Author Summary Small non-coding RNAs play critical roles during develop- ment 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-interfer- ing 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 pachy- tene piRNAs disrupts germ cell development and results in defects in post-meiotic genome integrity. Therefore, mutations in MOV10L1 and other piRNA pathway compo- nents may contribute to male infertility in humans. 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]. Appar- ently, 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 piR- NAs 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. Results MOV10L1 Is Essential for Spermiogenesis MOV10L1, a putative RNA helicase, interacts with all three mouse Piwi proteins, and is an essential component of the piRNA Pachytene piRNA Biogenesis Requires MOV10L1 pathway [22]. To explore the biogenesis and function of pachytene piRNAs, we disrupted MOV10L1 function specifically during and after male meiosis using Cre-mediated inactivation of a conditional Mov10l1 allele (Mov10l1fl) (Figure 1A and Figure S1) at the following stages: after postnatal day 7 (Neurog3-Cre) [41], at the pachytene stage (Hspa2-Cre) [42], and in post-meiotic spermatids (Prm-Cre) [43]. Cre-mediated recombination of the conditional Mov10l1 allele deletes the RNA helicase domain, producing a truncated protein termed MOV10L1D. In male Mov10l1fl/- Neurog3-Cre mice resulting from intercrosses of Mov10l1fl/fl mice with Neurog3-Cre mice [41], Cre-mediated disruption of Mov10l1 was first detected in testes at postnatal day 9 (leptotene/zygotene spermatocytes), with a decrease in the abundance of the full-length MOV10L1 protein in the mutant testes compared with the wild type (Figure S2A). Mov10l1fl/- Neurog3-Cre males were sterile, with substan- tially smaller testes (140610.5 mg/pair at 2–4 months of age) compared to age-matched wild-type mice (189618.4 mg/pair) (Student’s t test, p,0.0008). In contrast to seminiferous tubules from wild-type mice (Figure 1B), tubules from Mov10l1fl/- Neurog3-Cre mutant mice lacked elongated spermatids, while earlier germ cell stages including pachytene spermatocytes and round spermatids were present (Figure 1C). Acrosome staining with the anti-ACRV1 antibody identified spermiogenic arrest at the step 4 spermatid stage. Therefore, very different to the meiotic arrest observed in male germ cells with ubiquitous deletion of Mov10l1 [22,23], postnatal disruption of Mov10l1 mediated by Neurog3-Cre causes post-meiotic spermiogenic arrest (Figure 1C), revealing that MOV10L1 plays an essential role during the post- meiotic stages of spermatogenesis. To distinguish consequences of inactivation of MOV10L1 during the pachytene stage from those resulting from disruption at earlier stages such as in differentiating spermatogonia, we generated Mov10l1fl/- Hspa2-Cre mice in which Cre is expressed specifically in spermatocytes, particularly pachytene cells (Figure 1A) [42]. Deletion of MOV10L1 in Mov10l1fl/- Hspa2- Cre mice occurred by postnatal day 14, apparent from a decrease in the abundance of the full-length MOV10L1 protein in the mutant testes (Figure S2B). Notably, Mov10l1fl/- Hspa2-Cre males were also sterile. Although testes (159624 mg/pair) from 2–3 month old Mov10l1fl/- Hspa2-Cre mice were slightly smaller than those from Mov10l1+/2 males (182626 mg/pair) (Student’s t test, p,0.2), histological analysis revealed spermiogenic arrest at the round spermatid stage (Figure 1D). The most advanced spermatids in Mov10l1fl/- Hspa2-Cre males were late round spermatids at step 8. The arrest of spermiogenesis at early and late round spermatid stages in Mov10l1fl/- Neurog3-Cre and Mov10l1fl/- Hspa2-Cre mutant mice, respectively, demonstrates that MOV10L1 is required for the differentiation of post-meiotic germ cells. The temporal delay in the spermiogenic arrest in Mov10l1fl/- Hspa2- Cre testes is likely due to the late onset of Hspa2-Cre expression, which may allow residual MOV10L1 to persist longer. The round spermatid arrest in Mov10l1fl/- Neurog3-Cre and Mov10l1fl/- Hspa2-Cre testes could be due to disruption of MOV10L1 function during the pachytene stage of meiosis, or at early spermatid stages. To define the requirement for MOV10L1 more precisely, we disrupted Mov10l1 with Cre recombinase under the control of the protamine 1 (Prm) promoter, which is only expressed in post-meiotic spermatids [43]. Mov10l1fl/- Prm-Cre males exhibited normal fertility but a slight reduction in testis weight (Table S1). Histological analysis of testes from Mov10l1fl/- Prm-Cre males revealed normal spermiogenesis (Figure S3). These genetic studies demonstrate that disruption of MOV10L1 function at the pachytene stage causes spermiogenic arrest. PLOS Genetics | www.plosgenetics.org 2 November 2012 | Volume 8 | Issue 11 | e1003038