Localização da NA+, K+-ATPase em espermatozoides ovino in natura, pós-descongelação e capacitados in vitro e determinação dos efeitos de seu bloqueio sobre as características espermáticas

Abstract

Aiming to localize the Na+, K+-ATPase in ram sperm (raw, post-thaw and capacitated in vitro), and to evaluate the influience of ouabain addition on kinematic sperm parameters, two experiments were performed. Exp.1: semen samples (four rams, seven replicates) were harvested, diluted (Tris-gema, final concentration 200 x 106 sperm/mL) and frozen at -196 C°. Exp. 2: sêmen samples from four rams (five replicates) were diluted in TALPm (50 x106 sperm/mL) and aliquot for two precedures: in vitro capacitation (addition of 2,5% of estrous sheep serum - ESS) or ouabain addition (10-4 M), in order to block the Na+,K+-ATPase. Semen aliquots from both experiments were evaluated using CASA system for kinematic parameters and with aid of flow cytometry it was determined localization of Na+, K+-ATPase (Bodipy FL Ouabaína), evaluated plasmatic (IP) and acrosomal membrande (FITC-PNA) integrity, viability and stability of sperm plasma membrane (YO-PRO e M540). During Exp. 2, it was also evaluated the mitochondrial membrane potential (JC-1). For Exp. 2, all analysis were performed at 0h and 3h of incubation time (36 °C). These are the results: 1) The Na+, K+-ATPase was localized on middle piece of sperm flagellum, despite sample status (raw, post-thaw and capacitated); 2) the freezing-thawing proccess reduced (P<0,05) MT, MP and VAP and increased BCF. There was also a reduction (P<0.05) on percentage of sperm stained with Bodipy FL Ouabain, with intact plasmatic and acrossomal membranes, and percentage of sperm with stabilized plasma membrane; 3) Throught incubation time, the Na+, K+-ATPase blocking reduced (P<0.05) MP, VCL and VSL, whereas on control group reduced (P <0.05) MT, VCL and ALH; 4) The addition of ESS to TALPm reduced (P<0.05) MP, VCL and ALH, and on control group reduced (P<0.05) MT and MP, with no difference between treatments for kinematic parameters. Therefore, despite of treatment, there was an increasing (P<0.05) on percentage of sperm with damages plasma membrane, intact acrosome and no stain for Na+, K+-ATPase. In conclusion, despit of sperm phisiological status, Na+, K+-ATPase ramains on middle piece of ram sperm flagellum and its inhibition have no influence on sperm motility parameters.