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Figures
- Fig. 1.
Mouse mature IZUMO1 and fragments. The region of each fragment is shown by the position of the aligned blank bar, with the N- and C-terminal amino acid residue numbers inside the bar. The N-terminal amino acid sequence of native IZUMO1 was confirmed to be Cys-Ile-Lys-Cys-Asp. The fragments that were used in this work were numbered after the removal of signal peptides (from the first Met to the 21st Pro) from the pre-protein. Gray circles and filled bar in mature IZUMO1 indicate the cysteines and transmembrane region, respectively. There are two clusters of cysteines: at the distal N-terminal end and around residues 114-144. We prepared IZUMO1PFF and IZUMO1PFF-core with and without a His-tag and linker. In addition, the fragments with an additional cysteine and linker at the C terminus were also made for the conjugation of a fluorescent dye. IZUMO1234-298, IZUMO15-56 and IZUMO126-113 have an additional nine amino acids containing a His-tag and linker.
- Fig. 2.
Establishment of inhibitory monoclonal antibodies to IZUMO1 in sperm-egg fusion in vitro. (A) All antibodies were immunoreactive against the 56 kDa protein IZUMO1 in wild-type spermatozoa but not in Izumo1-null spermatozoa. Solubilized sperm protein (10 μg) was subjected to sodium dodecyl sulfate PAGE (SDS-PAGE), western blotting and detection with anti-IZUMO1 monoclonal antibodies under non-reduced conditions. Basigin (BSG) is shown in the bottom panel as a control. (B,C) The inhibitory effects of these antibodies were investigated in in vitro fertilization and the sperm-egg fusion assay. The in vitro fertilization rate was assessed after two-cell development (n=5) (B). Average numbers of fused spermatozoa were observed 30 minutes after insemination (n=3) (C). Total numbers of examined eggs are shown at the bottom of each graph. All analyses were performed in the presence of 10 μg/ml of control IgG, Mab34, Mab120 and Mab125 antibodies. Values are presented as means±s.e.m.
- Fig. 3.
Interaction of antibodies against the IZUMO1 fragments. (A-D) Surface plasmon resonance (SPR) spectra of Mab34 (A), Mab120 (B), Mab125 (C) and OBF13 (D) with 20 μg/ml of IZUMO1PFF (molar concentration is 1.5 μM, assuming the monomer in solution; left panels), IZUMO1Ig domain (2.0 μM; middle) and IZUMO1234-298 (2.3 μM; right) were measured.
- Fig. 4.
Binding of IZUMO1 fragments to the egg surface. (A-C) The binding of IZUMO1PFF (A), IZUMO1PFF-core (B) and IZUMO15-56 (C) to the wild-type egg surface. (D,E) Differential interference contrast (DIC) (D) and confocal (E) images were taken of the egg with fluoresceinated IZUMO1PFF. The egg plasma membrane and nucleus are stained by fluoresceinated IZUMO1 fragments (3 μM) and Hoechst 33342, respectively.
- Fig. 5.
Effects of IZUMO1 fragments on sperm-egg fusion in vitro. (A,B) The penetration of spermatozoa was examined in the presence of 8 μM IZUMO1PFF (A) and control samples without fragments (B). Fused spermatozoa were stained using Hoechst 33342 that was preloaded into the egg. The arrowheads and asterisks indicate fused spermatozoa and metaphase II-arrested chromosomes, respectively. (C) The average number of fused spermatozoa was determined in the presence (8 μM) and absence of fragments. The number of spermatozoa that were fused to an egg was counted using ∼50 eggs in each group (n=3). Values are presented as mean±s.e.m.
- Fig. 6.
Interaction of IZUMO1 fragment with Cd9-/- egg surface. (A-D) Binding of IZUMO1PFF (A,C) and IZUMO15-56 (B,D) to the Cd9-/- (A,B) and wild-type (C,D) egg surface. The eggs were stained with 3 μM Alexa Fluor 546-conjugated IZUMO1 fragments.
- Fig. 7.
A series of biophysical measurements of IZUMO1 fragments. (A-D) Circular dichroism (CD) spectra (A), guanidine hydrochloride (GdnHCl)-induced unfolding curves (B), sedimentation equilibrium curves (C) and pair-distance distribution p(r) functions (D) of IZUMO1 fragments. (A) CD spectra of IZUMO1PFF (black), IZUMO1PFF with His-tag and linker (broken black), IZUMO1PFF-core (red) and IZUMO15-56 (blue) were measured For CD measurements, the protein concentration was 10 μM. IZUMO15-56 has a His-tag and a linker at the C-terminal end. (B) The unfolding curves of IZUMO1PFF (open circle) and IZUMO1PFF-core (closed circle) were detected by ellipticity using a 1 mm cuvette and a protein concentration of 10 μM. The left and right axes are for IZUMO1PFF and IZUMO1PFF-core, respectively. (C) Sedimentation equilibrium experiments of IZUMO1PFF. The red line is the linear fitting result of the data, indicating that the apparent molecular weight was 25,030. The deviation between the empirical data and the fitted line was plotted in the upper panel. Because the calculated molecular mass of monomeric IZUMO1PFF is 12,916, IZUMO1PFF is predicted to form a dimer. The theoretical lines for a monomer (broken black line) and a trimer (solid black line) are also shown for comparison. The nonlinear fitting analysis gave results that were consistent with those of the linear fitting analysis, in which the differences between these two methods were about 1%. (D) p(r) functions of IZUMO1PFF-core (black) and lysozyme (red) were computed by the GNOM program (Svergun, 1992) using their small angle X-ray scattering (SAXS) profiles.
- Fig. 8.
Influence of helix-breaking mutation on IZUMO1PFF. (A-E) Effects of proline mutation on CD spectrum (A), affinity to the egg surface (B-D) and inhibitory activity in sperm-egg fusion in vitro (E). (A) CD spectra of IZUMO1PFF (black) and IZUMO1PFF-Pro (broken black line) were measured buffer using a 1 mm cuvette and a protein concentration of 10 μM. (B-D) The binding of IZUMO1PFF (B), IZUMO1PFF-Pro (C) and IZUMO15-56 (D) to the wild-type egg surface was investigated. The egg plasma membrane and nucleus are stained by fluoresceinated IZUMO1 fragments (3 μM) and Hoechst 33342, respectively. (E) The average number of fused spermatozoa in the presence (8 μM) and absence of fragments was calculated. The number of spermatozoa that fused to an egg was counted using ∼50 eggs in each group (n=3). Values are presented as mean±s.e.m.
- Fig. 9.
Interactions between egg and cultured cells transfected with Izumo1 cDNA. (A) Mouse wild-type eggs and Cos-7 cells that were transfected with mouse Izumo1 cDNA were incubated for 3 hours at 37°C. The fluorescent signal from anti-IZUMO1 monoclonal antibodies that were conjugated with Alexa Fluor 546 was shown in red on the bright-field image. (B-D) Magnified image (2.5×) of the region surrounded by the white square in A.
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