knockout embryos. SYCP3 and SMC1b are involved in the synaptonemal complex formation, are germ cell–specific, and implicated in human aneuploidy. Sycp3 deficient females are subfertile, but one third of the embryos die in utero due to aneuploidy [29]. Smc1b encodes a meiosis specific component of the cohesin complex, important in sister chromatid cohesion and recombination [58]. Smc1b deficient females are sterile, like Hormad12/2 females, and meiosis continues until metaphase II [59]. However, unlike Smc1b2/2, Hormad12/2 ovaries do not lose oocytes by 6 months of age, and postnatal oocyte counts do not differ significantly from the wild type. Sycp32/2 ovaries show significant decline in the primordial follicle pool at postnatal day 8, indicating accelerated apoptosis of oocytes as germ cell clusters break down to form primordial follicles [52]. DSBs, as indicated by the assembly of the histone variant cH2AX, form in Smc1b2/2 and Sycp32/2 meiocytes and may lead to higher rate of oocyte loss in these mutants [47,52]. Hormad1 deficient ovaries do not display loss of primordial follicles. Hormad12/2 ovaries may not be losing oocytes at an accelerated rate due to our observations that DSBs, as assessed by cH2AX staining, are substantially reduced in Hormad1 mutants. Similar to Hop1 mutants in yeast, lack of HORMAD1 may derepress Dmc1-independent inter-sister repair pathway, resulting in efficient DNA break repairs [21]. The reduced DSBs in Hormad12/2 meiocytes, and de-repression of Dmc1- independent inter-sister repair pathways, may not affect regular apoptotic pathways that eliminate substantial number of oocytes during germ cell clusters breakdown to form primordial follicles at the time of birth, resulting in no decrease in Hormad12/2 primordial oocyte numbers. Materials and Methods Animal breeding All murine experiments were carried out on the 129S7/ SvEvBrd x C57BL/6 hybrid background. Litters were weaned at 3 weeks and breeding pairs set up at 6 weeks of age. One mating pair was placed per cage and inspected daily for presence of litters. All experimental and surgical procedures complied with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Agricultural Animal Care and Use of Committee of Baylor College of Medicine and the Institutional Animal Care and Use of Committee of University of Pittsburgh. Targeting construct, generation of transgenic mice, and genotyping Targeting construct, electroporation and ES cell selection was done as previously described [60–61]. Exons 4 and 5 of the mouse Hormad1 gene were replaced with a neomycin resistance gene flanked by 5.3 kb (59) and 6.2 kb (39) homologous sequences (Figure S1A). Targeted ES cells were verified by Southern blotting, and microinjected into the C57BL/6 blastocysts to produce chimeric mice that carried the mutation into the germ line. These mice were mated with C57BL/6 wild-type mice to generate Hormad1 heterozygous animals that were subsequently crossed to produce F2 offspring for functional analysis. PCR genotyping was performed using the following primers: WT-forward (59- TCAA- GACCAACCTGGGCTAC -39) and WT-reverse (59- CCA- TGTGGGTTGTAGGGAGT -39) to amplify a 196-nucleotide wild-type band, and KO- forward (59- TCAAGACCAAC- CTGGGCTAC -39) and KO- reverse (59- GGGGAACTTCCT- GACTAGGG -39) to amplify a 505-nucleotide mutant band (Figure S1A). HORMAD1 Is Essential for Male and Female Meiosis Histology and immunohistochemistry analysis Testes were fixed in Bouin’s solution (Sigma-Aldrich, MO, USA) and ovaries were fixed in 10% buffered formalin or 4% paraformaldehyde. Fixed tissues were embedded in paraffin, serially sectioned (5 mm) and stained with hematoxylin and eosin or with Periodic Acid Schiff (PAS). At least five pairs of testes and ovaries of each genotype were subjected to gross and microscopic analyses for each time point. Germ cell cysts, primordial, primary, and secondary follicles were defined as described [45]. We used antibodies against SOHLH1 [45], PLZF ( sc-22839, Santa Cruz, CA,USA), DDX4 (ab13840, Abcam, MA, USA), ATM phospho- S1981 (05-740, Millipore, CA, USA), and HORMAD1 proteins. Affinity purified, anti-HORMAD1 guinea pig and rabbit anti- bodies, were generated against part of HORMAD1 protein (amino acids 23-373) at Cocalico Biologicals (Lancaster, PA, USA). Meiotic chromosome analysis and immunostaining Oocytes and spermatocytes for chromosome analyses were prepared essentially as described previously [62–63]. Ovaries and testes were incubated in trypsin-EDTA solution at 37uC for 15 min and washed briefly in PBS. Trypsinized ovaries and testes were pipetted repeatedly and centrifuged, followed by resuspen- sion in PBS. Cell suspensions were placed on poly-L-lysine-coated slides containing 120 mM sucrose solution and 0.05% Triton X- 100. The slides were fixed in 2% paraformaldehyde and 0.02% SDS for 1 hour at room temperature, washed in distilled water and air dried, and stored at 280uC before use. Immunostaining was preformed as described [62–63]. Slides were incubated with the primary antibody overnight at 4uC, washed with PBS and incubated with Alexa-488 and Alexa-595 (Invitrogen, CA, USA) secondary antibody (1:300 dilutions) for 1 hour at room temperature. After washing with PBS, slides were mounted using VECTASHIELD medium with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, CA, USA). SYCP2 rabbit and guinea pig immunoaffinity-purified antibody [48], DMC1 (sc- 8973, Santa Cruz, CA, USA), RAD51 (ab1837, Abcam, CA, USA) and RPA (ab87272, Abcam, CA, USA) were used. Dr. Christer Ho¨o¨g (Karolinska Institutet, Sweden) kindly provided SYCP1 and SYCP3 rabbit immunoaffinity-purified antibodies. Dr. William R. Brinkley (Baylor College of Medicine, TX, USA) kindly provided anti-CREST human serum. Quantification of the immunofluorescence signal Wild-type and mutant oocyte and spermatocyte spreads were stained at the same time with the same mixture of antibodies. In each experiment, when comparing wild type and mutants, imaging of the cells was performed on the same day with the same microscope and camera settings. PerkinElmer Volocity software 5.3 was used to control for possible changes in illumination during the course of imaging and measurement of the immunofluorescence. We measured total immunofluorescence of cH2AX in identical-sized rectangles that were placed over the cell boundaries. Fifty individual leptotene stage spreads from wild type, mutant spermatocytes and oocytes were subjected to immunofluorescence analysis. A two-tailed non-parametric Wil- coxon–Mann–Whitney two-sample rank-sum test was used for sample comparison. Electron microscopy analysis Testes were dissected into ,0.25 cm pieces and fixed in 1x PBS with 2% formaldehyde and 3% glutaraldehyde (Ted Pella Inc., CA, USA), for 2 hr at room temperature. Samples were treated PLoS Genetics | www.plosgenetics.org 16 November 2010 | Volume 6 | Issue 11 | e1001190