Extrinsic cholinergic innervation of rat ovary regulates the molecular mechanisms of apoptosis under the influence of nitric oxide

DELSOUC MB, ; VALLCANERAS S; DELGADO SM; CASAIS M

Congreso; Sociedad de Biología de Cuyo XXXIII Reunión Científica Anual. Mendoza); 2016

Extrinsic Cholinergic innervation of rat ovary regulates the molecular mechanisms of apoptosis under the influence of nitric oxideDelsouc MB, Vallcaneras S, Delgado SM, Casais M. Laboratorio de Biología de la Reproducción, FQByF, UNSL. IMIBIO-SL, CONICET.E-mail: bdelsouc@gmail.com Using the ex vivo coeliac ganglion-superior ovarian nerve-ovary (CG-SON-O) system of Holtzman rats in the first proestrous,we previously reported that Acetylcholine (Ach) in CG increases the levels of nitric oxide (NO) and causes oxidative stress in the O.The aim of this work was to determine whether the effects caused by the ganglionic cholinergic stimulus promote apoptosis in the O. The CG-SON-O systemwas incubated at 37°C for 180 min in Krebs-Ringer bicarbonate buffer with 2%albumin. The CG and the O were placed in separate compartments, connected by the SON. Cholinergic stimulation of the CG was achieved by 10-6M Ach [(Ach)CG]. To analyze the influence of NO in altered ovarian function by the extrinsic cholinergic input, 400µM Aminoguanidine (AG), a selective inhibitor of inducible NO synthase, was added in both compartments: ovarian [(AG)O] and ganglionic [(AG)CG], separately, with and without ganglionic cholinergic stimulus (Ach)CG. At the end of the experiments, ovaries were cleaned and flash-frozen in liquid nitrogen. The gene expression of Bax, Bcl-2, Fas and Fas ligand (FasL) was evaluated by RT-PCR and correlated with the apoptotic index. Statistical comparisons were performed by one way ANOVA followed by the Tukey´s test. A probability of p<0.05 was used to denote statistical significance. (Ach)CG increased the expression of Bax, Fas and FasL (pro-apoptotic genes), and decreased the expression of Bcl-2 (anti-apoptotic gene), thus shifting the Bax/Bcl-2 ratio in favor of apoptosis (p<0.05). The opposite effect was obtained with (AG)O (p<0.05). In addition, (Ach+AG)CG decreased Fas expression (p<0.05). In conclusion, the results suggest that cholinergic stimulation of CG promotes the extrinsic and intrinsic pathways of apoptosis in the O as a result of oxidative stress mediated by NO.