Abstract Piwi-interacting RNAs are a diverse class of small non-coding RNAs implicated in the silencing of transposable elements and the safeguarding of genome integrity. In mammals, male germ cells express two genetically and developmentally distinct populations of piRNAs at the pre-pachytene and pachytene stages of meiosis, respectively. Pre-pachytene piRNAs are mostly derived from retrotransposons and required for their silencing. In contrast, pachytene piRNAs originate from ∼3,000 genomic clusters, and their biogenesis and function remain enigmatic. Here, we report that conditional inactivation of the putative RNA helicase MOV10L1 in mouse spermatocytes produces a specific loss of pachytene piRNAs, significant accumulation of pachytene piRNA precursor transcripts, and unusual polar conglomeration of Piwi proteins with mitochondria. Pachytene piRNA–deficient spermatocytes progress through meiosis without derepression of LINE1 retrotransposons, but become arrested at the post-meiotic round spermatid stage with massive DNA damage. Our results demonstrate that MOV10L1 acts upstream of Piwi proteins in the primary processing of pachytene piRNAs and suggest that, distinct from pre-pachytene piRNAs, pachytene piRNAs fulfill a unique function in maintaining post-meiotic genome integrity.

Author Summary Small non-coding RNAs play critical roles during development and in disease. The integrity of the germline genome is of paramount importance to the wellbeing of offspring and the survival of species. Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs abundantly expressed in the gonad. Compared to microRNAs and small-interfering RNAs (siRNAs), the biogenesis and function of piRNAs remain poorly understood. Here we have identified MOV10L1, a putative RNA helicase, as a master regulator of piRNA biogenesis in mouse. We find that production of pachytene piRNAs requires MOV10L1. Blockade of pachytene piRNAs disrupts germ cell development and results in defects in post-meiotic genome integrity. Therefore, mutations in MOV10L1 and other piRNA pathway components may contribute to male infertility in humans.

Citation: Zheng K, Wang PJ (2012) Blockade of Pachytene piRNA Biogenesis Reveals a Novel Requirement for Maintaining Post-Meiotic Germline Genome Integrity. PLoS Genet 8(11): e1003038. https://doi.org/10.1371/journal.pgen.1003038 Editor: John C. Schimenti, Cornell University, United States of America Received: March 16, 2012; Accepted: September 5, 2012; Published: November 15, 2012 Copyright: © 2012 Zheng, Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health/National Institute of Child Health and Human Development grant R01HD069592. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Piwi-interacting RNAs (piRNAs) are a diverse class of gonad-specific small interfering RNAs that bind to members of the Piwi subfamily of Argonaute proteins. One common function of piRNAs in all species studied so far is the silencing of transposable elements, which is essential for the protection of genome integrity during germ cell development [1]–[3]. Distinct from miRNAs and siRNAs in origin, length, structure, and biogenesis, piRNAs are generated by dicer-independent processing of long precursor transcripts, however, the precise mechanisms of their biogenesis remain largely unclear [4], [5]. In mice, the Piwi family has three members: Miwi (Piwil1), Mili (Piwil2), and Miwi2 (Piwil4). These Piwi genes exhibit different developmental expression patterns in testis. While Miwi2 is expressed in fetal and perinatal germ cells [6], the expression of Miwi is restricted to pachytene spermatocytes and round spermatids in adult testes [7]. Mili is expressed from the fetal germ cell stage onwards through the round spermatid stage [8]. Two developmentally distinct populations of piRNAs are expressed in mouse male germ cells at the pre-pachytene and pachytene stages. Pre-pachytene piRNAs are mostly derived from transposable elements and are associated with MILI and MIWI2 in fetal and perinatal male germ cells [6], [9], [10]. Pachytene piRNAs originate from ∼3000 genomic clusters [11] and bind to both MILI and MIWI [12]–[17]. Interestingly, more than 90% of MILI- and MIWI-bound pachytene piRNAs shared identical 5′end sequences [18]. As a result, most MILI- and MIWI-bound pachytene piRNAs map to the same genomic clusters [18]. The biogenesis of piRNAs involves primary and secondary processing mechanisms [1], [2]. Pre-pachytene piRNAs derive from precursor transcripts that are cleaved into putative primary piRNA intermediate molecules by a yet unknown primary processing mechanism, followed by loading onto MILI for further processing. In embryonic germ cells, the endonuclease (slicer) activity of MILI is required for the secondary piRNA processing mechanism, which amplifies MILI-bound piRNAs through an intra-MILI ping-pong loop and generates all MIWI2-bound secondary piRNAs [19]. In this feed-forward ping-pong model, Piwi proteins with piRNAs complimentary to retroelement-derived transcripts drive transcript cleavage and piRNA amplification [6], [9], [10], [19]. In contrast, the biogenesis of pachytene piRNAs only engages the primary processing mechanism, i.e. the presumptive cleavage by an unknown nuclease and eventual processing of the precursor transcript into mature piRNAs [5], [17], [20], [21]. Therefore, pachytene piRNAs provide a simple and ideal system for dissecting the mysterious primary processing mechanism in mammals [11], [13]–[16]. We and others previously demonstrated that MOV10L1, a putative RNA helicase, interacts with all mouse Piwi proteins and is required for biogenesis of pre-pachytene piRNAs [22], [23]. MOV10L1 homologues are evolutionarily conserved among insects (Armi in Drosophila melanogaster), plants (SDE3 in Arabidopsis thaliana), and vertebrates (MOV10 and MOV10L1). Arabidopsis SDE3 is required for post-transcriptional gene silencing [24]. Drosophila Armi is essential for the maturation of RISC (RNA-induced silencing complex) and miRNA-mediated silencing [25], [26]. Armi is also relevant to the piRNA pathway, evident from the loss of specific piRNAs and the activation of retrotransposons in armi mutants [27], [28]. Specifically, Armi plays an essential role in the primary piRNA processing pathway [29]. In contrast to Drosophila and Arabidopsis with a single Mov10l1 homologue, the vertebrate genome encodes two genes (Mov10 and Mov10l1), which apparently arose by gene duplication. MOV10 is ubiquitously expressed and associates with Ago proteins, forming part of the purified human RISC [30], [31]. Depletion of MOV10 in cultured cells leads to reduced miRNA-mediated silencing [30]. We initially identified MOV10L1 as a putative RNA helicase that is specifically expressed in mouse germ cells [32], [33]. Disruption of Mov10l1 leads to meiotic arrest, de-repression of transposable elements, and depletion of both MILI- and MIWI2-associated perinatal piRNAs [22], [23]. Apparently, MOV10 and MOV10L1 function in the miRNA and the piRNA pathway, respectively, due to specialization after gene duplication during vertebrate evolution. The existing piRNA pathway mouse mutants either fail to deplete all pachytene piRNAs or exhibit meiotic arrest prior to the pachytene stage, leaving the biogenesis and role of pachytene piRNAs largely unexplored. Inactivation of either Mili or Miwi2 causes postnatal meiotic arrest at the leptotene/zygotene stage in the male germline [8], [34]. Similarly, other piRNA pathway mutants, such as Ddx4 (Vasa), Mael, Gasz, Tdrd9, Mov10l1, and Mitopld, also exhibit early meiotic arrest in males [22], [35]–[40]. Inactivation of Miwi leads to spermiogenic arrest at the round spermatid stage [7]. However, MILI-associated pachytene piRNAs are abundant in Miwi-deficient testes [17], [18]. Therefore, a mouse mutant containing pachytene spermatocytes, but lacking all pachytene piRNAs (both MILI- and MIWI-bound piRNAs) has not been available to specifically study the function of pachytene piRNAs. In this study, we have specifically and completely depleted the pachytene piRNA population in the male germline of Mov10l1 mutant mice, uncovering a novel function for pachytene piRNAs in maintaining post-meiotic genome integrity.

Discussion We have identified MOV10L1 as the only factor known to date that is required for the production of all pachytene piRNAs in mouse. As the biogenesis of pachytene piRNAs only involves the primary processing pathway, our conditional Mov10l1 mutants provide a unique opportunity to delineate this enigmatic component of piRNA biogenesis in mammalian species. Presumably, long piRNA precursor transcripts are first cleaved into intermediate molecules, and then processed into mature piRNAs (Figure 6D). Observations that the Drosophila Armi-Piwi-Yb complex is associated with a population of 25–70 nt piRNA intermediate-like (piR-IL) molecules support this hypothesis [20]. Furthermore, recent biochemical studies using silkworm ovarian cell lysate have shown that intermediate piRNA molecules with 5′ U are specifically loaded onto Piwi proteins and then trimmed from the 3′end to generate mature piRNAs [21]. Here, we show that, in the mouse male germline, postnatal disruption of Mov10l1 does not affect the expression of Piwi proteins (MILI and MIWI) but causes a complete loss of pachytene piRNAs, demonstrating that MOV10L1 functions upstream of Piwi proteins in the piRNA biogenesis pathway. Consistent with its homology to Drosophila Armi [25], [26], MOV10L1 is therefore a master regulator of piRNA biogenesis in mouse. This notion is further supported by the dramatic accumulation of pachytene piRNA precursors in the Mov10l1 mutant testes. As MOV10L1 interacts with Piwi proteins, we postulate that MOV10L1 may facilitate the loading of intermediate piRNA molecules onto the Piwi proteins in mouse (Figure 6D). In spermatocytes, proteins of the piRNA pathway such as MILI, MIWI, TDRD1, MAEL, and GASZ, localize to the nuage - inter-mitochondrial cement [7], [8], [36], [37], [44], [51]. However, the functional significance of the physical association of nuage with mitochondria in germ cells is poorly understood. MitoPLD, a mitochondrial signaling protein, is essential for nuage formation and piRNA production, suggesting an important role for mitochondria in these mechanisms [39], [40]. In this study, we find an unusual polar congregation of piRNA pathway proteins (such as MILI, MIWI, TDRD1, and GASZ). Similar to wild-type MOV10L1, truncated MOV10L1Δ is distributed diffusely through the cytoplasm of pachytene spermatocytes; therefore the polar coalescence of the other piRNA pathway components in MOV10L1-deficient pachytene cells is likely caused by the absence of pachytene piRNAs. However, as the association of Piwi-MOV10L1 is disrupted in the Mov10l1 mutant, it is also possible that the localization of Piwi proteins and their interacting partners has become perturbed as a consequence of this disruption. The unusual polar congregation of piRNA pathway proteins with mitochondria in Mov10l1 mutant spermatocytes suggests that MOV10L1 and/or pachytene piRNAs are essential for nuage formation and proper mitochondria distribution. Consistent with such a role, we find that the chromatoid body, a prominent nuage in spermatids, is fragmented in pachytene piRNA-deficient mutant cells. This previously unknown role in organelle distribution shows that pachytene piRNAs are intricately integrated in the inter-dependent relationships among piRNA production, nuage formation, and mitochondria organization that are essential for male germ cell maturation. A recent study has shown that MIWI is an RNA-guided RNase with slicer activity that directly cleaves transcripts of the LINE1 retrotransposon [18]. Miwi-deficient and MiwiADH (slicer inactive) mutant testes, in which MIWI is either absent or lacks slicer activity, exhibit substantial accumulation of LINE1 transcripts and protein. In Mov10l1fl/- Neurog3-Cre testes, however, LINE1 RNA levels are not affected. One possible explanation for these differential effects on LINE1 abundance could be that, in Mov10l1fl/- Neurog3-Cre testes, MIWI is catalytically intact and may function as a slicer through pachytene piRNA-independent mechanisms. Moreover, MIWI directly binds to spermiogenic mRNAs, independent of piRNAs [17]. Although previous genetic studies of piRNA pathway mutants show that perturbation of pre-pachytene piRNAs causes meiotic arrest and de-repression of LINE1 and IAP retrotransposons, the functions of pachytene piRNAs have remained elusive. Our study on the role of Mov10l1 and the piRNA pathway during later stages of meiosis and spermiogenesis demonstrates that pachytene piRNAs fulfill distinct and essential functions during post-meiotic stages of male germ cell development. Most importantly, the massive DNA damage observed in piRNA-deficient round spermatids in the absence of de-repression of LINE1 and IAP transposable elements suggests that the integrity of the post-meiotic germ cell genome remains highly prone to damage, and that pachytene piRNAs fulfill a protective role at this stage by yet undefined mechanisms.

Materials and Methods Ethics Statement Mice were maintained and used for experimentation according to the guidelines of the Institutional Animal Care and Use Committee of the University of Pennsylvania. Mice and Antibodies Neurog3-Cre, Hspa2-Cre, and Prm-Cre mice were purchased from the Jackson Laboratory (Stock numbers: Neurog3-Cre, 006333; Hspa2-Cre, 008870; Prm-Cre, 003328). Mov10l1fl/fl mice were generated previously [22]. Genotyping for Mov10l1 and Cre alleles was performed separately on genomic DNA isolated from tails. The anti-MOV10L1 antibody was generated previously [22]. Other antibodies used were MILI (Abcam), MIWI (Abcam, or gifts from R. Pillai), GASZ (M. M. Matzuk), LINE1 ORF1p (S. L. Martin), IAP (B. R. Cullen), TDRD1 (S. Chuma), TOP2B (Santa Cruz Biotechnology), PRM2 (SHAL), and ACTB (Sigma-Aldrich). Immunoprecipitation and Detection of piRNAs Mouse testicular extract preparation, immunoprecipitation, and 5′ end-labeling of piRNAs were performed as described previously [22]. Antibodies were described previously [22]. Northern Blot Analysis of piRNAs Northern blot analyses were performed as previously described with modifications [14]. Total RNAs were isolated from mouse testes using Trizol reagent, separated by 15% denaturing polyacrylamide gel, and electro-blotted onto GeneScreen Plus hybridization membrane. Membranes were UV crosslinked and hybridized with 32P end-labeled oligonucleotide probes in Ultrahyb Oligo Buffer (Ambion Cat#8663) at 42°C. Probes for detecting pachytene piRNAs, a pre-pachytene piRNA, or microRNA were perfectly complementary to their sequences: probe-piR1: AAAGCTATCTGAGCACCTGTGTTCATGTCA; probe-piR2: ACCAGCAGACACCGTCGTATGCATCACACA; probe-piR3: ACCACTAAACATTTAGATGCCACTCTCA; probe-let7g: TACTGTACAAACTACTACCTCA; pre-pachytene piRNA probe (derived from sense SINE B1): 5′-TGGCTGTCCTGGAACTCACTYTGT [10]. After hybridization, membranes were washed three times at 42°C in 2×SSC buffer containing 0.5% SDS, or stripped by boiling in 0.1×SSC containing 0.1% SDS. Membranes were exposed to a phosphor imager screen for autoradiography. Histological, Immunofluorescence, and Electron Microscopy (EM) Analyses For histology, testes were fixed in Bouin's solution overnight, processed, sectioned, and stained with hematoxylin and eosin. Immunofluorescence was performed on frozen sections of testes fixed in 4% paraformaldehyde as previously described [52]. EM followed a standard protocol used at the Electron Microscopy Resource Laboratory of the University of Pennsylvania. Expression Analysis of piRNA Precursor Transcripts PCR primers for piRNA precursor transcripts were chosen from genomic clusters to which each piRNA was mapped [10], [14], [50]. PCR primers and PCR product sizes are listed in Table S2.

Acknowledgments We gratefully acknowledge gifts of antibodies: P. Prabhakara Reddi (ACRV1), Ramesh S. Pillai (MIWI), Shinichiro Chuma (TDRD1), Bryan R. Cullen (IAP), Sandy L. Martin (L1 ORF1p), and Martin M. Matzuk (GASZ). We thank Lan Ye for qPCR and Duonan Yu for small RNA Northern blot analysis. We thank Sigrid Eckardt for editing the manuscript. We thank Ramesh Pillai, Zissimos Mourelatos, Anastasios Vourekas, and Fang Yang for comments on the manuscript.

Author Contributions Conceived and designed the experiments: KZ PJW. Performed the experiments: KZ. Analyzed the data: KZ PJW. Wrote the paper: PJW.