with 0.5% uranyl acetate and osmium tetraoxide, dehydrated with ethanol, and embedded in LX-112 medium (Ladd Research Industries, VT, USA). The samples were polymerized in a 70uC oven for 2 days. Ultrathin sections (70 –100 nm) were cut in a Leica Ultracut microtome (Leica, IL, USA), stained for 5 min in 1% aqueous uranyl acetate and 2 min in 1% aqueous lead citrate at room temperature in a Leica EM Stainer, and examined by a JEM 1010 transmission electron microscope (JEOL, MA, USA) at an accelerating voltage of 80 kV. Digital images were obtained using AMT Imaging System (Advanced Microscopy Techniques Corp, MA, USA). Western blot analysis and phosphatase treatment Western blot analysis was performed essentially as described previously [64]. Postnatal day 5, 8, 14, 18 and 21 testes were collected and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA with or without protease inhibitor cocktail (Roche)). Total protein (20 mg) was loaded onto the SDS–PAGE gel, transferred to nitrocellulose membrane, and immunoblotted with HORMAD1 rabbit antibody (1:5000 dilutions). Varying amounts of protein phosphatase I (Sigma P7937, MO, USA) were incubated with 20 mg of 18 day old wild-type total testes lysates, for 30 min on ice. Phosphatase treated testes lysates were electrophoresed, trans- ferred to nitrocellulose membrane and immunoblotted with anti- HORMAD1 rabbit antibody. RNA isolation, RT-PCR, quantitative real-time PCR (Q- PCR), and microarray analysis RT-PCR and quantitative real time PCR were performed as described previously [45,65]. We used previously published oligonucleotide sequences corresponding to Rnh2, Mov10l1, Pramel3, Usp26, Fthl17, Tex11, Tex13 [66], and Hormad1 (forward 59 CTGCTGACACCAAGAAAGCA 39- and reverse 59- CC- TGGTGGTTGGTAATCTGG -39) and Hormad2 (forward 59 GCTCATCAGGGGCTAGACTG 39- and reverse 59- TGG- TTCGCTGACCTTCTTCT -39) primers. Q-PCR was per- formed using the SyberGreen PCR Master Mix (Bio-Red, CA, USA) and probe set specific for each gene under investigation. Each sample was analyzed in triplicate from at least three independent wild type and Hormad12/2 testes cDNA samples. The relative amount of transcripts was calculated by the DDCT method as described by Applied Biosystems, and normalized to b- actin. The average and standard errors were calculated for the triplicate measurements, and the relative amount of target gene expression for each sample was plotted. Student’s t test was used to compute the P values. Significance was defined as a P value,0.05. RNA expression arrays on wild-type and Hormad12/2 2 week old testes were performed on the Illumina BeadChip MouseWG-6 2.0 arrays and analyzed as previously described (GEO accession numbers, GSE21524) [67–68]. Superovulation and oocyte collection in mouse Mouse oocytes and pre-implantation embryos were collected by using standard protocols for timed mating [69]. Briefly, 4 to 6 weeks female mice were superovulated by injection of 5 IU of pregnant mare serum gonadotropin (PMSG) (Prospec, Rehovot, Israel) and 48 h later 5 IU of human chorionic gonadotropin (hCG) (Sigma, MO, USA). Females were placed individually with 10-week old males immediately after the injection of hCG. Collection of one cell, two cell, four cell, eight cell, and blastocyst stages was performed at 24 h, 42 h, 68 h, and 96 h after hCG injection, by flushing the oviducts and uterine horns with HEPES- HORMAD1 Is Essential for Male and Female Meiosis buffered media under the microscope. Removal of the cumulus cells was achieved in HEPES-buffered media containing 1 mg/ml hyaluronidase (Sigma, MO, USA). FISH analysis Oocytes and embryos were exposed to a hypotonic solution containing 0.1% sodium citrate, 0.1% bovine serum albumin for about 15 min and gently transferred onto the slides. Cells were fixed with fresh methanol: acetic acid (3:1) solution dispensed carefully over the surface of the slide. The slides were air-dried and dehydrated at room temperature in a series of 70%, 85% and 100% ethanol solutions before FISH analysis. Twenty BAC (bacterial artificial chromosomes) clones from a mouse RPCI-23 library representing sequences unique to specific chromosomes were selected from the UCSC Genome browser (http://genome.ucsc.edu) and NCBI (http://www.ncbi.nlm.nih. gov) databases (Table S1). Probe DNA extraction was performed according to the standard alkaline lysis method and labeled by standard nick translation with Spectrum Orange- or Spectrum Green-dUTP using a commercially available kit (Abbott/Vysis, IL, USA). Sequential fluorescence in situ hybridization experiments using a mixture of three probes were performed according to manufacturer’s instructions (Abbott/Vysis, IL, USA). Each mixture contained a red, green and yellow (combined green/ red) fluorescent probes. Probes were applied to slides, hybridized for 20 hr at 37uC, washed with 0.46SSC/0.3% NP-40 for 2 minutes at 73uC and with 26SSC/0.1% NP-40 for 1 minute at room temperature, and counterstained with DAPI. Digital FISH images were captured by a Power Macintosh G3 System using MacProbe software version 4.4 (Applied Imaging, CA, USA). Supporting Information Figure S1 Generation of Hormad12/2 Mice. (A) Disruption of the Hormad1 locus. The targeting construct and schematized genomic locus of Hormad1 are shown. The neo cassette replaced HORMA domain encoding highly conserved exons 4 and 5, and introduced an Xba I restriction enzyme site into the locus. This Xba I site was used as a diagnostic for Southern blot analysis of ES cells electroporated with the targeting vector (data not shown). Arrows indicate genotyping primers used to distinguish wild-type and mutant alleles in the transgenic animals. (B) RT-PCR analysis of Hormad1 knockout mice shows lack of transcript corresponding to Hormad1 in the knockout animals. A faintly visible lower molecular weight band in the knockout (2/2), corresponds to a mutant transcript without exons 4 and 5. Removal of exons 4 and 5 causes a frameshift mutation. (C) Total protein was isolated from testes of 2 week old wild-type (+/+), Hormad12/2 (2/2) and Hormad1+/2 (+/2) mice and Western blot analysis was performed with anti-HORMAD1 specific antibody. No significant amount of HORMAD1 was detected. The b-actin signal serves as a loading standard. Found at: doi:10.1371/journal.pgen.1001190.s001 (0.18 MB TIF) Figure S2 Adult testes are atrophied in Hormad12/2 Mice. (A) Reduced testicular size and weight in Hormad12/2 (KO) as compared to the wild-type (WT) testes from 4 week old male siblings. (B) Ratio of testes/body weight from wild-type (WT) and Hormad12/2 (KO) testes. Error bars represent the standard error of mean. Student’s t test was used to calculate P values. Found at: doi:10.1371/journal.pgen.1001190.s002 (0.18 MB TIF) Figure S3 Excess apoptosis and increased apoptotic index in Hormad12/2 testes. (A-E) TUNEL assay in 1, 2, 3, 4, and 6 week old wild-type mouse testes. (F-J) TUNEL assay in 1, 2, 3, 4, and 6 PLoS Genetics | www.plosgenetics.org 17 November 2010 | Volume 6 | Issue 11 | e1001190