HORMAD1 Is Essential for Male and Female Meiosis Figure 7. HORMAD1 disrupts cH2AX, BRCA1, and ATR localization to the XY chromosomes. Chromosome spread assay was performed on wild-type and Hormad12/2 leptotene (A-D), zygotene (E-H) and pachytene (I-L) spermatocytes to determine effects of HORMAD1 deficiency on cH2AX, BRCA1 and ATR localization to the sex chromosomes. (A-D) Immunofluorescence assay with anti-SYCP2 (Green) or anti-HORMAD1 (Green) and anti-cH2AX (Red). (E-H) Immunofluorescence assay with anti-SYCP1 (Green) or anti-HORMAD1 (Green) and anti-cH2AX (Red) antibody. (C and D) At the leptotene stage, cH2AX and SYCP2 localization to chromatin can be detected in Hormad12/2 spermatocytes, however, cH2AX is substantially reduced. (F and H) cH2AX staining localizes preferentially to the sex chromosome in the wild-type pachytene stage spermatocytes but no preferential localization to the sex chromosomes was observed in the Hormad12/2 spermatocytes, which is not surprising since sex body does not form in Hormad1 mutants. (G) Arrow head indicates truncated axial fiber. (I, M-M’’) BRCA1 and HORMAD1 co-localize to the sex chromosomes, but Hormad1 deficiency disrupts BRCA1 localization (J). Similarly, ATR co-localizes with HORMAD1 to the XY chromosomes (K, N-N’’), and Hormad1 deficiency disrupts ATR localization (L). These results indicate that HORMAD1 is upstream of the currently known critical components of the meiotic sex chromosome inactivation complex, cH2AX, BRCA1 and ATR. DNA was stained with DAPI (Blue), and arrows indicate sex chromosomes. Scale bars: 10 mm. doi:10.1371/journal.pgen.1001190.g007 chromosome inactivation [39]. Meiotic sex chromosome inactiva- tion also involves ATR and BRCA1 dependent phosphorylation of H2AX [18,40]. Interestingly, Hormad1 deficiency, similar to H2ax deficiency, abolishes the formation of the sex body (Figure 7G and 7H, and data not shown). The lack of sex body formation is most likely due to the disruption of Hormad12/2 spermatocytes prior to the pachytene. We examined the cH2AX localization in the wild- type and Hormad12/2 spermatocytes. Chromatin in Hormad12/2 spermatocytes stained with anti-cH2AXantibodies, but no prefer- ential localization to the sex chromosomes was observed (Figure 7G and 7H). BRCA1 and HORMAD1 have recently been shown to co-localize in the sex body [18]. We determined whether BRCA1 localization is dependent on HORMAD1. BRCA1 protein could not be localized in Hormad12/2 spermatocytes (Figure 7I–7J, 7M). This finding is unlike H2ax knockout, where BRCA1 is still detected on the sex chromosomes despite the lack of the sex body [40]. We also examined ATR localization in wild-type and Hormad12/2 testes. In wild-type testes, HORMAD1 and ATR co- localized in the sex body, but we could not detect ATR in Hormad12/2 spermatocytes (Figure 7K–7L, 7N). Above data suggest that HORMAD1 may be involved in the recruitment of BRCA1, ATR and cH2AX to the sex chromosome. PLoS Genetics | www.plosgenetics.org 8 November 2010 | Volume 6 | Issue 11 | e1001190