Mechanism Of Lysine Succinylation Of Lactate Dehydrogenase C In Regulating Human Sperm Motility

Background: Male infertility has become a global issue that attract more and more attention.Low sperm motility(asthenozoospermia)is a common cause of male infertility.It is of great theoretical and clinical significance to reveal its mechanism.It takes a lot of energy for sperm motility,and glycolysis plays an important role of energy synthesis in sperm.Lactate dehydrogenase C(LDHC)is a glycolytic enzyme which is specifically expressed in testis and sperm.Knockout of Ldhc significantly reduced sperm ATP content and sperm motility,even causing male infertility.However,there is less research about the regulatory mechanism of LDHC activity.It was found that lactate dehydrogenase activity was regulated by post-translational protein modifications(PTMs).Our previous study found LDHC was modified by lysine succinylation(Ksucc),a novel PTM participating in many cellular processes and diseases.However,the role of LDHC-Ksucc in regulating LDHC activity and sperm motility remains to be elucidated.Objective: The study aimed to elucidate how Ksucc regulates LDHC activity and sperm motility and the role of LDHC-Ksucc in the development of asthenozoospermia.The results will provide a new molecular target for clinical diagnosis and treatment of asthenozoospermia.Methods:1)The differentially modified Ksucc sites in LDHC between normozoospermic and asthenozoospermic sperm were screened by immunoaffinity approach coupled to nanoliquid chromatography tandem mass spectrometry(LC-MS/MS)and parallel reaction monitoring(PRM).The antibody against differentially modified LDHCKsucc was synthesized and was used to semi-quantify LDHC-Ksucc level in 25 normozoospermic and 25 asthenozoospermic sperm by western blots.Spearman correlation is used to evaluate the correlation between LDHC-Ksucc levels and sperm progressive motility.2)Lactate dehydrogenase(LDH)activity assay kit is used to detected the LDH activity of normozoospermic and asthenozoospermic sperm.The Spearman correlation is used to evaluate the correlation between LDH activity and LDHCKsucc levels or sperm progressive motility.3)The LDHC activities of wildtype LDHC,LDHC(Lysine mutated to Arginine),and LDHC(Lysine mutated to Glutamate)were examined to evaluate the effect of succinylated LDHC on its enzyme activity.4)The interaction and colocalization between carnitine palmitoyltransferase 1A(CPT1A)and LDHC in human sperm were detected by immunoprecipitation and immunofluorescence,respectively.In vitro experiment of succinylation and LDHC activity assay were used to evaluate whether CPT1 A succinylated LDHC and regulate its enzyme activity.In addition,knockout of Cpt1 a in GC-1 cell line for further verification.5)The protein level of CPT1 A in 25 normozoospermic and 25 asthenozoospermic sperm was semi-quantified by western blots.Spearman correlation is used to evaluate the correlation between CPT1 A protein level and sperm progressive motility,LDHC-Ksucc levels or LDH activity.In addition,the ATP concentration was detected in asthenozoospermic sperm with low CPT1 A protein level.Results:1)The level of succinylated lysine317 in LDHC(LDHC-K317succ)was identified to be decreased in asthenozoospermic sperm compared with normozoospermic sperm by sperm succinylome and PRM technique.2)The decreased level of LDHC-K317 succ in asthenozoospermic sperm was confirmed in 50 sperm samples.3)The LDHC(Lysine mutated to Glutamate)activity was higher than wildtype LDHC,while LDHC(Lysine mutated to Arginine)activity was lower than wildtype LDHC,indicating that K317 succ is essential for enzyme activity of LDHC.4)CPT1A and LDHC interacted with each other and colocalized in the human sperm tail.CPT1 A mediated the succinylation of LDHC-K317 and maintained the LDHC activity in vitro.Knockdown of Cpt1 a decreased LDHC-K317 succ level and LDH activity in CC-1 cell line.These results imply that CPT1 A mediates the succinylation of LDHC-K317 and maintains the activity of LDHC.5)The protein level of CPT1 A was significantly decreased in asthenozoospermic sperm compared with normozoospermic sperm.CPT1 A protein level is positively corelated with sperm progressive motility,LDHC-K317 succ level and LDH activity.In addition,the ATP concentration was reduced in asthenozoospermic sperm with low CPT1 A protein level.These results suggested that LDHCK317 succ is important in the development of asthenozoospermia.Conclusion: This study identified the first asthenozoospermia-related succinylation event(LDHC-K317succ)in human sperm.CPT1 A mediates the occurrence of LDHCK317 succ,activates LDHC,regulates glycolytic-dependent ATP synthesis,and thus maintains sperm motility.Abnormalities in this regulatory pathway may lead to asthenozoospermia.