Study On Enhancing Glycolysis Process And Promoting Self-renewal Of Bovine Spermatogonial Stem Cells

Spermatogonal stem cells（SSCs）are a type of stem cells located on the basement membrane of the seminiferous tubules of testis tissue that can stably perform self-renewal and differentiate into mature sperm.They are the basis of spermatogenesis.For livestock animals,the cultivation and transplantation of SSCs may improve the production efficiency of animals and provide a new way for the preparation of transgenic animals.The isolation and culture of SSCs is an important basis for its application.However,due to the low content in testicular tissue and no stable in vitro culture system,the ability of domestic animal SSCs to maintain their own dryness in vitro is extremely poor.Some studies believe that glycolysis is of great significance to the maintenance of the self-renewal ability of SSCs.In this experiment,the testis tissue of 3-8 months old calves was used as the experimental material.The bSSCs were separated and purified by the differential adhesion method,and then the aerobic glycolysis ability was enhanced by adding glycolysis promoters or hypoxic culture.Analysis it promotes the phenomenon and possible mechanism of bSSCs’self-renewal ability.The results showed that after adding the glycolysis intermediate product Phosphoenol pyruvate（pep）（1 mmol/m L）or the pyruvate dehydrogenase kinase activator ps48（25μmol/m L）to promote glycolysis,The three key enzymes of glycolysis,HK,PFK,PK,and the contents of lactate and lactate dehydrogenase were significantly up-regulated;the ability of bSSCs to form intact clones and cell viability in the in vitro culture process were significantly higher than those of the control group;RT-q PCR and Western Blot results showed that the expression levels and protein expression levels of genes related to self-renewal and pluripotency in bSSCs such as Gfrα-1,Ddx4,Plzf,Pgp9.5,c-Myc and Cxcr4 were significantly higher than those of the control group（P<0.05）.When treated with 2-deoxyglucose（2dg）,which inhibits glycolysis,the effect on bSSCs is opposite to that of pep or ps48.Compared with normoxic conditions（21%O₂）,hypoxic（10%O₂）conditions have the same effect on bSSCs culture as pep or ps48treatment,with enhanced glycolysis capacity,SSCs self-renewal ability and increased pluripotency-related gene expression.After culturing bSSCs under conditions of enhanced glycolysis for more than 2 months,the cells were in good condition,and there was no significant difference in the expression of bSSCs marker genes,indicating that the glycolysis process is conducive to the self-renewal of bSSCs.In summary,the glycolysis process is required for the long-term culture of bSSCs in vitro.Adding glycolysis promoters or hypoxic culture enhances the glycolysis process,promotes the self-renewal ability of SSCs,and enhances the expression of stemness-related genes.The glycolysis process plays an important role in maintaining the self-renewal ability of SSCs.