Effects Of α-ketoglutarate On Glucose Metabolism And Antioxidation Ability Of Capacitated Boar Sperm

In vitro fertilization in mammals(IVF)is an important biotechnology for animal reproduction,and the current low fertilization efficiency in boars seriously affects the efficiency of IVF embryo production.In order to further improve the success rate of IVF and increase fertility in boars,sperm capacitation as a key step in the technical aspects of IVF may serve as an effective breakthrough.Researches have found that α-ketoglutarate(AKG)has the functions of regulating energy metabolism,antioxidation and improving cell vitality,but no one has investigated the role of AKG as an important intermediate of energy supply and antioxidant in the process of sperm capacitation.Since sperm require a large amount of energy supply during capacitation,it is crucial to investigate the positive effects of AKG in the process of boar sperm capacitation to improve the IVF rate.In this study,AKG was added to boar sperm capacitation solution to detect its glucose metabolism and antioxidation effects during boar sperm capacitation.The specific tests were as follows:Experiment 1: Effect of AKG on in vitro capacitation status of boar sperm.Sperm of landrace boars were divided into 7 groups: fresh sperm group,capacitation control group(0 mmol/L AKG)and capacitation AKG treatment group(5,15,30,45,60 mmol/L AKG).After 2 h of capacitation incubation,the percentage of capacitation sperm motility,viability,acrosome integrity,and plasma membrane integrity were not changed by adding any concentration of AKG compared to the capacitation control group(P > 0.05).The wobble coefficient,curve line velocity,average path velocity,straight line velocity and amplitude of lateral head displacement of capacitated sperm in AKG treatment groups were significantly higher than those in control group(P < 0.05).Except for 5 mmol/L,the protein tyrosine phosphorylation level and tyrosine kinase activity of the capacitated sperm treated with other concentrations of AKG were significantly higher than those of the control group(P < 0.05).In brief,AKG,as an additive drug for sperm capacitation in vitro,has no side effects.And it can increase the sperm motility and capacitation.Experiment 2: Effect of AKG on glucose metabolism and capacitation pathway of capacitated boar sperm.After the capacitation promotion of AKG was verified,the mechanisms involved in regulating the glucose metabolism and capacitation pathway of sperm were further examined.Compared with the capacitation control group,AKG at concentrations of 15 mmol/L,30 mmol/L and 45 mmol/L significantly increased the content of ATP and lactic acid in capacitated sperm(P < 0.05),while the pyruvate contents of capacitated sperm treated by all concentrations of AKG were not significantly different(P > 0.05).When AKG concentration was 45 mmol/L and 60 mmol/L,the activities of phosphofructose kinase and lactate dehydrogenase were significantly lower than those of the control group(P < 0.05).15 mmol/L AKG was used as the optimal concentration for subsequent experiments.Sperm mitogen-activated protein kinase(MAPK)activity was significantly higher in the AKG group than in the fresh sperm(P < 0.05).The phosphorylation level of AMP-activated protein kinase(AMPK)in the AKG group was significantly higher than that in the capacitation control group(P < 0.05).The activities of glutamate dehydrogenase,glutamine synthetase and the content of peroxisome proliferator-activated receptor γ of capacitated sperm in AKG group were not significantly different from those in the capacitation control group(P > 0.05).The sperm glucose transport protein(GLUT1)in AKG group was significantly higher than that in the capacitation control group(P < 0.05).In summary,AKG can affect the energy metabolism of capacitated sperm,and the content of ATP and lactic acid was the highest when AKG concentration was 15 mmol/L.AKG can inhibit the glycolytic enzyme activity of capacitated sperm while it promoted the activity of capacitation-related enzyme MAPK.Accordingly,it increased the content of GLUT1 to transport glucose into sperm and significantly affected the phosphorylation level of Thr172-AMPK located in the acrosome,which in turn enhanced the occurrence of boar sperm capacitation.Experiment 3: Effect of AKG on oxidation resistance of capacitated sperm.The total antioxidation capacity and activities of superoxide dismutase(SOD),catalase and glutathione peroxidase of capacitated sperm in AKG group were significantly higher than those in the capacitation control group(P< 0.05).The expression of SOD1 and SOD2 of capacitated sperm in AKG group was significantly higher than that in capacitation control group(P <0.05).The content of 4-hydroxynonenal,8-hydroxy-2-deoxyguanosine,Kelch-like ECH-associated protein and nuclear factor E-2-related factor 2 in the capacitated sperm of AKG group did not significantly differ from that of the capacitated sperm in the control group(P > 0.05),while the content of lipid peroxide malondialdehyde(MDA)was significantly lower than that of the capacitated sperm in the control group(P < 0.05).In a word,AKG can protect sperm from oxidative stress by significantly increasing the antioxidation capacity of capacitated boar sperm,promoting the expression of SOD1 and SOD2,and reducing the content of MDA.In conclusion,AKG can promote the motility rate and protein tyrosine phosphorylation of capacitated sperm while it increases the level of the glucose metabolism and the activities of tyrosine kinase and MAPK of capacitated sperm.And it improves AMPK phosphorylation and GLUT1 transport protein to positively regulate the capacitation of boar sperm.At the same time,AKG can increase the antioxidation capacity of capacitated sperm and positively influence the capacitation process.