The Molecular Signal And Mechanism Of Anti-Human Sperm Antibodies With Application In Biological Targeting

Objective:Bioinformatics analysis of special human sperm membrane protein was chosen as antigen,and Artificial polypeptides with immunogenicity were synthesized to immunize animals and obtain antibodies.Conjugated antisperm antiodies with Glod Nanoparticles（GNPs）,a new type of spermicide of antisperm antibody Immuno-targeting GNPs was prepared in the study.Meanwhile,the study of mechanism of the anti-sperm antibody working on human mature spermatozoa was completed by means of analysis of the resultes of difference of mRNA profile from processing the Hight-throughput sequencing in order to provide the theoretical basis for the study of immunological infertility and male contraception.Methods:Using the NCBI and Uniprot database with key words a local database in human sperm membrane protein was established and special human sperm membrane protein was filtered by SignalP 4.1 Server、TMHMM and ExPASY-ProtScale software.The polypeptide synthesized was cross-linked KLH（Keyhole Limpet Hemocyanin）in order to increase the immunogenicity of the polypeptide.After the polypeptide was immunized New Zealand rabbits,the rabbit blood serunl was collected and the polyclone antibodies in the rabbit serunl were purified by ammonium sulfate precipitation.The Antibody titers were measured by ELISA and antibody specificity was verified by immunofluorescence and Western blot.GNPs were prepared with a reduction of chloroauric acid by sodium citrate.Immuno-targeting GNPs were linked anti-sperm antibody to observe the spermicidal effect in vitro.mRNA was extracted from human sperm incubated with SPACA₃,SPAG₈ and OR1D2 antibodies,respectively.The quality of a RNA library and the result of whole genome sequencing for Agilent 2500 were tested.All of the differentially expressed genes,GO cluster and KEGG pathway were analyzed by bioinformatics softwares.Finally,the results were verified by RT-PCR.Results:1.Mining and analysizing of protein in the local human sperm membrane protein database,the proteins of SPACA₁,SPACA₃,SPAG₈ and OR1D2 as the specificity molecules of the anthropogenic sperm membrane protein were selected for follow-up experiments.2.The highest antibody titers of SPACA₁,SPACA₃,SPAG₈ and OR1D2 were 1:1600,1:1600,1:3200 and 1:6400,respectively.The resulut of Immunofluorescence assay showed that SPACA₁ protein was mainly distributed in the acrosome area of the sperm head,SPACA₃ protein distributed in the acrosome area of sperm head and tail,SPAG₈ protein mainly in the sperm head,neck and tail,and OR1D2 protein mainly in acrosome area and equatorial segment and throughout the sperm tail.The resulut of Western blot showed SPACA₁ protein was located at 35KD;SPACA₃ protein at24KD and 14KD;SPAG₈ protein at 50KD;OR1D2 protein was in 26KD and 32KD,respectively.3.Characterization of 20 and 40nm GNPs was utilized by TEM,UV absorbable spectroscopy and particle size instrument.The carrying capacity of antibody in Immuno-Nano GNPs linked SPACA₃,SPAG₈ and OR1D2 antibody was identified by Western blotting.The result of spermicidal test in vitro,showed that the minimum spermicidal concentration of Immuno-Nano GNPs linked SPACA₃,SPAG₈ and OR1D2 antibody was 5×10^-9 mol/L,2.5×10^-9 mol/L and 1.25×10^-9 mol/L,respectively.4.The quality of whole genome sequencing for Agilent 2500 was evaluated in the study.The main peak length of cDNA library was 466bp,and Q30 ratio was greater than 90%.Compared with the control group,there were 146,110 and 90 genes up-regulated in SPACA3,SPAG8 and OR1D2 groups,and 159,256 and 192 genes were down-regulated,respectively.After analysis of differential gene expression,there were 9 up-regulated and 9 down-regulated in SPACA₃,SPAG₈ and OR1D2groups,respectively.GO cluster analysis obtained 48,52 and 51 categories in SPACA₃,SPAG₈ and OR1D2 groups,respectively.KEGG pathway analysis obtained62,117 and 126 pathways in SPACA₃,SPAG₈ and OR1D2 groups,respectively.The RT-PCR expression of PNRC2 and JPH2 was consistent with the sequencing results.Conclusion:1.The method for designing membrane protein antigen polypeptide by bioinformatics analysis was set up and the antibodies were successfully prepared depending on the method for the follow-up experiment in the study in vivo.2.With recombinant protein A-mediated technigue,the Immuno-targeting GNPs linked antibodies having the spermicidal effect were prepared in the study.3.The differential gene expression of mature spermatozoa was affected by antisperm antibodies.Up-regulated genes include JPH2,AC005863.1,AC084149.2,HNRNPUL2-BSCL2,AC108938.5,RP11-386G11.5,RPPH1,CTC-344H19.6 and PRH1-PRR4,and down-regulated genes include PNRC2,ZBTB9,SEC13P1,KRT16P6,FABP5P14,ATP6V1G2-DDX39B,AC005519.4,RP13-1032I1.10 and RP5-850E9.3.Both up and down-regulated genes may play an important role in the processing of anti-sperm antibodies.It was posssible that they were the potential markers for immune infertility diagnosis,prognosis and treatment.In addition,through the GO cluster and KEGG pathway analysis anti-sperm antibody has significant effects on reproductive development-related activities and metabolic pathways.Antisperm antibody has an effect on Metabolic pathways（hsa01100）in mature sperm.The mechanism of anti-sperm antibody in sperm may be due to multi gene involvement and multi gene regulation.