Transcriptional Alterations of Genes Related to Fertility Decline in Male rats Induced by Chronic Sleep Restriction

The adverse effects of sleep disorders on male fertility are increasingly concerning. In this study, a rat model of chronic sleep restriction (CSR) was established using the modified multiplatform method. The effects of CSR on the fertility of male rats were evaluated first based on sexual behavior. Serum hormones, including testosterone (T), prolactin (PRL), luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and sperm parameters (concentration, viability, motility, deformation rate) were measured, and testicular histology was analysed by hematoxylin and eosin staining. The transcriptional differences between CSR rats and control rats were detected by RNA sequencing (RNA-Seq), and DNA methylation was then detected by bisulfite sequencing. After the differently expressed genes of CSR rats were sequenced and screened, some representative up- and down-regulated genes were randomly sampled to verify the sequencing results by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Finally, functional annotations were completed, including gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomic (KEGG) pathway analyses. The results showed that the sexual behavior of CSR rats did not change when compared with control group rats. The sperm concentration, viability and motility of the CSR rats decreased significantly, while the sperm malformation rate increased significantly. In the KEGG pathway analysis database, some specific differentially expressed genes were screened, which are involved in metabolic pathways, inflammation-related pathways, the renin-angiotensin system, etc. However, the aforesaid differentially expressed genes in the testes were not related to their DNA methylation status. CSR could significantly reduce the fertility of male rats, and one of its mechanisms occurs by altering gene expression in the testes, which has nothing to do with their DNA methylation. It was suggested that CSR could cause male infertility by significantly altering the testicular transcriptome.睡眠障碍对男性生育力的不良影响日益受到关注。本研究采用改良多平台法建立慢性睡眠限制（chronic sleep restriction, CSR）大鼠模型。首先基于性行为评估慢性睡眠限制对雄性大鼠生育力的影响；检测血清睾酮（testosterone, T）、催乳素（prolactin, PRL）、黄体生成素（luteinizing hormone, LH）及卵泡刺激素（follicle-stimulating hormone, FSH）等激素水平，以及精子参数（浓度、活率、活力、畸形率）；通过苏木精-伊红染色分析睾丸组织学结构。采用RNA测序（RNA sequencing, RNA-Seq）检测慢性睡眠限制大鼠与对照组大鼠的转录组差异，随后通过亚硫酸氢盐测序检测DNA甲基化水平。对慢性睡眠限制大鼠的差异表达基因进行测序筛选后，随机选取部分代表性上调及下调基因，通过实时定量反转录聚合酶链反应（real-time quantitative reverse transcription polymerase chain reaction, qRT-PCR）验证测序结果。最后完成基因本体（gene ontology, GO）及京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomic, KEGG）通路注释分析。结果显示，慢性睡眠限制大鼠的性行为与对照组相比无显著变化；但其精子浓度、活率及活力均显著降低，精子畸形率显著升高。在KEGG通路分析数据库中，筛选得到部分参与代谢通路、炎症相关通路、肾素-血管紧张素系统等的特异性差异表达基因。然而，睾丸中上述差异表达基因与其DNA甲基化状态无关。研究表明，慢性睡眠限制可显著降低雄性大鼠的生育力，其机制之一是通过改变睾丸基因表达实现，且该过程与DNA甲基化无关。本研究提示，慢性睡眠限制可通过显著改变睾丸转录组引发男性不育。