Study On The Mechanism Of PGAM1-mediated Glycolytic Pathway In Sertoli Cells Of Tibetan Sheep To Regulate The Proliferation And Differentiation Of Spermatogonial Stem Cells

Tibetan sheep is one of the three major sheep breeds in China.They are found all over the Qinghai-Tibet Plateau and surrounding areas.They are the most important means of production and living for local herdsmen.They have the characteristics of strong adaptability,resistance to rough feeding,low fertility,and late sexual maturity.Among them,fecundity is one of the most important economic traits in sheep production,and early breeding of excellent rams is particularly important for genetically improving the fecundity and economic benefits of mutton sheep populations.Testicular development and spermatogenesis are the determinants of ram fecundity.The process of spermatogenesis is regulated by a variety of cells in testicular tissue.The glycolysis process of Sertoli cells can provide energy substrates for the development of spermatogenic cells.Phosphoglycerate mutase 1(PGAM1)is a key enzyme in the glycolysis process of Sertoli cells(SCs).Therefore,exploring the specific function of PGAM1 in Sertoli cells and the regulation mechanism of spermatogenic cells is a necessary condition for studying the molecular regulation mechanism of spermatogenesis,a complex biological process.This is of great significance for exploring the regulation mechanism of ram testicular development and spermatogenesis,as well as the prevention and diagnosis of reproductive disorders in breeding rams.In this study,the Sertoli cells of Tibetan sheep were cultured by in vitro cell culture technology to explore the specific function of PGAM1 in Sertoli cells,and then a co-culture system of Sertoli cells and Spermatogonial stem cells(SSCs)was constructed.Transcriptome and targeted metabolome(energy metabolism)sequencing techniques were used to explore the effect of PGAM1 on SSCs via the SCs glycolytic pathway.The main findings of the study are as follows:1.Tibetan sheep SCs were isolated by the combined digestion method of two enzymes,purified by Percoll discontinuous density gradient centrifugation combined with differential adhesion method,and cultured in vitro.si RNA and overexpression vector that interfered with PGAM1 gene were designed and synthesized,and the function of PGAM1 gene in primary Tibetan sheep SCs was studied.It was found that the cell viability of SCs in pc DNA3.1(+)-PGAM1 group was significantly higher than that in control group(P<0.05),the apoptosis rate decreased,the expression of proproliferation genes was significantly up-regulated and the expression of pro-apoptotic genes was significantly down-regulated(P<0.05),while the results in the si-PGAM1 group were just the opposite.The expression,key enzyme activities and main products of the pc DNA3.1(+)-PGAM1 group were significantly higher than those in the control group(P<0.05),and the LDH activity,ATP content,Pyruvate content and lactate secretion were also significantly higher than those in the control group(P<0.05),and the results in the si-PGAM1 group were just the opposite.It indicated that PGAM1 could promote the proliferation of sheep SCs and inhibit their apoptosis by regulating the expression of proliferation and apoptosis-related genes.At the same time,PGAM1 generates lactic acid and energy required for the growth and development of SSCs by regulating the expression of downstream genes of glycolysis,providing energy substrates for the development of SSCs,thereby ensuring the smooth progress of spermatogenesis.2.The targeted mi RNAs(mi R-3614-5p)of PGAM1 were predicted by bioinformatics software,and wild-type recombinant vectors(PGAM1-WT)and mutant recombinant vectors(PGAM1-MUT)were constructed using dual luciferase.The reporter gene was used to verify its targeting relationship.RT-q PCR and WB were used to detect that overexpression of mi R-3614-5 inhibited the expression of PGAM1 in SCs,while interfering with mi R-3614-5p promoted the expression of PGAM1,so PGAM1 was mi R-3614-Functional targets of 5p.In addition,RT-q PCR,flow cytometry,CCK-8,ELSA and other analyses found that mi R-3614-5p inhibited the proliferation of SCs and promoted their apoptosis by targeting PGAM1.It is suggested that the mi R-3614-5p/PGAM1 axis regulates the energy substrates required by SSCs at least in part by regulating the glycolytic metabolic pathway of SCs.3.Based on the regulatory effect of PGAM1 on the glycolytic metabolism of SCs,a SCs-SSCs co-culture system was constructed using mice as model animals,overexpressing and interfering with the PGAM1 gene,and performing transcriptome sequencing.458(117 down-regulated,341 up-regulated)and 409 DEGs(110 downregulated,299 up-regulated)were identified.Nine DEGs were randomly selected and verified by RT-q PCR,indicating that the results obtained by RNA-seq were reliable.KEGG pathway analysis was performed on these DEGs and found that they were mainly involved in cytokine-cytokine receptor interaction,cell adhesion molecules,PI3K-Akt signaling pathway,MAPK signaling pathway,as well as fatty acid,cholesterol,glycerophospholipid,glutamate,glutathione Signaling pathways such as the metabolism of glycerol.Further analysis of these DEGs revealed that GDNF,FGF2,SCF,SETDB1 and other genes were key genes regulating the proliferation and selfrenewal of SSCs.4.The SCs-SSCs co-culture system was constructed with mice as model animals,and the PGAM1 gene was overexpressed and interfered with,and the targeted metabolome(central carbon metabolism)was sequenced.A total of 11 differential metabolites were identified in the PGAM1 gene overexpression group.a total of 16 differential metabolites were identified in the PGAM1 gene interference group.KEGG pathway analysis of these differential metabolites found that they were mainly involved in glyoxylate and dicarboxylic acid metabolism,cofactor biosynthesis,amino acid biosynthesis,alanine,aspartate and glutamate metabolism,purine metabolism,etc.Further screening of glutamate,glutamine,threonate,leucine,alanine,lysine,serine,succinate,fumarate,phosphoenolpyruvate,ATP,ADP and AMP may be a potential biomarker for regulating the proliferation and differentiation of SSCs through glycolytic metabolism.Taken together,based on the in vitro culture of Tibetan sheep SCs,this study investigated the function of PGAM1 gene,its targeting mi RNA and its regulatory process.The regulation of PGAM1 gene on SCs proliferation,apoptosis and glycolysis pathway was clarified.The SCs-SSCs co-culture system was further established.Through RNA-seq and energy metabolism sequencing technology,some key genes and metabolic small molecules that affected the proliferation and differentiation of SSCs through the PGAM1 glycolysis metabolic pathway were screened.This study will provide a new scientific basis for the study of spermatogenesis in sheep,as well as a theoretical reference for related researches on mammalian spermatogenesis and male sterility.