Since ATR, BRCA1 and cH2AX are involved in the transcriptional silencing of sex chromosomes, we examined whether Hormad1 deficiency affects transcriptional repression. Previous studies have shown that H2ax and Brca1 deficiencies individually, lead to the over-expression of genes exclusively expressed from the X or Y chromosome [40]. We examined whether X-linked germ cell–specific genes were over-expressed in Hormad12/2 testes compared to wild-type testes. We performed quantitative real-time PCR on select autosomal genes (Hormad2, Rnh2 and Mov10l1) as well as X-chromosome derived genes (Usp26, Fthl17, Pramel3, Tex11, and Tex13) on wild-type and Hormad12/2 testes (Figure 8A–8H). Rnh2 and Mov10l1 are germ cell–specific transcripts, derived from autosomes, and were not differentially expressed between wild-type and Hormad12/2 testes (Figure 8B and 8C). In contrast, all of the germ cell–specific transcripts transcribed from the X chromosome were significantly elevated in Hormad12/2 testes over the wild-type. These include Usp26 (4.5 fold increase), Fthl17 (6.5 fold increase), Pramel3 (3.5 fold increase), Tex11 (2.2 fold increase), and Tex13 (2.8 fold increase) (Figure 8D–8H). Moreover, RNA expression microarray analyses comparing two week old Hormad1 deficient testes with corresponding wild-type, indicate that almost 20% of the up- regulated genes derive from the X chromosome (Figure 8I). Our results are remarkably similar to transcriptional de-repression observed in H2ax and Brca1 mutants [39–40], and indicate that HORMAD1 is a germ cell and meiosis specific factor critical in meiotic sex chromosome inactivation and transcriptional silencing. Hormad1 deficiency abrogates ATM autophosphorylation HORMAD1 is a likely mammalian counterpart to the yeast HORMA domain meiotic protein, Hop1. Hop1 is phosphorylated by Mec1/Tel1, the budding yeast homologue to the mammalian ATR and ATM kinases, and this phosphorylation is thought to play an important role in inter-homologous recombination [21]. We therefore examined HORMAD1 expression in Atm deficient mice as well as ATM expression in Hormad12/2 animals. ATM is a serine/threonine-specific protein kinase that has been associated with cell cycle regulation, apoptosis, and response to DNA damage repair. ATM kinase activation is associated with increased auto- phosphorylation of ATM at multiple sites including serine 1981 [41]. We examined HORMAD1 protein expression in testes between postnatal days 5–21 (Figure 9A). Eight to ten day old testes contain spermatogonial cells as well as preleptotene/ leptotene spermatocytes [34]. At postnatal day 14–18, testes contain pachytene spermatocytes and visible sex bodies [34]. HORMAD1 is known to be phosphorylated [17] (Figure 9B). Western blot analysis on testes extracts detected phospho- HORMAD1 beginning at postnatal day 14 (Figure 9A). Phos- pho-HORMAD1 protein was decreased in postnatal day 18 and 21 wild-type testes (Figure 9A). Phospho-HORMAD1 appearance correlates temporally with sex body formation. HORMAD1 phosphorylation was not affected by Atm deficiency (Figure 9A). These results indicate that ATM is not responsible for HOR- MAD1 phosphorylation. We also examined HORMAD1 locali- zation in Atm2/2 spermatocytes. We observed HORMAD1 localization to chromosomal axes in Atm deficient spermatocytes, as previously described by others [17] (Figure 9C and 9D). Since synaptonemal complexes do not form in Atm mutants, HOR- MAD1 association with unsynapsed chromosomes does not require ATM. The anti-ATM phospho-S1981 antibody did not detect phosphorylated ATM in Hormad12/2 testes (Figure 9E and 9F). These results suggest that HORMAD1 is upstream of ATM auto-phosphorylation, and therefore likely upstream of ATM kinase activation. HORMAD1 Is Essential for Male and Female Meiosis Hormad1 deficiency does not affect gross ovarian development We have previously shown that Hormad1 RNA expression in the ovary was confined to the germ cell [16]. Meiosis I in the female gonad commences circa E13.5 and most oocytes arrest at the dictyate stage by the time of birth. Antibodies against HORMAD1 recognized HORMAD1 protein at E14.5 (leptotene) and E18.5 (arrest in diplotene) oocytes (Figure 10A and 10B), but little HORMAD1 protein was detected in the newborn ovary oocytes (Figure 10C), at the time when oocytes are arrested in diplotene. Deficiency in genes critical in meiosis can disrupt early ovarian development, as is the case for Dmc1, Msh5, Spo11 and Atm [42–44]. We therefore examined ovarian development in Hormad12/2 females. Antibodies against germ cell–specific transcriptional regulator SOHLH1 [45] stained wild-type and knockout oocytes throughout embryonic gonadal development with no significant differences noted (Figure 10A–10F). Moreover, the numbers of primordial, primary and secondary follicle counts did not significantly differ between the mutant and wild-type ovaries at post-natal day 8 (Figure S4). These results indicate that Hormad1 deficiency does not affect embryonic ovarian development, germ cell cyst breakdown, and primordial follicle formation. We also examined the histology of wild-type and Hormad12/2 ovaries between 2 and 30 weeks of life. Mice reach sexual maturity around 6 weeks of life, and mouse ovaries at this time consist of all of the follicular types including corpora lutea, an indication that the ovaries are ovulating. Postnatal Hormad12/2 ovaries were grossly indistinguishable from wild-type mice between 2 and 30 weeks of life, with abundant corpora lutea in the Hormad12/2 ovaries indicating that the normal process of oocyte maturation was not disrupted, and ovulation has occurred (Figure 11A–11H). We induced superovulation in knockout and wild-type mice with exogenous gonadotropins to determine whether subtler ovarian defects contributed to infertility in Hormad12/2 mice. Hormad12/2 females super-ovulated 28611 eggs (n = 22), while wild-type animals superovulated 29614 eggs (n = 13). We therefore did not observe significant difference between the number of eggs superovulated from wild-type versus knockout mice. These results indicate that ovarian development is grossly normal in Hormad12/2 mice, and that ovarian defects are unlikely to account for observed infertility. Hormad1 deficient eggs fertilize and embryonic development arrests at blastocyst stage due to aneuploidy We studied early embryonic development of fertilized Hor- mad12/2 oocytes because ovarian defects were unlikely to explain observed infertility. We recovered embryos from wild-type male matings with Hormad12/2 females at E0.5, E1.5, E2.5, and E3.5. Comparable numbers of morphologically indistinct 1-cell zygotes were recovered from oviducts of control and mutant female mice at E0.5 (Figure 12A–12C), and little difference was noted at the 2- cell stage, except for an increased number of 1-cell embryos in the knockouts, indicating a lag in the progression from the 1-cell to 2- cell stage (Figure 12D–12F). By E2.5, the number of normal appearing 8-cell stage embryos was significantly less in Hormad12/2 fertilized eggs as opposed to the wild-type (Figure 12G–12I), and no morphologically normal blastocysts were observed in the Hormad12/2 fertilized eggs at E3.5. It is interesting to note that at E3.5, a significant number of 4 and 8 cell stage embryos were observed in the knockout while only blastocysts were observed in the wild-type E3.5 embryos (Figure 12J–12L). We also tested, using PLoS Genetics | www.plosgenetics.org 9 November 2010 | Volume 6 | Issue 11 | e1001190