DNA strand exchange [30–31]. These early steps in recombina- tion are critical for establishing the physical connections between homologous chromosomes during meiosis. Hop1 has been implicated in modulating the formation and processing of double stranded breaks [19]. We examined formation of DMC1, RAD51, and RPA foci in zygotene stage Hormad12/2 spermatocytes. There is a dramatic decrease in the number of DMC1 foci as compared to the wild-type (Figure 5A and 5B). We counted a total of 98.9628.2 DMC1 foci in the wild-type spermatocytes (n = 50) and 9.2863.9 DMC1 foci in the Hormad12/2 spermatocytes (n = 45). The number of RAD51 foci was also decreased from 189.3631.8 in the wild-type spermatocytes (n = 50), to 69.3634.5 in the Hormad12/2 spermatocytes (n = 40) (Figure 5C and 5D). Following double-strand breaks formation by SPO11, RPA is recruited together with RAD51 to the single stranded DNA regions [32]. We counted a total of 194.5664.5 RPA foci in the wild-type spermatocytes (n = 30) as compared to 70.1638.5 foci in the Hormad12/2 spermatocytes (n = 20) (Figure 5E and 5F). These results indicate that early meiotic recombinational events were disrupted and not surprisingly, MLH1, a protein that forms foci in later stages of recombination and required for the formation of most of the crossovers (chiasmata) observed in mice [33], was dramatically reduced in Hormad12/2 spermatocytes (data not shown). HORMAD1 Is Essential for Male and Female Meiosis We also examined DMC1, RAD51 and RPA foci formation in female meiocytes at embryonic day 15.5 (E15.5). Embryonic ovaries contain zygotene to early pachytene oocytes at E15.5 [34]. We counted a total of 208.76117.1 DMC1 foci in the wild-type E15.5 oocytes (n = 50), and 79.1681.5 foci in the Hormad12/2 oocytes (n = 30) (Figure 6A and 6B), a total of 197.9646.0 RAD51 foci in the wild-type oocytes (n = 50) versus 85.1637.6 foci in the Hormad12/2 oocytes (n = 40) (Figure 6C and 6D) and a total of 317.166135.3 RPA in the wild-type oocytes (n = 50) and 51.7648.8 in the Hormad12/2 oocytes (n = 50) (Figure 6E and 6F). DMC1, RAD51 and RPA foci are therefore, similar to our observations in spermatocytes, significantly decreased in Hormad1 deficient female meiocytes. These results indicate that homologous recombination is significantly affected in Hormad12/2 mammalian germ cells, as previously reported for HOP1 [35]. We also observed effects of Hormad1 deficiency on cH2AX staining (a phosphorylated form of histone H2AX), a well known surrogate marker for DSB formation [36]. In the leptotene stage, phosphorylation of H2AX is induced by SPO11 catalyzed DSBs in meiotic DNA, and cH2AX appears as large, cloud-like patterns thatdisappearat the pachytene stage [37]. At the leptotene stage, cH2AX staining in Hormad12/2 spermatocytes was significantly decreased (76% decrease in signal Figure 5. Hormad 2/2 spermatocytes are defective in early recombination. (A-B’) Immunofluorescence assay with anti-SYCP2 (Green) and anti-DMC1 (Red) antibody in zygotene spermatocytes. (C-D’) Immunofluorescence assay with anti-RAD51 (Red) and anti-SYCP2 (Green) antibody in zygotene spermatocytes. (E-F’) Immunofluorescence assay with anti-RPA (Green) and anti-SYCP2 (Red) antibody in zygotene spermatocytes. DMC1 catalyzes DNA strand exchange during recombination, and DMC1, RAD51 and RPA foci mark early recombination events. DMC1, RAD51 and RPA foci are drastically decreased in Hormad12/2 spermatocytes. Scale bars: 10 mm. doi:10.1371/journal.pgen.1001190.g005 PLoS Genetics | www.plosgenetics.org 6 November 2010 | Volume 6 | Issue 11 | e1001190