Histological Study Of Testes And Identification Of Genes Associated With Spermatogenesis In Tibetan Sheep And Hu Sheep

Testes are highly differentiated tissues in male mammals that ensure normal reproduction by producing sperm and secreting androgens.This process is influenced by many factors,but is fundamentally regulated by a large number of protein-coding genes.Therefore,it is important to study the transcription and functional characteristics of genes involved in testicular development and spermatogenesis of sheep.At present,there have been no reports on testicularly differentially expressed genes among sheep breeds with different fecudity,so it is significant to carry out this research.In addition,the mechanism of the fecundity difference between Tibetan sheep and Hu sheep can also be explored at the molecular level.In this study,the physical morphology,histological parameters and the contents of reproductive hormone(FSH and T)in testis of Tibetan sheep and Hu sheep were compared by means of testicular morphological index determination,HE staining,Masson staining and ELISA.The combined analysis of transcriptome sequencing(RNA-Seq)and bioinformatics was used to identify the differentially expressed genes,new differentially expressed transcripts and variable shear events during spermatogenesis of Tibetan sheep and Hu sheep.The main results are as follows:1.Compared with Hu Sheep,Tibetan sheep had more connective tissue,sparser collagen fibers,and significantly more blood vessels in the interstitial tissue.The diameter,area and thickness of spermatogenic tubules of Tibetan sheep were significantly lower than those of Hu sheep(P <0.05),and the number of sertoli cells and spermatogenic cells at all levels were significantly lower than those of Hu sheep(P <0.01).2.A total of 466 differentially expressed genes(DEGs)were identified in the testes of Tibetan sheep and Hu sheep,among which 226 DEGs were up-regulated and 240 DEGs were down-regulated.These differentially expressed genes were mainly enriched in GO terms such as "cell process","cell" and "binding",and significantly enriched in signaling pathways such as "protein digestion and absorption","c AMP","adhesion plaque" and "p53".Seven functional genes,including COL1A1,COL1A2,GAMT,SOX9,BCL2,CLCA2 and GGT5 were screened out.3.A total of 155 new differentially expressed transcripts were identified in Tibetan sheep and Hu sheep,103 of which were up-regulated and 52 down-regulated.These differentially expressed new transcripts were significantly enriched in GO terms such as "cell process","membrane part" and "binding",and "calcium signaling pathway" and "nuclear factor-κB inflammatory signaling pathway" were also significantly enriched.4.The variable shear(AS)genes in testicular tissue of Tibetan sheep and Hu sheep were 85.00% and 86.31%.The main type of AS was transcription start site variable shear(TSS),which accounted for 44.90% in Tibetan sheep and 45.52% in Hu sheep.5.RT-q PCR was used to verify 10 DEGs and 10 differentially expressed new transcripts,and Western blot(WB)was used to verify two differentially expressed genes(BCL2 and MMP2)The expression results were consistent with those of RNA-Seq.The results showed that the physical morphology,histological parameters and main reproductive hormone contents of testis of Tibetan sheep were significantly different from those of Hu sheep,which may be one of the germplasm characteristics formed in Tibetan sheep in plateau environment.The differentially expressed genes in testicular tissue of Tibetan sheep and Hu sheep are mainly involved in biological processes such as "reproductive process","biological adhesion","reproduction","growth","cell connectivity" and "transporter activity",and some signal pathways such as "protein digestion and absorption","c AMP signal pathway","adhesion " and "p53".The differentially expressed new transcripts are mainly involved in the biological processes such as "cell process","membrane part" and "binding",and the signaling pathways such as "calcium signaling pathway" and "nuclear factor-κB inflammatory signaling pathway".These may be related to the regulation of testicular development and spermatogenesis in sheep.