Regulation And Mechanism Of Lipid Hydrolases On Epididymal Sperm Maturation And Motility In Kunming Mice

2-arachidonic glycerol（2-AG）is one of the main endocannabinoids in many mammals.Recent studies have found that 2-AG plays a key regulatory role in the hyperactivation of human sperm by binding to CatSper or not which depends on the decomposition of 2-AG by lipid hydrolase ABHD2.However,the breakdown of 2-AG in mouse sperm does not seem to be carried out by ABHD2.Previous studies in our laboratory have found that monoacylglycerol lipase MAGL,lipid hydrolases ABHD12and ABHD6 exist in mouse sperm,and they can hydrolyze 2-AG in mouse sperm.However,the activity（phosphorylation/dephosphorylation）form of lipid hydrolase itself and its regulatory mechanism remain unknown.Phosphatase PP1γ2 is a key molecule in the regulation of sperm functions such as epididymal sperm maturation and sperm motility.However,the regulation mechanism of its activity is not completely clear,and whether it is related to the above-mentioned lipid hydrolases is also unknown.At the same time,how CatSper and other calcium channels in sperm such as pmca4(plasma membrane Ca²⁺-ATPase 4)and TRPV1（transient receptor potential vanilloid-1）play the role of regulating sperm motility,and whether these hydrolases are involved in regulating sperm motility and their mechanisms need to be explored.Therefore,Kunming mice were used as experimental materials in this study.Phosphorylated and non-phosphorylated forms of ABHD12,ABHD6 or MAGL protein in caput and caudal epididymal mouse spermatozoa were analyzed by Western blotting,protein co-immunoprecipitation,Ca²⁺fluorescence imaging and sperm phosphatase activity analysis.The effects of different calcium channel inhibitors and MAGL/ABHD12 inhibitors on the motility of caudal epididymal mouse sperm,whether MAGL or ABHD6/ABHD12 is associated with PP1γ2,and the regulation mechanism of PP1γ2 activity have also been analyzed.The results showed that:（1）After incubation with ABHD6/12 and MAGL antibodies,the content of 2-AG in caudal epididymal mouse sperm was significantly increased compared with the control group（P<0.05）;ABHD6/12 and MAGL exist in different phosphorylation/dephosphorylation forms in caudal and caput epididymal mouse sperm,respectively..Moreover,the content of 2-AG in caput epididymal mouse sperm was significantly higher than that in caudal epididymal sperm,indicating that the three lipid hydrolases could hydrolyze 2-AG in mouse sperm,and the effects of ABHD12 and MAGL were stronger than that of ABHD6,the phosphorylation/dephosphorylation of lipid hydrolase in caudal epididymal mouse sperm may be the different active forms of 2-AG hydrolase.（2）The motility of caudal epididymal mouse sperm was significantly decreased（P<0.05）after co-incubation with ABHD12 inhibitor DO-264 or MAGL inhibitor JZL-184,and the concentration of calcium ions was also significantly decreased（P<0.05）.When DO-264 and JZL-184 of IC50 were added to sperm suspension at the same time,the two inhibitors showed strong antagonism（CI>1）,indicating that ABHD12 and MAGL regulate sperm motility by hydrolyzing endogenous cannabinoid 2-AG,opening the calcium ion channel CatSper and allowing calcium ions to enter the sperm.Moreover,both lipid hydrolases may act simultaneously on2-AG,and there may be a competitive relationship on the hydrolysis of 2-AG.（3）The motility of caudal epididymal mouse sperm was significantly decreased（P<0.05）after co-incubation with Catsper inhibitor HC-056456,TRPV1 inhibitor Capsazepine or PMCA4 inhibitor Caloxin1b1,and the concentration of calcium ions was also significantly decreased（P<0.05）When the maximum concentration of each inhibitor was used,CatSper inhibitor HC-056456 significantly reduced calcium ion concentration in sperm.Moreover,the average fluorescence intensity of HC-056456group was significantly lower than that of Capsazepine,a TRPV1 inhibitor,and Caloxin1b1,a PMCA4 inhibitor groups（P<0.05）,indicating that CatSper,TRPV1and PMCA4 were all involved in the regulation of calcium ion concentration in mouse sperm,CatSper is the most important calcium channel in mouse sperm.TRPV1and PMCA4 also play a role,but with limited regulatory ability.（4）Interaction between SDS22 and p17 was found in caput epididymal sperm.When SDS22 was bound to p17,PP1γ2 with high activity decreased sperm motility.There was no interaction between SDS22 and p17 in caudal epididymal sperm,so SDS22 inhibited the activity of PP1γ2 and sperm motility was improved.In the present study,the existence forms of MAGL,ABHD6 and ABHD12 in mouse spermatozoa were clarified,and the activity forms,action modes and regulatory mechanisms of these three lipid hydrolases were preliminarily investigated.The important calcium channels in spermatozoa and the regulatory effects of calcium ions on mouse sperm motility were investigated,and the activity regulatory mechanism of PP1γ2 was explored.This study enriched the content of motility regulation in mammalian sperm.It can also provide new ideas for understanding the pathogenesis of male infertility and developing new treatment methods,which has important theoretical and practical significance.