DEV030858 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Characterization of the RIM-BP3 antibodies. As the SH3 and FNIII domains of RIM-BP3 show high similarity with the other two paralogs, RIM-BP1 and RIM-BP2, we chose an N-terminal region (RIM-BP3 N), the linker region between FNIII and SH3 domains (RIM-BP3 C) of less sequence conservation, and the SbcC domain as antigen regions to raise rabbit polyclonal antibodies (Fig. 1A). The three antibodies were affinity purified with immobilized antigens and their specificities were verified by immunoblotting using extracts of 293T cells transfected with pFlag-CMV-2-RIM-BP3 (+). Western blotting with commercial anti-Flag antibody confirmed the expression of epitope-tagged RIM-BP3. All three antibodies recognize a specific band of about 190 kDa as does the anti-Flag antibody (the full-length RIM-BP3 protein has a calculated molecular mass of around 180 kDa). The detection of α-tubulin served as a loading control.

• Supplemental Figure S2 -

  Fig. S2. Loss of the RIM-BP3 protein in elongate spermatids of the RIM-BP3P ^Pknockout mouse. Testicular germ cells were prepared and immunostained with the anti-RIM-BP3 SbcC antibody (green). The slides were counterstained with DAPI to visualize the nucleus (blue). Staining signals were detected only in spermatids from the wild-type mouse (Fig. 2). Scale bar: 10 µm.

• Supplemental Figure S3 -

  Fig. S3. HE-stained cross-sections of seminiferous tubules. Cross-sections of testes from the wild-type (+/+) and RIM-BP3 knockout (-/-) mice were stained with Hematoxylin and Eosin. The developmental stages of seminiferous epithelium are marked on the left. Scale bar: 40 µm. In the seminiferous tubules of the mutant mouse, no change is obvious with the spermatogonia, spermatocytes and somatic cells. Malformed elongated spermatids are visible at a higher magnification at phase XII (D, inset).

• Supplemental Figure S4 -

  Fig. S4. Hook1 distribution on the malformed manchette in RIM-BP3 -deficient spermatids. Testicular germ cells were prepared from adult wild-type (WT) and RIM-BP3-deficient (KO) mice, and immunostained with monoclonal anti-α-tubulin (red) and polyclonal anti-Hook1 (green) antibodies. The slides were counterstained with DAPI to visualize the nucleus. A schematic diagram of the manchette development in wild-type and mutant spermatids is shown on the right. The broken lines represent either the longitudinal axis of a wild-type spermatid nucleus or the longitudinal axis of the caudal region of a RIM-BP3P ^Pdeficient spermatid. The manchette of mutant spermatids is indistinguishable from that of the wild-type at step 9. At step 11/12, while the normal manchette forms an asymmetric trapezoid, the manchette of mutant spermatids is still in a symmetric conical shape. Despite the abnormal manchette development, the localization of Hook1 on the manchette is unchanged in mutant spermatids.