Effect Of Transient Scrotal Hyperthermia On Human Sperm:An ITRAQ Based Proteomic Analysis

OBJECTIVE Through this prospective study,we aimed to find out the key sperm proteins which caused spermatogenesis disorder by the differential proteomics methods.METHODS The ten volunteers received testicular wild warming in a 43°C water bath for 30 min each day and lasted 10 days.Semen samples were taken before and after bathing sixth week when the sperm density and mobility were lowest.By the iTRAQ and 2DLC-MS/MS technique,and Quick GO,KEGG Pathway databases,the enrichment analysis of molecular functions,biological processes and metabolic pathways were obtained.With the help of STRING database,the network diagram of the differential proteins were acquired.Then the expression of ODF2,ODF1 and ACTRT2 protein in mice sperm were verified by Western blot,and the function of them were further explored by immunofluorescence and histochemistry.RESULTS 1.A total of 3446 trusted proteins were identified by FDR ? 1.0%,and one or more peptides with more than 95% confidence.2.61 differentially expressed proteins were found,of which 28 proteins were up-regulated,and 33 proteins were down-regulated.3.The 61 kinds of differential proteins were involved in 367 biological processes,including sexual reproduction,fertilization,gametogenesis,sperm motility,cell development,sperm differentiation,RAN positioning and so on;they were also mainly involved in the molecular functions of nucleic acid binding,dynamic protein binding,non-folded protein binding,activation of nucleotide activity and others;With KEGG metabolic pathway annotation classification,there are 19 different proteins involved in mRNA splicing,carbohydrate digestion and absorption,glucose production,fructose,and mannose metabolism,Thyroid hormone synthesis and other metabolic pathways.4.The expression of ODF2,ODF1 and ACTRT2 in mouse sperm by the Western blot were consistent with the results of proteomics.5.The proteins,ODF2 and ODF1,were located in the tail of mouse sperm,and did not change the position after heat shock treatment.In mouse testes,ACTRT2 and ODF1 were localized in spermatocytes,sertoli cells and round spermatozoa.ODF2 was localized in round spermatozoa and sperm,without the change of location after heat shock too.CONCLUSION 1.According to the proteomics result,we found that 26 proteins of the 61 differential proteins formed two independent interactions,and the remaining 35 proteins did not participate any interaction.The 61 proteins were mainly composed of intracellular organelles,ribosome protein complex and flagellum,participating in sexual reproduction,fertilization,sperm motility and cell development and other biological processes,consisting of nucleic acid and protein binding molecular function;but the effect of the heat shock on sperm still need to be further studied 2.The down-regulation of ODF2,ODF1 and ACTRT2 protein is related to the spermatogenesis,which is expected to be a new target for the diagnosis and treatment of male infertility.