The Preliminar Study On The Main Energy Supply Pathway And The Effect Of Glucose On The Growth Characteristics Of In Buffalo Cumulus Cells

Glucose,which directly or indirectly involved in the metabolic process,is an important nutrient in animals' body.Studies have demonstrated that glucose is an major energy source for mammalian follicular development,oocyte maturation and embryonic development,and it is involved in the molecular regulation of cumulus expansion,oocyte maturation and early embryonic development.In this study,we explored the main pathway of energy utilizing pathway of glucose in buffalo cumulus cells at first.Based on that,relationship between the effects of glucose on the growth characteristics of buffalo cumulus cells and the cell glycolysis were investigated,so as to further optimize the cumulus cells culture conditions and to establish a foundation for improving of the culture system of the buffalo oocytes in vitro maturation.Firstly,the oxidative phosphorylation pathway inhibitors,pentose phosphate pathway inhibitors and glycolytic pathway inhibitors were added to the medium of the second generation buffalo cumulus cells in vitro,and the changes of cells capacity were measured by ATP bioluminescence assay after relating glucose metabolic pathway were inhibited.The results showed when the oxidative phosphorylation pathway was inhibited,the growth of buffalo cumulus cells are normal and the cells capacity was no significant change(P>0.05).As the pentose phosphate pathway was inhibited,the buffalo cumulus cells productivity was reduced,but the cells still maintain normal growth status.Then,when the glycolysis pathway was inhibited,the growth status of buffalo cumulus cells was affected to varying degrees,and showed very significant change(P<0.05).A gradient change was found between the buffalo cumulus cells productivity and the degree of inhibition of the glycolysis pathway.After the glycolysis pathway or the pentose phosphate pathway was inhibited respectively,the cells production capacity was significantly reduced(P<0.05).However,the degree of decrease in cells productivity after inhibiting the glycolysis pathway was higher than that of pentose phosphate pathway(61.46%vs 24.7%).Secondly,the effects of glucose on the growth characteristics of buffalo cumulus cells were studied,including cell growth tendency,cell proliferation rate,cell proliferation level,apoptosis,cell utilization and cytotoxicity.The buffalo cumulus cells were treated with different concentrations of glucose(0 g/L,1 g/L,4.5 g/L and 9 g/L)for 48 h.The results showed that there was no significant difference in the morphology of buffalo cumulus cells treated with different concentrations of glucose,and the cell growth curves showed a "S" type.However,the buffalo cumulus cells grown relatively slow when the cells were cultured in the medium containing 0 g/L glucose.For the 1g/L and 4.5 g/L glucose treated group,the growth trends of the cells were similar between two groups.However,compared with the control group,the doubling time of the two groups was shortened and the proliferation ability of cells was significantly improved(P<0.05),but the utilization rate of glucose in the 4.5 g/L treatment group was significantly higher than that of the 1 g/L treatment group(P<0.01).The addition of 9 g/L glucose in the cells culture medium had a stimulating effect on the proliferation of cumulus cells in a certain period of time,and the effect subsequently disappeared with the prolongation of the treatment time and the proliferation of cell was inhibited.The mRNA expression of glycolysis related genes in buffalo cumulus cells was detected by fluorescence quantitative PCR.The results showed that the expression levels of Hexokinase(HK),Phosphofructokinase(PFK)and Lactate dehydrogenase(LDH)mRNA in 4.5 g/L treatment group were significantly higher than those in 0 g/L,1 g/L and 9 g/L treatment groups.These above results demonstrated that:(1)The glycolytic pathway and pentose phosphate pathway are the main energy supply pathway of glucose metabolism in buffalo cumulus cells in vitro,and the cell capacity by the glycolysis pathway is more prominent,but buffalo cumulus cells rarely rely on oxidative phosphorylation pathway to produce capacity.(2)The suitable glucose concentration(4.5 g/L)is conducive to promote the proliferation of buffalo cumulus cells and reduce the cell apoptosis,moreover,it can enhance the cell glycolysis and promote cell uptake of glucose via increasing the expression of HK,PFK and LDH genes in cells.