Release of ovoperoxidase from sea urchin eggs hardens the fertilization membrane with tyrosine crosslinks

Abstract

One feature of fertilization is the alteration of the vitelline layer, by components released from the e, to produce an elevated, covalently crosslinked, hard, insoluble, fertilization membrane. The following evidence indicates that crosslinking and hardening are caused by the production of diand trityrosyl residues, by oxidation of protein-bound tyrosyl residues in thepresence of a peroxidase. Hardening of the fertilization membrane, as evidenced by its loss of solubility in 50 mM dithiothreitol, is inhibited by compounds known to inhibit many peroxidases. A peroxidase, here called the ovoperoxidase, is released from eggs at fertilization. This enzyme is, inhibited by the same compounds that inhibit hardening and at similar concentrations. Inhibitors of the ovoperoxidase and the hardening reaction include KCN, 3-amino-1,2,4-triazole, NaN3, phenylhydrazine, K4Fe(CN)o, sodium sulfite, and glycine ethyl ester. In addition, tyramine and N-acetyltyrosine both inhibit hardening, but O-methyltyrosine does not. Dityrosyl and trityrosyl residues are found in acid hydrolysates of isolated, hardened fertilization membranes. These residues have been identified by cellulose phosphate column chromatography, thin-layer chromatography, and amino acid analysis. The amino acid data have been used to estimate that there is one dityrosine crosslink per 55,000 daltons of protein.We suggest that, by catalyzing the crosslinking of tyrosyl residues, the ovoperoxidase leads to the production of a hard fertilization membrane that blocks the entry of additional sperm. Because peroxidases are spermicidal, a secondary function of the enzyme could be to kill sperm in the vicinity of the fertilized egg. Fertilization involves the union of two haploid genomes to make a new diploid individual. To ensure that only one sperm fertilizes an egg, many eggs become modified after penetration by the first sperm. In the case of the sea urchin Strongylocentrotus purpuratus, an immediate block to further sperm entry (polyspermy) is caused by a depolarization of the plasma membrane of the egg (1). After depolarization, the egg surface is rearranged by the cortical reaction (reviewed in refs. 2 and 3), in which secretory vesicles beneath the plasma membrane of the unfertilized egg release their contents in a massive exocytosis. A trypsin-like protease is released to cleave sperm receptors from the egg and thus serve as a second block. to polyspermy (3-5). During the cortical reaction, the vitelline layer that surrounds the egg elevates and is converted into a fertilization membrane. The fertilization membrane becomes altered in its morphology, solubility, and permeability in a discrete, multistep, assembly process that occurs in fixed sequence (6). This elevated structure is another block to sperm entry.The fertilization membrane is formed from the vitelline layer in a reaction with components released from the cortical granules (2, 3, 6). The structure thus produced is resistant to physical deformation (7), solubilization by protein denaturants (...

Mammalian cortical granules: Contents, fate, and function

“…Cherr et al (1988) also show gold u In sea urchins, hardening of the fertilization enveloDe (analogous to the ZP) is catalyzed by a CG ovoper-oxidase and is associated with a slow block to polyspermy (Foerder and Shapiro, 1977;Hall, 1978). Mammalian CG (mouse) have also been shown cytochemically to contain an ovoperoxidase by staining them with 3,3'-diaminobenzidine (DAB) in the presence of H,02 .…”

“…Since ZP hardening is inhibited by the tyrosine analogues tyramine and N-acetyl-L-tyrosine (8 l% and 73%, respectively), the ovoperoxidase probably functions in hardening of the ZP by cross-linking tyrosine residues . The hardening of the fertilization envelope in echinoderm embryos establishes a slow block to polyspermy (Foerder and Shapiro, 1977;Hall, 1978), and ZP hardening could serve a similar function in mammals. It has also been suggested that ZP hardening in mammals protects the developing pre-implantation embryo (Cran, 1989).…”

Oviduct-specific glycoprotein and heparin modulate sperm–zona pellucida interaction during fertilization and contribute to the control of polyspermy

“…We examined whether the oviductal factor responsible for ZP changes resistance to proteolysis and spermoocyte interaction matched any of the previously described factors, such as proteinases (24,25), a peroxidase (26), and a disulfide bond-forming reagent (27) that have been described as factors responsible for cortical granule effects. When pig IVM oocytes were incubated for 30 min in bOF with a mixture of proteinase inhibitors, the ZP did not dissolve even after 206 min in pronase solution, compared with 1 min in control oocytes.…”

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c‐type cytochrome biogenesis synthesise the methylamine‐dehydrogenase polypeptides but cannot assemble the tryptophan‐tryptophylquinone group

“…A possible role for the mauG gene product at the stage of cross-linking of the two methylamine-dehydrogenase Lchain tryptophan residues is suggested by the involvement of peroxidases in the formation of dityrosine cross-links between proteins in the sea urchin egg fertilisation envelope (Foerder and Shapiro, 1977;Hall, 1978), the embryo envelope of the brine shrimp Artemia salina (Roels, 1971) and the bristle cuticle of lepidopteran larvae (Locke, 1969). Furthermore, the reaction cycle of yeast cytochrome c peroxidase involves the one electron oxidation of an active site byptophan residue to a tryptophan radical (Sivaraja et al, 1989); this implies that the putative mauG gene product, functioning as a peroxidase, may be able to utilise H,O, to initiate a free-radical-mediated cross-linking of the two Lchain tryptophan residues forming the TTQ prosthetic group.…”