The adverse effects of sleep disorders on male fertility are of increased concern. 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 differentialy expressed genes of CSR rats were sequenced and screened, 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, as well as others. 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 is not related to their state of DNA methylation. The results of this study suggest that CSR could cause male infertility by significantly altering the testicular transcriptome.Abbreviations: CSR: chronic sleep restriction; SD: sleep deprivation; RNA-Seq: RNA sequencing; NGS: next generation sequencing; qRT-PCR: real-time quantitative reverse transcription polymerase chain reaction; KEGG: Kyoto encyclopedia of genes and genomic; NO: nitric oxide; INOS: Inducible nitric oxide synthase; Il6: interleukin-6; Tnf: tumour necrosis factor alpha; Hsd11b1: hydroxysteroid 11-beta dehydrogenase 1; Dnmt3a: DNA methyltransferase 3Ax; PSD: paradoxic sleep deprivation; DNMTs: DNA methyltransferases family; REM: rapid eye movement sleep; PGD: preimplantation genetic diagnosis; PGS: preimplantation genetic screening; ECS: expanded carrier screening; T: testosterone; FSH: follicle stimulating hormone; LH: luteinizing hormone; PRL: prolactin; BC group: Blank Control group; MC group: Model Control group; Hist1h2ba: histone cluster 1 H2ba; Lgr4: leucine-rich repeat-containing G protein-coupled receptor 4; Atrn: attractin ; Ogg1: 8-oxoguanine DNA glycosylase; SNVs: single nucleotide variants ; HPG axis: hypothalamic-pituitary-adrenal axis; Star protein: steroid acute regulatory protein; Dmac2l: distal membrane arm assembly complex 2 like; Esr1: estrogen receptor 1; MAPK pathways: mitogen-activated protein kinase pathways; Sos2: SOS Ras/Rho guanine nucleotide exchange factor 2; Jak2: Janus kinase 2; Pik3cb: phosphatidylinositol-4,5-bisphosphate 3-kinase, and catalytic subunit beta; Kras: KRAS proto-oncogene and GTPase; RRBS: reduced representation bisulfite sequencing; DEGs: differently expressed genes; SPF: Specific Pathogen Free; HE: hematoxylin & eosin; DMR: differentially methylated region; GO Analysis: Gene Ontology analysis; SINE: short interspersed nuclear elements; LINE: long interspersed nuclear elements; LTR: long terminal repeats.

中文翻译：

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慢性睡眠受限诱导雄性大鼠生育能力下降相关基因的转录变化。

睡眠障碍对男性生育能力的不利影响日益引起人们的关注。在这项研究中，使用改良的多平台方法建立了慢性睡眠受限（CSR）大鼠模型。首先基于性行为评估了CSR对雄性大鼠生育力的影响。测量包括睾丸激素（T），催乳激素（PRL），促黄体激素（LH）和促卵泡激素（FSH）在内的血清激素和精子参数（浓度，生存力，运动性，变形率），并通过苏木精和曙红染色。通过RNA测序（RNA-Seq）检测CSR大鼠和对照组大鼠之间的转录差异，然后通过亚硫酸氢盐测序检测DNA甲基化。对CSR大鼠差异表达基因进行测序和筛选后，随机采样代表性的上调和下调基因，以通过实时定量逆转录聚合酶链反应（qRT-PCR）验证测序结果。最后，完成了功能注释，包括基因本体论（GO）和《京都基因与基因组百科全书》（KEGG）途径分析。结果表明，与对照组相比，CSR大鼠的性行为没有改变。CSR大鼠的精子浓度，活力和运动能力明显下降，而精子畸形率则明显增加。在KEGG途径分析数据库中，筛选了一些特定的差异表达基因，这些基因与代谢途径，炎症相关途径，肾素-血管紧张素系统以及其他有关。然而，睾丸中的上述差异表达基因与它们的DNA甲基化状态无关。CSR可以显着降低雄性大鼠的生育能力，其机制之一是通过改变睾丸中的基因表达而实现的，这与它们的DNA甲基化状态无关。这项研究的结果表明，CSR可能通过显着改变睾丸的转录组而导致男性不育。SD：睡眠不足；RNA-Seq：RNA测序；NGS：下一代测序；qRT-PCR：实时定量逆转录聚合酶链反应；KEGG：《京都基因与基因组百科全书》；NO：一氧化氮；INOS：诱导型一氧化氮合酶；Il6：白介素6；Tnf：肿瘤坏死因子α；Hsd11b1：羟基类固醇11-β脱氢酶1；Dnmt3a：DNA甲基转移酶3Ax; PSD：自相矛盾的睡眠剥夺；DNMT：DNA甲基转移酶家族；REM：快速眼动睡眠；PGD​​：植入前遗传学诊断；PGS：植入前基因筛查；ECS：扩展的载体筛选；T：睾丸激素；FSH：促卵泡激素；LH：促黄体激素；PRL：催乳素；BC组：空白对照组；MC组：模型控制组；Hist1h2ba：组蛋白簇1 H2ba；Lgr4：富含亮氨酸的重复序列的G蛋白偶联受体4；Atrn：吸引蛋白；Ogg1：8-氧鸟嘌呤DNA糖基化酶；SNV：单核苷酸变异体；HPG轴：下丘脑-垂体-肾上腺轴；星蛋白：类固醇急性调节蛋白；dmac2l：远侧膜臂复合体2样；Esr1：雌激素受体1；MAPK途径：有丝分裂原激活的蛋白激酶途径；Sos2：SOS Ras / Rho鸟嘌呤核苷酸交换因子2; Jak2：Janus激酶2；Pik3cb：磷脂酰肌醇-4,5-双磷酸3-激酶和催化亚基β；Kras：KRAS原癌基因和GTPase；RRBS：亚硫酸氢盐还原表示法；DEGs：表达不同的基因；SPF：无特定病原体；HE：苏木精和曙红；DMR：差异甲基化区域；GO分析：基因本体分析；SINE：短散布的核元素；LINE：散布着长核的元素；LTR：长终端重复。差异甲基化区域 GO分析：基因本体分析；SINE：短散布的核元素；LINE：散布着长核的元素；LTR：长终端重复。差异甲基化区域 GO分析：基因本体分析；SINE：短散布的核元素；LINE：散布着长核的元素；LTR：长终端重复。