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The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility

Nature Genetics volume 52, pages 728–739 (2020)Cite this article

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Abstract

Pachytene PIWI-interacting RNAs (piRNAs), which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like microRNAs, cleave targets like short interfering RNAs or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 (pi6) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6−/− fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving messenger RNAs encoding proteins required for sperm function. pi6 also participates in a network of piRNA–piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10, as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability.

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Fig. 1: pi6em1/em1, pi6em2/em2 and pi17−/− promoter deletion in mice.
Fig. 2: Reduced fertility in pi6em1/em1 males by natural mating.
Fig. 3: Fertilization defects of pi6em1/em1 and pi6em2/em2 sperm revealed by IVF and ICSI.
Fig. 4: Impaired sperm capacitation in pi6 mutant sperm.
Fig. 5: Embryos derived from pi6em1/em1 sperm fail to develop.
Fig. 6: mRNAs encoding proteins required for sperm capacitation and zona pellucida binding are direct targets of pi6 piRNAs.
Fig. 7: pi6 piRNAs and piRNAs from other loci form a network to repress mRNA expression and facilitate piRNA biogenesis.

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Data availability

All sequencing data are available through the National Center for Biotechnology Information Sequence Read Archive using accession number PRJNA634688. Source data are provided with this paper.

Code availability

The code used for identifying piRNA-directed cleavage sites is available at https://github.com/weng-lab/GTBuster. All other codes used in this study are described in the Methods and Nature Research Reporting Summary. Source data are provided with this paper.

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Acknowledgements

We thank P. Cohen, K. Grive and E. Crate at Cornell University for generously sharing protocols and advice on germ cell sorting and meiotic chromosome studies; H. Florman, P. Visconti and M. Gervasi for sharing protocols and advice on sperm studies; the UMMS Transgenic Animal Modeling Core for advice on fertility test and embryo phenotype; the UMMS FACS core for advice on and help with germ cell sorting; the UMMS EM Core (supported by National Center for Research Resources Award SI0OD021580) for advice on and help with sperm transmission electron microscopy; and members of our laboratories for critical comments on the manuscript. This work was supported in part by National Institutes of Health grants GM62862 to P.D.Z. and P01HD078253 to P.D.Z. and Z.W.

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Author notes

  1. Yu Fu

    Present address: Oncology Drug Discovery Unit, Takeda Pharmaceuticals, Cambridge, MA, USA

Authors and Affiliations

  1. Howard Hughes Medical Institute and RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA

    Pei-Hsuan Wu, Katharine Cecchini, Deniz M. Özata, Amena Arif, Cansu Colpan, Ildar Gainetdinov & Phillip D. Zamore

  2. Bioinformatics Program, Boston University, Boston, MA, USA

    Yu Fu

  3. Program in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA, USA

    Yu Fu, Tianxiong Yu & Zhiping Weng

  4. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA

    Amena Arif, Cansu Colpan & Zhiping Weng

  5. School of Life Sciences and Technology, Tongji University, Shanghai, China

    Tianxiong Yu

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Contributions

P.-H.W., K.C., Y.F., Z.W. and P.D.Z. conceived and designed the experiments. P.-H.W., K.C., D.M.Ö., A.A. and C.C. performed the experiments. Y.F., T.Y., I.G. and P.-H.W. analyzed the sequencing data. P.-H.W., Y.F. and P.D.Z. wrote the manuscript.

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Correspondence to Pei-Hsuan Wu, Zhiping Weng or Phillip D. Zamore.

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Extended data

Extended Data Fig. 1 Confirmation of mutant founder genotypes.

a, Genotyping of mutant founders by PCR. Mutant founders were generated by injecting sgRNAs and Cas9 mRNAs into C57BL/6 one-cell zygotes, which were transferred to surrogate mothers and screened after birth. Gel images were cropped for clarity (see also Source Data). Genomic sequences of pi6 promoter region in pi6em1 (b) and pi6em2 (c) mouse lines. The presence of both deleted and undeleted PCR products indicate a heterozygous mutant founder that carries just one CRISPR-edited chromosome. d, Genomic sequences of pi17 promoter region in pi17–/– mouse lines. Dashes, genomic sequences deleted by CRISPR; dots, unaltered sequence omitted for clarity.

Extended Data Fig. 2 pi6em1/em1 adult male phenotype.

a, Number of litters produced in 6 months by 2–8 month-old males. b, Body and testis weight of 2–4 month-old pi6em1/em1 and pi6em2/em2 males. Each dot represents an individual mouse. Vertical lines denote median; boxes report 75th and 25th percentiles; whiskers indicate the maximal and minimal values. c, Representative spermatozoa from C57BL/6 and pi6em1/em1 males. d, Representative patterns of meiotic chromosome synapsis in pi6em1/em1 pachytene spermatocytes. SYCP1, Synaptonemal complex protein 1; SYCP3, Synaptonemal complex protein 3. e, Quantification of patterns of meiotic chromosome synapsis depicted in (d) from C57BL/6 (n = 4) and pi6em1/em1 (n = 4) males.

Extended Data Fig. 3 Abundance of transposons in pi6em1/em1 and pi6em2/em2 germ cells.

a, Proportions of the whole genome or piRNA sequences composed of repetitive sequences. b, Abundance of mature piRNAs from the top five major pachytene piRNA-producing loci in indicated cell types measured by small RNA-seq. Each dot represents the abundance of unique-mapping reads in one C57BL/6 (n = 3) or pi6em1/em1 (n = 3) male. Vertical black lines denote median; boxes report 75th and 25th percentiles; whiskers indicate the maximal and minimal values. c, Abundance of transposon-derived RNAs in mouse germ cells. Each dot represents the mean of four (wild-type and pi6em1/em1) or three (pi6em2/em2) biologically independent RNA-seq experiments. Gray dots indicate change in abundance <2-fold and/or FDR > 0.05 determined by DESeq2 (see also Methods).

Extended Data Fig. 4 Pregnancy rate of surrogate mothers in IVF and ICSI experiments.

Percent of pregnant surrogate mothers in IVF (a) and ICSI (b).

Extended Data Fig. 5 Transcripts directly cleaved by pi6 and pi10-qC2-545.1 piRNAs.

a, Strategy to identify piRNA-directed cleavage sites. b, pi6-dependent cleavage sites in mRNAs or pachytene piRNA precursors from pi10-qC2-545.1 and pi10-qA3-143.1 showing inferred base pairing with the corresponding pi6 piRNA guides. An exemplary piRNA guide is shown where more than one piRNA can direct the same cleavage. c, Cleavage sites in pi6 precursors explained by pi10-qC2-545.1 piRNAs. An exemplary piRNA guide is shown.

Extended Data Fig. 6 Transcriptome changes in pi6em1/em1 and pi6em2/em2 cells.

a, Expression of mRNAs measured by qRT-PCR using oligo dT(20) to prime cDNA synthesis and PCR primers spanning pi6 piRNA-directed cleavage sites (gene names in red) or designed to detect full-length RNA (gene names in black). Pou2f2 mRNA abundance in spermatids was below the limit of detection by qRT-PCR. b, Abundance of piRNA precursors from the top five major pachytene piRNA-producing loci in indicated cell types measured by RNA-seq. For (a) and (b), thick vertical lines denote median, boxes report 75th and 25th percentiles, and whiskers indicate the maximal and minimal values. Each dot represents an individual mouse.

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Wu, PH., Fu, Y., Cecchini, K. et al. The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility. Nat Genet 52, 728–739 (2020). https://doi.org/10.1038/s41588-020-0657-7

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