The Role Of VAMP4 In Sperm Motility

It is generally accepted that vigorous sperm motility is a central component for successful natural fertilization. Both the long-distance journey thro?gh the female reproductive tract and the subsequent penetration of the extracellular matrices surrounding the egg rely on the motility of the spermatozoa. A high percentage of poorly motile or immotile spermatozoa in semen is obviously detrimental to male fertility. Altho?gh sperm motility if so important to fertilization, the molecular basis of these events is not fully understood.In our laboratory, our previous construction of a large-scale haploid proteome using the advanced hybrid linear ion trap(LTQ)-Orbitrap mass spectrometer with high accuracy identified VAMP4, a member of VAMP family, which constructs SNARE complex with other proteins.So far, there is no study of the function of VAMP4 in sperm.In somatic cell, most energy consumes comes from glucose, which is mainly taken thro?gh the VAMP related glucose transporter (GLUT)-mediated endocytosis. Motility of sperm need large amount of energy, and glucose is one of the most important resources for energy. Altho?gh some researches on the localizations of the GLUT family proteins in sperm have been reported, it is still not known whether the glucose uptake involves the GLUT-mediated endocytosis mechanism similar to that in somatic cells.To study the function of VAMP4 in sperm, we first examined the localization and expression pattern of VAMP4 in mouse sperm. Our data demonstrate that VAMP4 is expressed in mouse testis and sperm, and it was immunolocalized to the head and the tail of sperm. Immunoreactivity in mouse sperm tail was mainly seen in the midpiece region and weekly seen in the principal piece region. The antibody inhibition assay indicated that the motility was significantly decreased in sperm incubated with VAMP4 antibody compared with that in sperm treated with IgG. The penetration assay of mouse sperm thro?gh zona-free mouse eggs was performed. And the group with un-capacitated sperm cultured with VAMP4 antibody showed significantly decreased rates of fertilization compared with control IgG antibody.In the transmission electron microscopic study, we found a lot of small vesicles in the sperm midpiece, and it was not reported before. Using immune electron microscopic study, we found that VAMP4 is located in the vesicle, indicating the possible role of VAMP4 in endocytosis in sperm tail.Subsequently, we investigated the function of VAMP4 in glucose uptake in sperm. Antibody inhibition assay showed that the glucose uptake significantly decreased in sperm treated with the VAMP4 antibody, not only in experiments by measuring the glucose in supernatant but also in experiments by measuring the ability in taking [1-3H] 2-Deoxy-D-glucose of sperm. Besides, the ATP level of the sperm cultured with VAMP4 antibody has significant decreased.Asthenozoospermia is a disease in which sperm motility is affected. We found that the expression of VAMP4 protein was greatly diminished in some cases of asthenozoospermia using Western blot analysis.In conclusion, our study showed the possibility for the first time that the glucose uptake, which was necessary for the sperm motility, might be thro?gh the VAMP4-related GLUT-mediated endocytosis.