Biogenesis and function of tRNA fragments during sperm maturation and fertilization in mammals (ICSI)

Parental dietary conditions can influence the metabolic traits of offspring. In mice, paternal consumption of low protein diet alters cholesterol and lipid metabolism of progeny. Here, we examine RNA species expressed in male reproductive tissues of mice. Protein restriction leads to altered levels of multiple small RNAs in mature sperm, as well as throughout the male reproductive tract, with decreased levels of let-7 family members and increased levels of 5’ fragments of tRNA-Gly isoacceptors. Intriguingly, tRNA fragments are scarce in the testis, but their levels increase in sperm during post-testicular maturation in the epididymis. We find that epididymosomes – extracellular vesicles which fuse with sperm during epididymal transit – exhibit RNA payloads closely matching those of mature sperm, and can deliver tRNA fragments to immature sperm in vitro both in mouse and in bull. Finally, we show that tRNA-Gly-GCC fragments play a role in repressing genes associated with the endogenous retroelement MERVL, both in ES cells and in preimplantation embryos. Our results shed light on small RNA biogenesis during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the early embryo. Zygotes were generated by ICSI from oocytes/females fed a Control diet and sperm/males fed either a Control or Low Protein diet. The sperm was isolated from either the Rete testis or the Cauda epididymis and injected either as a whole sperm or just the sperm head. Following fertilization by ICSI the zygotes developed for 28 hours (2C stage) and were harvested for single-embryo RNA-Seq.

亲本的膳食状态可对子代的代谢表型产生影响。在小鼠模型中，父本摄入低蛋白饮食会改变子代的胆固醇及脂质代谢水平。本研究针对小鼠雄性生殖组织中的RNA表达物种展开分析。蛋白质限制会使成熟精子及整个雄性生殖道内的多种小RNA水平发生改变，具体表现为let-7家族成员的表达量下调，而tRNA-Gly同工受体（tRNA-Gly isoacceptors）的5'端片段水平上调。
值得注意的是，tRNA片段在睾丸中含量极低，但在精子经附睾完成睾丸后成熟的过程中，其水平会显著升高。本研究发现，附睾小体（epididymosomes）——一种在精子经附睾转运过程中与精子发生融合的细胞外囊泡——其携带的RNA载荷与成熟精子的RNA谱高度匹配，且能够在体外将tRNA片段递送至未成熟精子中，该现象在小鼠和公牛中均存在。
最后，本研究证实，tRNA-Gly-GCC片段能够在胚胎干细胞（Embryonic Stem Cells, ESCs）及植入前胚胎中，抑制与内源性逆转录元件MERVL相关的基因表达。本研究结果阐明了精子睾丸后成熟过程中小RNA的生物发生机制，并将tRNA片段与早期胚胎中活跃的内源性逆转录元件的调控过程建立了关联。
本研究通过卵胞浆内单精子注射（Intracytoplasmic Sperm Injection, ICSI）技术构建受精卵：供体卵母细胞/雌鼠饲喂对照饮食，供体精子/雄鼠则分别饲喂对照饮食或低蛋白饮食。精子分别从睾丸网或附睾尾中分离，注射时可采用完整精子或仅使用精子头部。经ICSI受精后，受精卵将体外培养28小时至二细胞（2C）阶段，随后收集样本用于单胚胎RNA测序分析。