Hydrogen peroxide production, chemiluminescence, and the respiratory burst of fertilization: Interrelated events in early sea urchin development

Abstract

After fertilization of the sea urchin, Strongylocentrotus purpuratus, a crosslinked fertilization membrane is formed; the crosslinks (dityrosine residues) are synthesized in a reaction catalyzed by an ovoperoxidase that is released from the cortical granules during fertilization. The substrate for ovoperoxidase activity, hydrogen peroxide, is generated by the egg coincident with the "respiratory burst" that follows parthenogenetic activation by the divalent ionophore A23187 or fertilization. This burst of oxygen consumption may be almost quantitatively accounted for by hydrogen peroxide evolution, as measured by the peroxidase-catalyzed quenching of scopoletin fluorescence. Neither the burst of oxygen consumption nor hydrogen peroxide production occurs when the inhibitor of cortical granule discharge, procaine, is present at fertilization.Fertilization or parthenogenetic activation with A23187 also is associated with a burst of light emission. This chemiluminescence is inhibited in vivo by inhibitors of the ovoperoxidase, such as 3-amino-1,2,4-triazole, phenylhydrazine, sulfite, or azide. A crude ovoperoxidase preparation catalyzes hydrogen peroxide-dependent chemiluminescence that is similarly inhibited. Thus, the bursts of oxygen uptake, peroxide production, and chemiluminescence appear to be several manifestations of the peroxidative system released at fertilization. This system may additionally be responsible for spermicidal activity and thus may act as a component of the block to polyspermy. One of the early biochemical observations on the activation of egg metabolism at fertilization was that of Otto Warburg (1), who noted a marked increase in oxygen consumption on fertilization of sea urchin eggs. This observation has been confirmed with eggs of several different species (2). Oxygen consumption incb eases in an initial burst to reach a maximum at about 2 min post-fertilization and then falls to a plateau level slightly higher than that present before fertilization (3, 4). The metabolic basis for this respiratory burst is not known.In a study of fertilization-induced alterations in the egg of the sea urchin, Strongylocentrotus purpuratus, we found (5) that the glycoprotein coat on the egg surface is converted into a rigid, relatively impermeable "fertilization membrane" by the formation of crosslinks between tyrosine residues. The formation of the di-and trityrosine structures is catalyzed by an ovoperoxidase that is released from cortical granules in a massive exocytosis which occurs within seconds of fertilization (6). The occurrence of such a peroxidase-mediated reaction in the vicinity of the fertilized egg implies that the substrate for that enzyme, hydrogen peroxide (H202), is either formed or liberated by the egg with kinetics appropriate to its involvement in the crosslinking reaction. In this paper, we show that the egg does produce H202 at the appropriate time, and, additionally,The costs of publication of this article were defrayed in part by the payment of page charges. This article mu...

Fertilization and Nicotinic Acid Adenine Dinucleotide Phosphate Induce pH Changes in Acidic Ca2+ Stores in Sea Urchin Eggs

“…Importantly, pH L changes were clearly independent of exocytosis/endocytosis and cytosolic pH changes. Moreover, acridine orange is unlikely to be responding to the oxidative burst at fertilization because this initiates much later, 2-3 min after insemination (19,(22)(23)(24)(25), and direct addition of H 2 O 2 (10 M to 1 mM) to eggs did not induce a pH L ASH (data not shown). Taken together, our data strongly support an alkalinization of cortical vesicles.…”

Generation of O−2 and tyrosine cation‐mediated chemiluminescence during the fertilization of sea urchin eggs

“…The generation of H202 in the egg is probably an NAD(P)H-O2 oxidoreductase activity which was found in the egg cortex and completely accounted for ovoperoxidase [4]. The natural chemiluminescence associated with fertilization of sea urchin eggs has been reported by Foerder et al [2], but its properties remain unknown. We report here that Or and excited species involved in tyrosine cation radicalmediated reactions are generated during the fertilization of sea urchin eggs.…”

“…One of the substrates of ovoperoxidase appears to be H202 which is synthesized by the egg in a burst after fertilization [2,3]. The generation of H202 in the egg is probably an NAD(P)H-O2 oxidoreductase activity which was found in the egg cortex and completely accounted for ovoperoxidase [4].…”

Mitochondrial Activation and Nitric Oxide (NO) Release at Fertilization in Echinoderm Eggs

“…Therefore, it is assumed that mitochondrial activity should increase accompanying the sequential phenomena, called "egg activation," that comprise changes in intracellular concentration of Ca 2+ ([Ca 2+ ] i ), pH, nitric oxide (NO) release (ΔNO), other second-messenger signals, and cytoplasmic changes, leading to activation of many important enzymes and factors for development. In sea urchins, early studies on respiration demonstrated that a burst of respiration started immediately after fertilization and reached the peak level within 3 min (Foerder et al 1978 ;Fujiwara and Yasumasu 1997 ;Warburg 1908 ). One third of the respiration at fertilization should be responsible for mitochondrial respiration.…”