The role of INOS/NO/CGMP pathway on in vitro maturation of bovine oocytes-cumulus complexes in presence of follicular wall hemisections

The role of nitric oxide (NO) in the mammalian oocyte maturation and the mechanisms involved in this process has not been completely understood. NO is produced by nitric oxide synthases (NOS) and stimulates the soluble guanylate cyclase enzyme (sGC) to produce cGMP. This nucleotide controls the action of phosphodiesterase 3A, which has the function of metabolizing cAMP to the inactive form, leading to the resumption of meiosis. Aiming to better understand these events, we used two active substances in this pathway: aminoguanidine (AG), which inhibits the inducible NOS isoform (iNOS); and 1H- [1,2,4] oxadiazol [4,3-a] quinoxalin-1-one (ODQ), which acts by inhibiting the sGC. Groups of 20 COC (120 COC/treatment) were cultured with eight follicular wall hemisections (HS) in an incubator at 38.5°C and 5% CO2 in 200 µl of maturation medium (TCM-199/BSA) supplemented with AG (1 and 100 mM) and ODQ (10-3 M). The controls consisted of COCs cultured in the presence (C -) and absence of HS (C +). Oocyte nuclear maturation state was assessed by staining with 0.2% acetic orcein after 22h of IVM. The intracellular concentrations of cGMP (30 COC/treatment) and cAMP (10 COC/treatment and 50 oocytes/treatment) were measured at 0, 1, 3, 6, 9h for COCs and 0, 3, 6, 9h in the oocytes with an enzyme immunoassay. The results were analyzed with the Tukey test (P 0.05), while 100 mM AG inhibited resumption of meiosis and progression to MII. The addition of ODQ stimulated meiosis resumption compared to C -. However, it inhibited the progression to MII (21.9 ± 3.5%; P<0.05). cGMP concentrations decreased over time in all experimental groups. The results observed in group with 1 mM AG do not differ from those observed in group C -. However, the addition of 100 mM AG and 10-3 M of ODQ decreased the concentration of cGMP, except at 3 and 6h. The group treated with ODQ had the lowest concentrations (P<0.05). The concentration of cAMP in COCs increased over time in all treatments (P<0.05). For this reason, this nucleotide was only measured in oocytes, where its concentration was descending. The C+ group had the lowest (P<0.05) concentration of cAMP, while groups treated with 100 mM AG and 10-3 M of ODQ had higher concentrations of cAMP (P<0.05). The results suggest that the activity of the iNOS/NO/cGMP pathway is important for maintaining the COC in the germinal vesicle stage (GV) of meiosis, and that progression to the MII is modulated by cGMP and cAMP concentrations in the COC/oocyte.