Application Of Screening Of Sterility Associated Proteins Using Co-immunoprecipitation Technology And Mass Spectrometric Analysis And Preliminary Verification

Infertility affects about 15%-20% of reproductive-age couples worldwide,and it is not clear about etiology of infertility,diagnosis and treatment.To reveal the mechanisms of immunological infertility,the method of co-immunoprecipitation(CO-IP)and liquid chromatogram mass/mass(LC-MS/MS)was used to screen sperm membrane proteins which interacting with antisperm antibodies(ASA).We used software analyze the biological function of identified proteins.These proteins may be valuable as an indicator in the clinical diagnosis and monitoring treatment of infertility.The disease group from cryptogenic infertile patients were screened ASA positive by ELISA and conformed with mixed antiglobulin reaction(MAR).The healthy controls were serum samples which ASA-negative and already possessed healthy offspring.Spermatozoa samples from 48 donors with normal sperm parameters.The purified human sperm membrane proteins were then mixed with serum from disease group(positive for ASA)and control group(not containing ASA).The binding proteins of antisperm antibodies were enriched using CO-IP assay.The immunoprecipitates were separated on Sodium dodecyl sulfate-polyactylamide gel(SDS-PAGE),then the binding proteins were cut from the gel and analyzed by LC-MS/MS after the enzymolysis.These proteins could be identified as definition,biological function(s)and subcellular localization with Uniprot database,GO and pathway annotation analysis with David datebase.The serum samples from infertile persons(39 females and 17 males)were screened ASA positive by ELISA and conformed with MAR.The healthy controls(17 females and 13 males)were ASA-negative in ELISA.One hundred and seven proteins were obtained from the study and these could be divided into three groups,13 antigens detected by control serum samples only,14 antigens recognised by both infertile patients and control sera and 80 antigens specific for patients with ASA.Using software analyze biological function of these identified proteins.Fifteen novel sperm membrane proteins discovered with co-immunoprecipitation coupled LC-MS/MS analysis could be referred as male immunoinfertility-related antigens.These 15 proteins are sperm cation channel protein 1(CatSper1),sperm cation channel protein 3,sperm cation channel protein 4,sperm associated antigen 9,apolipoprotein A-I(APOA-I),dynein heavy chain 14,cylicin-2,izumo sperm-egg fusion protein 4,thioredoxin domain-containing protein 2,IQ domain-containing protein H,IQ domain-containing protein F1,spermatogenesis-associated protein 5,spermatogenesis-associated protein 5-like protein 1,sperm acrosome membrane-associated protein 1,E3 ubiquitin-protein ligase RNF 114.Two of all identified proteins(APOA-I and CatSper1)were validated using Western immunoblotting technique and Immunofluorescence analysis.After the sperm treated with the anti-APOA-I antibody and anti-CatSper1 antibody,using transmission electron microscope to observe sperm microstructure changes and computer-assited semen analysis to observe changes of sperm motility.Fifteen proteins discovered with CO-IP technology and LC-MS/MS analysis could be referred as male immunoinfertility-related antigens and they may hold the great importance in revealing the secret of immunological infertility.Two of all identified proteins(APOA-I and CatSper1)were proved to be contained in human sperm protein.After the sperm treated with the anti-Apolipoprotein A-I antibody and anti-CatSper1 antibody,the results of the images of electron microscope show changes of sperm head membrane and tail mitochondria microstructure and computer-assited semen analysis show the reduction of sperm motility.The identified protein may be helpful to understand the molecular basis of fertilization,better diagnose/treat immunoinfertility and develop immunocontraceptive methods.