Vesikulärer Transfer ("Transsekretion") von Membrankomponenten auf bovine Nebenhodenspermien

During the passage through the male reproduction tract spermatozoa interact with factors, which amongst others are released from the epididymis. The outcome of the spermatozoa modification is essential for the maturation to full functional germ cells. During the differentiation in the epididymis the epididymosomes (apocrin secreted epididymal vesicles) play a crucial role. Previous work of our group demonstrated a switch of the plasma membrane Ca2+-ATPase isoform 4 (PMCA4) splice variants from PMCA4b in bovine caput spermatozoa membrane to PMCA4a in bovine cauda spermatozoa membrane. In addition the Ca2+-ATPase activity of spermatozoa increased significantly during the transit through the epididymidis. The possibility of a transfer of PMCA4a from epididymosomes to spermatozoa was postulated (Sanchez-Luengo et al. 2004, Brandenburger et al. 2011). The aim of the present study was analyze the fusogen properties of epididymosomes closely. Therefore, epididymal vesicles were isolated from different epididymal regions. After this, epididymosomes were characterized morphologically as well as biochemically. Furthermore, the vesicle transfer of membrane components (lipids, proteins) was analyzed, especially the potential transfer of PMCA to bovine epididymal spermatozoa. Electron microscopical analyses showed, that cauda epididymosomes are greater than caput epididymosomes. Lipid analyses of caput and cauda epididymosomes proved an increase of the cholesterol/phospholipid rate with a decrease of cholesterol and phospholipid concentration. The phospholipids Phosphatidylethanolamine (PE), Phosphatidylcholine (PC) and Sphingomyeline(SM) were predominantly detected, whereby the concentration in caput and cauda epididymosomes was not significantly modified. For protein analyses, the author focused on PMCA. By means of Westernblot analyses, it could be shown, that cauda vesicles exhibit more PMCA4a than caput vesicles. Also the Ca2+-ATPase activity increased significantly from caput to cauda epididymosomes. To determine in in vitro experiments, the fusogen properties of bovine epididymosomes with spermatozoa, bovine epididymosomes were labeled with the fluorescent dye Octadecylrhodamin B (R18). Caput spermatozoa showed a higher fusion rate than cauda spermatozoa. The optimal pH value for the fusion was in the acid area (pH 5 - 6). Lipid analyses of epididymal spermatozoa proved a decrease of the cholesterol/phospholipid rate from caput to cauda spermatozoa. This could also be achieved after in vitro fusion of caput spermatozoa with epididymosomes. Therefore, it is postulated, that this modification is a result of the interaction from spermatozoa with epididymosomes. In vivo it was documented that the concentration of PE, PC and SM decreased in spermatozoa during passing through the epididymis, whereby in vitro no alterations were etermined. After labeling the epididymosomes with Biotin, a transfer of biotin-labeled proteins of epididymosomes on epididymal spermatozoa membrane could be shown by in vitro fusion. In fact the Ca2+-ATPase activity increased significantly after in vitro fusion of epididymosomes with spermatozoa. Nevertheless, transfer of PMCA4a could not be clarified definitively by immuncytology, because the antibody against PMCA4a reacted deficient on spermatozoa smear. Using the antibody against PMCA4 a slight increase of fluorescence was shown in the middle piece of spermatozoa tail. Furthermore, previous work of our group showed that the Ca2+-ATPase activity of epididymal spermatozoa is stimulated organ-specific by the seminal vesicle major protein PDC-109 respectively (no significant stimulation in heart and kidney) (Sanchez-Luengo et al. 2004). In this study, this stimulation could also be documented for epididymosomes and spermatozoa after in vitro fusion. It is well-known that PDC-109 binds on phospholipids and that the PMCA activity can be affected by phospholipids. Further analyses have to display potential correlation between lipid modification and the increase of Ca2+-ATPase activity in spermatozoa after in vitro fusion. In summary, the trans secretion (cell-to-cell transfer) between epididymosomes and spermatozoa resulted in a disposition of lipids and proteins with influencing Ca2+-ATPase-activity.