Proteomic Analysis Of White Yak Testis In Different Developmental Stages And Reproductive Candidate Gene HSP60 Biology Research

The Tianzhu White yak(Bos grunniens)is a special yak breed in our country,it is a major production and living materials for the local herdsmen,the primitive breeds has low fertility,which breeded naturally for 1 child in 2 years or 2 child in 3 years,so the production of white yak were severely restricts.Testicular as the main reproductive organs affects the reproduction of the male animal,its main function is to secrete hormones,produce sperm,these physiological processes need a variety of precision between protein expression and regulation.Therefore,the white yak testicles as the material,the differentially expressed proteins in the testis of the different development stages white yak can explain the molecular mechanism of spermatogenesis and provide reference for the white yak reproductive performance.This study aimed to explore the protein expression profiles of white yak testis at different sexual developmental stages(1,2,4,and 8 year of age).The protein profiles of yak testis were determined using two-dimensional electrophoresis and MALDI-TOF-TOF,and analyse the function and biological process and metobolic pathways on the different expression proteins.The candidate heat shock protein 60 gene(HSP60)was cloned and located on Hypothalamic-Pituitary-Testicular Axis.And analyzed the effect of cell proliferation on sertoli cells by HSP60,and the study's main results are as follows:1?Mapped the proteome of white yak testis,437 protein spots were analyzed using the PD Quest 8.0.1 software.Mass spectrometry was performed to identify those significantly differential expressed proteins,29 protein spots showed more than 1.5-fold differences(P<0.05)in at least one time point and were successfully identified.2 proteins were upregulated with age,5 proteins were downregulated with age,3 proteins were upregulated before 4 years and downregulated subsequently,15 proteins were upregulated before 2 years and downregulated later,and 4 proteins fluctuated with age.2?All the identified proteins were annotated by Blast2 GO.And discover that the identified proteins were mainly involved in “cellular process and single-organism process and metabolism process”,“organelle and cell component and extracellular domain”,“binding and catalytic activity”.Sublocation shows that 18 proteins at cytoskeleton,6 proteins at nucleus,3 proteins at mitochondrial,2 proteins at extracelluar matrix.3?The western blotting was used to confirm the results.the changing pattern of HSP60 and RGN were the same as 2-DE.It shows that the differentially expressed proteins results reliable.4?The candidate gene HSP60 was cloned,and the results showed that the full length c DNA of the HSP60 was 2 300 bp,with a ORF length of 1 722 bp,which encoded 572 amino acids,the theoretical molecular weight and isoelectric point of the protein were 60.977 k Da and 5.69,the HSP60 protein was predicted to be a type of soluble and non-transmembrane protein.The amino acid sequence similarity analysis indicated among the White yak and Bos taurus,Bos indicus,ovis aries,Pantholops hodgsonii,Camelus ferus,Ceratotherium simum simum,Oryctolagus cuniculus,Pan troglodytes were 100%?99%?99%?99%?99%?99%?98% and 98%,respectively.its show that HSP60 was high homology.5?The expressing and location of HSP60 show that HSP60 expressed broadly in hypothalamus,pituitary,testis.The expression of HSP60 in hypothalamus and pituitary were significantly higher than testis,but no significant difference between the hypothalamus and pituitary.In addition,the immunohistochemical staining analysis revealed that HSP60 expressed broadly in paraventricular nucleus magnocellular,paraventricular nucleus parvocellular and neurokeratin network in White yak hypothalamus,glandular cells in pituitary,spermatogonium,spermatocyte,sertoli cells and leydig cells in testis.It indicate that HSP60 take part in the regulation of HPT-axis and spermatogenesis.6 ? The sertoli cells were isolated for study the effect of HSP60 on cell proliferation,the over-expression vector p IRES2-EGFP-HSP60 and si RNA-HSP60 was synthesised.After transient transfection,the expression of HSP60 m RNA was up-regulated in over-expression groups and down-regulated in silent groups at 24?48?72h.The cell proliferation was detected by MTS.The results showed that the over-expression groups were significantly higher than the negative control(NC)at 24?48?72h.The silence groups were lower than NC at 24?48?72h,but the difference was not significant.The expressing of cyclin D1 gene and PCNA gene were detected by q PCR.The results showed that in over-expression groups the expression of cyclin D1 gene at 48 h was significantly higher than NC and PCNA gene at24?48?72h were significantly higher than NC.In silence groups,the expression of cyclin D1 gene and PCNA gene at 24?48?72h were lower than NC,but the difference were not significant.This study suggests that the regulation of HSP60 gene in white yak sertoli cell of cell proliferation was positive.According to the results of proteomics screening,the heat shock protein 60 in Tianzhu white yak is potentially reproductive physiological regulation,and HSP60 gene was cloned and located,the fusion protein His-HSP60 was gotten by prokaryotic expression in vitro,and study the cell proliferation of sertoli cells with the HSP60,the result show that the sertoli cells proliferation were regulated at vigorous stage of sexual function of white yak by HSP60,and influence the development of testis.This study provides the theoretical foundation for the effect of HSP60 on White yak testis development and spermatogenesis.