Elsevier

Developmental Biology

Volume 92, Issue 1, July 1982, Pages 227-239
Developmental Biology

Full paper
A protein from abalone sperm dissolves the egg vitelline layer by a nonenzymatic mechanism

https://doi.org/10.1016/0012-1606(82)90167-1Get rights and content

Abstract

Unfertilized abalone eggs (Haliotis rufescens) possess an elevated fibrous glycoproteinaceous vitelline layer (VL) about 0.6 μm in thickness. Sperm bind to the VL by the tip of a large unreacted acrosome granule. After binding, the tip of the granule opens and the soluble contents are released onto the VL. A hole about 3 μm in diameter then forms in the VL in the area of the discharging acrosome. Ultrastructural observations show the hole to be filled with attenuated VL fibers. The sperm then swims through the hole and interacts with the egg plasma membrane. The soluble contents of abalone acrosomes can be obtained by induction of the acrosome reaction in high-calcium seawater. Two major proteins of subunit molecular weights 13,000 (13K) and 15,000 (15K) are found in the supernatant after removal of the reacted sperm by centrifugation. Gel analysis of whole sperm shows these two proteins are the major components of the cell. The 13K protein can be purified on the basis of its solubility at lower ionic strength. This protein is a potent solubilizer (lysin) of egg vitelline layers. Characterization of the 13K lysin yields an isoelectric point of about 9, basic amino acids accounting for 19.6% of its weight, a negative PAS reaction, a nondenatured-molecular-weight estimate of 17,000, the presence of exposed hydrophobic regions, and a lack of enzyme activity. The lytic action of the 13K protein is rapidly inactivated by boiling, showing that the native conformation is necessary for activity. The lysin does not degrade the macromolecular components of the VL. It does not produce reducing sugars, peptides, lysophosphatides, or SH groups. A turbidometric assay for lysin activity was developed using isolated VLs and 13K lysin. When lysin is added to VLs in seawater the dissolution action occurs for only 15–30 sec before abruptly stopping. Mixing various amounts of lysin with a constant amount of VLs shows that the lysin dissolves VLs by a stoichiometric, noncatalytic (nonenzymatic) mechanism. For example, about 11 μg of lysin are required for the complete dissolution of 63 μg of VL protein (the VL is 36% protein). An identical conclusion was reached by K. Haino-Fukushima (1974, Biochim. Biophys. Acta, 352, 179–191) working with an 8.8K lysin of another archeogastropod, Tegula pfeifferi. Isolated abalone VLs are composed of about five major glycoproteins ranging in molecular weight from 32 to 44K. High ionic strength such as 2 M KCl does not solubilize VLs, but agents which destroy hydrophobic bonds between macromolecules, such as NaSCN, dimethylsulfoxide, and heat, are VL solubilizers. Exposed hydrophobic portions of the lysin might bind to the hydrophobic regions of VL glycoproteins and competitively dissociate the VL fibers from each other, thus, destroying the VL's structural integrity. Stoichiometric mechanisms for making holes in egg investments may be more biologically attractive than enzymatic mechanisms. A stoichiometric reaction would be quickly self-limiting and nondegradative to other cell surface components.

References (53)

  • C.A. Lewis et al.

    Morphology of abalone spermatozoa before and after the acrosome reaction

    J. Ultrastruct. Res

    (1980)
  • A.C. Lopo et al.

    Radioiodination and characterization of the plasma membrane of sea urchin sperm

    Dev. Biol

    (1980)
  • O.H. Lowry et al.

    Protein measurement with the Folin phenol reagent

    J. Biol. Chem

    (1951)
  • T. Moriya et al.

    Characterization and partial purification of arylsulfatase from the seminal plasma of the sea urchin, Strongylocentrotus intermedius

    Arch. Biochem. Biophys

    (1980)
  • M. Schliwa

    Proteins associated with cytoplasmic actin

    Cell

    (1981)
  • G.W. Schwert et al.

    A spectrophotometric determination of trypsin and chymotrypsin

    Biochim. Biophys. Acta

    (1955)
  • R.G. Spiro

    Analysis of sugars found in glycoproteins

  • V.D. Vacquier

    The appearance of β-1,3-glucanohydrolase activity during the differentiation of the gut of sand dollar plutei

    Dev. Biol

    (1971)
  • S.K. Wada et al.

    Studies on the acrosome. V. An egg-membrane lysin from the acrosomes of Mytilus edulis spermatozoa

    Exp. Cell. Res

    (1956)
  • L. Warren

    The thiobarbituric acid assay of sialic acids

    J. Biol. Chem

    (1959)
  • K. Weber et al.

    The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis

    J. Biol. Chem

    (1969)
  • C.W. Wrigley

    Gel electrophoresing

  • C.R. Austin

    Spermatozoa and ova: The role of membranes in the fertilization process

  • M. DuBois et al.

    Colorimetric method for determination of sugars and related substances

    Anal. Chem

    (1956)
  • G. Fairbanks et al.

    Electrophoretic analysis of the major polypeptides of the human erythrocyte membranes

    Biochemistry

    (1971)
  • D.S. Friend et al.

    Acrosome disruption in sperm: Freeze-fracture of altered membranes

    J. Cell Biol

    (1974)
  • Cited by (0)

    This work was supported by NOAA, National Sea Grant College Program, Department of Commerce, under Grant 04-8-M01-189, the State Resources Agency, Project R/A-39 and by NIH Grant HD-12986 to V.D.V.

    View full text