| Mammalian spermatozoa are not able to fertilize after ejaculation. They acquire this ability only after residing in the female uterine tract for a finite period of time that varies depending on the species. The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation. Capacitation is associated with changes in membrane properties, enzyme activities and motility that prepare the sperm for the acrosome reaction and for penetration of the egg vestments prior to fertilization. Hernandez and his colleague found the localization of ENaC-αto the flagellar mid piece and of ENaC-δto the acrosome in mouse sperm (Hernandez-Gonzalez EO et al., 2006). By the methods of single cell fluorescence analysis and electrophysiology, they found that the two EnaCs participate in the regulation of the capacitation-associated hyperpolarization through producing a depolarization that was potently inhibited by amiloride and its analog EIPA, high pH, and by the incubation of mouse sperm under capacitating conditions.Our study investigated the regional distribution of epithelial Na+ channel (ENaC) alpha-subunit in the testis and sperm of rat and human. ENaC-αprotein was found on immunoblots of sperm and testis membrane preparations and indicated localization to the spermatocyte, spermatid, and sperm by Immunostaining. Taken together, these data indicate that ENaC-αis expressed in spermatocyte and distributed to the flagellar mid piece of sperm finally. The dynamic distribution changes of ENaC-αin spermatogenesis also suggest that ENaC-αis likely to participate in the regulation of sperm motility by someway. Sperm motility assays showed a significant difference between samples with EIPA and those without in human sperm. 1. Western-blot detecting ENaC-αsubunit in the testis and sperm of rat and humanThe main goal of this portion is to study the expression of ENaC-αprotein in the testis and sperm of rat and human.Western-blot is the classical method used in the assessment of a protein in tissue or cultivated cells. Proteins of the samples are obtained from tissue or cultivated cells, subjected to SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Electrophoretic transfer of proteins to nitrocellulose filter and immunodetection of the goal protein are carried out by special antibody and the second antibody(alkaline phosphatase labelled). Using the ECL kit, immunoblots were developed with the film.We confirmed the presence of ENaC-αin the testis and sperm of rat and human by western blot analysis using polyclonal antibodies against the ENaC-αsubunit. Antibody detected bands with a Mr of ~75 KDa in testis and sperm.2. Indirect immunofluorescence and immunohistochemistry detecting ENaC-αdistribution in the testis and sperm of rat and humanAs discribed above, ENaC-αsubunit is expressed in the testis and sperm of rat and human. Indirect immunofluorescence and immunohistochemistry detecting ENaC-αdistribution in the testis and sperm of rat and human were applied for finding the distribution of ENaC-αin the testis and sperm.As a result, we found that that ENaC-αexpress in leydig cell, vascular endothelial cell(VEC), Ap type spermatogenous cell, spermatocyte, spermatid and sperm. The sample of human testis is not good enough for use in Indirect immunofluorescence and immunohistochemistry, no sperm is found. But the positive staining was found in spermatocyte of the human sample.ENaC-αdistribution character in the sperm of rat and human were detected too. ENaC-αwas locallized on the midpiece of flagella of the sperm of rat and human.3. Assessment of sperm motilityMammalian sperm are not able to fertilize after ejaculation. They acquire this ability only after residing in the female uterine tract for a finite period of time that varies depending on the species. The molecular, biochemical, and physiological changes that occur in sperm while in the female tract are collectively referred to as capacitation. Capacitation is associated with changes in membrane properties, enzyme activities and motility that prepare the sperm for the acrosome reaction and for penetration of the egg vestments prior to fertilization. Hernandez and his colleague found the localization of ENaC-αto the flagellar mid piece and of ENaC-δto the acrosome in mouse sperm (Hernandez-Gonzalez EO et al., 2006). By the methods of single cell fluorescence analysis and electrophysiology, they found that the two EnaCs participate in the regulation of the capacitation-associated hyperpolarization through producing a depolarization that was potently inhibited by amiloride and its analog EIPA, high pH, and by the incubation of mouse sperm under capacitating conditions.Our study investigated the regional distribution of epithelial Na+ channel (ENaC) alpha-subunit in the testis and sperm of rat and human. ENaC-αprotein was found on immunoblots of sperm and testis membrane preparations and indicated localization to the spermatocyte, spermatid, and sperm by Immunostaining. Taken together, these data indicate that ENaC-αis expressed in spermatocyte and distributed to the flagellar mid piece of sperm finally. The dynamic distribution changes of ENaC-αin spermatogenesis also suggest that ENaC-αis likely to participate in the regulation of sperm motility by someway. Sperm motility assays showed a significant difference between samples with EIPA and those without in human sperm.4. SummarySummarizing the findings of other researchers and ours, ENaC-αis closely relevant to the male fertility and may take part in the regulation of sperm mortility. Here, for the first time, we raise and confirm a new action of EIPA on promoting male fertility, which provide an indirect evidence that the open ENaC-αmay inhibits sperm motility by some way. Meanwhile, we further investigate the mechanism of ENaC-αinhibiting sperm motility in the aspect of pH change in the liquid environment surrounding sperm. The role of ENaC-αin regulation of sperm motility might also be the target of antifertility medicine for male contraception, and may provide a way to evaluate male sperm functions in clinics.
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