PDILT associates with testis-specific chaperones and ADAM3. (A) Immunoprecipitates (IPs) with anti-PDILT antibody from testis lysates (100 μg) were probed with antibodies against CANX, CALR, CLGN, and CALR3. Testis lysates (10 μg) were loaded as the input control. PDILT associated with CLGN and CALR3, but not with CANX and CALR. Asterisk indicates an IgG signal recognized by the secondary antibodies. (B) IPs with anti-PDILT antibody from testis lysates (100 μg) were probed with antibodies against ADAM1B, ADAM2, ADAM3, and PDILT. PDILT only associated with ADAM3. (C) Heterodimerizations of ADAM1A/ADAM2 and ADAM1B/ADAM2 were probed by anti-ADAM2 antibody under nonreducing conditions (). Both complexes were absent in Clgn^−/− testis but were not impaired in Pdilt^−/− testis. (D) Western blot analysis of sperm lysates. ADAM3 was not detected in Pdilt^−/− sperm, whereas other sperm fertilizing proteins were present. (E) Immunofluorescence staining of sperm with antibodies against ADAM2 and ADAM3. ADAM3 was not detected in Pdilt^−/− sperm, whereas ADAM2 remained.

ADAM3 requires PDILT/CALR3 to be folded properly and transported onto the sperm membrane. (A) Western blot analysis of testis and epididymal sperm lysates. ADAM3 was not observed in caput epididymal sperm of Pdilt^−/− mice. Because testis and caput epididymis contain fewer sperm, 20 μg of extracts were loaded, whereas 6 μg was loaded for corpus and cauda epididymis. Asterisk indicates an IgG signal recognized by the secondary antibodies. (B) Live germ cells collected from testes of the different mutant mice were trypsinized and then analyzed by Western blotting. The lysate prepared from 5 × 10⁵ cells was loaded in each lane. ADAM2 was not digested in the Clgn^−/− germ cells, whereas ADAM3 was not digested in the Calr3^−/− and Pdilt^−/− germ cells. (C) Membrane proteins from testicular germ cells were separated into detergent-depleted or -enriched fractions with Triton X-114. The proteins were separated by SDS/PAGE under reducing (R) or nonreducing (NR) conditions, then subjected to Western blotting analysis. The control proteins ZPBP1 (that localizes to the acrosomal matrix) and IZUMO1 (that contains a transmembrane domain) were distributed in detergent-depleted and -enriched phases, respectively. (D and E) Immunoprecipitates (IPs) with anti-CLGN, CALR3, or PDILT antibodies from testis lysates of wild-type or each mutant mouse (100 μg) were examined by Western blotting with the anti-ADAM3 antibody.

Impaired sperm migration from the uterus into the oviduct in Pdilt^−/− mice. (A) Pregnancy rate (pregnancy/vaginal plug formation) presented as a percentage. Pdilt^−/− males copulated normally but failed to induce pregnancy. Four Pdilt^−/− males were examined. The number of males used and the total number of plugs observed are indicated in parentheses. (B) Uterus and oviduct collected from females mated with Pdilt^+/− and Pdilt^−/− males carrying Acr3-EGFP and CAG/su9-DsRed2-tagged sperm 2 h after coitus (). Although heterozygous mutant sperm were observed in the oviduct and the UTJ, Pdilt^−/− sperm were not observed in the UTJ. (C and D) Artificial sperm deposition into oviduct. Capacitated spermatozoa were directly deposited into the oviduct with a fine glass needle under a stereomicroscope. The eggs were collected 24–27 h after the sperm deposition and the two-cell stage embryos were judged as fertilized. (E) Newborn pups developed from the eggs fertilized with Adam3^−/− sperm after artificial sperm deposition into oviduct.

Sperm unable to bind to ZP can fertilize cumulus-intact eggs. (A and B) Pdilt^−/− sperm were not able to fertilize eggs in vitro. Heterozygous-type sperm successfully bound to the egg ZP, but Pdilt^−/− sperm failed to adhere despite frequent collisions (Movie S1). The average fertilization rate from three independent experiments is presented. (C) Pdilt^−/− sperm can fertilize cumulus-intact and ZP-intact eggs in vitro.