PDILT cooperates with the testis-specific calreticulin-like chaperone, calsperin (CALR3), in the endoplasmic reticulum and plays an indispensable role in the disulfide-bond formation and folding of ADAM3. Pdilt(-/-) mice were male infertile because ADAM3 could not be folded properly and transported to the sperm surface without the PDILT/CALR3 complex.
Fig. 1 Analysis of PDILT protein in testes extracts from wild-type and Pdilt knock-out mice by western blotting with anti-PDILT antibody. Sperm was lyzed in lysis buffer containing 1% Triton-X100 and extracts were prepared as supernatants of lysates after centrifugation. Samples were reacted with anti-PDILT antibody at 1/1,000 dilution. ƒ¿-Tubulin was used as a control.
Fig.2 Western blotting analysis of PDILT expression in various tissues with anti-PDILT antibody.
Proteins were extracted from various tissues with lysis buffer containing Triton X-100 and subjected to western blot analysis. Tissue proteins (30 ug) and sperm protein (10 ug) were reacted with anti-PDILT antibody at 1/1,000 dilution.
Fig.3 Immunoprecipitation of PDILT protein from mouse testis extracts.
Extracts (100 µg) of wild-type (+/+) and Pdilt (-/-) mouse testes were immunoprecipitated with anti-PDILT antibody at 1/100 dilution and the precipitates were analyzed by western blotting using the same antibody at 1/1,000 dilution. 1. Input. 2. Wild-type. 3. Pdilt (-/-)
Fig.4 Immunofluorescence staining of mouse testicular sections with anti-PDILT antibody. Testes were collected from adult mice and were fixed in 4% paraformaldehyde/PBS, cryopreserved in garaded 10-30% sucrose, and embedded in a TissueTek OCT compound (Sakura Finetechnical, Tokyo). Frozen sections (5 µm) were mounted on APS-coated glass slides. After washing and blocking, the slides were reacted with anti-PDILT antibody at 1/100 dilution. As a secondary antibody, Alexa Fluor 546 conjugated anti-rabbit IgG antibody was used. PDILT was detected only in spermatid .
# Tokuhiro K., et al. Protein disulfide isomerase homolog PDILT is required for quality
control of sperm membrane protein ADAM3 and male fertility . Proc Natl Acad Sci U S
A. 2012 Mar 6;109(10):3850-5. PMID: 22357757
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.