Several recent studies link parental environments to phenotypes in subsequent generations. Here, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA levels in mature sperm, with decreased let-7 levels and increased levels of 5’ fragments of glycine tRNAs. tRNA fragments are scarce in testicular sperm, but are gained as sperm mature in the epididymis. Epididymosomes – vesicles that fuse with sperm during epididymal transit – carry RNA payloads matching those of mature sperm, and deliver RNAs to immature sperm in vitro. Functionally, tRNA-Gly-GCC fragments repress genes associated with the endogenous retroelement MERVL, both in ES cells and embryos. Our results shed light on small RNA biogenesis, and its dietary regulation, during post-testicular sperm maturation, and link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.

The biogenesis of the RNA payload of mature sperm is of great interest, because RNAs delivered to the zygote at fertilization can affect early development. Here, we tested the hypothesis that small RNAs are trafficked to mammalian sperm during the process of post-testicular maturation in the epididymis. By characterizing small RNA dynamics during germ cell maturation in mice, we confirm and extend prior observations that sperm undergo a dramatic switch in the RNA payload from piRNAs to tRNA fragments (tRFs) upon exiting the testis and entering the epididymis. Small RNA delivery to sperm could be recapitulated in vitro by incubating testicular spermatozoa with caput epididymosomes. Finally, tissue-specific metabolic labeling of RNAs in intact mice definitively shows that mature sperm carry RNAs that were originally synthesized in the epididymal epithelium. These data demonstrate that soma-germline RNA transfer occurs in male mammals, most likely via vesicular transport from the epididymis to maturing sperm.

The most fundamental process for the perpetuation of a species is the transfer of information from parent to offspring. Although genomic DNA contributes to the majority of the inheritance, it is now clear that epigenetic information −information beyond the underlying DNA sequence − is also passed on to future generations. However, the mechanism and extent of such inheritance are not well-understood. Here, I review some of the concepts, evidence, and mechanisms of intergenerational epigenetic inheritance via sperm small RNAs. Recent studies provide evidence that mature sperm are highly abundant in small non-coding RNAs. These RNAs are modulated by paternal environmental conditions and potentially delivered to the zygote at fertilization, where they can regulate early embryonic development. Intriguingly, sperm small RNA payload undergoes dramatic changes during testicular and post-testicular maturation, making the mature sperm epigenome highly unique and distinct from testicular germ cells. I explore the mechanism of sperm small RNA remodeling during post-testicular maturation in the epididymis, and the potential role of this reprograming in intergenerational epigenetic inheritance.

A number of molecular pathways play key roles in transmitting information in addition to the genomic sequence-epigenetic information-from one generation to the next. However, so-called epigenetic marks also impact an enormous variety of physiological processes, even under circumstances that do not result in heritable consequences. Perhaps inevitably, the epigenetic regulatory machinery is highly responsive to metabolic cues, as, for example, central metabolites are the substrates for the enzymes that catalyze the deposition of covalent modifications on histones, DNA, and RNA. Interestingly, in addition to the effects that metabolites exert over biological regulation in somatic cells, over the past decade multiple studies have shown that ancestral nutrition can alter the metabolic phenotype of offspring, raising the question of how metabolism regulates the epigenome of germ cells. Here, we review the widespread links between metabolism and epigenetic modifications, both in somatic cells and in the germline.

SummaryRNAs present in mature mammalian sperm are delivered to the zygote at fertilization, where they have the potential to affect early development. The biogenesis of the small RNA payload of mature sperm is therefore of great interest, as it may be a target of signaling pathways linking paternal conditions to offspring phenotype. Recent studies have suggested the surprising hypothesis that the small RNA payload carried by mature sperm may include RNAs that were not synthesized during testicular spermatogenesis, but that are instead delivered to sperm during the process of post-testicular maturation in the epididymis. To further test this hypothesis, we characterized small RNA dynamics during testicular and post-testicular germ cell maturation in mice. We show that purified testicular germ cell populations, including mature testicular spermatozoa, carry extremely low levels of tRNA fragments (tRFs), and that tRFs become highly abundant only after sperm have entered the epididiymis. The process of small RNA delivery to sperm can be recapitulated in vitro, as caput epididymosomes deliver small RNAs including tRFs and microRNAs to mature testicular spermatozoa. Finally, to definitively identify the tissue of origin for small RNAs in sperm, we carried out tissue-specific metabolic labeling of RNAs in intact mice, finding that mature sperm carry small RNAs that were originally synthesized in the somatic cells of the epididymis. Taken together, our data demonstrates that soma-germline small RNA transfer occurs in male mammals, most likely via vesicular transport from the epididymis to maturing sperm. CC-BY-NC 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/194522 doi: bioRxiv preprint first posted online Sep. 27, 2017; INTRODUCTIONInheritance of phenotypic changes in the absence of any change in DNA is known as epigenetic inheritance, and is essential for cell state inheritance during mitosis. Epigenetic information is not only inherited during cell division, but at least some epigenetic information can be transmitted from one generation to the next in multicellular organisms, a mechanistically complex process that requires epigenetic information to be maintained throughout the disruptive processes of gametogenesis, fertilization, and early embryonic development. Although decades of study of cell state inheritance and other cases of epigenetic inheritance have identified several major molecular conduits of epigenetic information -chromatin structure, small RNAs, DNA modifications, prions, and transcription factors -much remains to be understood regarding the establishment and erasure of epigenetic information during gametogenesis and early development.Small (<40 nt) RNAs are one of the best-established carriers of epigenetic information, and play key roles in epigenetic inheritance paradigms in C. elegans (Fire et al