Figure 9. HORMAD1 phosphorylation is unaffected by Atm deficiency while Hormad1 deficiency disrupts ATM autopho- sphorylation. (A) Western blots analyses with anti-HORMAD1 specific antibodies show that HORMAD1 protein and its phosphorylated form (*) appear circa post-natal day 14. Atm1 deficient testes (Atm2/2) express both forms of HORMAD1 and HORMAD1 is therefore unlikely to be ATM1 substrate. (B) The presumed phosphorylated form of HORMAD1 (higher molecular weight band indicated by asterisk) decreases in intensity after protein phosphatase I (PPI) treatment. (C and D) Chromosome spread assay in wild-type (Atm+/+), and Atm deficient spermatocytes (Atm2/2). HORMAD1 preferentially localizes to unsynapsed regions of chromosome axes (C), and HORMAD1 antibody stains unsynapsed chromosome axes in Atm2/2 spermatocytes (D) more intensely than in the wild-type spermatocytes. Scale bars: 10 mm. (E and F) Immunohistochemistry analysis in 6 week old wild-type and Hormad12/2 testes with anti-phospho ATM-S1981 antibody. Phospho- ATM S1981 was detected in wild-type zygotene, but not detected in Hormad12/2 zygotene spermatocytes. Scale bars: 50 mm. doi:10.1371/journal.pgen.1001190.g009 Chicago Sky Blue 6B dye (Sigma, MO, USA) injection into the tail vein [46], whether blastocysts derived from Hormad12/2 fertilized eggs could implant. We did not detect implantation of Hormad12/2 fertilized eggs (Figure 12M). These results indicate that a defect in early embryogenesis led to premature loss of embryos. Hormad1 deficiency causes aneuploidy and early embryonic demise We hypothesized that aneuploidy is the major cause of embryo wastage in Hormad12/2 females. Unlike meiosis in testes, errors during oocyte meiosis have milder effect on oocyte loss and HORMAD1 Is Essential for Male and Female Meiosis apoptosis, as has been observed in Sycp3 and Smc1b knockouts [27,29,47]. The presence of growing oocytes in the Hormad12/2 ovaries, as opposed to early germ cell loss observed in testes during pachytene stage of meiosis, indicates greater tolerance of Hormad1 deficiency in the oocytes as compared to spermatocytes. The whole range of ovarian follicle types are present in the Hormad12/2 ovaries, including primordial follicles, primary, secondary and antral follicles. In contrast to other critical genes during meiosis, such as Dmc1, Msh5, Spo11 and Atm that cause early ovarian failure due to rapid loss of oocytes following disruption of meiosis I [42–44], Hormad1 deficiency does not activate apoptotic pathways and does not lead to gross premature loss of oocytes. We used chromosome specific mouse BAC (bacterial artificial chromosome) clones to perform fluorescent in situ hybridization (FISH) on germinal vesicle (GV) oocytes, 2 cell and 8 cell stage embryos to determine whether aneuploidy is significantly higher in Hormad12/2 embryos. Wild- type GV oocytes are arrested in meiosis I and contain bivalent chromosomes that consist of four chromatids. Examination of 58 GV oocytes from three independent knockout animals and 112 GV oocytes from three wild-type animals with BAC probes specific for chromosomes 19, 18 and X, revealed no significant differences, with presence of four chromatids for each of the chromosome examined as expected (Figure 13A–13C). Following fertilization and completion of meiosis II, each cell in the developing embryo should contain two chromosomes except for the sex chromosomes. We examined by FISH, mouse chromo- somes 6 and X in the 2-cell wild-type and Hormad12/2 embryos. Sixty three cells examined for chromosome 6 in the Hormad12/2 2- cell embryos revealed that 22% of the cells had 4 signals corresponding to chromosome 6, 35% had 3 signals corresponding to chromosome 6, 24% had 1 signal, and 5% had no signal. Wild- type 2-cell embryos were also examined by chromosome specific FISH, and 44 cells examined out of 46 showed only 2 signals, as expected. In 2-cell Hormad12/2 embryos, out of sixty three cells examined for chromosome X, 44% showed four, three or no signal. Wild-type 2-cell stage embryos, as expected, showed only 2 or 1 signal, consistent with either XX or XY sex of the cell. These results indicate widespread hypo and hyperdiploidy in 2-cell embryos (Figure 13D–13F). We also examined cells from the 8-cell stage embryos. A total of 84 cells from the Hormad12/2 embryos were examined with FISH probes specific for chromosomes 11, X and 2. Hormad12/2 cells from the 8-cell stage, hybridized with BAC specific to chromosome 11, showed 4 signals in 10% of the cells, and 3 signals in 55% of the cells. Therefore, a total of 65% of the cells were either monosomic or trisomic for chromosome 11. In the wild-type 8 cell stage embryo, a total of 96 cells were scored for chromosome 11, and only 2 out of 96 (2%) cells showed a single signal, while 94 out of 96 cells (98%) showed two signals as expected. Similar results were obtained for chromosome 2 (Figure 13G–13I). Our results indicate widespread aneuploidy involving different chromosomes in both 2-cell and 8-cell embryos. Such aneuploidy causes early embryo demise and failure of implantation. We also visualized second metaphase (M2) spindle using anti-b- tubulin antibodies in Hormad12/2 oocytes. During M2, sister chromatids migrate to the opposite pole to form functional, haploid gametes. Sister chromatids are bi-oriented on the spindle and congregate in the center of the spindle prior to separation (Figure 13J). Each sister kinetochore in a pair is attached to the opposite spindle, and such arrangement generates sufficient tension to cause proper segregation. M2 in Hormad12/2 oocytes is grossly disrupted with mis-orientation of chromatid attachment to the spindle observed in all M2 oocytes examined (Figure 13K) PLoS Genetics | www.plosgenetics.org 11 November 2010 | Volume 6 | Issue 11 | e1001190