Sperm plasma membrane

About: Sperm plasma membrane is a research topic. Over the lifetime, 1016 publications have been published within this topic receiving 49964 citations.

Identification and expression analysis of Drosophila melanogaster genes encoding β-hexosaminidases of the sperm plasma membrane

Abstract: Sperm surface beta-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two beta-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for beta-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the alpha-subunit of the mammalian lysosomal beta-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the beta-subunit of all known beta-N-acetylhexosaminidases, which we have named beta(1) and beta(2), respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an alphabeta(2) structure, and DmHEXB, with a beta(1)beta(2) structure. Immunofluorescence demonstrated that all the gene products localized to the sperm plasma membrane. Although none of the genes was testis-specific, fdl was highly and preferentially expressed in the testis, whereas Hexo1 and Hexo2 showed broader tissue expression. Enzyme assays carried out on testis and on a variety of somatic tissues corroborated the results of gene expression analysis. These findings for the first time show the in vivo expression in insects of genes encoding beta-N-acetylhexosaminidases, the only molecules so far identified as involved in sperm/egg recognition in this class, whereas in mammals, the organisms where these enzymes have been best studied, only two types of polypeptide chains forming dimeric functional beta-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.

Epididymal specific, selenium-independent GPX5 protects cells from oxidative stress-induced lipid peroxidation and DNA mutation

Abstract: STUDY QUESTION Can selenium (Se) independent, epididymal-specific glutathione peroxidase 5 (GPX5) protect CHO-K1 cells from oxidative damage and, more specifically, from lipid peroxidation and DNA mutation? SUMMARY ANSWER CHO-K1 cells expressing GPX5 have increased resistance to oxidative challenge and, more specifically, decreased levels of lipid peroxidation and decreased levels of the downstream DNA lesion 8-oxo-7,8-dihydroguanine (8-oxodG) compared with control cells. WHAT IS KNOWN ALREADY GPX5 associates with sperm during transit of the epididymis, and has been postulated to protect sperm from peroxide-mediated attack. However, its function as an active glutathione peroxidase has been questioned due to substitution of the classical selenocysteine residue at its active site. Indirect evidence for a functional role for GPX5 has been provided by in vivo studies, in particular from the GPX5 knockout mouse whereby offspring sired by GPX5(-/-) males have a higher rate of spontaneous abortion and developmental defects, attributed to increased oxidative injury (8-oxodG) to sperm DNA, but only when the GPX5(-/-) males are over 1 year of age. Interestingly, we have previously shown severely reduced levels of GPX5 in humans. STUDY DESIGN, SIZE, DURATION To look more directly at its role in protection against oxidative damage, we have used an in vitro system, generating a CHO-K1 mammalian cell line expressing recombinant rat GPX5. PARTICIPANTS/MATERIALS, SETTING, METHODS We have used the recombinant CHO-K1 cells to determine whether GPX5 is able to protect these cells from an administered oxidative challenge, using a range of approaches. We compared the viability of GPX5-expressing cells with control cells by both MTT and trypan blue exclusion assays. We next investigated whether GPX5 protects the cells specifically from lipid peroxidation, by using the fluorescent reporter molecule C11-BODIPY(581/591), and thus from downstream DNA mutation, by comparing levels of the DNA lesion 8-oxodG. We also investigated whether GPX5 can be transferred to rat sperm via epididymosomes. MAIN RESULTS AND THE ROLE OF CHANCE GPX5-expressing CHO-K1 cells had increased viability compared with control cells following oxidative challenge (P < 0.005). We also found that GPX5-expressing CHO-K1 cells had significantly lower levels of C11-BODIPY(581/591) oxidation, and hence lipid peroxidation, compared with control cells. Levels of 8-oxodG DNA damage were also markedly lower in the nuclei of GPX5-expressing cells than in control cells. Finally, we showed that GPX5 can be transferred to rat sperm via epididymosomes. LIMITATIONS, REASONS FOR CAUTION GPX5 is not active in glutathione peroxidase assays using H₂O₂ as the substrate. However, the related non-mammalian Se-independent GPXs show preference for electron donors other than glutathione, with a number utilizing thioredoxin as a reducing equivalent. Hence, the in vitro activity of GPX5 needs to be assessed using a range of alternative substrates and electron donors. GPX5 is secreted by the epididymis and associates with the sperm plasma membrane. We showed that this transfer can occur via epididymosomes; however, the mechanism for transfer and the identity of a potential binding partner in the sperm membrane needs to be determined. Finally, our study utilized an in vitro system that needs to be translated to human sperm. WIDER IMPLICATIONS OF THE FINDINGS Our study supports an important role for GPX5 as an antioxidant, possibly acting as a phospholipid hydroperoxidase and participating in the maintenance of cell and DNA integrity.

Voltage-operated calcium channels in male germ cells

Abstract: The acrosome reaction is a key event in fertilization. Current models for induction of the acrosome reaction incorporate a necessary influx of Ca 2+ , which is mediated by agonist-induced gating of ion channels in the sperm plasma membrane. The difficulty of applying electrophysiological techniques to spermatozoa has severely hampered studies on the expression of functional ion channels in these cells. However, during the last few years, a combination of molecular and physiological techniques (applied to immature spermatogenic cells) has elucidated both the expression of Ca 2+ channels in male germ cells and their role in induction of the acrosome reaction. It now appears that a range of voltage-operated Ca 2+ channels, similar to those that occur in somatic cells, is expressed in spermatozoa. Male rodent germ cells express a low-voltage activated (T-type) channel that is regulated by membrane potential and provides the primary Ca 2+ influx mechanism in zona pellucida-stimulated spermatozoa. In human spermatozoa, similar channels are apparently expressed, but their function in induction of the acrosome reaction has yet to be established. A range of other, high voltage-activated channels also appear to be present in rodent and human spermatozoa, but their roles are not yet known. In this review, the structure and characteristics of voltage-operated Ca 2+ channels are outlined and the evidence for their expression and function in male germ cells is assembled and discussed.