Pachytene piRNA Biogenesis Requires MOV10L1 Figure 3. Northern blot analysis of individual pachytene piRNAs in conditional Mov10l1 mutant testes. RNAs were prepared from adult testes and hybridized with radiolabeled probes specific for pachytene piR1, piR2, piR3, and a pre-pachytene piRNA. Hybridization with a let7g miRNA probe served as a loading control. The three individual pachytene piRNAs were absent in Mov10l1fl/- Neurog3-Cre testes, sharply reduced in abundance in Mov10l1fl/- Hspa2-Cre testes, and present in Mov10l1fl/- Prm-Cre testes, while the level of the pre-pachytene piRNA was not affected in either of these mutant testes. doi:10.1371/journal.pgen.1003038.g003 Polar Conglomeration of piRNA Pathway Components with Mitochondria in Pachytene piRNA-Deficient Spermatocytes We next examined the consequences of the loss of pachytene piRNAs on the localization of piRNA pathway components such as MILI, MIWI, TDRD1, and GASZ. In wild-type pachytene spermatocytes, these proteins localize to cytoplasmic nuage granules (also called inter-mitochondrial cement) (Figure 4A, 4C, 4E, 4G) [7,8,37,44]. Strikingly, in Mov10l1-deficient pachytene spermatocytes, these four proteins congregated to one extremely large novel perinuclear polar ‘‘granule’’ (Figure 4B, 4D, 4F, and 4H). Further analyses revealed immunoreactivity of the polar granule to a cocktail of antibodies against mitochondrial proteins (OXPHOS), demonstrating co-localization of mitochondria with MILI in polar granules of Mov10l1-deficient pachytene spermato- cytes (Figure S4). Electron microscopy (EM) analysis confirmed that in Mov10l1-deficient pachytene spermatocytes, mitochondria form a single cluster (Figure 4J), in contrast to their random distribution in wild-type pachytene cells (Figure 4I). Consistent with a recently described role for MitoPLD, a mitochondrial surface protein, in the piRNA pathway [39,40], these data strongly suggest a novel but yet unknown role for mitochondria in the biogenesis of pachytene piRNAs and/or a function for pachytene piRNAs in the cytoplasmic organization and distribution of mitochondria and piRNA pathway protein components. We next examined the status of chromatoid bodies, which are large and dynamic ribonucleoprotein aggregates prominent in haploid spermatids. Chromatoid bodies contain various RNA regulatory proteins as well as piRNA pathway components, but their precise function remains unclear [45]. Wild-type round spermatids contained one prominent chromatoid body, visualized by EM as a multi-lobular electron-dense nuage (Figure S5). In Mov10l1-deficient spermatids, however, the chromatoid body was fragmented (Figure S5). A similar fragmentation of chromatoid bodies has been observed in other mouse mutants of RNA processing pathway proteins with male infertility phenotype, PLOS Genetics | www.plosgenetics.org 5 November 2012 | Volume 8 | Issue 11 | e1003038