Voltage-dependent activation of frog eggs by a sperm surface disintegrin peptide.

Fertilin, a sperm protein of the metalloprotease/disintegrin/cysteine-rich (MDC) family, plays a critical role in sperm-egg binding in mammals. Peptides corresponding to the disintegrin domain of fertilin and antibodies against fertilin have been shown to inhibit mammalian sperm-egg binding and fusion. A protein from the same family, xMDC16, was recently cloned from frog (Xenopus laevis) testis and was found to be involved in frog sperm-egg binding. Here we report that xMDC16 is localized predominantly on the posterior surface of egg jelly-activated sperm, and peptides from the disintegrin domain of this protein activate eggs when applied near the egg surface. Egg activation was dependent on (1) specific amino acid residues (KTX); (2) the presence of divalent cations, but not external Ca2+ alone; and (3) voltage across the egg plasma membrane. This is the first demonstration of egg activation in vertebrates by the surface application of a peptide derived from a sperm surface protein, supporting a model for egg activation that involves a signal transducing receptor for sperm in the egg's plasma membrane.

Identification of metalloprotease/disintegrins in Xenopus laevis testis with a potential role in fertilization.

Proteins containing a membrane-anchored metalloprotease domain, a disintegrin domain, and a cysteine-rich region (MDC proteins) are thought to play an important role in mammalian fertilization, as well as in somatic cell-cell interactions. We have identified PCR sequence tags encoding the disintegrin domain of five distinct MDC proteins from Xenopus laevis testis cDNA. Four of these sequence tags (xMDC9, xMDC11.1, xMDC11.2, and xMDC13) showed strong similarity to known mammalian MDC proteins, whereas the fifth (xMDC16) apparently represents a novel family member. Northern blot analysis revealed that the mRNA for xMDC16 was only expressed in testis, and not in heart, muscle, liver, ovaries, or eggs, whereas the mRNAs corresponding to the four other PCR products were expressed in testis and in some or all somatic tissues tested. The xMDC16 protein sequence, as predicted from the full-length cDNA, contains a metalloprotease domain with the active-site sequence HEXXH, a disintegrin domain, a cysteine-rich region, an EGF repeat, a transmembrane domain, and a short cytoplasmic tail. To study a potential role for these xMDC proteins in fertilization, peptides corresponding to the predicted integrin-binding domain of each protein were tested for their ability to inhibit X. laevis fertilization. Cyclic and linear xMDC16 peptides inhibited fertilization in a concentration-dependent manner, whereas xMDC16 peptides that were scrambled or had certain amino acid replacements in the predicted integrin-binding domain did not affect fertilization. Cyclic and linear xMDC9 peptides and linear xMDC13 peptides also inhibited fertilization similarly to xMDC16 peptides, whereas peptides corresponding to the predicted integrin-binding site of xMDC11.1 and xMDC11.2 did not. These results are discussed in the context of a model in which multiple MDC protein-receptor interactions are necessary for fertilization to occur.

Sources of Energy for Increased Metabolic Demand During Metamorphosis of the Abalone Haliotis rufescens (Mollusca).

Pelagic, lecithotrophic (nonfeeding) larvae of the red abalone (Haliotis rufescens) settle and subsequently metamorphose into benthic juveniles capable of feeding on particulate food. Thus, metamorphosis must be fueled by either endogenous reserves or a nonparticulate food source such as dissolved organic material (DOM) in seawater. The metabolic rates (measured as oxygen consumption) of abalone larvae were found to increase by an average of 3- to 5-fold from the larva to early juvenile stage. The total cost of development from embryo to juvenile measured for three cultures ranged from 41.6 mJ to 55.0 mJ. Meeting this cost would require 1.3 to 1.7 {mu}g of biomass (ash-free dry mass), which is similar to the initial biomass of the spawned oocyte at 1.36 +/- 0.04 {mu}g (mean of four cultures). However, there was no net loss of biomass during development from the oocyte to the juvenile. The uptake of alanine and glucose from seawater by larvae and juveniles could provide one-third of the organic material required to supply metabolism, even if the transporters were only operating at 20% of their maximum capacity throughout development. For larvae undergoing metamorphosis (between 6- and 9-days-old) the proportion of total metabolic demand supplied using aerobically catabolized biomass was only 39%. The higher metabolic rates of metamorphosis are met only in part by consuming stored endogenous reserves. Concomitant with an increase in mass-specific metabolic rate during metamorphosis, the maximal capacity (Jmax) for the transport of dissolved alanine from seawater increased 3-fold, from 61.2 +/- 1.9 (SE) to 182.0 +/- 49 pmol alanine individual-1 h-1. The majority (range: 61% to 100%) of the energy requirements of larval and early juvenile development of H. rufescens could be supplied by input of DOM from the environment. Measurements of transport rates of amino acids and sugars by these animals, and calculations of the energy input from these substrates, indicate that the cumulative transport of DOM from seawater during development to the early juvenile stage could supply an amount of energy equivalent to the initial maternal endowment of energy reserves to the oocyte of this lecithotrophic species.

Evidence for both tyrosine kinase and G-protein-coupled pathways leading to starfish egg activation.

To investigate possible pathways leading to egg activation at fertilization, the ability of exogenously introduced tyrosine kinase and G-protein-coupled receptors to mimic events of fertilization was examined. Oocytes of the starfish Asterina miniata were injected with RNA for a chimeric receptor consisting of the extracellular domain of the beta form of the mouse platelet-derived growth factor (PDGF) receptor and the transmembrane/intracellular domain of the human fibroblast growth factor (FGF) receptor, or with RNA for the rat serotonin 1c receptor. These oocytes were cultured for 1 to 3 days and then matured with 1-methyladenine. In response to PDGF or serotonin, the injected eggs underwent responses like those at fertilization: cortical granule exocytosis, a rise in intracellular free calcium, and DNA synthesis. Some of these artificially activated eggs cleaved, and some of the PDGF-activated eggs were observed to form larvae. A PDGF/FGF receptor with a point mutation which eliminated its ability to interact with phospholipase C-gamma did not cause fertilization-like responses. Thus components of a signaling pathway involving phospholipase C-gamma, characteristic of tyrosine kinase receptors, as well as components of a pathway involving a G-protein and phospholipase C-beta, characteristic of G-protein-coupled receptors, appear to be present in starfish eggs. Either or both could function in egg activation at fertilization.

Energy Metabolism and Amino Acid Transport During Early Development of Antarctic and Temperate Echinoderms.

The rates of oxygen consumption by embryos of antarctic echinoderms (Acodontaster hodgsoni, Odontaster validus, Psilaster charcoti, and Sterechinus neumayeri) were compared to the biomas (ash-free dry organic weight) of the egg of each species. These species could survive for months to years (range: 10 months to 5 years) by relying solely on the reserves present in the egg. However, certain species did not use any of the egg's reserves during early development. Embryonic stages of O. validus (a species with planktotrophic larvae) did not decrease in lipid, protein, or total biomass during the first 35 days of development. During the first 42 days of development, embryos of A. hodgsoni (a species with lecithotrophic development) used protein as an energy source. For both species lipid composed 40 to 50% of egg biomass, but was not used as an energy reserve. Larvae of O. validus have a high-affinity transport system for amino acids dissolved in seawater (K1 = 1.3 {mu}M for alanine). The rate of alanine transport from a low concentration (50 nM) could supply 32% of the larva's metabolic needs. This is a 10-fold higher input to metabolism than was determined (3% at 50 nM) for larvae of a temperate asteroid, Asterina miniata. Larvae of antarctic echinoderms live in an environment where the food supply is low for most of the year. Relative to their metabolic rates, antarctic larvae have larger energy stores and planktotrophic larvae have higher nutrient transport capacities when compared to larvae from temperate regions. These physiological differences allow antarctic larvae to survive for long periods without particulate food.

Activation by serotonin of starfish eggs expressing the rat serotonin 1c receptor.

Starfish oocytes were injected with mRNA for the serotonin 1c receptor or with rat brain poly A+ mRNA, incubated to allow expression of the membrane protein, then matured to eggs by addition of 1-methyladenine. Applying serotonin to these eggs caused cortical granule exocytosis like that occurring at fertilization. Because the serotonin 1c receptor specifically activates a G-protein, these results provide support for the hypothesis that sperm activate eggs by way of a receptor-G-protein interaction. The starfish oocyte may be a generally useful system for expression of exogenous mRNA for membrane proteins.

Pertussis toxin inhibits 1-methyladenine-induced maturation in starfish oocytes.

Starfish oocytes injected with pertussis toxin (3-6 micrograms/ml) or its catalytically active A-subunit (1 microgram/ml) did not undergo germinal vesicle breakdown in response to 1-methyladenine (1-10 microM). The pertussis block could be bypassed by transfer of cytoplasm that contained maturation-promoting factor (MPF). After insemination, pertussis-blocked, MPF-rescued oocytes underwent cortical vesicle exocytosis and cleavage. These results suggest the involvement of a pertussis sensitive G-protein in the pathway coupling 1-methyladenine action at the cell surface to the reinitiation of meiosis.

G-proteins and egg activation.

G-proteins are present in eggs, and experiments in which GTP-gamma-S, GDP-beta-S, cholera toxin and pertussis toxin have been injected into eggs have indicated the involvement of G-proteins in egg activation at fertilization and in oocyte maturation. Eggs into which serotonin or muscarinic acetylcholine receptors have been introduced by mRNA injection produce fertilization-like responses when exposed to serotonin or acetylcholine; since these neurotransmitter receptors act by way of G-proteins, this observation further supports the conclusion that a G-protein is involved in the fertilization process.