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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.

Yuan R, Primakoff P, Myles DG - J. Cell Biol. (1997)

Bottom Line: This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain.Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin alpha, fertilin beta, cyritestin, ADAM 4, and ADAM 5.Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology, University of California, Davis 95616, USA.

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.

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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.
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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.

Mentions: Detergent extracts were prepared from three cell populations: testicular cells (TC), testicular sperm (TS), and epididymal sperm (ES). All extracts were electrophoresed by SDS-PAGE, and then subjected to immunoblot analysis with the anti-peptide antibodies. We used antibodies directed against the COOH-terminal cytoplasmic tail (Table I), because the processing of guinea pig fertilin β and meltrin α occurs in the NH2-terminal extracellular domain at the junction of the metalloprotease and disintegrin domains (Blobel et al., 1992; Yagami-Hiromasa et al., 1995). If a similar pattern of processing occurs for mouse fertilin β and cyritestin, then the COOH-terminal antibodies should be able to recognize both the precursors and processed forms. Using the mβ-CT1 antibody, we found two bands in the testicular cell extract with molecular mass of 101 and 88 kD (Fig. 4 A). At the testicular sperm stage, there were two bands observed: one at 101 kD and the other at 55 kD. At the epididymal sperm stage, there was only one band that ran at 55 kD (Fig. 4 A). To test the specificity of these bands, 50 μg/ml of the same peptide used for generating the antibody was added during the first antibody incubation in the immunoblot. The peptide inhibited binding of mβ-CT1 to all the bands in all three cell populations (Fig. 4 A′).


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.

Yuan R, Primakoff P, Myles DG - J. Cell Biol. (1997)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139869&req=5

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.
Mentions: Detergent extracts were prepared from three cell populations: testicular cells (TC), testicular sperm (TS), and epididymal sperm (ES). All extracts were electrophoresed by SDS-PAGE, and then subjected to immunoblot analysis with the anti-peptide antibodies. We used antibodies directed against the COOH-terminal cytoplasmic tail (Table I), because the processing of guinea pig fertilin β and meltrin α occurs in the NH2-terminal extracellular domain at the junction of the metalloprotease and disintegrin domains (Blobel et al., 1992; Yagami-Hiromasa et al., 1995). If a similar pattern of processing occurs for mouse fertilin β and cyritestin, then the COOH-terminal antibodies should be able to recognize both the precursors and processed forms. Using the mβ-CT1 antibody, we found two bands in the testicular cell extract with molecular mass of 101 and 88 kD (Fig. 4 A). At the testicular sperm stage, there were two bands observed: one at 101 kD and the other at 55 kD. At the epididymal sperm stage, there was only one band that ran at 55 kD (Fig. 4 A). To test the specificity of these bands, 50 μg/ml of the same peptide used for generating the antibody was added during the first antibody incubation in the immunoblot. The peptide inhibited binding of mβ-CT1 to all the bands in all three cell populations (Fig. 4 A′).

Bottom Line: This protein family has been named ADAM because all members contain a disintegrin and metalloprotease domain.Full length cDNA clones have been isolated for five ADAMs expressed in mouse testis: fertilin alpha, fertilin beta, cyritestin, ADAM 4, and ADAM 5.Characterization of these two ADAM family members showed that they are both processed during sperm maturation and present on mature sperm.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Biology, University of California, Davis 95616, USA.

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.

Show MeSH
Related in: MedlinePlus