HORMAD1 is located along the axial elements (Figure 2A, 2C, and 2E). Recent studies also show that HORMAD1 localizes to the axial elements [17–18]. We also studied whether absence of SYCP2 affected HORMAD1 localization along the chromosomes. HORMAD1 localization is independent of major germ cell– specific components of the axial elements of the synaptonemal complex because neither Sycp2 (Figure 3A–3F) nor Sycp3 mutation affected HORMAD1 localization to the axial elements [18]. We examined localization of germ cell–specific synaptonemal complex proteins SYCP1, SYCP2 and SYCP3 in Hormad12/2 testes. SYCP1 is a major component of the transverse filaments and is known to form fibrillar structures in Sycp3 mutant spermatocytes [27]. However, the fibers are truncated, contain axial gaps, and do not associate with the centromeres of the meiotic chromosomes. We also observed truncated fibers with anti-SYCP1 antibodies in Hormad12/2 mice (Figure 2B). HOR- MAD1 is therefore not necessary for SYCP1 binding to the chromatin. We also examined localization of SYCP2 and SYCP3 proteins in Hormad12/2 mice. The localization of SYCP2 and SYCP3 to the chromatin was not significantly affected by the lack of HORMAD1 (Figure 2D and 2F). These results indicate that HORMAD1 is not necessary for SYCP2 and SYCP3 localization to the axial elements. HORMAD1 Is Essential for Male and Female Meiosis The deficiency in synaptonemal complex proteins such as SYCP3 is known to affect chromosome synapsis [27]. In order to determine the effect of HORMAD1 deficiency on chromosome synapsis during meiosis I, we utilized CREST sera. CREST sera labels centromeres and allows the determination of the pairing status during meiosis [28]. In the wild type spermatocytes, prior to the synapsis, 40 centromeres are usually observed in the leptotene stage. The number of visible centromeres become reduced as the synapsis of homologues progresses. At the completion of the synapsis in pachytene, 20 centromeric foci are usually observed corresponding to 19 autosomal homologues and partially paired X-Y chromosomes. We examined CREST foci formation in Hormad12/2 spermatocytes. Examination of over 100 Hormad12/2 spermatocytes and oocytes in meiosis I, revealed greater than 20 centromeric foci in both male and female germ cells, most containing 40 CREST foci (Figure 4A, 4D, and data not shown). These results indicate that Hormad1 deficient germ cells cannot complete homologous chromosome pairing, and Hormad1 is therefore critical for chromosome synapsis during meiosis. Our experimental evidence strongly suggests that HORMAD1 localizes to the axial core and is yet another critical component of the synaptonemal complex. To determine the effect of Hormad1 Figure 2. HORMAD1 co-localizes with SYCP2 and SYCP3, but not with SYCP1. Chromosomal spread assay in wild-type (+/+) and Hormad12/2 (2/2) zygotene stage spermatocytes. (A–B) Immunofluorescence staining with anti-SYCP1 (Green) and anti-HORMAD1 (Red) antibody. HORMAD1 preferentially localizes to unsynapsed regions of chromosome axes (arrow head) and sex body (arrow) but does not co-localize with SYCP1 (A-A’’). Hormad1 deficiency does not affect SYCP1 localization to the axes (B). HORMAD1 co-localizes with SYCP2 on the unsynapsed chromosome axes (C-C’’), but HORMAD1 deficiency does not affect SYCP2 localization to the axes (D). SYCP3, another integral and critical component of the synaptonemal complex co-localizes with HORMAD1 on unsynapsed axes (E-E’’). Similar to SYCP1 and SYCP2, SYCP3 localizes to the axes despite HORMAD1 deficiency (F). DNA was stained with DAPI. Scale bars: 10 mm. doi:10.1371/journal.pgen.1001190.g002 PLoS Genetics | www.plosgenetics.org 4 November 2010 | Volume 6 | Issue 11 | e1001190