Cloning Of Tibetan Sheep PGAM1 And TPI1 Genes And Their Expression In Testis And Epididymis

Mammalian spermatogenesis is a complex and precise process,which is regulated by many genes and has an important impact on the reproductive performance of male animals.Phosphoglycerate mutase 1(PGAM1)and triosephosphate isomerase 1(TPI1)are key enzymes in glycolysis.Sertoli cells can provide energy substrate for the development of male germ cells through glycolysis.Therefore,exploring the expression and function of PGAM1 and TPI1 genes in male Tibetan sheep reproductive organs is of great significance to for in-depth understanding their regulatory mechanisms.In this experiment,the reproductive organs of Tibetan sheep at three critical developmental stages of before sexual maturity(3 months old),sexual maturity(1 year old)and adult(3 years old)were used as test materials.The complete CDS region sequences of PGAM1 and TPI1 genes were cloned for bioinformatics analysis.The expression levels of PGAM1 and TPI1 mRNA in the reproductive organs of Tibetan sheep were detected by qRT-PCR and Western blot methods.Immunohistochemical staining was used to detect the expression and location of PGAM1 and TPI1 proteins in the testis and epididymis tissues of Tibetan sheep at different ages.The regulatory effects of PGAM1 and TPI1 on testicular development and spermatogenesis in Tibetan sheep were studied.The main findings were as follows:1.The complete CDS region of the Tibetan sheep PGAM1 gene was 765 bp long,encoding 254 amino acids;protein structure prediction results indicated that PGAM1 was composed of α-helix(47.64%),extended strand(9.45%),β-turn(6.30%)and random coil(36.61%);the complete CDS region of the TPI1 gene was 861 bp long,encoding 286 amino acids;protein structure prediction results indicated that the TPI1 protein was composed of α-helix(47.55%),extended strand(16.78%),β-turn(5.59%)and random coil(30.07%).2.PGAM1 mRNA and protein were highly expressed in testis and epididymis of male Tibetan sheep at different development stages.PGAM1 protein mainly distributed in Sertoli cells and Leydig cells at different developmental stages,and also distributed in gonocytes and spermatogonia.In epididymal tissues,PGAM1 protein was distributed in the sperm tail of the epididymis after sexual maturity,pseudostratified columnar ciliated epithelium and loose connective tissue of epididymis at different developmental stages.The results demonstrate that PGAM1 may be mainly involved in regulating glycolytic metabolism pathway in Sertoli cells,also play a role in the migration and differentiation of gonocytes,and functional maintenance of Leydig cells.3.TPI1 mRNA and protein were expressed in testis and epididymis of male Tibetan sheep at different development stages,but mainly in testes and epididymides from 1-year-old and 3-year-old sheep.TPI1 protein was mainly distributed in Sertoli cells within testes at all stages of development,and sperm tails within epididymides from post-puberty(1-year-old and 3-year-old).In addition,weak TPI1 protein signals were present in Leydig cells,spermatogonia and spermatocytes from testes,and pseudostratified columnar ciliated epitheliums and loose connective tissues from epididymides.These results demonstrate that TPI1 gene may be involved in the regulation of glycolysis in Sertoli cells and sperm flagella,as well as the secretory function of pseudostratified columnar ciliated epithelium of epididymis,which in turn promotes the formation and maturation of sperms.The results suggest that PGAM1 and TPI1 gene both have high homology with other mammals.They may regulate the formation and maturation of spermatozoa mainly through glycolysis metabolic pathway,but their regulatory mechanisms need to be further explored.