布莱凯特黑牛大、小睾丸转录组分析及繁殖相关基因筛选OA北大核心CSTPCD

Testis size is significantly related to semen quality and count in mammals,and male fertility can be predicted by testicular size in adulthood.In order to explore key candidate genes affecting bull fertility in differently testicular sizes,peripheral blood and testis of Blackette black cattle(Bos taurus)of 12 months old were collected and divided big testis and small testis groups according to testicular weight,long diameter and short diameter for hormone determination,histological study of testis and transcriptomic sequencing in this study.The differential expression multiplier value|log2(Fold change)|≥1 and P-value≤0.01 were used as the conditions for selecting the differentially expressed genes(DEGs)and were subjected to Gene Ontology(GO)and KEGG analysis.Clone,qPCR,Western blot and immunohistochemistry(IHC)methods were used to detect and locate for candidate gene.The results showed that there were more testicular leydig cells and the development of spermatogenic tubules was better in big group.The concentrations of testosterone and estradiol of reproductive hormones in big testis were significantly higher than those in small testis group(P＜0.05).A total of 1 006 DEGs were obtained from the transcriptome results,297 genes were up-regulated and 709 genes were down-regulated in small testis.Functional analysis showed that the DEGs was enriched in cAMP and mitogen-activated protein kinase(MAPK)signaling pathways and played a role in reproduction and development;9 reproduction-related candidate genes were screened,such as dual specificity phosphatase 4(DUSP4),heat shock 70 kD protein 1-like(HSPA1L)and mitogen activated protein kinase kinase kinase kinase 4(MAP4K4).Further study on MAP4K4 gene and protein showed that the expression of MAP4K4 in big testis was significantly lower than that in small testis(P＜0.05).IHC found that MAP4K4 protein was highly expressed in sperm cells and spermatocytes and was mainly expressed in cytoplasm.Inhibition of MAP4K4 expression might promote spermatocyte proliferation and facilitate spermatoblast survival.This work provides reference for further understanding of the relationship between testis size and reproductive function in cattle.