Mitochondrial signals mediate paternal effects on offspring metabolism (Smart-Seq)

Spermatozoa deliver a complex and environment sensitive pool of small non-coding RNAs (sncRNA) to the oocyte at fertilisation [ref], which influences offspring development and adult phenotypic trajectories [refs]. Whether mature spermatozoa in the epididymis can directly sense the environment is still not fully understood [ref]. Here, we used two distinct paradigms of preconception acute High Fat Diet challenge to dissect epididymal vs spermatogenic contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNA fragments as sperm-born sensors. In human spermatozoa, we found mt-tsRNAs in linear association with BMI and showed that paternal overweight at conception is sufficient to double offspring obesity risk and compromise metabolic health. Using mouse genetics and metabolic phenotypic data, we show that alterations of mt-tsRNAs are downstream of mitochondrial dysfunction in mice. Most importantly, single embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tsRNAs at fertilisation and implied them in the control of early embryo metabolism. Our study supports the importance of paternal health at conception for offspring metabolism, propose mt-tsRNAs as sperm-born environmental effectors of paternal inheritance and demonstrate, for the first time in a physiological and unperturbed setting, father-to-offspring transfer of sperm mt-tsRNAs at fertilisation. Oocytes from C57BL/6N-mtST were fertilized with sperm from HFD or LFD treated C57BL/6N animals. Early 2cell embryos were collected after overnight incubation and snap frozen in lysis buffer according to SMARTseq2 protocol.精子（spermatozoa）会在受精过程中向卵母细胞递送一组复杂且对环境敏感的小非编码RNA（small non-coding RNAs, sncRNA）[参考文献]，该过程会影响子代发育与成年表型轨迹[参考文献]。目前学界尚未完全明确附睾内的成熟精子是否可直接感应环境变化[参考文献]。本研究采用两种不同的孕前急性高脂饮食（High Fat Diet, HFD）干预模型，解析附睾来源与生精过程来源的组分对精子小非编码RNA库及子代健康的贡献。研究发现，附睾成熟精子而非发育中的生殖细胞可对环境产生响应，并鉴定出线粒体tRNA片段作为精子源性的环境感应因子。在人类精子中，我们发现线粒体tRNA衍生片段（mitochondrial tRNA-derived small RNAs, mt-tsRNAs）的表达水平与身体质量指数（Body Mass Index, BMI）呈线性相关，同时证实父亲在受孕时的超重状态足以使子代肥胖风险翻倍，并损害子代代谢健康。借助小鼠遗传学与代谢表型数据，我们证明线粒体tRNA衍生片段的异常变化源于线粒体功能障碍的下游效应。最为关键的是，对基因杂交二细胞胚胎的单个胚胎转录组学分析显示，受精过程中存在精子向卵母细胞的线粒体tRNA衍生片段转运，且该片段可能参与调控早期胚胎代谢。本研究证实了受孕时父亲健康状态对子代代谢的重要性，提出线粒体tRNA衍生片段可作为父系遗传的精子源性环境效应因子，并首次在生理状态且未受干扰的实验体系中，证明了受精过程中精子线粒体tRNA衍生片段向子代的父系传递。本研究使用C57BL/6N-mtST品系的卵母细胞，与经高脂饮食或低脂饮食（Low Fat Diet, LFD）处理的C57BL/6N品系雄性的精子进行受精。过夜培养后收集早期二细胞胚胎，并依据SMARTseq2实验流程将其快速冷冻于裂解缓冲液中。