HORMAD1 Is Essential for Male and Female Meiosis Figure 3. HORMAD1 localization in Sycp2 mutant spermatocytes. Chromosome spread assay was performed on the wild-type (+/+) (A–C) and Sycp22/2 (D–F) zygotene spermatocytes with anti-HORMAD1 (green) and anti-SYCP2 (red) antibodies. The truncated SYCP2 protein (SYCP2t) in Sycp22/2 mice is still made and localizes to axial chromosomal cores[48]. SYCP2t lacks the domain necessary to bind SYCP3. Scale bars: 10 mm. doi:10.1371/journal.pgen.1001190.g003 deficiency on the structure of the synaptonemal complex, we visualized synaptonemal complexes during meiosis I in wild-type and Hormad12/2 spermatocytes using electron microscopy. In the wild-type, synaptonemal complexes were well visualized during the pachytene stage of meiosis (Figure 4B and 4C). Electron microscopy examination of one hundred and ten Hormad12/2 spermatocytes from three independent experiments revealed the lack of the typical tripartite synaptonemal complex structure (Figure 4E and 4F). Persistence of pre-synaptic number of centromeric foci (CREST staining) in Hormad12/2 spermatocytes, as well as non-visualization of the tripartite synaptonemal complex structure by electron microscopy, demonstrate that HORMAD1 is essential for chromosomal synapsis. Hormad1 deficiency disrupts localization of proteins important in early recombination Previous studies have indicated that Sycp3 deficiency has subtle effects on meiotic recombination [27]. Early recombination events do not seem to be disrupted in Sycp3, as similar number of DMC1 foci are present in Sycp3 mutant and wild-type meiosis [29]. DMC1 is a meiotic specific recombinase that together with ubiquitously expressed RAD51 catalyzes homologous pairing and Figure 4. HORMAD1 is required for chromosome synapsis during male meiosis. (A and D) Immunofluorescence with CREST (red) and anti- SYCP3 (green) antibodies. Anti-CREST antibody recognizes chromosome centromeres. Synapsed wild-type zygotene spermatocytes contain about 20 CREST foci (n = 50) (A-A’). However, Hormad12/2 spermatocytes contain approximately 40 CREST foci (n = 50), an indication that synapsis is disrupted (D-D’). Arrow indicates chromosome synapses in the wild-type. Scale bars: 10 mm. (B–C, E–F) Electron microscopy analysis of synaptonemal complexes in 2 week old wild-type and Hormad12/2 spermatocyte at different magnifications. The typical, tripartite structure of the synaptonemal complex consists of one central element (CE) that connects with two axial elements (AE) (C). We did not identify normal tripartite synaptonemal complex structure in Hormad12/2 spermatocytes. Scale bars: 1 mm. doi:10.1371/journal.pgen.1001190.g004 PLoS Genetics | www.plosgenetics.org 5 November 2010 | Volume 6 | Issue 11 | e1001190