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.

Gametogenesis is a thermosensitive process in numerous metazoans, ranging from worms to man. In Caenorhabditis elegans, a variety of RNA-binding proteins that associate with germ-line nuage (P granules), including the Piwi-clade argonaute PRG-1, have been implicated in maintaining fertility at elevated temperature. Here we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3), in promoting thermotolerant male fertility. A rescuing GFP::alg-3 transgene is localized to P granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, the 26G-RNAs which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for thermotolerant fertility in C. elegans and support a model in which AGOs and their small RNA cofactors function to promote robustness in gene-expression networks. Argonautes can be classified into three clades (2): (i) the AGO clade, which includes the human AGOs1-4, the Caenorhabditis elegans miRNA effectors ALG1/2, and all of the Arabidopsis thaliana AGOs; (ii) the Piwi clade, which more closely resembles Drosophila Piwi and includes the C. elegans piRNA AGO PRG-1; and (iii) an expanded family of worm-specific AGOs (WAGOs) that, with a few exceptions, lack residues in the PIWI domain thought to be necessary for slicer endonuclease activity.In C. elegans, null mutations are available for the entire family of over 24 AGO genes (3), and at least five different combinations of these mutants result in lethal or sterile phenotypes. alg-1/2 mutants display heterochronic defects and lethality that arise from loss of miRNA-mediated regulation of developmentally important transcripts (4). csr-1 mutants have severe chromosome segregation defects that result in embryonic lethality (3, 5), and prg-1 mutants have severe defects in the development of the germline (6-8). In addition, the simultaneous deletion of 12 WAGOs leads to a temperature-sensitive sterile phenotype, where mutants are viable and fertile at 20°C but sterile at 25°C (9).Here we describe two paralogous AGO-clade family members, alg-3 and alg-4, that are together critical for sperm development and function. Single mutants have only a minor reduction in brood size, but double alg-3/4 mutants exhibit drastically reduced brood sizes at elevated temperatures. We show that a rescuing GFP::ALG-3 protein is expressed in germ cells undergoing spermatogenesis and that, even though both male and hermaphrodite alg-3/4 mutants produce near wild-type numbers of spermatids, these spermatids exhibit severe defects in the activation process called spermiogenesis that converts spermatids into motile ameboid sperm.A class of 26nt small RNAs called the 26G-RNAs were first identified in deep-sequencing datasets in C. elegans as part of the previously described Dicer-ribonuclease-dependent endogenous small RNA pathway, called the ERI pathway, and a subset of these were noted to be enriched for spermatogen...

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.

SUMMARY During each life cycle germ cells preserve and pass on both genetic and epigenetic information. In C. elegans, the ALG-3/4 Argonaute proteins are expressed during male gametogenesis and promote male fertility. Here we show that the CSR-1 Argonaute functions with ALG-3/4 to positively regulate target genes required for spermiogenesis. Our findings suggest that ALG-3/4 functions during spermatogenesis to amplify a small-RNA signal that represents an epigenetic memory of male-specific gene expression. CSR-1, which is abundant in mature sperm, appears to transmit this memory to offspring. Surprisingly, in addition to small RNAs targeting male-specific genes, we show that males also harbor an extensive repertoire of CSR-1 small RNAs targeting oogenesis-specific mRNAs. Together these findings suggest that C. elegans sperm transmit not only the genome but also epigenetic binary signals in the form of Argonaute/small-RNA complexes that constitute a memory of gene expression in preceding generations.

The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) versus distal (cauda) epididymis and then characterized the development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, subsequently implant inefficiently, and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects but also suppressed the post-implantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs.