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. 2016 Jan 22;351(6271):391-396.
doi: 10.1126/science.aad6780. Epub 2015 Dec 31.

Biogenesis and Function of tRNA Fragments During Sperm Maturation and Fertilization in Mammals

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Free PMC article

Biogenesis and Function of tRNA Fragments During Sperm Maturation and Fertilization in Mammals

Upasna Sharma et al. Science. .
Free PMC article

Abstract

Several recent studies link parental environments to phenotypes in subsequent generations. In this work, we investigate the mechanism by which paternal diet affects offspring metabolism. Protein restriction in mice affects small RNA (sRNA) levels in mature sperm, with decreased let-7 levels and increased amounts of 5' fragments of glycine transfer RNAs (tRNAs). In testicular sperm, tRNA fragments are scarce but increase in abundance as sperm mature in the epididymis. Epididymosomes (vesicles that fuse with sperm during epididymal transit) carry RNA payloads matching those of mature sperm and can deliver RNAs to immature sperm in vitro. Functionally, tRNA-glycine-GCC fragments repress genes associated with the endogenous retroelement MERVL, in both embryonic stem cells and embryos. Our results shed light on sRNA biogenesis and its dietary regulation during posttesticular sperm maturation, and they also link tRNA fragments to regulation of endogenous retroelements active in the preimplantation embryo.

Figures

Fig.1
Fig.1. Dietary effects on small RNAs in sperm
(A) Size distribution of sequencing reads for cauda sperm small RNAs. (B-D) 5’ tRNA fragments are shown schematically, with arrows indicating dominant 3’ ends. (E) Dietary effects on sperm small RNA content. Scatterplot shows RNA abundance (ppm) for sperm isolated from Control (x axis, log10) compared to Low Protein sperm (y axis), with various RNA classes indicated. Multiple points for tRFs result from sequence differences between genes encoding a given tRNA isoacceptor. (F) Heatmap showing RNAs responding to diet across eight paired sperm samples.
Fig.2
Fig.2. tRNA cleavage predominantly occurs in the epididymis
(A) Sperm RNA payload diverges dramatically from testicular RNA. Scatterplot shows small RNAs in testis vs. sperm, as in Fig.1E. (B) Schematic of epididymis. Sperm exiting the testis enter the proximal (caput) epididymis, then proceed distally to corpus and cauda epididymis, and exit via the vas deferens. (C) tRFs are primarily generated in epididymis. Northern blots for 5’ ends of tRNA-Gly-GCC or tRNA-Val-CAC on RNA extracted from testis, caput, and cauda epididymis. Arrow indicates ~30-34 nt 5’ tRFs. 5S RNA serves as loading control. (D) Pie charts showing percentage of small RNAs mapping to the indicated features. (E) Scatterplot of small RNA abundance for sperm vs. epididymosomes. Sperm-enriched RNAs include piRNAs and fragments of mRNAs involved in spermatogenesis (eg Prm1), and represent RNAs synthesized during testicular spermatogenesis.
Fig.3
Fig.3. Changes in sperm tRF payload during epididymal transit
(A) Proximal-distal biases observed for RNAs in epididymis are recapitulated in sperm samples. (B) Proximal-distal biases for tRFs, aggregated by anticodon, in epididymis and sperm. (C) TaqMan of the indicated tRFs in caput sperm and reconstituted sperm, showing gain of tRFs relative to let-7 (t-test p=0.05 for Gly-GCC, 0.004 for Val-CAC). (D) Deep sequencing of reconstituted sperm. tRFs are aggregated by codon, and normalized to levels of tRF-Glu-CTC. Caput vs. cauda differences were broadly recapitulated in reconstitutions, with tRFs such as tRF-Val-CAC being delivered to caput sperm via fusion with cauda epididymosomes.
Fig.4
Fig.4. tRF-Gly-GCC regulates MERVL-driven transcripts
(A) Scatterplot shows mRNA abundance in ESCs transfected with GFP siRNA vs. LNA antisense to 5’ end of tRF-Gly-GCC. (B) Effect of tRF-Gly-GCC inhibition on MERVL-regulated genes is isoacceptor-specific. Affymetrix data for ESCs transfected with LNA antisense oligos, relative to matched GFP controls, showing genes changing 2-fold in 2 or more samples. (C) RNA-Seq data for ESCs transfected with GFP siRNA, or anti-tRF-Gly-GCC. (D) Genomic context of tRF-Gly-GCC target genes, showing nearby MERVL LTRs. (E) tRF-Gly-GCC inhibition affects MERVL targets in embryos. Averaged single embryo RNA-Seq data for control (n=28) or tRF-inhibited (n=27) 4-cell stage embryos. Among 2-fold upregulated genes, known MERVL targets(17) are indicated. (F) Single embryo data for two MERVL targets.
Fig.5
Fig.5. Paternal dietary effects on preimplantation development
(A) Embryos generated by IVF were cultured for varying times, then subject to single embryo RNA-Seq. (B) Single-embryo data for preimplantation embryos represented via PCA: first two principal components explain 74% of dataset variance. (C) mRNA abundance in 2-cell embryos generated via IVF using Control vs. Low Protein sperm (n=41 C and 39 LP). Cumulative distribution plots for tRF-Gly-GCC targets (p=4.5×10-7, KS test), other MERVL targets (17) (p=2.5×10-13), and all remaining genes, showing percentage of genes with the average Log2(LP/C) indicated on the x axis. Low Protein embryos exhibit a significant shift to lower expression of MERVL targets. Bottom panels show individual embryo data for two targets. (D) Small RNAs isolated from Control or Low Protein cauda sperm were microinjected into control zygotes. RNA-Seq (n=42 C and 46 LP embryos) reveals downregulation of tRF-Gly-GCC targets (p=4.8×10-14) driven by Low Protein RNAs. (E) Effects of synthetic tRF-Gly-GCC on 2-cell gene regulation, showing significant (p=0.0001) downregulation of target genes in embryos injected with tRF-Gly-GCC (n=26) vs. GFP controls (n=11). Inset shows effects of tRF-Glu-CTC (n=6). (F) Effects of epididymal passage on embryonic gene regulation. Intact sperm isolated from rete testis (n=12), or cauda epididymis (n=9), were injected into control oocytes, and mRNA abundance was analyzed as above.

Comment in

  • Biol Reprod. 2016 Apr;94(4):73

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