Effects And Mechanism Of Epididymis-specific Slc22a5 Deletion On Male Mice Reproduction

Objective:To observe the effects of epididymis-specific Slc22a5 deletion on male mice reproduction,changes in lipid composition of sperm membrane and expression of genes related to epididymal lipid metabolism.Methods:The epididymis-specific knockout Slc22a5 male mice were generated by crossbreeding Rnase10-iCre mice that Cre recombinase is specifically expressed in the epididymis.Slc22a5fl/fl,Rnase10iCre/wt male mice were divided into knockout group,and Slc22a5fl/fl,Rnasel0wt/wt male mice were divided into control.Mice in both groups were mated with wild-type female mice to test fertilization ability.Mice were anesthetized and executed,dissected and sampled,the expression of OCTN2 in the epididymis,epididymis index,and sperm quality(sperm motility,sperm concentration,and acrosome integrity)were examined.HE staining was performed on epididymis to observe histopathology changes.The lipid composition of sperm membrane was analyzed using liquid chromatography mass spectrometry.RNA from epididymal tissues was extracted and the expression of genes related to lipid metabolism was detected using PCR arrays.Differential expression genes were analyzed using Gene Ontology and STRING databases.Results:1.The expression of OCTN2 in the epididymis of control was 4.6-fold higher than the expression in the knockout group.2.Number of litters(14.17±1.47%vs.5.83±4.58%),sperm motility(68.33±5.61%vs.58.50±5.68%),and acrosome integrity(71.50±3.93%vs.62.17±8.66%)were significantly lower in knockout group compared with control(P<0.05),while the epididymis index(left epididymis index:(0.11±0.49%vs 0.11±0.27%;right epididymis index:0.11±0.33%vs 0.11±0.36%)and sperm concentration[(35.54±7.00)±106/100mg vs(31.04±16.11)×106/100mg]showed no significant difference(P>0.05).3.HE staining showed that the epithelial cells in caput,corpus and cauda of the epididymis of the knockout and control mice were well organized with regular cell arrangement,and no pathological changes such as cell flocculation and inflammatory cell infiltration were observed,and a numerous number of spermatozoa were seen in the lumen of epididymis.The morphology of epididymis was normal in both groups,and there was no significant difference.4.The lipid composition analysis of sperm membrane showed that 66 differential lipid coruponents were identified,of which 47 were up-regulated and 20 were down-regulated.The relative content of PUFA in the knockout group was lower than the ones in the control group.KEGG pathway analysis showed that differential lipid components were enriched in glycerophospholipid metabolism,linoleic acid metabolism,alpha-linolenic acid metabolism,glycosylphosphatidylinositol-anchor biosynthesis,glycerolipid metabolism and Arachidonic acid metabolism.5.The results of PCR array assay showed that 30 genes related to lipid metabolism were changed,the expression of Acox2,Acsm2,Akr1d1,Apoal,Apob,Apol8,Cdh13,Cyp11a1,Dkk1,Enpp7,Fabp9,Fgf10,Idi2,Lrp1b,Nr0b2,Scarf1,Sphk2,Wnt3a were upregulated,Acsm3,Acot8,Cebpa,Cebpd,Cfd,Elovl4,Fgf2,Gata3,Lep,Ncor were downregulated.GO enrichment analysis showed that Slc22a5 may be involved in cholesterol metabolism,adipocyte differentiation,lipid synthesis and lipid catabolism,fatty acid metabolism,Wnt signaling pathway,Notch signaling pathway and other biological processes,perform in fatty acid ligase,lipoprotein particle binding,protein-lipid complex binding,lipoprotein particle receptor binding and other biological functions.Based on the interaction network built on STRING database and Cytoscape software,Lep,Cebpa and Fgf2 might lays an important role in lipid metabolic regulation by Slc22a5.Conclusion:Epididymis-specific knockout of Slc22a5 resulted in decreased fertilization ability,reduced sperm motility and acrosome integrity,and modified the lipid composition of sperm membrane in male mice by a mechanism that may be related to altered epididymal lipid metabolism.