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

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.

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 pi6^em1 (b) and pi6^em2 (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. pi6^em1/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 pi6^em1/em1 and pi6^em2/em2 males. Each dot represents an individual mouse. Vertical lines denote median; boxes report 75^th and 25^th percentiles; whiskers indicate the maximal and minimal values. (c) Representative spermatozoa from C57BL/6 and pi6^em1/em1 males. (d) Representative patterns of meiotic chromosome synapsis in pi6^em1/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 pi6^em1/em1 (n = 4) males.

Extended Data Fig. 3. Abundance of transposons in pi6^em1/em1 and pi6^em2/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 uniquemapping reads in one C57BL/6 (n = 3) or pi6^em1/em1 (n = 3) male. Vertical black lines denote median; boxes report 75th and 25^th 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 pi6^em1/em1) or three (pi6^em2/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 pi6^em1/em1 and pi6^em2/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 qRTPCR. (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 75^th and 25^th percentiles, and whiskers indicate the maximal and minimal values. Each dot represents an individual mouse.

Figure 1.. pi6^em1/em1, pi6^em2/em2, and pi17^−/− promoter deletion in mice

Scissors indicate sites targeted by sgRNAs used to guide the Cas9-catalyzed promoter deletions. RNA-seq was used to measure the steady-state abundance of piRNA primary transcripts, and sequencing of NaIO₄ oxidation-resistant small RNA was used to measure the abundance of mature piRNAs in 17.5 dpp testes. See also Extended Data Figure 1 and Supplementary Table 1.

Figure 2.. Reduced fertility in pi6^em1/em1 males by natural mating

(a) Number of litters and pups per litter produced by male mice between 2–8 months of age. (b) Frequency and periodicity of litter production. Each bar represents a litter. (c) Number of embryos produced by males mated with C57BL/6 females. (d) Testis morphology analyzed by hematoxylin and eosin staining. (e) Concentration of sperm from the caudal epididymis of C57BL/6 (n = 13), pi6^+/em1 (n = 4), pi6^em1/em1 (n = 6), pi17^+/− (n = 7), and pi17^−/− (n = 4) males. In (a), (c), and (e), vertical black lines denote median; boxes indicate 75^th and 25^th percentiles; whiskers report the maximal and minimal values. Each dot represents an individual male. See also Extended Data Figure 2.

Figure 3.. Fertilization defects of pi6^em1/em1 and pi6^em2/em2 sperm revealed by IVF and ICSI

(a) Sperm function analyzed by in vitro fertilization (IVF) using oocytes with or without zona pellucida. Vertical black lines denote median; boxes indicate 75^th and 25^th percentiles; whiskers report the maximal and minimal values. Each dot represents the IVF result using sperm from an individual male. (b) Sperm function analyzed by intracytoplasmic sperm injection (ICSI). See also Extended Data Figure 3

Figure 4.. Impaired sperm capacitation in pi6 mutant sperm

(a) Strategy to measure sperm motility and acrosome reaction triggered with Ca²⁺ ionophore A23187. (b) Definition of path and progressive velocities. (c) Distribution of path and progressive velocities for sperm from C57BL/6 (n = 9), pi6^em1/em1 (n = 11), and pi6^em2/em2 (n = 2). Top: 10 μm/sec bins; bottom: bins correspond to immotile or slow, intermediate, and vigorous motility. (d) Distribution of progressive and hyperactivated sperm from C57BL/6 (n = 9), pi6^em1/em1 (n = 11), and pi6^em2/em2 (n = 2) mice determined by CASAnova. (e) (Top panel) Acrosome status of representative wild-type caudal epididymal spermatozoa. Green, peanut agglutinin to detect the acrosome; blue, DAPI to detect DNA. (Bottom panel) Acrosome reaction rates for C57BL/6 (n = 5) and pi6 mutant (n = 5) sperm. The results using pi6^em1/em1 and pi6^em2/em2 sperm for acrosome reaction were combined as indicated. In (c), (d), and (e), vertical lines denote median; boxes indicate 75^th and 25^th percentiles; whiskers report the maximal and minimal values. Each dot represents an individual male. See also Supplementary Movies 1–10.

Figure 5.. Embryos derived from pi6^em1/em1 sperm fail to develop

(a) Development of IVF-derived embryos. Red, number of embryos that developed to the stage appropriate for the elapsed time after fertilization. (b) Strategy for surgical transfer of fertilized two-cell embryos to surrogate mothers. (c) Percentages of IVF-derived two-cell embryos using sperm from C57BL/6 (n = 3), pi6^+/em1 (n = 3), or pi6^em1/em1 (n = 6) mice that developed to term. Each uterine cartoon represents one surrogate mother; colored circles depict embryos. The number of embryos transferred to each side of the oviduct is indicated. (d) Percentages of ICSI-derived two-cell embryos using sperm from pi6^+/em1 (n = 2) or pi6^em1/em1 (n = 2) mice that developed to term. See also Extended Data Figure 4

Figure 6.. mRNAs encoding proteins required for sperm capacitation and zona pellucida-binding are direct targets of pi6 piRNAs

(a) Strategy for purifying specific male germ cell types. (b) Scatter plots of steady-state transcript abundance in sorted testicular germ cells. Each dot represents the mean abundance of an mRNA measured using four (wild-type and pi6^em1/em1 cells) or three (pi6^em2/em2 cells) individual males. Differentially expressed transcripts (>2 fold-change and FDR <0.05) were identified using DESeq2 (see also Methods) and are indicated, (c) Ultrastructure of caudal epididymal sperm flagella and acrosomes from mice of indicated genotypes by transmission electron microscopy. See also Extended Data Figure 5 and 6, and Supplementary Tables 2, 3, and 4.

Figure 7.. pi6 piRNAs and piRNAs from other loci form a network to repress mRNA expression and facilitate piRNA biogenesis

(a) (Left panel) Cumulative abundance of pi6 and pi17 piRNA-directed, 3′ cleavage products in pi6^em1/em1 diplotene spermatocytes. (Right panel) Analysis of 5′ to 5′ distances for mature piRNAs derived from pachytene piRNA precursors cleaved by pi6 or pi17 piRNAs in diplotene spermatocytes. piRNA-directed cleavage sites were identified requiring uninterrupted base-pairing from position g2 to g16 between a pi6 (10 sites; top right panel) or pi17 (21 sites; bottom right panel) piRNA and the transcript, such that target cleavage between t10 and t11 would generate the 5′ monophosphorylated, 3′ cleavage fragment detected by degradome-seq. p value was computed using the Kolmogorov-Smirnov test, (b) Abundance of mature piRNAs measured by small RNA-seq. Each dot represents the abundance of uniquely mapping reads in one mouse. (c) Expression of piRNA precursors measured by RNA-seq. Each dot represents the abundance of transcripts in one mouse. In (b) and (c), horizontal black lines denote median; boxes report 75^th and 25^th percentiles; whiskers indicate the maximal and minimal values. (d) Pachytene piRNA-directed cleavage in pachytene piRNA precursors. The 22 loci producing the most abundant pachytene piRNAs plus pi10-qC2-545.1 and pi10-qA3-143.1 are shown. Each arrow points from the source of a piRNA towards the locus whose transcript it cleaves and the line thickness is proportional to the number of piRNA-directed cleavage sites shown in Supplementary Table 6. Thick lines link loci sharing a single divergently transcribed promoter. Plus and minus signs indicate the Watson and Crick strands, respectively. (e) A model for pi6 piRNA biogenesis and function. See also Extended Data Figures 5 and 6, and Supplementary Tables 4, 5, and 6.