Identification And Functional Research Of Lysine Crotonylation In Mice Sperm

Lysine crotonylation?Kcr?is a newly identified protein posttranslational modification?PTM?.It is involved in mouse spermatogenesis,but its sperm-special functions are poorly known.Mammalian spermatozoa are highly specialized cells with few transcriptional and translational activities,and thus their biological functions are usually regulated by PTMs.Phosphorylation is the most reported PTM that plays a necessary role in sperm capacitation.However,capable fertilization induced by the Ca²⁺ionophore A23187 occurs without activating phosphorylation,and thus far,new PTMs are undiscovered.Here,we used LC-MS/MS,western blot,immunofluorescence and bioinformatics to identify and functionally analyze the Kcr proteins in mouse sperm.Our results demonstrated that:1.Hyper-Kcr occurs during spermatogenesis,and it is still kept in mature sperm.The Kcr sites widely exist on histone and non-histone proteins,and 28 histone Kcr sites are identified on histone.Compared with histone Kcr sites in mouse MEF cell,most of that in sperm shift to C terminal,and the histone Kcr may be helpful to maintain the pyknotic structure of sperm head.2.The distribution and Motif of proteins Kcr in mouse sperm are different from those of Hela cell.Compared with Hale cell,Kcr in sperm hardly particates in the reported functions of DNA replication,gene expression and RNA splicing,but associates with reproduction and energy metabolism.All of the characteristics are accorded with the features that sperm are highly specialized cell without transcriptional activity.3.Kcr is a new PTM involves in A23187-induced sperm capacitation,and is regulated by intercellular Ca²⁺.Ca²⁺could increase Kcr levels through inhibiting the activity of HDACs,the enzymes of decrotonylated activity,instead of promoting the activity of CBP/P300,the enzymes of crotonylated activity.4.A23187-induced increasing Ca²⁺could increase Kcr levels of key enzymes involving in glycolysis,acetyl-CoA synthesis,citrate cycle and oxidative phosphorylation to inhibit the MMP,ATP production and phosphorylation.5.A23187 induces sperm motionless through Kcr,affecting the“power”,“structure”and“survival”of sperm.Power:increased Kcr levels could reduce energy production.Structure:tubulin Kcr and Kac may decrease the mobility of tubulin to inhibit the motility of sperm.Survival:the cytoprotective proteins,such as GST,could be crotonylated to increase their activity to keep sperm alive.These results present wide explorations of Kcr in mouse sperm,and connect Kcr with metabolism.Our research would provide a novel sight for therapy of metabilic diseases,and supply fundamental data for the further investigation of Kcr in mouse sperm.