JCS059949 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -
• Supplemental Figure S1 -

  Fig. S1. Phylogenetic tree for TSSK proteins from nine species. TSSK1 (or TSSK1B) and TSSK2 are found in all placental mammals included in this analysis Hs (Homo sapiens), Pt (Pan troglodytes), Mc (Macaca mulatta), Mm (Mus musculus), Rn (Rattus norvegicus), Bt (Bos taurus), Cf (Canis familiaris). Both proteins are also present in opossum (Md; Monodelphis domestica). For platypus (Oa; Ornithorhynchus anatinus), TSSK2 is present, but TSSK1 is missing in this tree, although we detected part of the gene encoding TSSK1 in an area of yet incomplete sequencing. We have not detected any TSSK1 or TSSK2 orthologs in Drosophila, zebrafish, chicken, and other non-mammalian species. TSSK1B is specific for primates. Platypus and opossum lack TSSK4, and platypus also lacks TSSK3. For chimpanzee, TSSK6 is not included in this tree, due to incomplete sequencing. The TSSK5 proteins show relatively large evolutionary distance from the other TSSKs, and a relatively high number of amino acid substitutions among species. The gene encoding TSSK5 contains the highest number of introns, 7 in mouse (supplementary material Table S1). Primates do not have TSSK5. In all non-primate mammalian species, including opossum, TSSK1 and TSSK2 are located in tandem.

• Supplemental Figure S2 -

  Fig. S2. Generation of the Tssk1/2 knockout. (A) Schematic presentation shows the wild type allele which contains Tssk1 and Tssk2 genes with a 3.06 kb intergenic region, and the targeting allele containing a floxed Tssk1-Tssk2-Neo locus. Positions of PCR primers used for genotyping are indicated with open arrowheads. 5′ and 3′ probes were used in Southern blotting for verification of homologous recombination (data not shown). (B) Genotypes of heterozygous and homozygous animals were verified by PCR, which yielded a 766 bp product from the wild type allele and a 363 bp product from the knockout allele.

• Supplemental Figure S3 -

  Fig. S3. Expression and activity of TSSK1, TSSK2, and TSKS. (A) Western blotting of TSSK1 and TSSK2. The expression of TSSK1 and TSSK2 is completely lost, in cytoplasmic fragments isolated from elongating spermatids from the −/− animals, and there is a marked loss in the +/− situation. (B) Western blotting of TSKS. Analysis of proteins from whole testis shows that expression of the testis-specific kinase substrate TSKS (and its isotype TSKS ISO1; Ensembl protein ID: ENSMUSP00000079122) is maintained at wild type level in the knockout (−/−). (C) In vitro kinase activity. Testis homogenates were incubated with ³²P, followed by immunoprecipation of proteins using anti-TSKS antibody. This shows that loss of TSSK1 and TSSK2 also results in loss of 32P incorporation into TSKS. The Western blot loading control shows immunoprecipitation of TSKS from +/+ and −/− testes.

• Supplemental Figure S4 -

  Fig. S4. mRNAs in wild type and Tssk1/2 knockout testes. (A-C) RNA in situ hybridization for adult wild type (+/+) testis, showing expression of Tssk1 (A), Tssk2 (B) and Tssk6 (C) mRNAs (blue signal), near the lumen of the tubules where the elongating spermatids are located. (D-F) In the knockout (−/−), the in situ signal for Tssk1 (D) and Tssk2 (E) mRNAs is completely lost, whereas expression of Tssk6 mRNA (F) is maintained. Scale bar: 100 µm in A-F.

• Supplemental Figure S5 -

  Fig. S5. TSSK1, TSSK2, and TSKS immunostaining of CB ring and satellite. (A-C) Immunostaining of adult wild type testis section with guinea pig anti-TSSK1(A), rabbit anti-TSKS (B), and the merged signals (C), showing colocalization of TSSK1 and TSKS on ring (open arrowhead) and satellite (closed arrowhead). (D-F) Immunostaining of adult wild type testis section with guinea pig anti-TSSK2(D), rabbit anti-TSKS (E), and the merged signals (F), showing colocalization of TSSK2 and TSKS on ring (open arrowhead) and satellite (closed arrowhead). (G-I) Immunostaining of adult Tssk1/2 knockout testis with anti-TSKS. In the knockout spermatids (−/−), TSKS is still present (also see the Western blot, supplementary material Figure S3B), but there is no progression to yield marked staining of ring and satellite. Ring formation seems to be initiated around step 9 spermatids (G; open arrowheads in inset), and a weak satellite-like signal is found at step 10-11 spermatids (H), but the cytoplasm of step 13 spermatids shows only dispersed cytoplasmic staining (I). Nuclei are stained with DAPI. +/+ is wild type and −/− is knockout. Scale bars: 20 mm.