Cry1 Deficiency Damages Mice Testicular Function

Objective:Cryptochrome 1(Cry1)gene is a member of the mammalian clock gene.It plays an important role in the process of circadian rhythm,immune response,and carbohydrate metabolism,however,its role in male reproduction remains unclear.In this study,we aims to investigate its roles in testicular functions using Cry1 knockout(KO)mice.Methods:1.DNA sequencing,immunofluorescence and Western blotting were employed to identify the Cry1 KO mice.2.HE staining was used to determine the effect of Cry1 KO on testis tissue architecture.Subsequently,TUNEL assay was performed to evaluate apoptosis in the testis.Sperm concentration in the cauda epididymis was determined by counting using a Neubauer chamber hemocytometer.Testosterone levels were measured using an ELISA kit according to the manufacturer's instructions.3.Transcriptome analysis was carried out using the digital gene expression(DGE)approach.4.Gene Ontology enrichment analysis was performed using the PANTHER classification system.Protein-protein interaction networks were created using the STRING v.10.0 analysis tool(http://string-db.org).DIANA-TarBase and TargetScan database were used to predict the target gene of miRNAs.Finally,miRNA-mRNA interaction networks were constructed using the Cytoscape v.3.5.1 platform.Results:1.Cry1 KO mice were generated using the CRISPR-Cas9 system.DNA sequencing and Western blotting analysis confirmed that the Cry1 gene was deleted in the KO mice.Furthermore,We examined the subcellular localization of CRY1 protein using immunofluorescent staining.The results showed that CRY1 was abundantly expressed in Sertoli cells,germ cells,and the interstitial area in WT mice.In contrast,there was no CRY1 signal in Cry1 KO mice.2.The results of HE staining and TUNEL assay showed that there was an increased number of degenerated and apoptotic germ cells in Cry1 KO mice.However,reduction in sperm count from caudal epididymis was observed in Cry1 KO as compared to WT mice(P < 0.05),indicating that Cry1 is required for spermatogenesis.ELISA results showed that loss of Cry1 has no effect on testosterone production compared with WT mice.3.High-throughput sequencing analysis identified 375 differentially expressed genes between Cry1 KO mice and WT mice.Bioinformaticsanalysis revealed that the differentially expressed genes were related to important biological processes including cell-cell communication,metabolism,chromatin reorganization,spermatogenesis,and the immune response.ConclusionThese results provide the first evidence of a correlation between dysregulation of Cry1 and male reproductive defects in mice,indicating that Cry1 plays a critical role in maintaining normal testicular function.