安诺优达基因科技（北京）有限公司Sperm tsRNAs contribute to intergenerational inheritanceof an acquired metabolic disorder
Qi Chen, Menghong Yan, Zhonghong Cao, et al.
2015，《Science》
电热暖水袋文章简介：
子代的代谢紊乱现象可以从父亲的饮食中觅得踪迹；但其机制至今仍不甚明了。本研究借助父代高脂肪饮食（HFD）小鼠模型进行研究，发现了一批长度为30~34 nt、来自tRNA5`端序列的精子tsRNA，这些
HFD精子tsRNA的表达谱以及RNA修饰谱均发生了显著的变化。将HFD父代精子tsRNA片段注射到正常小鼠的受精卵中，使得早期胚胎和出生后F1代胰岛相关代谢通路上的基因表达水平发生明显变化，并造成F1代小鼠代谢紊乱。但这些变化却与基因的CpG岛DNA甲基化程度并不相关。因此，精子tsRNAs可以看作一种父代表观遗传的因素，并可以调节饮食诱导代谢紊乱的跨代遗传。 文章单位：Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
二氯丙醇
影响因子：33.611
文章主要内容
1.1 单细胞测序方法：
采用Smart-seq2技术进行反转录，20 ng的cDNA用于单细胞转录组的文库构建。 1.2测序平台及策略：IlluminaHiSeq 2500，PE125
2. 研究结果
2.1父代获得性代谢紊乱性状通过精子RNA向F1代传递
研究通过从第五周开始持续喂食F0代雄性老鼠6个月高脂食物（HFD），获得了父亲饮食诱导代谢紊乱的老鼠模型。将HFD和ND组的精子总RNA分别注射进正常的受精卵中，发现来源HFD组精子的后代葡萄糖耐受性受损。small RNA-seq分析了两组精子sncRNA的表达谱，结果显示成熟的精子携带一类small RNA子集，这类高度富集的small RNA长度在30-40 nt 之间，来自tRNA的5`端，命名为tRNA衍生的小RNA (tsRNA)，而且在HFD处理下，tsRNA
安诺优达基因科技（北京）有限公司比miRNA更敏感。
图1 父代获得性代谢紊乱性状通过精子RNA向F1代传递
2.2 精子tsRNA将父代获得性代谢紊乱传递给F1代
为了进一步研究到底是精子tsRNA还是其他精子RNA片段能诱导获得性性状的跨代传递，研究者分离出HFD组和ND组精子中片段大小不同的三类small RNA：30-40 nt（主要是tsRNA），15-25 nt（主要是miRNA）和大于40 nt的RNA，并将这三类RNA片段注射到正常的受精卵中。结果发现，注射15-25 nt的RNA以及注射大于40 nt的RNA片段都没有造成F1子
安诺优达基因科技（北京）有限公司代代谢紊乱，然而注射了30-40 nt的RNA与ND组相比，HFD组的雄性子代在葡萄糖耐受测试
中表现出葡萄糖不耐症。这些结果证明精子30-40 nt的RNA片段（主要是tsRNA）是重现F1
监视设备代获得性代谢紊乱表型效果所必须的。
图2 精子tsRNA将父代获得性代谢紊乱传递给F1代
2.3 高脂肪饮食父亲小鼠精子的tsRNA使得F1代早期胚胎和胰岛的基因表达差异化
为了揭开注射精子tsRNA的F1子代葡萄糖不耐症的潜在原因，研究者分离了F1代的胰岛
氧化挂具并进行RNA-Seq和RRBS。RNA-Seq分析显示，相对于ND组，HFD组中差异基因主要在代谢
通路（包括酮代谢、碳水化合物代谢、单糖代谢）表达下调。而全基因组RRBS分析显示在
ND组和HFD组中获得的DMR相关基因与变化的转录本没有共性基因，这说明差异DNA甲基
化并不是F1代转录本活性变化的直接原因。
为了证明另外的重要可能性，即注射精子tsRNA到受精卵中可能造成胚胎期转录本发生
级联变化并最终指导F1代胰岛中基因表达的改变，研究者收集注射了ND和HFD精子tsRNA
图像型火焰探测器后的8细胞时期胚胎和胚囊进行比较转录组学分析。发现早期胚胎转录本的变化可能对下游
造成了严重的影响，导致F1代胰岛中基因表达的重编码，最终造成代谢紊乱。信号通路分
析揭示这些差异表达的基因具有调节多样化细胞事件的功能（如：细胞凋亡、自噬，氧化压
力，葡萄糖摄入等）。这些证据都表明了精子tsRNA可能通过转录级联（下调表达基因）来
影响从胚胎期到成年的代谢基因表达水平，并最终影响F1代表型。
安诺优达基因科技（北京）有限公司
图3 来自高脂肪饮食父亲小鼠精子的tsRNA使得F1代早期胚胎和胰岛的基因表达差异化
铝合金精密铸造
（A）F1代胰岛差异表达基因的热图；（B）在8细胞时期和胚囊时期分别注射对照组和高脂诱导组精子tsRNA 后差异表达基因的散点图；（C）在8细胞期和胚囊期显著变化的基因个数的韦恩图；（D）精子tsRNA倾向与启动子区域而不是mRNA编码区域匹配；（E）在韦恩图中，蓝表示的是8细胞时期表达的与tsRNA 匹配的基因个数，红表示的是注射对照组和高脂诱导组精子tsRNA后显著变化的基因个数。
3. 结论
本研究从精子RNA角度为研究获得性性状的跨代遗传现象开拓了全新的领域，未来关于精子tsRNA及其修饰谱在早期胚胎发育调节中的作用机制将是生殖发育领域内亟待解决的关键问题。
4. 参考文献
1. Chen Q, Yan M, Cao Z, Li X, Zhang Y, Shi J, Feng GH, Peng H, Zhang X, Zhang Y et al: Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder. Science 2015.
REPORTS Cite as: Chen et al., Science 10.1126/science.aad7977 (2015).
Increasing lines of evidence from worms to mammals sug-gest that parental environmental exposure can affect the germline and influence future generations through epige-netic mechanisms (1, 2). Specifically, diet-induced metabolic changes in mammals are inherited from father to offspring (3, 4), suggesting sperm mediated epigenetic inheritance (5). DNA methylation is affected (6, 7), yet a causal relationship in transgenerational inheritance has not been established. Small non-coding RNAs (sncRNAs) regulate DNA methyla-tion, histone modifications, and mRNA transcription (8), and can induce non-Mendelian transgenerational inher-itance in mammals (9–13). Altered sperm miRNA
profiles have been observed following paternal exposure to diet or trauma (14, 15); however, since mammalian sperm harbors a diversity of sncRNAs (16), the specific population of RNAs that mediate intergenerational epigenetic memory remains unknown (17, 18). Here, we report that a subset of sperm tRNA-derived small RNAs (tsRNAs), mainly from 5′tRNA halves and about 30-34 nucleotides (19), showed alterations in expression profile and RNA modifications after paternal high-fat diet exposure and transmitted certain metabolic disorders from father to offspring.
To establish a model of intergenerational transmission of paternal diet-induced metabolic disorder (4, 7, 14), we con-tinuously fed F0 male mice with a high-fat diet (HFD, 60% fat) or a normal diet (ND, 10% fat) for 6 months beginning at 5 weeks of age. As expected, males fed a HFD became obese, glucose intolerant, and insulin resistant, whereas the ND group did not (fig. S1). The sperm heads of ND and HFD mice were injected into normal mouse oocytes, and the em-bryos were transferred into surrogate mothers. Male off-spring resulting from the HFD and ND sperm were fed a ND and exhibited no obvious differences in body weight within 16 weeks (fig. S2A), However, offspring produced by the HFD sperm exhibited onset of impaired glucose toler-ance and insulin resistance as early as 7 weeks of age (fig. S2, B and C), which became more severe at 15 weeks as re-vealed by a glucose tolerance test (GTT) and an insulin tol-erance test (ITT) (fig. S2, D and F). Although the e
mbryo manipulation procedures may induce epigenetic alterations, our parallel sperm head injection experiments have elimi-nated the potential influence of male-female contact and semen factors during natural mating (20), suggesting that the sperm itself contains sufficient information to transmit acquired metabolic disorder to offspring.
To assess whether sperm RNAs can induce intergenera-tional phenotypes (15), we purified total RNAs from sperm of both HFD and ND mice and injected them into normal zygotes (RNA injection was normalized to approximately the amount of 10 sperm). Again, the male offspring from both groups exhibited similar body weight growth (Fig. 1A), whereas the offspring from HFD group developed impaired glucose tolerance, showing significantly higher blood glu-cose and serum insulin levels during GTT than ND group at both 7 and 15 weeks of age (Fig. 1, B and C and fig. S3). However, the insulin sensitivity of the HFD group’s off-
Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder
Qi Chen,1,2*† Menghong Yan,3† Zhonghong Cao,1,4† Xin Li,1† Yunfang Zhang,1,4† Junchao Shi,1,4† Gui-hai Feng,1 Hongying Peng,1,5 Xudong Zhang,1,4 Ying Zhang,1 Jingjing Qian,1,4 Enkui Duan,1* Qiwei Zhai,3* Qi Zhou1*
1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. 2Department of Physiology and Cell Biology, School of Medicine, University of Nevada, Reno, NV 89512 USA. 3Key Laboratory of Nutrition and Metabolism, Chinese Academy of Sciences Center for Excellence
in Molecular Cell Science, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China. 4University of Chinese Academy of Sciences, Beijing 100049, China. 5Beijing Royal Integrative Medicine Hospital, Beijing University of Chinese Medicine, Beijing, China.
*Corresponding author. E-mail: vada.edu (Q.C.); duane@ioz.ac (E.D.); qwzhai@sibs.ac (Q.Zha.); qzhou@ioz.ac (Q.Zho.)
†These authors contributed equally to this work.
Increasing evidence indicates that offspring metabolic disorders can result from the father’s diet, but the mechanism remains unclear. Here, in a paternal high-fat diet (HFD) mouse model, we show that a subset
of sperm tRNA-derived small RNAs (tsRNAs), mainly from 5′ tRNA halves and ranging in size from 30 to
34 nucleotides, exhibit changes in expression profiles and RNA modifications. Injection of sperm tsRNA fractions from HFD male into normal zygotes generated metabolic disorders in the F1 offspring and altered gene expression of metabolic pathways in early embryos and islets of F1 offspring, which was unrelated to DNA methylation at CpG-enriched regions. Hence, sperm tsRNAs represent a type of paternal epigenetic factor that may mediate intergenerational inheritance of diet-induced metabolic disorder.
First release: 31 December 2015 (Page numbers not final at time of first release) 1
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