Pachytene piRNA Biogenesis Requires MOV10L1 Figure 2. Mov10l1 is required for biogenesis of pachytene piRNAs. (A) Depletion of pachytene piRNAs in mutant testes from Mov10l1fl/- Neurog3-Cre adult mice. Total RNAs were 32P-end-labelled and separated by denaturing polyacrylamide gel electrophoresis. 18S and 28S ribosomal RNAs served as a loading control. (B) MILI is devoid of pachytene piRNAs in Mov10l1fl/- Neurog3-Cre testes. MILI was immunoprecipitated from testicular extracts. One-tenth of the immunoprecipitated material was used for detection of associated piRNAs, and the remainder was used for Western blotting (WB) analysis of MILI. (C) MIWI immunoprecipitation on Mov10l1fl/- Neurog3-Cre and wild-type (+/+) testes, and serial dilutions (1:2) of MIWI IP complexes. (D) Depletion of pachytene piRNAs in mutant testes from Mov10l1fl/- Hspa2-Cre adult mice. (E) MILI is loaded with pre- pachytene piRNAs in postnatal day 10 Mov10l1fl/- Neurog3-Cre testes. doi:10.1371/journal.pgen.1003038.g002 disruption of Mov10l1 at post-natal day 9, we anticipated that the production of pre-pachytene piRNAs would not be affected in Mov10l1fl/- Neurog3-Cre testes. To test this hypothesis, we performed immunoprecipitation of postnatal day 10 testis lysates with anti-MILI antibodies. Postnatal day 10 testes do not contain pachytene spermatocytes, and express only MILI but not other Piwi proteins (Figure 1A). Consequently, all MILI-bound piRNAs in postnatal day 10 testes are pre-pachytene piRNAs [10]. We found that pre-pachytene piRNAs were present in postnatal day 10 Mov10l1fl/- Neurog3-Cre testes (Figure 2E). Furthermore, Northern blot analysis showed that abundance of a specific pre- pachytene piRNA was not reduced in adult testes from Mov10l1 mutant mice, regardless of whether deletion had been mediated by Neurog3-Cre, Hspa2-Cre, or Prm-Cre (Figure 3). These data demonstrate that pre-pachytene piRNA production is not affected in the Mov10l1 conditional mutant testes. PLOS Genetics | www.plosgenetics.org 4 November 2012 | Volume 8 | Issue 11 | e1003038