The action of L-arg/no in the in vitro bovine sperm capacitation does not occur via cGMP signaling pathway

Nitric oxide (NO) participates in intra- and intercellular signaling and it is formed by a catalytic reaction from Larginine (L-arg). In sperm, NO has been related to improvement in motility and capacitation, which favors the oocyte fertilization. Its biological effects can be induced by cyclic guanosine monophosphate (cGMP). It’s not clear if there is any relationship between NO, increased level of sperm cGMP and sperm capacitation in bovines so far. Thus, the aim of this study is to investigate whether sperm capacitation is related to the L-arg/NO/cGMP signaling pathway. Two experiments were performed as follows: 1) Evaluation of sperm capacitation; and 2) Measurement of the concentration of cGMP in sperm after capacitation. Cryopreserved sperm from three Nellore bulls (Bos indicus) were used with four replicates for each bull (n=12) for experiment 1 with in vitro capacitation induced in 200 μL of capacitating medium supplemented with 20 μg/mL heparin (control) plus treatments, as follows: L-arg) 1mM L-arg (precursor of NO); PTIO) 1mM PTIO (chelator of NO); L-arg+PTIO) 1 mM L-arg and 1mM PTIO. Experiment 2 was carried out with three replicates for each bull (n=9) and one more group was included for treatment: BrcGMP+PTIO) 5mM 8-Br-cGMP (cGMP analogue) and 1mM PTIO. Sperm capacitation was evaluated by fluorescent labeling of chlortetracycline (CTC), and the measurement of the concentration of cGMP was performed by ELISA. Data were subjected to analysis of variance (ANOVA) and means compared by Tukey test at 5% probability. In experiment 1, L-arg had a higher percentage (P 0.05). These results suggest that during in vitro sperm capacitation: 1) the increase of NO by the addition of L-arg improved sperm capacitation and 2) the action of L-arg/NO in the in vitro bovine sperm capacitation does not occur via cGMP signaling pathway