Novel Synaptonemal Complex Protein SYCE3 Figure 1. Identification and characterization of mouse SYCE3. (A) Multiple alignment of full-length SYCE3 protein sequences of various vertebrates. A predicted tyrosine phosphorylation site is marked in green, and a conserved serine phosphorylation site in red (calculated for the mouse protein sequence). (B) Tissue-specific expression pattern of SYCE3 mRNA as shown by RT-PCR analysis with SYCE3 specific oligonucleotides. (C) Temporal expression pattern of SYCE3 during mouse spermatogenesis analyzed by RT-PCR using RNA of testicular cells from mice of different ages (day 8–25) and Western blot analysis using 56105 cells of the same mice separated on a 16%/6 M urea tricine-SDS gel and detection of SYCE3 with affinity purified guinea pig anti-SYCE3 antibody. doi:10.1371/journal.pgen.1002088.g001 protein (see materials and methods). Western blot analysis of total protein fractions of pubertal mouse testes of different ages revealed a single band with the expected mass (12 kDa) from day 12 onwards, validating the fact that SYCE3 is first expressed with the onset of meiosis (Figure 1C). In addition, SYCE3 restriction to meiotic cells was confirmed by immunofluorescence analysis on frozen mouse testis sections: SYCE3 expression is confined to spermatocytes and completely absent in spermatogonia, sperma- tids and spermatozoa (Figure S1B). SYCE3 Selectively Localizes to the Central Element of the Synaptonemal Complex Using immunocytochemistry on spread mouse spermatocytes, we investigated the subcellular localization of SYCE3 in meiotic cells. For proper staging of meiotic cells we used SYCP3 as a marker for AEs/LEs (Figure 2A). Fluorescence staining of SYCE3 showed that it specifically localizes to the synapsed regions of homologous chromosomes, whereas SYCP3 was present in AEs and LEs. SYCE3 was first detectable during zygotene when synapsis is initiated and staining persisted on synapsed regions of homologous chromosomes until diplotene. The localization to synapsed regions was verified by double-labeling experiments performed with SYCE3 and TF-protein SYCP1 (a marker for synapsed regions) in which a virtually identical staining pattern was observed (Figure 2B). In pachytene oocytes SYCE3 also localized to synapsed chromosomes (Figure 2C). For a detailed analysis of SYCE3 localization within the SC we performed immuno-gold electron microscopy on testis sections using an affinity-purified SYCE3 antibody and an antibody against the coiled-coil region of SYCP1 as a control (Figure 3A). In preparations incubated with SYCE3 antibodies, gold particles exclusively localized to the CE of the SC. This is in strong contrast to the findings when using an antibody against the coiled-coil region of SYCP1, where - as expected - TFs between CE and LEs are labeled (see also [29]). For a quantitative analysis of immunogold data we sub-divided the distance between the SC center and the outer edge of LEs into 7 equal sections and counted the immuno-gold particles in each section (see [36]). In the case of SYCE3 the bulk of gold particles (n = 304) localized to the two sections adjacent to the SC center. In contrast, gold particles corresponding to the SYCP1 coiled-coil domain (n = 135) largely localized to the area between LEs and CE (see also [25]; Figure 3B). Since three CE-specific proteins had been discovered previously (SYCE1, SYCE2 and Tex12; [28,29]), we became interested in the localization of SYCE3 in respect to the other CE-specific proteins. As previously described, SYCE1 is distributed rather continuously in pachytene along synapsed areas of homologous chromosomes, whereas SYCE2 and Tex12 localize in a more punctuated pattern. Co-localization of SYCE3 and SYCE1 on spread mouse spermatocytes revealed that in both zygotene and pachytene cells these two proteins co-localize in a rather continuous pattern along the synapsed chromosomes (Figure 4, see inset). In contrast, double-labeling experiments with SYCE3 and SYCE2 showed that in zygotene and pachytene cells SYCE3 does not necessarily co-localize with SYCE2 (Figure 4, see inset). Here, SYCE3 was distributed in a more continuous pattern whereas SYCE2 appeared more punctuated. Lateral Element Assembly As Well As Central Element Components SYCE1, SYCE2, and Tex12 Are Not Required for SYCE3 Chromosome Loading To investigate the possible dependence of SYCE3 localization on the presence of other SC-proteins, we performed immunocy- tochemistry on spread spermatocytes of mice deficient for SYCP1 [27], SYCP3 [20], SYCE1 [30], SYCE2 [31] and Tex12 [32]. In the absence of SYCP3, double-labeling for SYCE3 and SYCP1 showed that SYCE3 mimics SYCP1 localization, indicating that SYCE3 is loaded only to synaptic chromosome regions (Figure 5B). In Sycp12/2 mice, SYCE3 is completely absent from the AEs (Figure 5A). In the absence of CE protein SYCE1, on the other hand, SYCE3 localizes to the AEs in a weak discontinuous pattern which is independent of whether AEs are in close apposition or not (Figure 5C, and insets therein). In mice lacking CE protein PLoS Genetics | www.plosgenetics.org 3 May 2011 | Volume 7 | Issue 5 | e1002088