Efect of inhibition of L-ARG/INOS/NO pathway by aminoguanidine on in vitro maturation of bovine oocytes-cumulus complexes (COCS) in the presence of follicular wall hemisections

The knowledge of the mechanisms involved in oocyte-cumulus complex (COC) maturation is important for the progress of reproductive biotechnologies and assisted reproduction techniques. Nitric oxide (NO) is involved in the process of in vitro maturation of bovine COCs (Viana, K. S. et. al., Anim. Reprod. Sci. 102, 217-22, 2007). The synthesis of NO occurs from L- arginine, which is catalysed by three isoforms of nitric oxide synthase (NOS) (Ignarro, L.J., 2000, Nitric Oxide, Biology and pathobiology, 845p). Studies support that NO derived from inducible nitric oxide synthase (iNOS) affects oocyte maturation (Matta, G. S. C. et.al, 2009 Anim. Reprod. Sci. 111, 189- 201). This study aimed to evaluate the role of iNOS in the maturation of COCs by adding the selective inhibitor aminoguanidine (AG) to the culture medium. Groups of 20 COCs (120 COCs/treatment) were cultured with eight follicular wall hemi-sections (HS) at 38.5°C and 5 % CO2 in 200 ml of maturation medium (TCM 199/BSA) supplemented with different AG concentrations (1, 10, 50 , 100 and 150 mM). Controls consisted of COCs cultured in the presence (control -) or absence of HS (control +). Nuclear maturation was assessed by staining with 2% acetic orcein and plasma membrane integrity of cumulus cells by propidium iodide staining after 22h of culture. A randomized design with 6 replicates (120 COCs/treatment) and 7 treatments (1, 10, 50, 100 and 150 mM AG and two controls) was used. The percentage of cellular integrity was subjected to ANOVA and multiple linear regression, nuclear maturation was assessed by ANOVA and means were compared by the Tukey test (P < 0.05). The integrity of cumulus cells of the group of oocytes cultured without HS (control +) (85.9 ± 2.3 %) differed in relation to the control group - (71.2 ± 3.7 %), and to groups treated with 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 addition of 150 mM AG promoted the lowest number of viable cells (48.3 ± 3.3%; P <0.05). The presence of HS (Control -) decreased the percentage of oocytes that reached metaphase II (MII) (41.0 ± 4.0%) compared to the control group + (78.5 ± 3.9%). However, the addition of 100 and 150 mM AG blocked the progression of meiosis to MII compared to other treatments and controls (P <0.05). These results suggest that activity of iNOS is directly linked to cumulus cells integrity and resumption and progression of meiosis to MII.