ttgcttcgctctctcttgcttcttgctctcttttcctgaagatgtaagaataaagctttgccgcaga- agATTCTGGTCTGTGGTGTTCTTCCTG Forward primer IAP-bisF2 from: TTGTGTTTTAAGTGG- TAAATAAATAATTTG Reverse primer IAP-bisR2 from: CAAAAAAAACACA- CAAACCAAAAT L1Md-A2 (prebisulfite conversion): chrX:154580101- 1544585723 (L1 beginning at 154580138)AAGTTACAAA- TAATTTTCTGGGGCCcggatctggggcacaagtcccttccgctcgactcgtg- actcgagccccgggctaccttgccagcagagtcttgcccaacacctgcaagggtccacaca- ggactccccgcgggaccctaagacctctGGTGAGTGGATCACAGTGC- CTGCCC Forward primer methyl L1-F: AAGTTATAAATAATT- TTTTGGGGTT Reverse primer methyl L1-R: AAACAAACACTATAATC- CACTCACC Small RNA Isolation, Sequencing, and Bioinformatics Analysis Fifteen mg of total RNA from WT and Gasz2/2 mouse testes were gel-fractionated to isolate 18–40 nt small RNAs, followed by 39 and 59 adapter ligation, and product amplification by RT-PCR as per the small RNA kit (FC-102-1009, Illumina) protocol. Finally, the small RNA library was sequenced using a Solexa/ Illumina GA-1 Genome analyzer. Small RNA sequences were analyzed through a high-throughput computational pipeline. For each sample, all sequence reads were aligned to a reference set of miRNAs (miRNA pipeline) and all currently identified piRNAs. The reads are also mapped to the reference mouse genome (NCBI Build 37, UCSC mm9) using the Pash software package [65,66], and uploaded to Genboree platform (www.genboree.com) to identify snoRNAs, scRNAs and repeat-associated small RNAs. We performed a local Smith-Waterman alignment of each unique sequence read against each of the mature microRNAs in miRBase version 11.0, allowing for a 3 base overhang on the 59 end and a 6 base overhang on the 39 end. The alignments were scored such that a matching or overhanging base counts as 2 points and mismatches as 21. Each unique sequence read which achieves a per-base alignment score of 2 (i.e., a perfect match) was associated with each mature microRNA for which it achieved that score. The read counts of all redundantly aligning reads to multiple hairpins in the genome were equally apportioned to each mature microRNA to which they align. For repeat-associated small RNAs, each mapping is associated with LINEs, SINEs, DNA and RNA repeat subtypes. Reads were also mapped to consensus mouse and mammalian repeats from Repbase [67], and rasiRNAs [11] using Blat [68] with a sensitive setting only requiring one 8mer seed filtered such that$90% of the reads mapped. All sequences corresponding to repeat-associated piRNAs and puta- tive novel piRNAs have been deposited at piRNABank (http:// pirnabank.ibab.ac.in/) [69]. Supporting Information Figure S1 Histological analysis of 5-day-old testes. Histological analysis of testes of Gasz+/2 and Gasz2/2 mice. Seminiferous tubules for both genotypes contain spermatogonia and juvenile Sertoli cells. G, spermatogonia; Mit, mitotically dividing sper- matogonia; Ser, Sertoli cells. [Scale bar: 20 mm] Found at: doi:10.1371/journal.pgen.1000635.s001 (7.69 MB TIF) Figure S2 The XY body fails to appear in Gasz2/2 testes due to elevated DNA damage. (A–C) cH2AX localizes in WT testes to perinuclear chromatin of early germ cells [arrow in (A)] and the XY body in pachytene spermatocytes [arrowhead in (B)] while GASZ Is Essential for Male Meiosis there is increased non-XY body staining in Gasz2/2 [arrow in (C)]. [Scaling: 5,0006magnification] Found at: doi:10.1371/journal.pgen.1000635.s002 (2.87 MB TIF) Figure S3 Immunohistochemical and TUNEL analysis of juvenile Gasz+/2 and Gasz2/2 testes. (A–D) Immunohistochemical analysis of testes of Gasz+/2 and Gasz2/2 mice using antibodies to SYCP3 and H1.T. SYCP3, a marker for all primary spermato- cytes [70], labeled fewer spermatocytes in Gasz2/2 than Gasz+/2 testes from 12-day-old mice. H1.T is a testis-specific histone H1 expressed at low levels in early spermatocytes, peaking in late pachytene spermatocytes, and continued expression in round spermatids [71]. While numerous H1.T-positive spermatocytes were detected in Gasz+/2 testes, few H1.T-positive cells were observed in Gasz2/2 testes composed of predominantly early spermatocytes and rare atypical ‘‘pachytene’’ spermatocytes. (E–J) TUNEL analysis was performed on the Gasz+/2 and Gasz2/2 testes. In Gasz+/2 testes, TUNEL-positive germ cells were rare, predominantly affecting spermatogonia with the exception of 21- day-old testes when there is a normal developmental peak in germ cell apoptosis [72]. In Gasz2/2 testes at postnatal days 10 and 21 and in the adult, there is enhanced germ cell apoptosis. By their size, location, and abundance, the most dying cells appear to be spermatocytes. Consistent with pachytene spermatocyte loss being restricted to the stages I-VI of the cycle of the seminiferous epithelium (Figure S5), after 14 days of age we observed more inter-tubule variation including tubules lacking TUNEL positive cells. [Scale bars: 100 mm (A–D) and 50 mm (E–J)] Found at: doi:10.1371/journal.pgen.1000635.s003 (9.80 MB TIF) Figure S4 GASZ localizes to perinuclear cytoplasmic granules. Immunolocalization of GASZ in adult testes using anti-GASZ antibody. Immunostaining is detectable in spermatogonia [G in (B,F)], preleptotene spermatocytes [PL in (C)], pachytene sper- matocytes [P in (A–D)], and round spermatids [R in (A–C)]. Staining of leptotene [L in (D)] and zygotene spermatocytes [Z in (E)] as well as elongating [E in (E–F)] and condensing [C in (A–C)] spermatids was negligible. The most intense staining was detected in middle to late pachytene spermatocytes where GASZ displays a granular distribution pattern in the perinuclear region of the cytoplasm. Only dying germ cells are immunoreactive in Gasz2/2 seminiferous tubules (G). STAGE I, V, VIII, IX, X, XII in (A–F) designates the corresponding stage seminiferous tubule. Found at: doi:10.1371/journal.pgen.1000635.s004 (9.51 MB TIF) Figure S5 Diagramatic summary of Gasz2/2. Loss of pachytene spermatocytes in Gasz2/2 testes correlates with the stages of the seminiferous epithelium where GASZ immunostaining is most intense (green bars). Pachytene spermatocytes in stages I–VI (yellow) can be seen undergoing apoptosis. The most mature germ cells in stage VII–XII seminiferous tubules are early spermato- cytes. All germ cells absent from Gasz2/2 testes are shown in red. The diagram is modified from [73]. Found at: doi:10.1371/journal.pgen.1000635.s005 (0.70 MB TIF) Figure S6 IWI and GASZ co-localize in late pachytene spermatocytes. Staining is shown for GASZ [(A) in green], MIWI [(B) in red], and merge (C). GASZ and MIWI co-localize in some granules in pachytene spermatocytes (arrows). GASZ does not co- localize with MIWI in the chromatoid body (arrowheads). [Scaling: 5,0006magnification] Found at: doi:10.1371/journal.pgen.1000635.s006 (2.48 MB TIF) Figure S7 MVH levels are reduced in Gasz spermatocytes. Immunofluorescent analysis of Gasz+/2 (A) and Gasz2/2 (B) testes. MVH prominently stains spermatocytes in Gasz+/2 testes [asterisks in PLoS Genetics | www.plosgenetics.org 12 September 2009 | Volume 5 | Issue 9 | e1000635