A role for the disintegrin domain of cyritestin, a sperm surface protein belonging to the ADAM family, in mouse sperm-egg plasma membrane adhesion and fusion

Abstract

Sperm-egg plasma membrane fusion is preceded by sperm adhesion to the egg plasma membrane. Cell-cell adhesion frequently involves multiple adhesion molecules on the adhering cells. One sperm surface protein with a role in sperm-egg plasma membrane adhesion is fertilin, a transmembrane heterodimer (alpha and beta subunits). Fertilin alpha and beta are the first identified members of a new family of membrane proteins that each has the following domains: pro-, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane, and cytoplasmic domain. This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain. Previous studies indicate that the disintegrin domain of fertilin beta functions in sperm-egg adhesion leading to fusion. Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin alpha, fertilin beta, cyritestin, ADAM 4, and ADAM 5. The presence of the disintegrin domain, a known integrin ligand, suggests that like fertilin beta, other testis ADAMs could be involved in sperm adhesion to the egg membrane. We tested peptide mimetics from the predicted binding sites in the disintegrin domains of the five testis-expressed ADAMs in a sperm-egg plasma membrane adhesion and fusion assay. The active site peptide from cyritestin strongly inhibited (80-90%) sperm adhesion and fusion and was a more potent inhibitor than the fertilin beta active site peptide. Antibodies generated against the active site region of either cyritestin or fertilin beta also strongly inhibited (80-90%) both sperm-egg adhesion and fusion. Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm. Indirect immunofluorescence on live, acrosome-reacted sperm using antibodies against either cyritestin or fertilin beta showed staining of the equatorial region, a region of the sperm membrane that participates in the early steps of membrane fusion. Collectively, these data indicate that a second ADAM family member, cyritestin, functions with fertilin beta in sperm-egg plasma membrane adhesion leading to fusion.

Figure 1

Sequence alignment of disintegrin domain active sites of snake disintegrins and guinea pig fertilin β with mouse ADAM family members. The sequences shown are the 13 amino acids that form the RGD-containing loop of two snake venom disintegrins, kistrin and bitistatin, and the corresponding 14 amino acids of guinea pig fertilin β and mouse ADAM family proteins. Italicized residues are those which align with RGD, and the underlined sequences are the peptide sequences tested in the adhesion and fusion assay.

Figure 2

Effects of ADAM active site peptides on sperm–egg binding and fusion. Both experimental (A) and control (B) peptides were tested at 500 μM. FI, fertilization index; FR, fertilization rate. The number of eggs tested for each peptide was between 43 and 82. For the controls without peptides, 292 eggs were tested; the average number of sperm bound per egg was 11.6; the FR was 88.4%, and the FI was 1.17. The sequences of each peptide are under their names. Error bars represent SEM. □, Percentage inhibition of binding; ▪, percentage inhibition of FI; ▨ , percentage inhibition of FR.

Figure 2

Effects of ADAM active site peptides on sperm–egg binding and fusion. Both experimental (A) and control (B) peptides were tested at 500 μM. FI, fertilization index; FR, fertilization rate. The number of eggs tested for each peptide was between 43 and 82. For the controls without peptides, 292 eggs were tested; the average number of sperm bound per egg was 11.6; the FR was 88.4%, and the FI was 1.17. The sequences of each peptide are under their names. Error bars represent SEM. □, Percentage inhibition of binding; ▪, percentage inhibition of FI; ▨ , percentage inhibition of FR.

Figure 3

Dose-dependent inhibition of sperm–egg binding (A), FI (B), and FR (C) with fertilin β and cyritestin peptides. Peptide concentrations tested ranged from 100 to 500 μM. Error bars are SEM.

Figure 3

Dose-dependent inhibition of sperm–egg binding (A), FI (B), and FR (C) with fertilin β and cyritestin peptides. Peptide concentrations tested ranged from 100 to 500 μM. Error bars are SEM.

Figure 3

Dose-dependent inhibition of sperm–egg binding (A), FI (B), and FR (C) with fertilin β and cyritestin peptides. Peptide concentrations tested ranged from 100 to 500 μM. Error bars are SEM.

Figure 4

Expression of fertilin β and cyritestin during sperm maturation. Cells at different developmental stages were run on reducing SDS-PAGE and blotted with affinity-purified anti-peptide antibodies (10 μg/ml). About 10⁶ cells were loaded per lane. A, fertilin β; B, cyritestin. Antibodies for the blots were fertilin β, mβ-CT1; cyritestin, mCyri-CT1 (TC, TS, ES), and mCyri-CT2 (ES′). The panels on the left (A and B) show the test immunoblots. The panels on the right (A′ and B′) show control blots done in the same way as the tests, except 50 μg/ml of peptide was added during the incubation with anti-peptide antibody. TC, testicular cell; TS, testicular sperm; ES, epididymal sperm.

Figure 4

Expression of fertilin β and cyritestin during sperm maturation. Cells at different developmental stages were run on reducing SDS-PAGE and blotted with affinity-purified anti-peptide antibodies (10 μg/ml). About 10⁶ cells were loaded per lane. A, fertilin β; B, cyritestin. Antibodies for the blots were fertilin β, mβ-CT1; cyritestin, mCyri-CT1 (TC, TS, ES), and mCyri-CT2 (ES′). The panels on the left (A and B) show the test immunoblots. The panels on the right (A′ and B′) show control blots done in the same way as the tests, except 50 μg/ml of peptide was added during the incubation with anti-peptide antibody. TC, testicular cell; TS, testicular sperm; ES, epididymal sperm.

Figure 5

Inhibitory effects on sperm–egg binding and fusion of antibodies to the active sites of fertilin β or cyritestin. Both preimmune and immune sera were tested at 1:50 dilution. FI, fertilization index; FR, fertilization rate. Approximately 30 eggs were tested with each antiserum. For the controls without serum, 41 eggs were tested; the average number of sperm bound per egg was 5.95; the FI was 1.12, and the FR was 85.1%. Error bars represent SEM. □, Percentage inhibition of binding; ▪, percentage inhibition of FI; ▨ , percentage inhibition of FR.

Figure 6

Indirect immunofluorescence of fertilin β and cyritestin on sperm surface. Live sperm were stained with antisera mβ-AS1 or mCyri-AS1 at dilution 1:50 followed by the Fab fragment of rhodamine-conjugated goat anti–mouse IgG. The images are representative of the major pattern observed in each sperm population. Micrographs are a combined transmission image (green) and rhodamine-stained image (red). Fertilin β, A, acrosome intact sperm; C, acrosome reacted sperm. Cyritestin, B, acrosome intact sperm; D, acrosome reacted sperm.