The Study Of Molecular Mechanisms Underlying The Effects Of Tyrosine Phosphorylation Of Dihydrolipoamide Dehydrogenase On Mouse Sperm Motility

In mammalian sperm,as a mitochondrial matrix enzyme,DLD is a protein marker for sperm reproductive ability with the changes of its expression affecting the fertilization potential directly or indirectly.In addition,the post-translational modifications of proteins have become the research hotspot of mammalian sperm reproductive markers.However,few researches have studied the tyrosine phosphorylation of DLD as well as the relationships between tyrosine phosphorylation of DLD and intracellular energy metabolism in sperm.In particular,whether reversible changes of tyrosine phosphorylation of DLD would alter sperm reproductive capacity and the underlying mechanisms have not been reported.Therefore,the heavy metal ions and small molecule antioxidants were used as the induction factors to induce the reversible changes of tyrosine phosphorylation of DLD in the present study.Tyrosine phosphorylation of DLD was taken as starting point to study the relationships between DLD tyrosine phosphorylation,energy metabolism and sperm motility and also to investigate the molecular mechanisms by which the changes of tyrosine phosphorylation of DLD alter sperm functions.This study provides some insights into the molecular mechanisms by which changes of protein post-translational modifications affect the reproductive capacity of sperm.The main work of this research is as follows:The effects of different concentrations of Cadmium（Cd）on mouse sperm protein tyrosine phoshorylation were analyzed by immunoblotting technique（WB）.The results showed that when Cd concentrations increased to 5μM,it could induce tyrosine phosphorylation of⁵5 kDa protein（s）.Meanwhile,the proteins that are tyrosine phosphorylated upon incubated with Cd was identified as DLD using TD-WB coupled with MS/MS.The tyrosine phosphorylation of DLD did not change after the addition of cAMP/PKA signal pathway inhibitor H-89 or Ca²⁺cascade signaling pathway regulators W7 and KN-93,which suggested that Cd-induced DLD tyrosine phosphorylation did not follow cAMP/PKA signal pathway and Ca²⁺cascade signal pathway.Meanwhile,DLD was not tyrosine phosphorylated in the presence of HCO₃^-and BSA,which indicated that tyrosine phosphorylation of DLD was not involved in mouse sperm capacitation.In addition,the DLD protein was enriched using DLD antibody together with immunoprecipitation technique and the tyrosine phosphory was subsequently detected.The results demonstrated that Cd could induce the tyrosine phosphorylation of DLD and BSA could prevent the tyrosine phosphorylation of DLD in the presence of Cd.These results further supported the previously indentified DLD protein.Additionally,the immunofluorescence（IF）results indicated that the fluorescence intensity in the middle piece of spermatozoa treated with Cd was significantly enhanced,while the fluorescence intensity in the mid-piece of sperm did not change significantly with the treatment of Cd and BSA.Coincidently,mitochondria were located in the middle piece of mouse spermatozoa and DLD is a mitochondrial protein,which further validated the correctness of the identified DLD.DLD is an NAD⁺-dependent oxidoreductase and also a part ofα-ketoglutarate dehydrogenase complex and pyruvate dehydrogenase complex in TCA.Meanwhile,the NADH generated from TCA was involved in mitochondrial respiration and the subsequently produced ATP regulated sperm motility.Protein tyrosine phosphorylation is an important covalent modification in regulateing enzyme activity.However,whether the tyrosine phosphorylation of DLD will affect intracellular metabolism and sperm functions has not been reported.The results showed that when DLD was tyrosine phosphorylated,its dehydrogenase activity was significantly reduced（P<0.05）.Meanwhile,intracellular NADH level,ATP level and sperm motility parameters including MOT、PRO、VCL and VSL were all dramatically decreased.Nevertheless,compared with the Cd group,the dehydrogenase activity of DLD,intracellular NADH level,ATP level and sperm motility were all significantly increased in BSA and Cd co-administrated group（P<0.05）.These results suggested that the tyrosine phosphorylation of DLD may firstly inhibit its dehydrogenase activity,then block TCA cycle,decrease mitochondrial energy metabolism and finally disrupt mouse sperm motility and reproductive capacity.Reversibility of protein modification is one of its main features;especially the reversible changes in enzymatic protein phosphorylation directly affect its physiological activity.Is there a reversibility of tyrosine phosphorylation of DLD induced by Cd?Thus,the antioxidants Vc and NAC were used as regulating factors to induce reversible changes in tyrosine phosphorylation of mouse sperm DLD to confirm the function changes of DLD after reversible tyrosine phosphorylation changes.The results showed that 20 mM Vc or 8 mM NAC could induce dephosphorylation of DLD to restore its dehydrogenase activity,intracellular NADH level,ATP level and sperm motility.These results suggested the tyrosine phosphorylation of DLD is reversible,and tyrosine phosphorylation/dephosphorylation may act as biomarkers for DLD inactivation/reactivation.Moreover,further results demonstrated that Vc and NAC caused-dephosphorylation of DLD was not due to their anti-oxidative capabilities,and it is may be attributed to the imbalance between the activity of tyrosine kinase and phosphatase caused by these antioxidants through binding with Cd ions.Taken together,the tyrosine phosphorylation of DLD could be induced by DLD without following cAMP/PKA signal pathway and Ca²⁺cascade signal pathway.And the tyrosine phosphorylation of DLD was not involved in mouse capacitation.Tyrosine phosphorylation could reduce the dehydrogenase activity of DLD,subsequently inhibit intracellular energy metabolism,ATP production and sperm motility.Under the induction of the small molecule antioxidant Vc and NAC,tyrosine phosphorylated DLD appeared dephosphorylation,which led to the reactivation of DLD activity.Tyrosine phosphorylation/dephosphorylation could regulate DLD activity,and hence act as biomarkers for DLD inactivation and reactivation.Tyrosine phosphorylation/dephosphorylation of DLD was involved in the regulation of mitochondrial energy metabolism,NADH level,ATP level and finally regulated sperm motility and functions.