283 ROLE OF iNOS/NO/cGMP PATHWAY ON IN VITRO MATURATION OF BOVINE COCs CO-CULTURED WITH HEMI-SECTIONS OF FOLLICULAR WALL

Abstract

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) works by stimulating the activity of the enzyme soluble guanylate cyclase (sGC) to synthesise cGMP, which has action in metabolism of PDE3A. Thus, NO controls the concentration of cAMP in the oocyte. The intraoocyte concentration of these cyclic nucleotides is directly linked to the control of maturation in rodents. The follicular wall hemi-sections (HS) in maturation medium partially inhibit nuclear maturation of oocytes in culture, which allows us to study the mechanism of meiosis resumption in bovines. The aim of this study is to evaluate the effect of iNOS and sGC inhibition in the nuclear maturation. Twenty cumulus-oocytes complexes (COC)/treatment were cultured with 8 HS of follicular wall at 38.5°C and 5% CO₂ in 200 µL of maturation medium (TCM 199/BSA) supplemented with different concentrations of aminoguanidine (AG), iNOS inhibitor (1,10, 50, 100, and 150 mM; n = 840), and the sGC inhibitor, 1H-[1,2,4] oxadiazole-[4,3-a] quinoxalin-1-one (ODQ; 10^–3, 10^–4, and 10^–5 nM; n = 600). The COC groups cultured in the presence (control – ve) or absence of HS (control + ve) were used as controls. The stage of nuclear maturation of oocytes was assessed by staining with 2% acetic orcein and plasma membrane integrity of cumulus cells assessed with propidium iodite (PI) and hoechst (H33342). Statistical analyses of the 6 replicates were performed by ANOVA followed by Tukey test (P < 0.05) using SAEG software (Fundação Arthur Bernardes-UFV-Viçosa, Brazil). The integrity of cumulus cells from the group of oocytes cultured without HS (control + ve; 85.9 ± 2.3%) differed from control – ve (71.2 ± 3.7%) and other treatments, 1, 10, 50, 100, and 150 mM AG, (57.8 ± 12.1; 66.3 ± 4.2; 58.2 ± 4.6; 55.3 ± 4.3; 48.3 ± 3.3, respectively; P < 0.05). The same occurred when ODQ was used, the control + ve showed the highest cellular integrity (81.1 ± 1.6), differing from the control – ve (68.1 ± 1.8) and treatment with 10^–5, 10^–4, and 10^–3 mM ODQ (72.0 ± 2.2; 64.6 ± 4.6; 49.6 ± 6.8, respectively; P < 0.05). The presence of HS (control – ve) decreased the percentage of oocytes that reached the metaphase II (MII) in both experiments (AG and ODQ; 41.0 ± 4.0; 39.1 ± 1.7, respectively) compared to control + ve (78.5 ± 3.9; 71.9 ± 16.6, respectively; P < 0.05). The addition of 100 and 150 mM AG inhibited the resumption of meiosis and progression to MII compared with other concentrations of AG and the controls + ve and – ve. The addition of ODQ stimulated resumption of meiosis, but at the concentration 10^–3 nM there was a decrease in the number of COC that reached MII (21.8 ± 3.4) compared to control + ve (71.9 ± 16.6) and – ve (39.1 ± 1.7) and the other treatments (10^–4 and 10^–5 nM; 33.0 ± 1.8; 35.7 ± 2.5, respectively; P < 0,05). Using the model of in vitro maturation in which partial inhibition of meiosis resumption occurs, the results of this experiment show that (1) the iNOS/NO/cGMP pathway modulates plasma membrane integrity of cumulus cells and (2) that the activity of iNOS/NO pathway is important for the maintenance of the COC at the stage of germinal vesicle (GV) and progression of meiosis to MII.

The authors acknowledge FAPERJ E-26/103.080/2011 for financial support.