摘要
目的 探索叶酸缺乏(Folate deficiency,FD)对小鼠睾丸发育和精子发生的影响,筛选叶酸影响精子发生中关键差异基因及信号通路.方法 建立C57BL/6小鼠FD模型(从胚胎时期至成年期).HE染色分析出生后不同阶段鼠龄睾丸组织及附睾组织结构,精子PAS染色进行形态学观察.利用转录组测序(RNA sequencing,RNA-Seq)技术筛选10周龄小鼠睾丸差异表达基因(Differentially expressed genes,DEGs)并利用GO数据库和KEGG数据库进行基因功能富集.结果 与对照组比较,FD组生精小管细胞数量减少,减数分裂延迟,附睾管中精子稀少.精子计数结果显示,FD组精子密度显著低于对照组(P＜0.05).RNA-Seq分析共得到210个DEGs.差异基因主要富集在胰岛素样生长因子结合、胞外分泌、蛋白质酪氨酸/苏氨酸磷酸酶活性、细胞增殖调控、多糖生物合成等方面.KEGG富集分析提示差异基因主要富集于甲状腺激素信号通路、Toll样受体信号通路、甾类激素生物合成等.其中Fgf22、Lrrc31和Inmt等基因表达异常可能参与FD导致的生精功能障碍.结论 FD影响生精小管的发育和精子发生,其潜在机制可能涉及多个差异表达基因及细胞增殖调控、多糖生物合成和甾类激素生物合成等通路.
Abstract
Objective To investigate the effect of folate deficiency(FD)on testis development and spermatogenesis in mice,and screen its related differential genes and pathways.Methods C57BL/6 mouse models with folate deficiency were established by feeding with FD diet from weaning to adult.The morphology of testis and epididymis tissues of the offspring male mice was observed by HE staining.Sperm morphology was analyzed by PAS staining.Differential genes in testicular tissue of 10-weeks-old mice were screened by transcriptome sequencing.GO and KEGG databases were utilized for gene enrichment analysis to obtain key genes and signal pathways.Results Compared with the control mice,FD mice presented a significant decrease in the number of spermatogenic tubule cells in testes and an obvious delay in meiotic onset,simultaneously,their epididymal ducts exhibited a significant decrease in the number of sperm(P＜0.05).Analysis of transcriptome profile showed 210 differentially expressed genes,which were enriched in terms of insulin-like growth factor binding,exocytosis,protein tyrosine/threonine phosphatase activity,regulation of cell proliferation,and proteoglycan biosynthetic process in GO analysis.KEGG analysis indicated DEGs were enriched in terms of thyroid hormone signaling pathway,Toll-like receptor signaling pathway,steroid hormone biosynthesis,etc.Abnormal expression of Fgf22,Lrrc31 and Inmt may be involved in FD-caused spermatogenic dysfunction.Conclusion FD exserts a negative impact on seminiferous tubules development and spermatogenesis,which may be associated with abnormal gene expression,as well as the obstruction of cell proliferation,polysaccharide biosynthesis,and steroid hormone biosynthesis.
基金项目
安徽省高等学校自然科学研究重点项目(KJ2021A0704)
国家级大学生创新训练项目(202110367050)
NETL
NSTL
万方数据