昼夜节律关键基因在睡眠行为改变导致的小鼠精子损害中的作用研究

Sperm number is key index of male fertility. Our previous researches in shift workers and other population suggested that the frequent change of sleep behaviors was associated with decreased sperm number in the population, but the underlying mechanism remains unknown. There are evidences suggesting that sleep behavior influences the expression of circadian genes including Clock, which were involved in the spermatogenesis process. But it is not validated whether the change of sleep behaviors induced sperm damage by disrupting the circadian genes expression. The plan of the present study is: 1)To modify the sleep of animals by controlling the light/dark cycle and feeding schedule, so that expression of circadian genes including Clock in the testes will be disrupted. 2) To measure the sperm biomarkers in testes and epididymis and evaluate the damage of spermatozoa as well as the stage in which the damage happens. 3) To modify the circadian gene expression in different directions and investigate whether the sleep-related sperm damage was influenced: to investigate the sperm biomarkers in the Clock knockout animals whose peripheral circadian rhythm are abolished in order to see whether the sleep change still induce sperm damage; and to attenuate the disruption of the circadian genes including Clock in the wild type animals by RNAi technique and investigate whether the spermatozoa damage was relieved, with expectation of clarifying the role of circadian genes in the sleep-related spermatozoa damage. This study will help to understand the mechanism of spermatozoa damage and to keep the male fertility in the population.

精子数量是影响男性生育的重要因素。我们前期发现倒班工人等人群睡眠行为的改变可能导致精子数量下降，其机制尚不清楚。近期有文献报道睡眠行为变化干扰昼夜节律关键基因Clock等的表达，而后者参与调控精子发生。但睡眠行为变化是否通过干扰昼夜节律关键基因表达而导致精子损害尚未证实。本课题拟开展以下研究：1）通过控制光照-黑暗周期和喂食时间的方法操纵小鼠睡眠行为改变，对昼夜节律关键基因Clock等表达造成干扰2）在此基础上通过生精上皮时相和精子数量等指标评价睡眠行为改变造成的精子损害并确认其发生阶段3）从反向和正向两方面调控昼夜节律基因表达并观察效应：在Clock基因敲除小鼠中观察睡眠行为变化是否仍导致精子损害；在野生型小鼠中以RNAi缓解Clock等表达所受干扰并观察精子损害是否改善，以明确昼夜节律关键基因在睡眠行为变化致精子损害中的作用，为阐明精子损害机制、维护男性生殖健康和行为干预提供理论依据。

本课题的研究内容包括三个方面：1）睡眠变化干扰昼夜节律的动物模型的构建；2）睡眠变化导致精子损害的效应观察；3）昼夜节律关键基因在睡眠变化导致的精子损害中的作用机制研究。主要研究进展包括1）建立了睡眠变化动物模型。建立了光照控制系统和转轮笼活动（睡眠）监测系统。通过对不同品系小鼠的比较发现，C57BL/6的活动（睡眠）行为的个体间差异更小，对光照干预的响应程度更好。设计了多种实验方案，可用于分析不同的研究问题。2）分析了睡眠变化对精液质量以及雄性授孕能力的影响，并初步鉴定了损害效应的发生阶段。绘制小鼠精液参数的昼夜节律曲线，发现节律干预后，小鼠精子总数较对照组出现明显降低，但精子总数的昼夜节律时相未受影响。若在节律干预后恢复35天（一个生精周期），则组间的精子总数差异消失，提示节律干预可能未对生精干细胞造成显著损害。生殖毒性试验分析发现，对照组雌鼠的受孕率为90.9%，而干预组雌鼠的受孕率仅为64.1%。对精子发生的各个阶段的生物标志物进行了分析发现，圆形精子和长形精子生物标志物（Tnp1；Prm2）的表达水平存在较明显差异；精母细胞阶段的生物标志物（Hspa2）的表达水平略有差异；而精原细胞阶段的生物标志物（Magea4）未见明显差别，提示损害效应可能起始于圆形精子阶段。3）分析了损害效应与生物钟基因的关系。表达组分析发现干预对小鼠睾丸组织基因表达组造成广泛影响。在被测序的29755个mRNA中，有22166个出现表达差异，Clock、Per3、Cry1、Sik1等65个生物钟基因呈现出组间差异，另有265个基因属于生殖过程通路。合成了生物钟关键调控基因Sik1的干扰序列，初步结果显示其可有效抑制Sik1的表达水平，有利于加速生物钟通路对光照变化的响应速度，并进一步观察其对节律干扰导致的小鼠精子损害效应的影响。采用Arntl突变小鼠模型B6.129-Arntltm1Bra/J接受节律干扰，发现干预组与对照组精液参数的差异无统计学意义，提示节律干预对小鼠精子的损害效应可能是通过生物钟通路实现的。