HORMAD1 Is Essential for Male and Female Meiosis Figure 6. Hormad 2/2 fetal oocytes are defective in early recombination. Chromosome spread assay in the wild-type and Hormad12/2 E15.5 fetal ovary. A-E represent zygotene stage, G and H represent leptotene stage. (A-B’) Immunofluorescence assay with anti-SYCP2 (Red) and anti-DMC1 (Green) antibody. (C-D’) Immunofluorescence assay with anti-SYCP2 (Red) and anti-RAD51 (Green) antibody. (E-F’) Immunofluorescence assay with anti-SYCP2 (Red) and anti-RPA (Green) antibody. (G-H’) Immunofluorescence assay with anti-SYCP2 (Green) and anti-cH2AX (Red) antibody. DMC1, RAD51, RPA and cH2AX signals were significantly decreased in Hormad12/2 fetal oocyte. DNA was stained with DAPI (Blue). Scale bars: 10 mm. doi:10.1371/journal.pgen.1001190.g006 intensity) as compared to the wild-type (Figure 7A–7D). cH2AX staining was also significantly decreased in Hormad12/2 fetal oocytes (71% decrease in signal intensity) (Figure 6G and 6H). These results suggest that similar to Hop1 mutants, DSBs do not efficiently form in Hormad1 mutants. HORMAD1 is a germ cell–specific component of the meiotic sex chromosome inactivation complex HORMAD1 protein localization along autosomes was previ- ously shown to be transient [17] (Figure 7B and 7F). HORMAD1 staining was highest along unsynapsed chromosome axis in the zygotene to pachytene stage and diminished significantly along autosomes in the pachytene [17–18] (Figure 7F and 7I). Interestingly, HORMAD1 localized strongly along desynapsed autosomes in diplotene meiocytes [17]. During the pachytene stage, HORMAD1 is faintly visible along the autosomes, but co- localizes strongly with cH2AX on the XY chromosomes, and specifically along the axial elements [17–18]. cH2AX is a phosphorylated form of histone H2AX, and a marker for DSBs [36,38]. H2AX is phosphorylated throughout the chromatin in leptotene spermatocytes and by the pachytene stage [17–18], cH2AX staining is undetectable on autosomes and restricted to the sex body (Figure 7E and 7F) [39]. H2ax knockout shows the essential role for H2AX in sex body formation and meiotic sex PLoS Genetics | www.plosgenetics.org 7 November 2010 | Volume 6 | Issue 11 | e1001190