ± 8.1%, 15.0 ± 8.9%; P < 0.05) cleavage and blastocyst development, respectively when compared with controls (89.1 ± 3.4%, 37.6 ± 7.3%, respectively), but did not differ, ( P > 0.05), from the group treated with 1 mM AG (80.9 ± 8.4%, 41.5 ± 10.5%, respectively). The results from the present study demonstrate that NO derived from iNOS affects the in vitro maturation of bovine COC, modulating the viability of cumulus cells and of oocyte, the progression of meiosis after GVBD, the migration of cortical granules, and cleavage and blastocyst development.