A variety of glycosylphosphatidylinositol （GPI）-linked proteins are acquired on spermatozoa from epididymal luminal fluids （ELF） during sperm maturation. These proteins serve roles in immunoprotection and in key s...A variety of glycosylphosphatidylinositol （GPI）-linked proteins are acquired on spermatozoa from epididymal luminal fluids （ELF） during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles （epididymosomes, EP） which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane （TM） proteins, including plasma membrane Ca^2＋-ATPase 4 （PMCA4）. Synthesized in the testis, PMCA4 is an essential protein and the major Ca^2＋ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4＇s interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca^2＋-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.展开更多
Prohibitin(PHB),an evoluti on arily con served mitochondrial inner membra ne protein,is highly expressed in cells that require strong mitoch on drial function.Recently,we dem on strated that the deleti on of Phb in sp...Prohibitin(PHB),an evoluti on arily con served mitochondrial inner membra ne protein,is highly expressed in cells that require strong mitoch on drial function.Recently,we dem on strated that the deleti on of Phb in spermatocytes results in impaired mitochondrial function.In addition,PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels,but a positive one with mitochondrial membrane potential and sperm motility.These results suggest that mitochondrial PHB expression plays a role in sperm motility.However,the mechanism of PHB-mediated regulation of sperm motility remai ns unk nown.Here,we dem on strate for the first time that PHB interacts with protei n kinase B(AKT)and exists in a complex with phospho-PHB(pT258)and phospho-AKT in the mitochondrial sheath of murine sperm,as determined using colocalization and coimmunoprecipitation assays.After blocking AKT activity using wortmannin(a phosphatidylinositol 3-kinase[PI3K]inhibitor),murine sperm have significantly(P<0.05)decreased levels of phospho-PHB(pT258)and the total and progressive motility.Furthermore,significantly(P<0.05)lower levels of phospho-PI3K P85 subunit a+γ(pY199 and pY46)and phospho-AKT(pS473;pT308)are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with no rmospermic subjects,which suggest a reduced activity of the PI3K/AKT pathway in these in fertile subjects.Importantly,these sperm from infertile subjects also have a significantly(P<0.05)lower level of phospho-PHB(pT258).Collectively,our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility,where PHB phosphorylation(pT258)level and PI3K/AKT activity are key regulatory factors.展开更多
Titanium dioxide(TiO,)nanoparticles(TNPs)are widely used commercially and exist in a variety of products.To determine if anatase TNPs(ATNPs)in doses smaller than previously used reach the scrotum after entry in the bo...Titanium dioxide(TiO,)nanoparticles(TNPs)are widely used commercially and exist in a variety of products.To determine if anatase TNPs(ATNPs)in doses smaller than previously used reach the scrotum after entry in the body at a distant location and induce sperm defects,100%ATNP(2.5 or 5 mg kg'body weight)was administered intraperitoneally to adult males for three consecutive days,followed by sacrifice 1,2,3,or 5 weeks later(long-)or 24,48 or 120 h(short-term exposure).Transmission electron microscopy revealed the presence of ANTP in scrotal adipose tissues collected 120 h postinjection when cytokine evaluation showed an inflammatory response in epididymal tissues and fluid.At 120 h and up to 3 weeks postinjection,testicular histology revealed enlarged interstitial spaces.Significantly increased numbers of terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling-positive(apoptotic)germ(P=0.002)and interstitial space cells(P=0.04)were detected in treated males.Caudal epididymal sperm from the short-term,but not a long-term,arm showed significantly(P<0.001)increased frequencies of flagellar abnormalities,excess residual cytoplasm(ERC),and unreacted acrosomes in treated versus controls(dose-response relationship).A novel correlation between ERC and unreacted acrosomes was uncovered.At 120 h,there were significant decreases in hyperactivated motility(P<0.001)and mitochondrial membrane potential(P<0.05),and increased reactive oxygen species levels(P<0.00001)in treated versus control sperm.These results indicate that at 4-8 days postinjection,ANTP induce structural and functional sperm defects associated with infertility,and DNA damage via oxidative stress.Sperm defects were transient as they were not detected 10 days to 5 weeks postinjection.展开更多
文摘A variety of glycosylphosphatidylinositol （GPI）-linked proteins are acquired on spermatozoa from epididymal luminal fluids （ELF） during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm-egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles （epididymosomes, EP） which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane-free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI-anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis-driven studies have shown that EP from several mammals carry transmembrane （TM） proteins, including plasma membrane Ca^2＋-ATPase 4 （PMCA4）. Synthesized in the testis, PMCA4 is an essential protein and the major Ca^2＋ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion-competent sites on membranes. On the basis of knowledge of PMCA4＇s interacting partners a number of TM and membrane-associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca^2＋-dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis-driven approach.
基金This project was funded by grants from the National Natural Science Foundation of China(No.81270738)and the Major State Basic Research Development Program of China(No.2014CB943104).
文摘Prohibitin(PHB),an evoluti on arily con served mitochondrial inner membra ne protein,is highly expressed in cells that require strong mitoch on drial function.Recently,we dem on strated that the deleti on of Phb in spermatocytes results in impaired mitochondrial function.In addition,PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels,but a positive one with mitochondrial membrane potential and sperm motility.These results suggest that mitochondrial PHB expression plays a role in sperm motility.However,the mechanism of PHB-mediated regulation of sperm motility remai ns unk nown.Here,we dem on strate for the first time that PHB interacts with protei n kinase B(AKT)and exists in a complex with phospho-PHB(pT258)and phospho-AKT in the mitochondrial sheath of murine sperm,as determined using colocalization and coimmunoprecipitation assays.After blocking AKT activity using wortmannin(a phosphatidylinositol 3-kinase[PI3K]inhibitor),murine sperm have significantly(P<0.05)decreased levels of phospho-PHB(pT258)and the total and progressive motility.Furthermore,significantly(P<0.05)lower levels of phospho-PI3K P85 subunit a+γ(pY199 and pY46)and phospho-AKT(pS473;pT308)are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with no rmospermic subjects,which suggest a reduced activity of the PI3K/AKT pathway in these in fertile subjects.Importantly,these sperm from infertile subjects also have a significantly(P<0.05)lower level of phospho-PHB(pT258).Collectively,our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility,where PHB phosphorylation(pT258)level and PI3K/AKT activity are key regulatory factors.
文摘Titanium dioxide(TiO,)nanoparticles(TNPs)are widely used commercially and exist in a variety of products.To determine if anatase TNPs(ATNPs)in doses smaller than previously used reach the scrotum after entry in the body at a distant location and induce sperm defects,100%ATNP(2.5 or 5 mg kg'body weight)was administered intraperitoneally to adult males for three consecutive days,followed by sacrifice 1,2,3,or 5 weeks later(long-)or 24,48 or 120 h(short-term exposure).Transmission electron microscopy revealed the presence of ANTP in scrotal adipose tissues collected 120 h postinjection when cytokine evaluation showed an inflammatory response in epididymal tissues and fluid.At 120 h and up to 3 weeks postinjection,testicular histology revealed enlarged interstitial spaces.Significantly increased numbers of terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labeling-positive(apoptotic)germ(P=0.002)and interstitial space cells(P=0.04)were detected in treated males.Caudal epididymal sperm from the short-term,but not a long-term,arm showed significantly(P<0.001)increased frequencies of flagellar abnormalities,excess residual cytoplasm(ERC),and unreacted acrosomes in treated versus controls(dose-response relationship).A novel correlation between ERC and unreacted acrosomes was uncovered.At 120 h,there were significant decreases in hyperactivated motility(P<0.001)and mitochondrial membrane potential(P<0.05),and increased reactive oxygen species levels(P<0.00001)in treated versus control sperm.These results indicate that at 4-8 days postinjection,ANTP induce structural and functional sperm defects associated with infertility,and DNA damage via oxidative stress.Sperm defects were transient as they were not detected 10 days to 5 weeks postinjection.
