(A)] versus low level staining of spermatogonia in Gasz2/2 testes [arrowheads in (B)]. [Scaling: 5,0006magnification] Found at: doi:10.1371/journal.pgen.1000635.s007 (1.67 MB TIF) Figure S8 Nuage marker mRNAs are modestly reduced in embryonic and juvenile Gasz2/2 testes. Quantitative RT-PCR analysis of MIWI2, MILI, MVH, TDRD1, MAEL, and MIWI in testes from e18.5, 7-, and 14-day-old mice (mean6SEM). (A) Gasz2/2 embryonic testes show no alteration of nuage mRNAs. (B) In the postnatal testis most nuage markers are not significantly reduced until post-natal day 14 (P14) with the exception of MIWI2 mRNA which was reduced at post-natal day 7 (P7). Found at: doi:10.1371/journal.pgen.1000635.s008 (0.43 MB TIF) Figure S9 Intermitochondrial cement is absent from Gasz2/2 gonocytes. Electron micrographs depicting a nuage localized to clustered mitochondria in Gasz+/2 [arrowheads in (A,C,E)] and the lack of a corresponding structure in Gasz2/2 newborn testes (B,D,F). Found at: doi:10.1371/journal.pgen.1000635.s009 (4.99 MB TIF) Figure S10 Length and nucleotide composition analysis of repeat-associated and unknown small RNAs. Comparison of small RNA length in control and Gasz2/2 testes from 10-day-old mice that mapped with $90% identity using Blat to consensus elements including all repeats (A), LTRs (B), LINE L1s (C), and SINEs (D). Developmental abundance of small RNA classes in Gasz2/2 testes and controls at postnatal days 7 (P7), 10 (P10), and 14 (P14) including SINE-associated small RNAs (E), repeat-associated small RNAs (19–23 nt), and other repeat-associated small RNAs. (H–I) Compositional analysis of the 1st and 10th nucleotides of repeat- associated piRNAs (25–29 nt) (H) and repeat-associated small RNAs (19–23 nt) (I). (E,F) Characterization of length of the unknown category of small RNAs (J), developmental abundance of the unknown small RNAs (19–23 nt) in Gasz2/2 testes and controls, and comparison of the nucleotide composition for 25– 29 nt versus the 19–23 nt classes (F). Found at: doi:10.1371/journal.pgen.1000635.s010 (1.22 MB TIF) Figure S11 Model for GASZ interaction with nuage proteins. A summary of reported interactions between nuage proteins suggests that they may form a protein network containing GASZ. Physical interactions between nuage proteins are depicted by connecting lines. Found at: doi:10.1371/journal.pgen.1000635.s011 (0.09 MB TIF) Table S1 Primers for quantitative RT-PCR assessment of nuage components. Found at: doi:10.1371/journal.pgen.1000635.s012 (0.02 MB XLS) Table S2 Non-repeat-associated piRNAs are reduced in Gasz2/2 testes. The average number of reads for piRNAs from two Gasz2/2 (KO) and control testes from postnatal day 7, 10, and 14 (P7, P10, and P14) mice are shown along with their percentage of the total reads from each testis. All piRNAs that were absent or reduced greater than three-fold in Gasz2/2 from controls are highlighted in gray. piRNAs with greater abundance on D7 and D14 were analyzed separately. piRNA ID is the accession assigned by piRNABank (http://pirnabank.ibab.in) followed by the Genbank References GASZ Is Essential for Male Meiosis accession. The fold-change represents the degree of reduction in Gasz2/2 from controls and was calculated for age-matched samples. Found at: doi:10.1371/journal.pgen.1000635.s013 (17.58 MB XLS) Table S3 miRNAs missannotated as piRNAs. Sequences annotated as piRNAs that increased in abundance in Gasz2/2 testes were compared to the map positions (UCSC mm9) of mouse pre-miRNAs. The majority of overlapping sequences mapped to a large microRNA cluster on the X chromosome. The ‘‘piRNA’’ reads observed and the similar pre-miRNA with mature miRNA sequences in bold are provided. Most of the sequences are identical with mature miRNAs or differ by 1–2 nt at the 39 end. We also observed several piRNAs mapping to the strand of the stem-loop opposite to known miRNAs to which we assigned putative designations. Novel testis piRNA t27 is described in [74]. Found at: doi:10.1371/journal.pgen.1000635.s014 (0.03 MB XLS) Table S4 A subset of repeat-associated small RNAs are reduced in Gasz2/2 testes. The average number of individual small RNAs from two Gasz2/2 (KO) and control testes from postnatal day 7 (P7), 10 (P10), and 14 (P14) mice that mapped with greater than 90% identity to individual repeat consensuses are shown along with their percentage of the total reads from each testis. Each repeat is classified according to a class of repeats (LINE, SINE, LTR) and families within that class. The fold reduction of piRNAs in the Gasz2/2 as compared to controls is shown with those elements reduced 10-fold highlighted in gray. Among those repeats displaying the most dramatic sustained reduction in Gasz mice included LTRs (LTR45, MER72B, MER87, RLTR10, LTR75, LTR90B, MT2_Mm, MamGyp-Int, MT-Int, MTB-Int, ORR1A0, ORR1A-Int, ORR1A1, and several IAPs) and LINE L1s (L1MD_A, L1Md_F3, L1Md_Gf, Lx2A1). SINE-associated small RNAs, processed by Dicer, are mostly unaffected in Gasz2/2 testes. SR (Simple Repeat); LC (Low Complexity); UNK (Unknown classification). Found at: doi:10.1371/journal.pgen.1000635.s015 (0.56 MB XLS) Acknowledgments We thank Paula Cohen (anti-SYCP3), Haifan Lin (anti-MIWI), Peter Moens (anti-H1.T), Shinichiro Chuma (anti-TDRD1, -TDRD6, and - TDRD7), Bryan R. Cullen (anti-IAP GAG), and Sandra L. Martin (anti- LINE L1 ORF1) for their antibody contributions; Deborah O’Brien, Mary Ann Handel, Howard Cooke, and Dirk De Rooij for helpful advice; David Bartel for his important suggestions on the SINE-associated small RNAs; Pengfei Liu for help in RNA isolation; and the Cullen Foundation for their generous support of the University of Houston’s Institute for Molecular Design (IMD) Illumina/Solexa Sequencer. Author Contributions Conceived and designed the experiments: LM GMB MPG AR KHB WY MMM. Performed the experiments: LM GMB MPG AR KHB DYH WY. Analyzed the data: GMB HZ RAH CC PHG. Contributed reagents/ materials/analysis tools: LM GMB KHB HZ RAH CC PHG. Wrote the paper: GMB MMM. 1. Lees-Murdock DJ, Walsh CP (2008) DNA methylation reprogramming in the germ line. Epigenetics: Official Journal of the DNA Methylation Society 3: 5–13. 2. Seki Y, Yamaji M, Yabuta Y, Sano M, Shigeta M, et al. (2007) Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice. Development 134: 2627–2638. 3. van der Heijden G, Bortvin A (2009) Transient relaxation of transposon silencing at the onset of mammalian meiosis. Epigenetics: Official Journal of the DNA Methylation Society 4: 76–79. 4. Tam OH, Aravin AA, Stein P, Girard A, Murchison EP, et al. (2008) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 7194: 534–538. PLoS Genetics | www.plosgenetics.org 13 September 2009 | Volume 5 | Issue 9 | e1000635