lysates were incubated overnight at 4uC with anti-GASZ, anti- MIWI, or no primary antibody, followed by incubation with protein G beads in 5% BSA to precipitate immune complexes. Co- immunoprecipitating proteins were detected by western blotting with anti-MVH (1:500; ab13840 Abcam Inc, Cambridge, MA), anti-MIWI (1:1000), anti-TDRD1 (1:1000), anti-MVH (1:500; Abcam Cambridge, MA), anti-GASZ (1:500), or b actin as above. Secondary antibodies used were horseradish peroxidase-labeled donkey anti-rabbit, goat anti-mouse, or goat anti-guinea pig (Jackson ImmunoResearch, West Grove, PA). Ten mg of WT and age-matched Gasz2/2 lysates were used as loading controls. Meiotic Marker and Apoptosis Evaluation To evaluate meiotic progression, slides were incubated with guinea pig anti-GASZ (1:500), rabbit anti-SYCP3 (1:500), or rabbit anti-H1.T (1:1000). Staining was visualized using biotiny- lated goat anti-rabbit or goat anti-mouse secondary antibodies at 1:200 and the Vectastain ABC Kit according to the manufacturer (Vector Laboratories, Burlingame, CA). TUNEL analysis was performed with three sections from five mice of each genotype for 10-, 21-day-old and 6-week-old mice by using the Chemicon ApopTag Fluorescein In Situ Apoptosis Detection Kit (S7110). Representative images were captured on a Ziess Axioskop (Carl Zeiss MicroImaging, Thornwood, NY). Yeast Two-Hybrid Screening Yeast two-hybrid screening was performed by using CLON- TECH Matchmaker Two-Hybrid Library Construction & Screening Kit. A yeast cDNA library was constructed from 17- day-old mouse testis cDNA. Full-length mouse Gasz cDNA, was subcloned into pGBKT7 vector, and was used as the bait construct for screening a yeast cDNA library constructed from 17-day-old mouse testis cDNA. After yeast mating, clones that grew on (SD Leu- Trp- Ade- His- X-a-Gal) selection plates were isolated, and candidate pGADT7-Rec-cDNAs were sequenced. Interactions were confirmed by mating mouse Gasz bait with prey constructs. To analyze how GASZ interacts with itself and other interacting proteins we generated prey constructs, by using pGADT7, expressing full-length GASZ, MILI, MIWI, and MVH constructs, as well as a RANBP9 construct lacking its proline-rich amino terminus (aa 1–50). Using the CLONTECH Matchmaker BioSensor Kit All constructs were confirmed by DNA sequencing. We made the mating culture by using full-length mouse Gasz bait mated with the prey constructs. Protein-protein interactions were quantified from the resultant fluorescent signal of an equal number of mated cells on a 96 well oxygen biosensor plate post culture for 24 hours using the CLONTECH Matchmaker BioSensor Kit. We added an equal number of mated cells to a 96 well oxygen biosensor plate, cultured the cells at 30uC for 24 hours, and quantified the resultant fluorescent signal. RT-PCR and QPCR Using the Superscript III Reverse Transcriptase Kit (Invitrogen, Rockville, MD) cDNA was synthesized from total mouse testis RNA from mice on embryonic day 18.5 and postnatal days 0, 7, and 14 primed with random hexamers following treatment with Turbo DNA-free (Ambion, Austin, TX). Gasz was PCR amplified using ACCGGTCCTCTCAGAAATTAAAA (forward, 111–133 of NM_023729.2) and ATTGGCGTCATAAGTCTCCTACA (reverse, 455–477 of NM_023729.2) primers. QPCR was performed in duplicate with primers specific to IAP and LINE1 [7] on six 14-day-old animals of each genotype using SYBR Green PCR Master Mix on a 7500 Real Time PCR System machine (Applied Biosystems). Nuage protein mRNAs were similarly GASZ Is Essential for Male Meiosis quantified using primers designed in Primer Express (Applied Biosystems, Foster City, CA) with the exception of those for Mvh [63] and are described in Table S1. Relative quantification was performed using Actb or Gapdh as appropriate. Cycle conditions were as follows: one cycle at 50uC for 2 min, followed by 1 cycle at 95uC for 10 min, followed by 40 cycles at 95uC for 15 s and 60uC for 1 min. The relative amount of transcripts was calculated by the DDCT method. SEM was calculated for the duplicate measure- ments, and the relative amount of target gene transcripts was plotted. Significance was determined using Student t-test. Electron Microscopic Analysis of Nuage Testes were removed from newborn mice and drop fixed in a modified Karnovsky’s mixture of 2.5% glutaraldehyde, 2% paraformaldehyde plus 2 mM CaCl2 in 0.1 M cacodylate buffer pH 7.4 overnight at 4uC. After primary fixation, the tissue was rinsed 3 times for 5 minutes in 0.1 M cacodylate buffer pH 7.4. The tissue was then post-fixed in 1% OsO4 in pH 7.4. The tissue was dehydrated in a gradient series from 30% to 100% ethanol. After ethanol dehydration, the tissue was given 3 changes of fresh propylene oxide for 20 minutes each. The tissue was infiltrated up to 1:1 propylene oxide plus Spurr’s Low Viscosity embedding resin. The tissue was placed in individual 00 BEEM capsules and polymerized at 70uC overnight. Thick sections were cut with a HistoDiatome knife on an RCM MT-6000XL ultra-microtome, stained with toluidine blue and examined on a light microscope for orientation. Thin sections were cut at 80 nm using a Diatome Ultra knife on the same ultra-microtome and picked up on 200 mesh copper grids. Thin sections were stained for 15 minutes in a saturated aqueous solution of uranyl acetate, counterstained for 6 minutes with Reynold’s lead citrate and examined on a Hitachi H7500 transmission electron microscope. Digital images were captured using Gatan Digital Micrograph software and a Gatan US1000 camera. Methylation Analysis of LINE L1 and IAP Total testicular genomic DNA was recovered from 3 pooled 14- day-old mice by phenol:chloroform extraction and ethanol precipitation. Aliquots of DNA were denatured for 15 minutes at 50uC in 0.3 M NaOH and bisulfite treatment was performed as described in [64]. Briefly, samples were incubated at 50uC overnight with sodium metabisulfite and hydroxyquinone and purified using a modified procedure for a Viral RNA Miniprep kit (Qiagen). Samples were desulfonated in NaOH for 15 minutes, neutralized with HCl, and purified as before. PCRs were performed using Platinum Taq Supermix (Invitrogen) or ExTaq (Takara) using primers described below from [11] to amplify twelve CG dinucleotides in IAP LTR1_Mm ERVK and from [7] to amplify eight CG dinucleotides in L1MD-A2. Each PCR was performed in 4 replicates, which were pooled and purified using a DNA clean-up and concentrator kit (Zymo Research). Amplicons were ligated into pGEM-T Easy vector (Promega) and sequenced. Capital letters in the reference sequences below denote sequences that are complementary to primer binding sites. These do not contain CG dinucleotides, and all cytosine residues are expected to convert during bisulfite treatment. Conversion of essentially all cytosines not part of CG dinucleotides confirmed the efficacy of bisulfite treament. IAP LTR1_Mm ERVK (prebisulfite conversion): chr10:83255039–83255316 (all IAP sequence)CTGTGTTC- TAAGTGGTAAACAAATAATCTGcgcatgtgccaagggtatcttatgac- tacttgtgctctgccttccccgtgacgtcaactcggccgatgggctgcagccaatcaaggagt- gacacgtccgaggcgaaggagaatgctccttaagagggacggggttttcgtttttctctctctc- PLoS Genetics | www.plosgenetics.org 11 September 2009 | Volume 5 | Issue 9 | e1000635