Pachytene piRNA Biogenesis Requires MOV10L1 mutant testes indicated that these round spermatids were not undergoing chromatin remodeling, excluding that cH2AX-posi- tivity was due to TOP2B activity (Figure 5E, 5G). Secondly, DNA damage might be induced by de-repressed transposable elements active in piRNA-deficient round spermatids. Genetic studies have shown that the piRNA pathway is required for silencing of retrotransposons such as LINE1 and IAP in pre-pachytene germ cells [19]. However, quantitative RT-PCR analysis revealed no de-repression of LINE1 (Figure 6B, 6C) or IAP in Mov10l1fl/- Neurog3-Cre testes, confirmed by immunofluorescent analyses of testis sections with anti-LINE1 and anti-IAP antibodies (Figure S6). Therefore, pachytene piRNAs are not required for silencing of LINE1 and IAP retrotransposons, although we cannot rule out the possibility that other transposable elements might be de- repressed in Mov10l1-deficient round spermatids. Notably, we did not observe cH2AX foci in round spermatids from Rnf17-deficient mice, in which spermatogenesis is also arrested at the round spermatid stage [49] but piRNA biogenesis does not appear to be severely affected (data not shown). These results suggest that the DSBs observed in round spermatids from Mov10l1fl/- Neurog3-Cre testes and Mov10l1fl/- Hspa2-Cre testes are not a direct consequence of their developmental arrest. Rather, these obser- Figure 6. MOV10L1 is essential for the primary processing of pachytene piRNA precursors. (A) The RNA helicase domain in MOV10L1 is required for interaction with MILI and MIWI. Input was 1/50th of extracts used for IP. TDRD1 is complexed with MILI and MOV10L1 and thus served as a positive control for immunoprecipitation. The same results were obtained from the repeat of IP experiments using independent samples. (B) Blockade of pachytene piRNA precursor processing in Mov10l1fl/- Neurog3-Cre testes. Total RNAs were pre-treated with DNase I. RT-PCR produced no products in controls without reverse transcriptase (data not shown). PCR primers and product sizes are listed in Table S2. (C) Quantitative RT-PCR analysis of piRNA precursor transcripts in adult testes. Numbers on the vertical axis represent fold increase of levels in mutant testis compared to levels in wild-type testes defined as 1. (D) Proposed model for the essential role of MOV10L1 in the primary processing of pachytene piRNAs. Single strand RNAs are transcribed from the piRNA clusters and are digested by an unknown nuclease into putative piRNA intermediate like molecules (piR ILs). Such piR ILs have been reported in Drosophila but not yet in mammals [20]. MOV10L1 and Piwi proteins select and bind to piR ILs for further processing into mature primary piRNAs through 39 trimming [21]. doi:10.1371/journal.pgen.1003038.g006 PLOS Genetics | www.plosgenetics.org 8 November 2012 | Volume 8 | Issue 11 | e1003038