Comparative Proteomic Analysis Of Prepuberty And Puberty Of Buffalo Testicular Sertoli Cells

Buffalo is one of the important economic species in the subtropical zone.It is an excellent germplasm resource for both milk and meat.The testis is the most important reproductive organ of male animals,and the Sertoli cells are the important component of the seminiferous tubules.Prepubertal（3-6 months）buffalo seminiferous tubules only contain Sertoli cells and spermatogonias,and there is no complete spermatogenesis function.At this stage,Sertoli cells mainly carry out their own proliferation.Pubertal（2-3 years old）buffalo seminiferous tubules are composed of Sertoli cells and various levels of spermatogenic cells,which carry out continuous spermatogenesis.At this stage,Sertoli cells mainly maintain the normal progress of spermatogenesis.In this study,a two-step enzymatic method combined with differential adherence method was used to separate and purify the cultured buffalo testicular Sertoli cells of prepuberty and puberty.Using iTRAQ（Isobaric tags for relative and absolute quantitation）label combined with liquid chromatography-tandem mass spectrometry（LC-MS/MS）technology to quantify and identify the proteomes of two buffalo testicular Sertoli cells at different developmental stages,and comparing the differences in composition and expression levels of proteins.In order to select important proteins related to the function of buffalo testicular Sertoli cells and spermatogenesis,and deepen the understanding of the growth and development mechanism of Sertoli cells,and provide a theoretical basis for further research on the effect of Sertoli cells on spermatogenesis.The results were shown as follows:1.Isolation,purification and identification of buffalo testicular Sertoli cells.In order to identify the specificity of WT1,immunohistochemical fluorescence analysis was performed on it.The result showed that the WT1 positive cells were distributed on the nucleus of the cells near the basement membrane in the seminiferous tubules of the testis,and the positioning result was consistent with the distribution of Sertoli cells in the testis.A two-step enzymatic digestion method was used to obtain the Sertoli cells-germ cells suspension liquid,and four differential adhesion methods were used to purify the Sertoli cells.It was found that the Sertoli cells were cultured with method a which is in 37℃,5%CO₂incubator for 4 hours and then suspended cells were removed,continuing to culture cells at 37℃for the best purification effect.In order to obtain high-purity Sertoli cells,the Sertoli cells combined with method a were subcultured,and immunofluorescence staining showed that the positive rate of WT1 of Sertoli cells in the 4th generation between prepuberty and puberty reached more than 95%.2.Using iTRAQ label combined with LC-MS/MS technology to quantify and identify buffalo testicular Sertoli cell proteins in the prepuberty and puberty.The results showed that a total of 1908 proteins were identified.Using the prepubertal buffalo testicular Sertoli cells as the control group,and the pubertal buffalo testicular Sertoli cells as the experimental group,a total of 411differentially expressed proteins（p<0.05,ratio≥1.3）were identified,including116 up-regulated proteins and 295 down-regulated proteins.GO analysis showed that differential proteins in biological process were significantly enriched in RNA processing,RNA splicing and m RNA metabolic process.Mainly located in intracellular non-membrane-bounded organelle,non-membrane-bounded organelle and organelle.The most significantly enriched domain is RNA recognition motif domain.KEGG analysis showed that the spliceosome signaling pathway was the most significantly enriched in differential proteins.STRING analysis showed that the interaction relationships between differential proteins were complex.Among them,10 proteins such as H2B played a central role in the protein interaction network.Using Western blot to verify the proteomics data.Western blot confirmed that histone H2B and Heat shock-related 70 KDa protein 2（HSPA2）changes in expression were consistent with the results of mass spectrometry;immunofluorescence analysis found that H2B was located in the nucleus of the Sertoli cells,and HSPA2 was located in the cytoplasm of the Sertoli cells,consistent with their functional positioning.In summary,this study successfully isolated buffalo testicular Sertoli cells.Using iTRAQ label combined with LC-MS/MS technology to establish the differential protein expression profile of buffalo testicular Sertoli cells during prepuberty and puberty for the first time.This study provided a new perspective to clarify the molecular mechanism of Sertoli cells in the process of spermatogenesis,and provided a reference for exploring the regulatory mechanism of Sertoli cells in the process of testicular development.