Novel Synaptonemal Complex Protein SYCE3 Figure 7. Initiation of synapsis is dependent on SYCE3. (A) Immunostaining of spread preparations of wild-type pachytene and Syce32/2 pachytene-like spermatocytes with SYCP3 (red) and SYCP1, SYCE1 or SYCE2 (green) acquired using a confocal microscope. In Syce32/2 spermatocytes, SYCP1 localizes in a weak discontinuous pattern at AEs independent of whether they are closely aligned or not. In contrast, SYCE1 and SYCE2 are completely absent from the AEs in SYCE3 deficient mice. Bars, 10 mm. (B) Electron micrographs showing synaptonemal complex assembly in wild-type and Syce32/2 spermatocytes. (LE) lateral elements, (CE) central element, (AE) axial elements. Bar, 200 nm. doi:10.1371/journal.pgen.1002088.g007 are presumed markers of future crossovers in the wild-type) is critically depending on its presence. SYCE3 Is Part of a Central Element Protein Complex We demonstrated that SYCE3 is important for fertility, initiation of synapsis and for a correct progression of meiotic recombination. Hence, we were interested in identifying binding partners of SYCE3 that would provide mechanistic insights into SYCE3 function. As the localization of the other CE components, SYCE1, SYCE2 and Tex12, is severely altered in Syce32/2 mice, they appear to be good candidates for being SYCE3 binding partners. Therefore, we performed co-transfection/immunopre- cipitation experiments in somatic cells that do not express meiosis- specific proteins according to the approach described by Stewart- Hutchinson et al. [48]. COS-7 cells were transfected with EGFP- or myc-tagged fusion constructs of SYCE1, SYCE2, Tex12, SYCP1 N-terminus, SYCP1 C-terminus (as a control) and SYCE3. We used either a myc or an EGFP-specific antibody for precipitation. We found that SYCE3 interacts with SYCE1 (Figure 10A, 10B), which is consistent with the highly similar spatial-temporal expression of these two proteins. Interestingly, we show that SYCE3 also binds to SYCE2 although these two proteins do not exactly colocalize in pachytene spermatocytes (Figure 10C, 10D). However, under these experimental conditions, we could not detect an interaction between SYCE3 and Tex12 or the N- or C-terminus of SYCP1 (Figure 10E, 10F, 10G, see also below). Discussion Meiotic chromosome synapsis is essential for proper meiotic progression as well as for male and female fertility. A critical step during this process is the assembly of the CE of the SC. In mammals, four proteins have been described which are essential for CE assembly: SYCP1, SYCE1, SYCE2 and Tex12. While PLoS Genetics | www.plosgenetics.org 8 May 2011 | Volume 7 | Issue 5 | e1002088