on the role of Mov10l1 and the piRNA pathway during later stages of meiosis and spermiogenesis demonstrates that pachytene piRNAs fulfill distinct and essential functions during post-meiotic stages of male germ cell development. Most importantly, the massive DNA damage observed in piRNA-deficient round spermatids in the absence of de-repression of LINE1 and IAP transposable elements suggests that the integrity of the post- meiotic germ cell genome remains highly prone to damage, and that pachytene piRNAs fulfill a protective role at this stage by yet undefined mechanisms. Materials and Methods Ethics Statement Mice were maintained and used for experimentation according to the guidelines of the Institutional Animal Care and Use Committee of the University of Pennsylvania. Mice and Antibodies Neurog3-Cre, Hspa2-Cre, and Prm-Cre mice were purchased from the Jackson Laboratory (Stock numbers: Neurog3-Cre, 006333; Hspa2-Cre, 008870; Prm-Cre, 003328). Mov10l1fl/fl mice were generated previously [22]. Genotyping for Mov10l1 and Cre alleles was performed separately on genomic DNA isolated from tails. The anti-MOV10L1 antibody was generated previously [22]. Other antibodies used were MILI (Abcam), MIWI (Abcam, or gifts from R. Pillai), GASZ (M. M. Matzuk), LINE1 ORF1p (S. L. Martin), IAP (B. R. Cullen), TDRD1 (S. Chuma), TOP2B (Santa Cruz Biotechnology), PRM2 (SHAL), and ACTB (Sigma-Aldrich). Immunoprecipitation and Detection of piRNAs Mouse testicular extract preparation, immunoprecipitation, and 59 end-labeling of piRNAs were performed as described previously [22]. Antibodies were described previously [22]. Northern Blot Analysis of piRNAs Northern blot analyses were performed as previously described with modifications [14]. Total RNAs were isolated from mouse testes using Trizol reagent, separated by 15% denaturing polyacrylamide gel, and electro-blotted onto GeneScreen Plus hybridization membrane. Membranes were UV crosslinked and hybridized with 32P end-labeled oligonucleotide probes in Ultrahyb Oligo Buffer (Ambion Cat#8663) at 42uC. Probes for detecting pachytene piRNAs, a pre-pachytene piRNA, or micro- RNA were perfectly complementary to their sequences: probe- piR1: AAAGCTATCTGAGCACCTGTGTTCATGTCA; probe-piR2: ACCAGCAGACACCGTCGTATGCATCA- CACA; probe-piR3: ACCACTAAACATTTAGATGC- CACTCTCA; probe-let7g: TACTGTACAAACTAC- TACCTCA; pre-pachytene piRNA probe (derived from sense SINE B1): 59-TGGCTGTCCTGGAACTCACTYTGT [10]. After hybridization, membranes were washed three times at 42uC in 26SSC buffer containing 0.5% SDS, or stripped by boiling in 0.16SSC containing 0.1% SDS. Membranes were exposed to a phosphor imager screen for autoradiography. Histological, Immunofluorescence, and Electron Microscopy (EM) Analyses For histology, testes were fixed in Bouin’s solution overnight, processed, sectioned, and stained with hematoxylin and eosin. Immunofluorescence was performed on frozen sections of testes fixed in 4% paraformaldehyde as previously described [52]. EM Pachytene piRNA Biogenesis Requires MOV10L1 followed a standard protocol used at the Electron Microscopy Resource Laboratory of the University of Pennsylvania. Expression Analysis of piRNA Precursor Transcripts PCR primers for piRNA precursor transcripts were chosen from genomic clusters to which each piRNA was mapped [10,14,50]. PCR primers and PCR product sizes are listed in Table S2. Supporting Information Figure S1 Breeding schemes for Mov10l1 conditional mutant mice. The breeding scheme for generating Mov10l1fl/- Neurog3- Cre mice is shown as an example. The same strategy was applied to generate Mov10l1fl/- Hspa2-Cre and Mov10l1fl/- Prm-Cre mice. Genotypes were identified by PCR analysis of tail genomic DNA. (TIF) Figure S2 Developmental stage-specific inactivation of Mov10l1 in the male germline. (A) Western blot analysis of Mov10l1fl/- Neurog3-Cre testicular extracts. Conditional disruption of MOV10L1 is associated with a decrease in the abundance of the full-length MOV10L1 and appearance of truncated MOV10L1D. Genotypes indicated were identified using tail genomic DNA. (B) Western blot analysis of Mov10l1fl/- Hspa2- Cre testes. (TIF) Figure S3 Histological analysis of testes from adult wild-type (A) and Mov10l1fl/- Prm-Cre (B) mice. Testis sections were stained with H&E as described in the Materials and Methods. In testes from Mov10l1fl/- Prm-Cre (B) mice, spermatogenesis appears to be normal. Abbreviations: Pa, pachytene spermatocytes; RS, round spermatids; ES, elongated spermatids. Scale bar, 25 mm. (TIF) Figure S4 Co-clustering of MILI with mitochondria in Mov10l1- deficient pachytene spermatocytes. Testis sections from adult wild- type (A) and Mov10l1fl/- Neurog3-Cre mice (B) were immuno- stained with anti-MILI antibody and a mixture of five monoclonal antibodies against mitochondrial components (OXPHOS cocktail, Mito Sciences). In wild-type pachytene spermatocytes, MILI and mitochondria were mostly dispersed throughout the cytoplasm (A), however, mitochondria clustered to the same polar cytoplasmic location as MILI in the Mov10l1-deficient pachytene spermato- cytes (representative pachytene cells were indicated by arrows in B). Co-localization in the pachytene cell (indicated by a large arrow) is shown in high magnification in the inset. Scale bar, 50 mm. (TIF) Figure S5 Fragmentation of chromatoid bodies in Mov10l1- deficient round spermatids. EM analysis of spermatids from wild- type and Mov10l1fl/- Neurog3-Cre testes showed multi-lobular and prominent chromatoid bodies in wild-type round spermatids (A, B), but fragmented and much less prominent chromatoid bodies in Mov10l1-deficient round spermatids (C, D). Scale bar, 2 mm. (TIF) Figure S6 Immunofluorescence analysis of LINE1 and IAP in testes from adult wild-type (A, D), Mov10l1fl/- Neurog3-Cre (B, E), and Mov10l12/2 (C, F) mice. Leydig cells reside in the interstitium and emit a strong autofluorescence signal. In Mov10l12/2 (ubiquitous knockout) testes, LINE1 (C) and IAP (F) are de-repressed in spermatocytes and spermatogonia, respectively [22], providing a positive control . In contrast, LINE1 (B) and IAP (E) are not de- repressed in Mov10l1fl/- Neurog3-Cre testes. Scale bar, 50 mm. (TIF) PLOS Genetics | www.plosgenetics.org 10 November 2012 | Volume 8 | Issue 11 | e1003038