高龄父亲精子miRNA表达谱异常及其在子代自闭症谱系障碍（ASD）发生中的作用和机制研究

Nowadays, the number of aged couples who seek to have a child increases significantly. Emerging evidence suggests that there is an increased risk of autistic spectrum disorders (ASD) in the offspring of aged father, and abnormal epigenetic mechanisms may contribute to the development of ASD. MicroRNAs (miRNAs) play an important role in epigenetic regulation. However, it is unknown if spermatozoa miRNA is the underlying mechanism of ASD in the offspring. In the pilot studies, we found that the profile of spermatozoa miRNAs in aged humans and mice was significantly altered compared with young controls by small RNA high-throughput sequencing; In mouse, the changes of neuropsychological behavior were also detected in the offspring of aged males and conceived by the injection of spermatozoa total-RNA from aged males into zygotes. Thus, we hypothesize that spermatozoa miRNAs of aged father may be involved in the development of ASD in offspring. This study aims to inject the miRNAs into mouse zygote and test the offspring by neuropsychological behavior, brain MRI and RNA Seq. In addition, RNA Seq、quantitative PCR and luciferase reporter gene system will be performed in blastocysts to explore the mechanisms. This study will determine if spermatozoa miRNAs of aged father result in ASD in the offspring and what are the mechanisms. Therefore, this study will provide a new theoretical basis for the prevention and treatment of ASD and show the evidence for further studies, which is of importance to improve the health outcomes in the offspring of aged fathers.

近年来有生育意愿的高龄夫妇数量不断增加。研究表明高龄父亲子代罹患自闭症谱系障碍（ASD）的风险增加，表观调节异常可能是发病机制之一；精子miRNA在表观调节中发挥重要作用，其是否参与调控子代ASD的发生尚未见报道。我们前期研究通过小RNA转录组测序发现人类及小鼠高龄父亲精子miRNA表达谱均异常；在小鼠模型中发现高龄父亲或受精卵胞浆注射高龄父亲精子总RNA的子代均检测到神经精神行为的改变，我们推测高龄父亲的精子miRNA可能参与介导子代ASD的发生。本研究将在小鼠模型进行受精卵胞浆注射高龄父亲异常表达的精子miRNA来获得子代，并通过行为学实验、脑部MRI及转录组学来鉴定和研究子代ASD；通过囊胚期转录组测序、定量PCR及荧光素酶报告基因系统等方法来探索miRNA在早期胚胎调控子代ASD发生的机制。本研究拓展了精子miRNA在表观遗传的新功能，也为该类疾病的防治提供新的理论依据和潜在靶点。

男性高龄与子代疾病风险增加显著相关，包括肌肉骨骼综合征、腭裂、急性淋巴细胞白血病、视网膜母细胞瘤、自闭症谱系障碍、精神分裂症等。有研究表明，高龄男性子代神经精神障碍发病率增高可能与精子发生过程中新生突变率增高有关。另一个影响因素可能是精子表观遗传修饰的改变。多项研究报道了男性精子DNA甲基化的改变与子代的神经精神疾病发生有关。另一种表观修饰来源于转运RNA的小RNA (transfer RNA-derived small RNAs, tsRNAs)同样被证实可以作为一种表观调控因子将父亲的代谢特征传递给后代。本次课题中，我们首先在小鼠实验中发现：1.高龄组自然交配的F1代焦虑样行为增高。2.小RNA测序显示雄性高龄改变了小鼠精子tsRNA表达谱。3.分离并注射小鼠精子tsRNA来源的高龄组子代小鼠焦虑样行为增高:与年轻组相比较，高龄组子代小鼠在旷场试验中心区域停留时间及高架十字迷宫实验开放臂上停留时间较短。4. 分别对两组F1代雄性小鼠大脑皮质和海马进行RNA测序，发现两组差异表达基因功能分析富集于多巴胺能突触和神经营养因子信号通路。5.囊胚期和2-细胞胚胎的RNA测序同样发现两组的差异基因富集于神经精神病学相关信号通路(神经营养蛋白信号和胆碱能突触信号通路)。6.在人类精子研究中发现，45-50岁男性与25-27岁男性的的精子tsRNA表达谱差异显著。GO分析表明，34个上调的精子tsRNA靶基因参与了神经发生、神经系统发育和神经元的生成。以上研究结果表明，精子tsRNA可作为一种表观遗传因子介导行为学特征的代际传递。本研究在小鼠模型中首次发现了精子tsRNA与子代神经精神类疾病的关系，为探索高龄男性子代神经精神类疾病高发提供新的思路，并且，精子tsRNA有望成为诊断和预防子代疾病的生物标记物。