| 【Objects】Bos grunniens(Yak)/ Bos cattle(cattle-yak)is a type of mammal that has long lived in the Qinghai-Tibet Plateau and has excellent adaptability to cold and oxygen deficiency.Cattle-yak is a hybrid of yak and cattle.The infertility of male catlle-yak is a problem that hinders the development of pastoral farming.As molecular chaperones,HSPs are widely involved in the growth,development,metabolism,reproduction and disease regulation of animal organisms while P53 plays an important role in the regulation of diseases and apoptosis.In this paper,different experimental techniques were used to explore the differences in the expression of HSPs in different organs of yak and cattle-yak,the effects of testicular development in different age groups,and the regulation of spermatogenesis by HSPs and P53 of yaks and cattle-yak.Therefore,our study aims at investigating the mechanisms of HSPs and P53 on male testicular development and sperm apoptosis as well as to provide reliable data and important theoretical basis for future research in yak/cattle-yak reproduction.【Methods】1.Based on the existing HSPs gene sequences of cattle or yak,primers were designed to clone the HSPs and P53 amino acid sequences of yak and cattle-yak.Bioinformatics software and network resources were used to perform bioinformatic analysis on the cloned genes and proteins,and differences of structural space were compared.2.Quantitative and qualitative detection of HSP genes and proteins in different organ tissues and testis of yak and cattle-yak using fluorescence quantitative RT-qPCR and Western blot(WB)methods.Data was analyzed using SPSS 21.0 software to determine significant differences.3.Immunohistochemical(IHC)and immunofluorescence(IF)techniques were used to localize and analyze the positive responses of HSPs in different organ tissues and testis of different age groups of yak and cattle-yak,and the optical density was measured using IPP6.0 software.The value helps to determine the strength of protein expression.【Results】1.The yak HSP40/60/90/110 gene sequence was cloned,and the evolutionary homology between HSP family members was different.There were significant differences in the expression of HSP70/90 genes and proteins in different organs of yaks,but the expression of HSP genes in different parts of cardiovascular was also significantly different(P <0.01).The HSP70/90 positive reaction was mainly observed in the lung terminal bronchiolar epithelium and cerebellar medulla,hepatocytes,cardiomyocytes,renal tubules and cerebellar Purkinje cells.2.There was significant difference in the expression of HSP70 in the uterus,ovary and fallopian tubes of yak during luteal phase,but there was no difference in the expression of HSP90.The expression of HSP70/90 was significantly higher in the lactating period of yak breast tissue than in the non-lactating period(P <0.01),and the expression of HSP70 was significantly higher than that of HSP90.The HSP70/90 positive reaction mainly concentrated on the intimal cells of the reproductive organs and the mammary epithelial cells of the mammary gland.3.The expression of HSP70/90 genes and proteins in different tissues and testis(at different developmental stages)of male yak and cattle-yak was significantly different,and the expression levels in young and adult testis were significantly higher than those in other age groups.HSP70 was highly expressed in the testis of yaks,but HSP90 was significantly higher in the testis of cattle-yak(P<0.01).HSP70/90 positive reactions are mainly present in epithelial cells,spermatogonia and Leydig cells.4.The expression of HSP27 and P53 genes and proteins were significantly different at different stages of testicular development in yak and cattle-yak,and they were both highest in adult testis.With age,HSP27 showed an upward trend in yak testicular tissue,while P53 showed up-regulated expression in cattle-yak testicular tissue.The HSP27 positive reaction was localized in yak stromal cells and spermatids in different developmental stages,whereas P53 was positively localized in primary spermatocytes and mesenchymal cells of cattle-yak.【Conclusion】1.Members of the HSP family belong to a class of conserved proteins in the process of biological evolution,and there are differences in their expression in different organs of yak.2.The expression of HSP70/90 in female yak reproductive organs has different effects on the lactation function of yak breast tissue.3.The expression of HSP70/90 in different organs of male yak was different.HSP70 was highly expressed in different stages of yak testicular development,while HSP90 was highly expressed in different stages of cattle-yak testicular development.Therefore,HSP70/90 has a close relationship with normal development of yak testes and male sterility in cattle-yak.4.HSP27 and P53 showed significant differences in gene and protein expression at different stages of yak and cattle-yak testicular development.HSP27 showed up-regulated expression with the development of yak testis,while P53 showed an upward trend with the development of cattle-yak sperm.5.HSP27/70 plays an important protective role in testis development and spermatogenesis of yaks.HSP90 and P53 can inhibit spermatogenesis and impede apoptosis of primary spermatocytes in cattle-yak,which is one of the causes of male infertility phenomenon in cattle-yak. |
