| The yak(Bos grunniens)is one of the unique rare cattle species in the Qinghai Tibet Plateau and its adjacent areas,which has special significance for the local economic development and the life of residents.However,the slow growth rate and low fecundity of yaks affect the industrial development and economic growth of local residents.Studies have shown that the growth,development and reproduction of mammals are controlled and regulated by hypothalamic pituitary gonadal axis(HPG)tissues and various factors secreted by them.The role of the HPG axis in determining survival and reproductive strategies is vulnerable to multiple signals,including insulin like growth factor 2(IGF2)and androgen receptor(AR).However,the expression of IGF2 and AR in the reproductive axis of male yak and the mechanism involved in regulating male reproductive metabolism are not completely clear.To explore the regulatory role of IGF2 and AR in the development of HPG shafting and testes in male yaks.In this study,the reproductive axis tissues of adult(4 years old)male yaks and testis tissues of yaks of different ages(2,4,6 and 8 years old)were collected to observe their histomorphological characteristics by hematoxylin eosin staining(H&E),and immunohistochemical staining(IHC),quantitative real time fluorescence PCR(q RT-PCR),western blotting(WB)and Immunofluorescence staining(IF)was used to study the distribution characteristics and expression patterns of IGF2 and AR proteins and genes in yak HPG axis tissues and testis tissues of different ages,and the correlation between IGF2 and AR and androgen metabolism was analyzed and prediction based on bioinformatics.The research results are as follows:1.The HPG axis of adult male yak has complete structure and clear cell morphology.The positive expression of IGF2 and AR protein was mainly distributed in pyramidal cells of hypothalamus,basophils of adenohypophysis,interstitial cells of testis,main cells and basal cells of epididymis.IGF2 and AR m RNA and protein were differentially expressed in HPG axis tissues of yaks,and the abundance of IGF2 and AR expression in gonad tissues was significantly higher than that in hypothalamus and pituitary.2.The seminiferous tubule lumen of 2～6 year-old yak testis becomes larger,more closely arranged,and the number of interstitial cells increases with age.The seminiferous tubule of 8 year-old yak testis becomes sparse,loose,and the number of interstitial cells decreases.IGF2 and AR immunohistochemical positive expression and IF signal in testis tissues of yaks of different ages are strongest in interstitial cells,and are associated with steroid synthase 3β-HSD co expression.The expression level of m RNA and protein increased gradually with testicular development and maturation(2～6 years old);When testicular function declined(8 years old),The expression of IGF2 and AR decreased at both transcription and translation levels.3.The enrichment analysis results of Gene Ontology and KEGG pathway showed that there were 42 GO items and 37 path secondary classification items(P < 0.05 and FDR <0.05)with significant differences in the participation of IGF2 and AR;It includes 7 items related to reproduction.Predictive analysis of IGF2 and AR transcriptional regulation found that AR has six binding sites on IGF2 gene promoter,suggesting that AR has the potential to directly regulate IGF2 transcription through these promoter binding sites.The co localization results of immunofluorescence showed that IGF2 and AR signals were co expressed in yak leydig cells.In conclusion,the expression of IGF2 and AR is positively related to the development and maturity of yak testes,and is regulated by HPG axis.The dynamic changes of IGF2 during testis growth and development may play an important role in AR mediated androgen metabolism and regulation of yak reproduction.The above results will provide data reference and theoretical basis for further research on the mechanism of IGF2 and AR in yak reproductive regulation. |
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