Analysis of the effect of mitric oxide synthase inhibition on mouse sperm employing a modified staining method for assessment of the acrosome reaction

Abstract

A commercially available staining kit (Spermac) was combined with swelling in a hypoosmotic medium (HOS) for simultaneous assessment of viability and acrosome reaction in mouse spermatozoa. We compared the results obtained with the combined technique (HOS-Spermac) with those obtained with currently used techniques: the chlortetracycline fluorescence assay and eosin exclusion. The results obtained with HOS-Spermac were the same as those obtained with the chlortetracycline fluorescence assay. Viability assessment with HOS-Spermac showed a good correlation with the percentage of spermatozoa showing eosin dye exclusion. Using this novel technique, we studied the effect of a nitric oxide synthase inhibitor (NG-nitro-L-arginine methyl ester, L-NAME) on the acrosome reaction. L-NAME produced a dose-dependent inhibition of spontaneous acrosome reaction and its inhibitory effect was specifically counteracted by L-arginine. We conclude that HOS-Spermac provides a simple and reliable tool for assessment of the acrosome reaction and that nitric oxide synthase participates in this important function of the spermatozoon.