Expression Of Prohibitin&UPS Associated Proteins In Sperm From Infertile Men

Part1Expression of Mitochondria Membrane Protein Prohibitin in Sperm from Infertile MenProhibitin (PHB), a highly conserved protein, is mainly expressed at the mitochondrial inner membrane in eukaryotic cells. It was well known that prohibitin participates in the morphogenesis of mitochondrial cristae, associates with the respiratory chain and defines functional subcompartments within mitochondria. We proposed that male infertility arised from poor sperm motility (asthenozoospermia, A) and/or low concentration (oligoasthenozoospermia, OA) may be associated with abnormalities in expression and functions of prohibitin in sperm mitochondria. Therefore, in the present study, sperms from male patients aged between30-40years old attempting intra-cytoplasmic sperm injection (ICSI)/in vitro fertilization (IVF) were collected. Their sperm motility was evaluated according to the2010World Health Organization (WHO) standards. Sperm mitochondrial membrane potentials (MITO) were also detected by flow cytometry after MitoTracker(?) Red probe staining. Finally, prohibitin protein expression was evaluated using immunofluorescence and Western blot. Here, we demonstrate that both total and progressive sperm motilities, and MITO are significantly lower in sperm from A and OA subjects than in normozoospermia (N) subjects; the decrease is more severe for OA than for A subjects. PHB expression is also significantly lower in sperm from A and OA subjects. Significantly positive correlations are found among PHB expression, MITO, and sperm motility in N, A, and OA subjects. Collectively, we concluded that PHB expression may be used as an indicator of sperm quality in infertile men. Part2Expression of UPS Associated Protein in Sperm from Infertile MenUbiquitin proteasome system (UPS) is one of the important pathways for proteolysis. It has been shown that the high ubiquitination level of human sperm closely correlates with the decline in sperm motility and concentration. However, the mechanism has not been elucidated yet. Therefore, in the present study, sperms from male patients aged between30-40years old attempting intra-cytoplasmic sperm injection (ICSI)/in vitro fertilization (IVF) were collected. Their sperm motility and concentration were evaluated according to the2010World Health Organization (WHO) standards. Western blot analysis was used to investigate expression of ubiquitin, polyubiquitinylated protein (FK1) and Lysine48-linkage specific polyubiquitin in sperm from poor sperm motility (asthenozoospermia, A) and/or low concentration (oligoasthenozoospermia, OA). Subsequencely, the differences of ubiquitin carboxy-terminal hydrolases-L3(UCH-L3) and20S proteasome in expression and enzyme activity were evaluated using Western blot and enzyme activity assay. The results of Western blot showed that ubiquitin, FK1and Lysine48-linkage specific polyubiquitin expression, especially FK1and Lysine48-linkage specific polyubiquitin expression, significantly increased in sperm from infertile men suffering from A or OA compared with those from normozoospermia (N) subject, with a higher degree of increase in OA patients. However,20S proteasome and UCH-L3expression decreased in sperm from infertile men suffering from A or OA compared with those from N subject. Enzyme activity assay showed that chymotrypsin-like activity of proteasome and catalytic activity of UCH-L3were significantly decreased in sperm from OA infertile men compared with N, however, no significant difference was found in those from A subjects. These results indicate that the high ubiquitination level in sperm from A or OA infertile men mainly displayed an increase in FK1, especially an increase in Lysine48-linkage specific polyubiquitin, which may be resulted from a decrease in the expression and enzyme activities of the20S proteasome and UCH-L3. Collectively, we concluded that the decrease in the expression and enzyme activities of the20S proteasome and UCH-L3could be one of the main cause of high ubiquitination level in sperm from A or OA infertile men.