Pachytene piRNA Biogenesis Requires MOV10L1 Figure 1. Post-natal disruption of Mov10l1 leads to post-meiotic spermiogenic arrest. (A) Timeline of mouse spermatogenesis, with blue lines and tan histograms representing developmental expression patterns of three mouse Piwi proteins (MIWI, MIWI2, MILI) and MOV10L1, and pre- pachytene and pachytene piRNA populations, respectively. Crosses on lines mark the time point of spermatogenic arrest in the respective mouse mutant (4 ubiquitous mutants and 2 Mov10l1 conditional mutants). Arrows on the timeline bar indicate the onset of Cre expression in the different Mov10l1 mutants generated. Disruption of Mov10l1 by Prm-Cre did not cause spermiogenic arrest (Table S1 and Figure S3). (B–D) Histology of testes from adult wild-type (B), Mov10l1fl/- Neurog3-Cre (C), and Mov10l1fl/- Hspa2-Cre (D) mice. H&E staining was performed on testis sections as described in the Materials and Methods. Abbreviations: Pa, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids. doi:10.1371/journal.pgen.1003038.g001 Blockade of Pachytene piRNA Biogenesis in the Mov10l1 Mutant Isolation and radiolabeling of total testicular small RNAs from adult Mov10l1fl/- Neurog3-Cre testes showed that mutant testes were devoid of pachytene piRNAs (Figure 2A). Immunoprecipi- tation experiments further revealed that both MILI- and MIWI- associated pachytene piRNAs were absent in the mutant (Figure 2B, 2C). As Mov10l1 mutant testes contained less MIWI protein than wild-type testes, we performed serial dilutions of immunoprecipitated complexes to rule out the possibility that the observed loss of MIWI-bound piRNAs was due to the detection limit of the assay. However, MIWI-associated piRNAs were detectable in wild-type testes even when MIWI protein was not detectable (Figure 2C, lane 4), indicating a specific depletion of pachytene piRNAs in the testes from Mov10l1fl/- Neurog3-Cre mice. Moreover, the abundance of pachytene piRNAs was sharply reduced in Mov10l1fl/- Hspa2-Cre testes (Figure 2D). In addition, Northern blotting showed that individual pachytene piRNAs (piR1, piR2, and piR3) were absent in testes from Mov10l1fl/- Neurog3-Cre mice and dramatically reduced in abundance in testes from Mov10l1fl/- Hspa2-Cre mice (Figure 3). As expected, the abundance of individual pachytene piRNAs was not affected in the testes from Mov10l1fl/- Prm-Cre mice (Figure 3). Therefore, MOV10L1 function is essential for the biogenesis of all pachytene piRNAs. Production of Pre-Pachytene piRNAs Is Not Affected in Mov10l1fl/- Neurog3-Cre Testes Pre-pachytene piRNAs are present in mitotic germ cells such as spermatogonia (Figure 1A). Because Neurog3-Cre initiated the PLOS Genetics | www.plosgenetics.org 3 November 2012 | Volume 8 | Issue 11 | e1003038