Pachytene piRNA Biogenesis Requires MOV10L1 Figure 4. Polar conglomerate of piRNA pathway protein components and mitochondria in Mov10l1-deficient pachytene spermatocytes. Adult testis sections were immunostained with antibodies (in green) against MILI (A, B), MIWI (C, D), TDRD1 (E, F), and GASZ (G, H). DAPI staining of DNA is shown in blue. (I) EM analysis of a wild-type pachytene spermatocyte. Note the random distribution of mitochondria (indicated by arrows). (J) EM image of a Mov10l1-deficient pachytene spermatocyte with a single mitochondrial cluster. Mito, mitochondria. Scale bars: A–H, 25 mm; I–J, 2 mm. doi:10.1371/journal.pgen.1003038.g004 implying importance of their structural integrity (Miwi, Tdrd5, and Tdrd6) [7,18,46,47]. Severe DNA Damage in piRNA-Deficient Round Spermatids The introduction of DNA double strand breaks (DSBs) into the germ cell genome takes place as part of the chromatin remodeling process occurring at the elongating spermatid stage (Figure 5A). This chromatin remodeling process is initiated by the replacement of canonical histones first with transition proteins and eventually by protamines. Concurrently, nucleosomal DNA supercoils must be resolved, presumably by topoisomerase IIB (TOP2B). TOP2B generates DNA double-strand breaks (DSBs), relaxes supercoils, and subsequently religates DNA ends [48]. DSBs trigger a DNA damage response, resulting in the phosphorylation of histone H2AX (cH2AX). In wild-type testis, histone H2AX phosphory- lation is therefore detectable in several germ cell stages that undergo changes in their chromatin configuration, including elongating spermatids (Figure 5B), but it is absent from round spermatids. Intriguingly, round spermatids from Mov10l1fl/- Neurog3-Cre testes exhibit a high degree of DNA damage visualized by cH2AX (Figure 5C). This could be due to a developmental progression of piRNA-deficient round spermatids to the ‘‘elongating’’ spermatid stage without apparent morpho- logical change. However, the absence of both TOP2B and PRM2 (protamine 2) in cH2AX-positive round spermatids from Mov10l1 PLOS Genetics | www.plosgenetics.org 6 November 2012 | Volume 8 | Issue 11 | e1003038