HORMAD1 Is Essential for Male and Female Meiosis Figure 1. HORMAD1 is required for spermatogenesis. (A–H). Periodic acid/Schiff reagent (PAS) stained cross sections from 6 weeks old wild-type testes (A-D) and Hormad1 2/2 (E-H) testes. Seminiferous tubule stages are shown above the panels. Lack of mature sperm and arrest in spermatogenesis is shown at different tubular stages. (I-P). Immunohistochemistry (IHC) of 6 weeks old wild type and Hormad12/2 testes with anti HORMAD1, SOHLH1, PLZF and DDX4 antibody. IHC with anti-HORMAD1 antibody shows HORMAD1 expression (brown) in wild-type stage IV pachytene, and stage XI zygotene spermatocytes (I), while no expression was observed in the knockout (M). SOHLH1 is expressed in differentiating spermatogonia and is present in wild-type and Hormad12/2 testes as shown by arrows (J and N). PLZF identifies differentiating and self-renewing spermatogonia and is expressed in both wild-type and Hormad12/2 testes as shown by arrows (K and O). DDX4 is an RNA binding protein specific for germ cells, and is under-expressed in Hormad12/2 spermatocytes (L and P). Arrows point to Spermatogonia (Sg) and Spermatocytes 1(Sc). Scale bars: 50 mm. doi:10.1371/journal.pgen.1001190.g001 Hormad12/2 testes (Figure S3A, S3B, S3C, S3D, S3E, S3F, S3G, S3H, S3I, S3J), and rising apoptotic index with age in the knockout as compared to the wild-type (Figure S3K). We counted leptotene, zygotene and pachytene spermatocytes in 6 week old wild-type and Hormad12/2 testes. Hormad12/2 testes showed declining number of spermatocytes beginning in stages II-III with 2868 spermatocytes as compared to 52612 in the wild-type (Figure 1A and 1E). No spermatocytes were noted in stages IV-IX in the Hormad12/2 testes (Figure 1F and 1G), and no significant difference was noted in the spermatocyte number in stages X-XII between the wild-type and knockout testes (Figure 1D and 1H). These results indicate that Hormad1 deficiency in the male gonad caused meiotic arrest at the pachytene stage. HORMAD1 is critical for chromosome synapsis Previous studies on HORMA domain proteins indicate their specific involvement in cell division. MAD2 is a ubiquitously expressed mammalian HORMA domain protein involved in both meiosis and mitosis [7], while yeast HOP1, RED1, nematode HIM3 and plant ASY1 genes are specifically involved in meiotic segregation, synapsis and recombination [2,10–11,13,15,22]. No mammalian counterparts to Hop1, Red1, Him3 and Asy1 have been functionally evaluated up to date. We previously hypothe- sized that HORMAD1 is a functional counterpart to Hop1, Him3 and Asy1 [16]. Critical components of the synaptonemal complex include meiosis specific SYCP1, SYCP2 and SYCP3 proteins. SYCP1 is a major component of the transverse filaments, while both SYCP2 and SYCP3 are components of the axial lateral elements [23–26]. To determine HORMAD1 localization during meiosis, and whether HORMAD1 localizes to the axial elements, or transverse filaments, we used antibodies against SYCP1, SYCP2 and SYCP3 to study their respective co-localization with HORMAD1. HORMAD1 co-localized with SYCP3 and SYCP2 but did not co-localize with SYCP1, which indicates that PLoS Genetics | www.plosgenetics.org 3 November 2010 | Volume 6 | Issue 11 | e1001190