Supporting Information Figure S1 SYCE3 is expressed in mouse testis. (A) Specificity of affinity purified anti-SYCE3 antibodies. Western blot analysis of Syce3+/+, Syce3+/2 and Syce32/2 testis tissue separated on a 16%/ 6 M urea tricin-SDS gel and detection of SYCE3 with an affinity purified rabbit anti-SYCE3 (left) and an affinity purified guinea pig anti-SYCE3 antibody (right). The same Western blots were stripped and incubated with a mouse anti-actin antibody as a loading control. (B) SYCE3 localization is restricted to meiotic cells. Immunolocalization of SYCE3 (green) on frozen sections of an adult wild-type mouse testis. DNA is labeled with Hoechst. (eSp) elongated spermatids, (Sc) spermatocytes, (Sg) spermatogo- nia, (Ser) Sertoli cells. Bar, 50 mm. (TIF) Figure S2 Generation and characterization of a Syce32/2 mouse. (A) Structures of the Syce3 gene located on chromosome 15, the replacement vector and the mutant containing the neomycin cassette. The location of the Southern blot probe and the lengths of expected fragments after BstEII digestion of wild- type and mutant samples is depicted below. (B) Long arm (F2) and short arm (F1) PCR of positively tested ES cell clone using external oligonucleotides. (C) Southern blot of a wild-type and positively tested ES cell clone with a SYCE3 specific external probe (left) and neomycin-specific probe (right). (D) Correct insertion of the replacement vector and genotyping of Syce32/2 mice was confirmed by Southern blot analysis using a SYCE3 specific external probe (left) and neomycin-specific probe (right). (E) Testes from Syce3+/+ (left) and Syce32/2 (right) littermates. Bar, 200 mm. (TIF) Figure S3 Loss of SYCE3 results in massive apoptotic events during spermatogenesis. (A) TUNEL assay on paraffin embedded testis sections from wild-type, heterozygote and homozygote Syce32/2 mice (day 30). TUNEL positive stained cells are labeled in green, DNA is shown in blue. Bar, 40 mm. (B) Light microscopic images of testis sections from paraffin embedded Syce32/2 mice showing stage X-II, IV and V–VI tubules. In stage X-II type A spermatogonia and zygotene or pachytene spermatocytes are present. Stage IV is characterized by intermediate spermatogonia and apoptotic pachytene spermatocytes. In type V–VI, B type References 1. Zickler D, Kleckner N (1999) Meiotic chromosomes: integrating structure and function. Annu Rev Genet 33: 603–754. 2. Page SL, Hawley RS (2004) The genetics and molecular biology of the synaptonemal complex. Annu Rev Cell Dev Biol 20: 525–558. 3. Yang F, Wang PJ (2009) The mammalian synaptonemal complex: a scaffold and beyond. Genome Dyn 5: 69–80. 4. Roeder GS (1995) Sex and the single cell: meiosis in yeast. Proc Natl Acad Sci U S A 92: 10450–10456. 5. Colaia´covo MP (2006) The many facets of SC function during C. elegans meiosis. Chromosoma 115: 195–211. 6. 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Novel Synaptonemal Complex Protein SYCE3 spermatogonia can be distinguished. (Ser) Sertoli cells, (P) pachytene cells, (A) type A spermatogonia, (aP) apoptotic pachytene cells, (In) intermediate spermatogonia and (B) type B spermatogonia. Bar, 10 mm. (TIF) Figure S4 SYCP1 localization in Syce12/2 and Syce22/2 spermatocytes. Immunofluorescence analysis of spread prepara- tions of wild-type and (A) Syce12/2 or (B) Syce22/2 mouse spermatocytes stained with SYCP1 and SYCP3. As previously described, SYCP1 localizes to Syce12/2 AEs in a weak discontinuous pattern (A and [30]), whereas SYCP1-staining is confined to sites of closer association of homologs in Syce22/2 spermatocytes (B, and [31]). These results clearly demonstrate that under our experimental conditions we can reproduce previously described weak immunofluorescence signals, consequently allow- ing a precise comparison of different CE-mutant phenotypes. Bar, 10 mm. 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