Aim： To perform screening, related to A-kinase anchoring proteins 4 （AKAP4） and tubulin proteins, in spermatozoa with absent or severely reduced motility in order to detect the status of the fibrous sheath and the ...Aim： To perform screening, related to A-kinase anchoring proteins 4 （AKAP4） and tubulin proteins, in spermatozoa with absent or severely reduced motility in order to detect the status of the fibrous sheath and the axonemal structure. Methods： An immunocytochemical study of tubulin, used as a positive control, and AKAP4 was carded out to detect the presence and the distribution of these proteins in different sperm samples. The morphological characteristics of sperm were studied by transmission electron microscope （TEM） and the results were elaborated using a formula reported in previous studies. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse partial sequences of the Akap4 and Akap3 genes. Results： Immunolabelling of tubulin and AKAP4 showed different patterns, which led us to divide the patients into groups. In group I, the absence of AKAP4 and tubulin was revealed, although these patients did not show alterations in the Akap4/Akap3 binding site. TEM evaluation highlighted that a high presence of necrosis was associated with total sperm immotility. In group Ⅱ, a regular AKAP4 and tubulin signal was present, although motility was reduced and TEM analysis revealed the presence of immaturity. In group Ⅲ, in which a weak AKAP4 label associated with normal tubulin staining and reduced motility was observed, a severe disorganization of the fibrous sheath was highlighted by TEM. Conclusion： While the role of AKAP4 in sperm motility is unclear, absent or weak AKAP4-1abelling seems to be associated with absent or weak sperm motility.展开更多
Calcium-binding tyrosine phosphorylation-regulated protein （CABYR） is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath （FS） protein involved in capacitation. A put...Calcium-binding tyrosine phosphorylation-regulated protein （CABYR） is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath （FS） protein involved in capacitation. A putative domain （amino acids 12-48） homologous to the regulatory subunit of type II cAMP-dependent protein kinase A （RII） dimerisation and A kinase-anchoring protein （AKAP）-binding domains of protein kinase A at the N-terminus suggests that CABYR may self-assemble and bind to AKAPs. Moreover, there is evidence that CABYR has limited interaction with AKAPs. However, further evidence and new relationships between CABYR and other FS proteins, including AKAPs, will be helpful in understanding the basic physiology of FS. In this study, a new strategy for co-immunoprecipitation of insoluble proteins, as well as the standard co-immunoprecipitation method in combination with mass spectrometry and western blot, was employed to explore the relationship between CABYR, AKAP3 and Ropperin. The results showed that AKAP3 was co.immunoprecipitated with CABYR by the anti-CABYR-A polyclonal antibody, and, conversely, CABYR was also co.immunoprecipitated with AKAP3 by the anti-AKAP3 polyclonal antibody. Another RIl-like domain containing protein, Ropporin, was also co-immunoprecipitated with CABYR, indicating that Ropporin is one of CABYR＇s binding partners. The interactions between CABYR, AKAP3 and Ropporin were confirmed by yeast two-hybrid assays. Further analysis showed that CABYR not only binds to AKAP3 by its RII domain but binds to Ropporin through other regions besides the RIl-like domain. This is the first demonstration that CABYR variants form a complex not only with the scaffolding protein AKAP3 but also with another Rll-like domain-containing protein in the human sperm FS.展开更多
The development of cancer is a complex process that requires the participation of many factors,including mutations in genes, regulation of signaling pathways, disruption of homeostasis, and failure of self-monitoring ...The development of cancer is a complex process that requires the participation of many factors,including mutations in genes, regulation of signaling pathways, disruption of homeostasis, and failure of self-monitoring mechanisms. Sufficient evidence has展开更多
The sperm DNA fragmentation index(DFI)is a metric used to assess DNA fragmentation within sperm.During in vitro fertilizationembryo transfer(IVF-ET),high sperm DFI can lead to a low fertilization rate,poor embryo deve...The sperm DNA fragmentation index(DFI)is a metric used to assess DNA fragmentation within sperm.During in vitro fertilizationembryo transfer(IVF-ET),high sperm DFI can lead to a low fertilization rate,poor embryo development,early miscarriage,etc.A kinase anchoring protein(AKAP)is a scaffold protein that can bind protein kinase A(PKA)to subcellular sites of specific substrates and protects the biophosphorylation reaction.Sperm protein antigen 17(SPA17)can also bind to AKAP.This study intends to explore the reason for the decreased fertilization rate observed in high sperm DFI(H-DFI)patients during IVF-ET.In addition,the study investigates the expression of AKAP,protein kinase A regulatory subunit(PKARIl),and SPA17 between H-DFI and low sperm DFI(L-DFI)patients.SPA17 at the transcriptional level is abnormal,the translational level increases in H-DFI patients,and the expression of AKAP4/PKARIl protein decreases.H,O,has been used to simulate oxidative stress damage to spermatozoa during the formation of sperm DFI.It indicates that H,O,increases the expression of sperm SPA17 protein and suppresses AKAP4/PKARIl protein expression.These processes inhibit sperm capacitation and reduce acrosomal reactions.Embryo culture data and IVF outcomes have been documented.The H-DFI group has a lower fertilization rate.Therefore,the results indicate that the possible causes for the decreased fertilization rate in the H-DFI patients have included loss of sperm AKAP4/PKARIl proteins,blocked sperm capacitation,and reduced occurrence of acrosome reaction.展开更多
文摘Aim： To perform screening, related to A-kinase anchoring proteins 4 （AKAP4） and tubulin proteins, in spermatozoa with absent or severely reduced motility in order to detect the status of the fibrous sheath and the axonemal structure. Methods： An immunocytochemical study of tubulin, used as a positive control, and AKAP4 was carded out to detect the presence and the distribution of these proteins in different sperm samples. The morphological characteristics of sperm were studied by transmission electron microscope （TEM） and the results were elaborated using a formula reported in previous studies. PCR was carried out on DNA extracted from peripheral blood lymphocytes to analyse partial sequences of the Akap4 and Akap3 genes. Results： Immunolabelling of tubulin and AKAP4 showed different patterns, which led us to divide the patients into groups. In group I, the absence of AKAP4 and tubulin was revealed, although these patients did not show alterations in the Akap4/Akap3 binding site. TEM evaluation highlighted that a high presence of necrosis was associated with total sperm immotility. In group Ⅱ, a regular AKAP4 and tubulin signal was present, although motility was reduced and TEM analysis revealed the presence of immaturity. In group Ⅲ, in which a weak AKAP4 label associated with normal tubulin staining and reduced motility was observed, a severe disorganization of the fibrous sheath was highlighted by TEM. Conclusion： While the role of AKAP4 in sperm motility is unclear, absent or weak AKAP4-1abelling seems to be associated with absent or weak sperm motility.
文摘Calcium-binding tyrosine phosphorylation-regulated protein （CABYR） is a highly polymorphic calcium-binding tyrosine- and serine-/threonine-phosphorylated fibrous sheath （FS） protein involved in capacitation. A putative domain （amino acids 12-48） homologous to the regulatory subunit of type II cAMP-dependent protein kinase A （RII） dimerisation and A kinase-anchoring protein （AKAP）-binding domains of protein kinase A at the N-terminus suggests that CABYR may self-assemble and bind to AKAPs. Moreover, there is evidence that CABYR has limited interaction with AKAPs. However, further evidence and new relationships between CABYR and other FS proteins, including AKAPs, will be helpful in understanding the basic physiology of FS. In this study, a new strategy for co-immunoprecipitation of insoluble proteins, as well as the standard co-immunoprecipitation method in combination with mass spectrometry and western blot, was employed to explore the relationship between CABYR, AKAP3 and Ropperin. The results showed that AKAP3 was co.immunoprecipitated with CABYR by the anti-CABYR-A polyclonal antibody, and, conversely, CABYR was also co.immunoprecipitated with AKAP3 by the anti-AKAP3 polyclonal antibody. Another RIl-like domain containing protein, Ropporin, was also co-immunoprecipitated with CABYR, indicating that Ropporin is one of CABYR＇s binding partners. The interactions between CABYR, AKAP3 and Ropporin were confirmed by yeast two-hybrid assays. Further analysis showed that CABYR not only binds to AKAP3 by its RII domain but binds to Ropporin through other regions besides the RIl-like domain. This is the first demonstration that CABYR variants form a complex not only with the scaffolding protein AKAP3 but also with another Rll-like domain-containing protein in the human sperm FS.
基金supported by the grants from the Outstanding Academic Leaders Plan of Shanghai [Grant No.2018BR07]the National Science Foundation of China [Grant No.81572061]+1 种基金Program for Young Medical Technicians(Clinical Examination)in Shanghai [2016]05Shanghai Health and Family Planning Commission Clinical Research Project for Health Industry [Grant No.20184Y0199]
文摘The development of cancer is a complex process that requires the participation of many factors,including mutations in genes, regulation of signaling pathways, disruption of homeostasis, and failure of self-monitoring mechanisms. Sufficient evidence has
基金This study was supported by Hebei Natural Science Foundation(H2022206019)Science and Technology(S and T)Program of Hebei(21377721D)+1 种基金Hebei Province Medical Technology Tracking Project(GZ2021028)Medical Science Research Project of Hebei Province(20170084,and 20211494).
文摘The sperm DNA fragmentation index(DFI)is a metric used to assess DNA fragmentation within sperm.During in vitro fertilizationembryo transfer(IVF-ET),high sperm DFI can lead to a low fertilization rate,poor embryo development,early miscarriage,etc.A kinase anchoring protein(AKAP)is a scaffold protein that can bind protein kinase A(PKA)to subcellular sites of specific substrates and protects the biophosphorylation reaction.Sperm protein antigen 17(SPA17)can also bind to AKAP.This study intends to explore the reason for the decreased fertilization rate observed in high sperm DFI(H-DFI)patients during IVF-ET.In addition,the study investigates the expression of AKAP,protein kinase A regulatory subunit(PKARIl),and SPA17 between H-DFI and low sperm DFI(L-DFI)patients.SPA17 at the transcriptional level is abnormal,the translational level increases in H-DFI patients,and the expression of AKAP4/PKARIl protein decreases.H,O,has been used to simulate oxidative stress damage to spermatozoa during the formation of sperm DFI.It indicates that H,O,increases the expression of sperm SPA17 protein and suppresses AKAP4/PKARIl protein expression.These processes inhibit sperm capacitation and reduce acrosomal reactions.Embryo culture data and IVF outcomes have been documented.The H-DFI group has a lower fertilization rate.Therefore,the results indicate that the possible causes for the decreased fertilization rate in the H-DFI patients have included loss of sperm AKAP4/PKARIl proteins,blocked sperm capacitation,and reduced occurrence of acrosome reaction.
