Author Summary The biology of germ cells is intimately intertwined with meiosis. Meiosis I is a unique biological event, when chromosomes pair, recombine, and segregate. The synap- tonemal complex is a protein lattice that enables chromosome pairing and recombination and is unique to meiosis I. Meiosis I requires a subset of factors that are unique to germ cells and meiosis. Germ cell–specific factors are known to play crucial roles during formation of the synaptonemal complex and include synaptonemal complex proteins SYCP1, SYCP2, and SYCP3, among others. We discovered a mouse HORMA domain contain- ing protein, Hormad1 (Nohma), which is germ cell–specific and essential for male and female fertility. Mice deficient in Hormad1 have severe defects in early recombination, synapsis, and segregation—functions attributed to yeast HORMA domain containing protein, Hop1. Moreover, Hormad1 is likely a germ cell–specific component of the meiotic sex chromosome inactivation and transcriptional silencing complex. and all are critical for meiosis. These HORMA proteins are: Hop1 [8] and Red1 [9] in yeast; Him-3 [10] in nematodes; and Asy1 [11] in plants. Him-3 localizes to the axial cores of both synapsed and unsynapsed chromosomes. C. elegans Him-3 mutants are deficient in chromosome pairing, synapsis, and the regulation of double strand break repair [10,12–13]. Synapsis in both male and female Asy1 mutants is disrupted [14–15]. In yeast, plants, and nematodes, HORMA domain proteins are critical components of the synaptonemal complex and essential for meiosis I. Others and we identified a previously uncharacterized gene that we named Nohma, later re-named to Hormad1 [16–18]. Hormad1 encodes a protein that contains a HORMA domain, and unlike Mad2, Hormad1 expression is germ cell–specific [16]. Mouse and human HORMAD1 are highly conserved and share 77% amino acid identity overall, and share 89% amino acid identity in the HORMA domain. Moreover, mouse and human HORMAD1 HORMA domains share 28% amino acid identity with Hop1 HORMA domain. Hop1 in yeast appears to bind near or at the sites of DSB formation and may modulate the initial DSB cleavage [19]. Hop1 mutants in yeast have reduced number of DSBs [20], and Hop1 may participate in recruiting DMC1, RAD51 and other proteins that are required for DNA repair during meiotic synapsis and recombination [19–20]. Phosphorylation of Hop1 by Mec1/ Tel1 yeast kinases is important for interhomologue recombination and prevents DMC1-independent repair of meiotic DSBs [21]. Here we report that HORMAD1 is likely the mammalian counterpart of Hop1, and that HORMAD1 deficiency disrupts mammalian synaptonemal complex formation, meiotic recombi- nation, and chromosome segregation. Results HORMAD1 is essential for spermatogenesis We previously showed that Hormad1 RNA expression in testes began at postnatal day 10, with little expression detected at birth or postnatal day 5 [16]. Hormad1 RNA expression pattern coincided with the onset of meiosis, and appearance of primary spermatocytes in the developing testes. In situ hybridization with anti-sense Hormad1 riboprobe revealed that Hormad1 expression was confined to germ cells, and specifically spermatocytes, with no signal detected in spermatogonia or sertoli cells [16]. We generated antibodies against HORMAD1 and studied its protein HORMAD1 Is Essential for Male and Female Meiosis localization pattern in testes. HORMAD1 localized exclusively in germ cells, specifically in zygotene, and early pachytene sper- matocytes as previously described for the RNA expression [16]. Since HORMAD1 protein showed localization consistent with its potential role in meiosis I and contains the HORMA domain, we disrupted the Hormad1 gene to examine its requirement for germ cell development and meiosis in mouse. Hormad1 is located on chromosome 3 and composed of sixteen exons. We deleted exons 4 and 5 (Figure S1A), and this mutation is predicted to remove 33 amino acids from the highly conserved HORMA domain and to cause a frame shift mutation. Small amounts of truncated Hormad1 RNA transcripts were detectable on RT-PCR, and Western blots on testes extracts showed absence of HORMAD1 protein in knockout mice as expected (Figure S1B and S1C). Female and male heterozygote matings produced expected Mendelian ratios, averaged 8.162 pups per litter (n = 20 breeding pairs) over a 6-month period, and remained fertile for at least 9 months. The litter size was statistically not significantly different from the wild-type average (8.462 pups per litter). Male and female mice heterozygous for the mutation (Hormad1+/2) were fertile with grossly normal male and female gonadal morphology and histology. However, both Hormad12/2 males and females were infertile with no pups produced over a period of 6 months from mating with wild-type female and male mice, respectively. While ovaries showed no gross morphologic differences between the knockout and wild-type mice, Hormad1 knockout adult testes were significantly smaller than the wild-type testes (Figure S2A). Testes in the 7-day-old Hormad12/2 mice were grossly normal and weighed 8.062.0 mg/pair and did not significantly differ from the wild-type, 9.063.0 mg/pair of testes. By 4 weeks of postnatal life, the knockout testes (4766 mg/pair) were 50% of the wild-type weight (9461.7 mg/pair), and by 8 weeks the knockout testes (6067.7 mg/pair) were 27% of the wild-type weight (22562.7 mg/pair) (Figure S2B). Histology at 6 weeks showed hypocellular seminiferous tubules with clumps of sertoli cells in the lumen. We observed spermatogonia and early spermatocytes, but no post-meiotic germ cells such as spermatids or spermatozoa (Figure 1A–1H). We therefore carefully examined spermatogenesis in Hormad12/2 mice. Spermatogenesis is a complex process that involves differentiating and proliferating self-renewing spermatogonia that differentiate into spermatozoa. Type A spermatogonia self-renew and can initiate differentiation into Type B spermatogonia which in turn differentiate into primary spermatocytes. Primary sper- matocytes undergo meiosis I to form secondary spermatocytes. Secondary spermatocytes enter meiosis II and divide to produce haploid spermatids. We examined Hormad1 knockout testes histology during gonadal development to determine the stage at which spermatogenesis is disrupted. Identical testes weights at postnatal day 7, and similar histology between Hormad12/2 and wild-type testes argue that pre-spermatogonia in Hormad12/2 testes proliferate into Type A spermatogonia without major disruption. Immunohistochemistry with antibodies directed against PLZF and SOHLH1, markers that identify self-renewing (PLZF) and differentiating spermatogonia (SOHLH1), showed the presence of both proteins in the wild-type as well as the knockout animals, confirming that spermatogonia are unaffected (Figure 1J, 1K, 1N, 1O). At postnatal day 10, testes contain preleptotene/ leptotene primary spermatocytes, and there was no gross difference between wild-type and Hormad12/2 testes. At 14 days, testes contain pachytene spermatocytes, and there was a significant difference between the wild-type and Hormad12/2 testes, with many apoptotic cells and few pachytene spermatocytes in PLoS Genetics | www.plosgenetics.org 2 November 2010 | Volume 6 | Issue 11 | e1001190