Lrg1 Deficiency Causes Abnormal Spermatogenesis And Steroidogenesis In Mice

Objective:Male infertility is most commonly caused by spermatogenetic failure,clinically noted as oligo-or azoospermia.Mammalian spermatogenesis is a highly-and intricately-regulated process that occurs in the seminiferous tubules of the testis,and which is modulated by intrinsic genetic mechanisms and extrinsic factors.Although research on male reproductive development and function has made great progress,our understanding of the molecular mechanisms of spermatogenesis remains limited.Leucine-rich-alpha-2-glycoprotein 1?LRG1?,a member of the membrane-associated leucine-rich repeat family,is a secreted glycoprotein that contains eight repeating consensus sequences with a leucine-rich motif.In our previous studies,we found that the expression of LRG1 in the testis was increased in cryptochrome 1 mutant mice,which show testicular dysfunction.Further,analysis of microarray data?GDS5172?revealed that Lrg1 trended toward increased expression in mouse testes upon treatment with JQ1,a selective small-moleculeinhibitor of the bromodomain,testis-specific?Brdt?.These results suggested that Lrg1 might play roles in testicular function;however,its role in spermatogenesis has not been studied.Thus,in the present study,we examined the localization of LRG1 in mouse testes,and explored the potential biological activities of Lrg1 in male reproduction in vivo by generating knockout?KO?mice.Methods:1.Western blot and immunofluorescence assay were performed to investigate the expression of LRG1 in testis.2.Lrg1 KO C57/BL6 mice were generated by Bab-BGI Biotechnology?Shenzhen,China?using the clustered regularly interspaced short palindromic repeats?CRISPR?/ CRISPR-associated?Cas?9 system.Lrg1 mutations were identified by DNA sequencing.3.Body weight,testis weight,epididymal weight,epididymal sperm counts,and sperm motility of WT mice and Lrg1 KO mice was measured.HE staining was used to observe the morphological changes of testis and sperm.4.Serum testosterone and LH levels of WT and Lrg1 KO mice were measured using enzyme-linked immunosorbent assay?6 animals in each group?.5.High-throughput RNA sequencing was used to identify differentially expressed genes.q RT-PCR was used to verify the resultsof transcriptome analysis.6.Bioinformatics analysisFunctional classifications of differently regulated genes in Lrg1 KO mice versus WT mice were obtained by Gene Ontology?GO?analysis using the PANTHER classification system.Protein-protein interaction?PPI?networks were created using the Search Tool for the Retrieval of Interacting Genes?STRING;V.10.0,http://string-db.org?database comprising known and predicted PPIs.The Molecular Complex Detection?MCODE?app was applied to identify core connected modules in the PPI network?degree cutoff =2,node score cutoff = 0.2,k-core = 2,and max.depth = 100?.TargetScan?http://www.targetscan.org/mmu₇1/?and DIANA-TarBase v.7.0?http://diana.imis.athena-innovation.gr/DianaTools?were used to predict gene targets of the deregulated mi RNAs.Further,we employed Cytoscape v.3.5.1 platform?http://www.cytoscape.org/?to construct the miRNA-mRNA interaction network.Results:1.LRG1 signal was detected in mouse testis spermatogonia and spermatocytes.2.Western Blot and immunofluorescence analysis of testicular tissue from WT and Lrg1 KO mice showed that the LRG1 was absent in KOmice,which confirmed the deletion of the Lrg1.3.Lrg1 knockout mice did not exhibit male infertility.However,compared with the WT control group,epididymal sperm counts and sperm motility of Lrg1 KO mice were significantly reduced.4.Lrg1 deficiency resulted in impaired testicular development and decreased testosterone production.5.Transcriptome analysis revealed that the altered gene expression induced by Lrg1 deficiency mainly involved meiosis,mitosis,chromosome segregation,spermatogenesis and metabolism processes.In addition,48 members of the C2H2-type zinc finger protein family and 17 miRNAs were identified as deregulated in Lrg1-deficient testis,indicating that the testicular transcriptional regulatory network was altered.Conclusions:Overall,our findings support the notion that disrupted Lrg1 signaling might cause abnormal spermatogenesis and steroidogenesis;thus,Lrg1 is essential for testis development and function.