David Epel

Chapter 7 Mechanisms of Activation of Sperm and Egg During Fertilization of Sea Urchin Gametes

Publisher Summary This chapter discusses the fertilization of the sea urchin egg. The metabolic and morphological changes of sea urchin gametes during fertilization are the best characterized of all embryos. In organization, this chapter explains the activation of both gametes, first considering the activation of the sperm upon its contact with egg jelly and the resultant cascade of the events leading to sperm–egg attachment and sperm–egg fusion. It then examines the consequences of sperm fusion with the egg that leads: (1) to the responses, excluding other sperm from fusing with the egg and (2) to the responses that result in the activation of embryonic development. The chapter describes the important roles played by intracellular calcium and cytoplasmic pH in turning on the cell metabolism. In sperm plasma membrane, the receptors interact with some component of the egg jelly or egg surface. This leads to increased Ca2+ content, the induction of membrane fusion, and the acrosomal exocytosis. At the same time, there is an acid efflux, polymerization of internal actin, and exposure of sperm lysins and sperm bindins that are attached to the newly formed membrane of the acrosomal process.

Embryo stability and vulnerability in an always changing world

Contrary to the view that embryos and larvae are the most fragile stages of life, development is stable under real-world conditions. Early cleavage embryos are prepared for environmental vagaries by having high levels of cellular defenses already present in the egg before fertilization. Later in development, adaptive responses to the environment either buffer stress or produce alternative developmental phenotypes. These buffers, defenses, and alternative pathways set physiological limits for development under expected conditions; teratology occurs when embryos encounter unexpected environmental changes and when stress exceeds these limits. Of concern is that rapid anthropogenic changes to the environment are beyond the range of these protective mechanisms.

Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm

The changes in the intracellular pH (pHi) of sea urchin sperm associated with motility initiation and acrosome reaction were investigated using uptake of two different probes; 9-aminoacridine and methylamine, as a qualitative index. Sperm suspended in Na+-free sea water were immotile and able to concentrate these amines 20-fold or greater indicating that pHi is more acidic than the external medium (pHo = 7.7). This uptake ratio was essentially constant over a wide range of probe and sperm concentrations. Discharge of the pH gradient with specific ionophores (nigericin, monensin, and tetrachlorosalicylanilide) or nonspecifically using low concentration of detergents (Triton X-100 and lysolecithin) all resulted in the release of the probes indicating they are indeed sensing the pH gradient across the sperm membrane. Addition of Na+ to sperm suspended in Na+-free sea water resulted in activation of motility with concomitant efflux of the probes indicating the alkalinization of pHi by 0.4-0.5 pH units. That this pHi change is the causal trigger of motility was suggested by experiments using NH4Cl and nigericin, which increased the pHi and resulted in activation of motility in the absence of Na+. When sperm were directly diluted into artificial sea water (motility activated), a slow reacidification of pHi was observed in one species of sea urchin (L. pictus) but not in the other (S. purpuratus). This acidification could be blocked by mitochondrial inhibitors, verapamil, or the removal of external calcium suggesting that the increase in metabolic activity stimulated by the influx of Ca2+ is responsible for the reacidification. Induction of acrosome reaction further alkalinized the pHi by about 0.16 pH units and was also followed by prolonged reacidification which correlated with the observed increase in Ca2+ uptake. Either mitochondrial agents or the removal of external Ca2+ could also block this pHi change suggesting a similar mechanism is involved.

An analysis of the partial metabolic derepression of sea urchin eggs by ammonia: The existence of independent pathways☆

Incubation of unfertilized eggs in ammonia has been reported to initiate such late responses to fertilization as K+-conductance, DNA synthesis, chromosome condensation and cytoplasmic mRNA polyadenylation. It does not initiate such early responses as Na+-influx and the cortical reactions. We have further analyzed this metabolic derepression and find that ammonia activation does not result in the early respiratory burst and also does not initiate the late activation of Na+-dependent amino acid transport. Protein synthesis is increased, similar to that following normal fertilization. This indicates that augmentation of protein synthesis is causally linked neither to the earlier Na+-influx, cortical reactions, and respiratory burst nor to the later activation of amino acid transport. The temporal correlation between activation of transport and increased protein synthesis is therefore coincidental. The association between increased protein synthesis and establishment of K+-conductance was analyzed by abolishing K+-conductance through acidification of the sea water. This did not affect protein synthesis, indicating that K+-conductance and protein synthesis are also not causally linked. There is also no obligate link between protein synthesis and chromosome condensation. Incubation in low concentrations of ammonia results in increased protein synthesis but not chromosome condensation. Higher ammonia concentrations cause chromosome condensation but with no further increase in rate of protein synthesis. This suggests a concentration-dependent hierarchy of activation. These results are consistent with the concept that the late fertilization changes are not causally linked and proceed independently of each other. As we have not been able to disassociate the early changes, they may be obligately linked and dependent on each other.

Protease released from sea urchin eggs at fertilization alters the vitelline layer and aids in preventing polyspermy

Abstract Sea urchin eggs release protease activity extracellularly from 30 to about 60 sec after insemination. This timing corresponds to the breakdown of the cortical granules. The evidence presented shows that the protease prevents polyspermy through its action on the vitelline layer. The protease ‘hardens’ the layer and makes it incapable of binding sperm. Sperm that are bound to the vitelline layer before cortical granule breakdown lose their attachment to the layer as the protease is excreted. By digesting the bonds between supernumerary sperm and the vitelline layer and by ‘hardening’ the layer the protease aids in establishing the block against polyspermy.

Calmodulin activates NAD kinase of sea urchin eggs: An early event of fertilization

NAD kinase, one of the first enzymes activated after fertilization of sea urchin eggs, is regulated by Ca2+ and calmodulin in vitro. The evidence is the requirement for low amounts of Ca2+ (Kd for Ca2+ of 4 x 10(-7) M) and the dissociation of a heat-stable activator from the enzyme which is similar to calmodulin on the basis of radioimmunoassay, activation of bovine brain phosphodiesterase and coelectrophoresis of a major protein of the activator fraction with bovine calmodulin. Also, the calcium stimulation of the enzyme is prevented by trifluoperazine, an inhibitor of calmodulin-associated reactions. In vivo studies show that the enzyme is activated by artificial parthenogenesis regimes that increase cytosolic Ca2+, but not by ammonia activation which only partially activates eggs and bypasses the Ca2+-rise step. These in vitro and in vivo studies indicate that calmodulin is part of the linkage between the rise in Ca2+ at fertilization and the turning on of egg metabolism.

NO is necessary and sufficient for egg activation at fertilization.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest—particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.

Nitromusk and polycyclic musk compounds as long-term inhibitors of cellular xenobiotic defense systems mediated by multidrug transporters.

Synthetic musk compounds, widely used as fragrances in consumer products, have been detected in human tissue and, surprisingly, in aquatic organisms such as fish and mollusks. Although their persistence and potential to bioaccumulate are of concern, the toxicity and environmental risks of these chemicals are generally regarded as low. Here, however, we show that nitromusks and polycyclic musks inhibit the activity of multidrug efflux transporters responsible for multixenobiotic resistance (MXR) in gills of the marine mussel Mytilus californianus. The IC10 (concentration that inhibits 10%) values for the different classes of musks were in the range of 0.09–0.39 μM, and IC50 values were 0.74–2.56 μM. The immediate consequence of inhibition of efflux transporters is that normally excluded xenobiotics will now be able to enter the cell. Remarkably, the inhibitory effects of a brief 2-hr exposure to musks were only partially reversed after a 24- to 48-hr recovery period in clean seawater. This unexpected consequence of synthetic musks—a long-term loss of efflux transport activity—will result in continued accumulation of normally excluded toxicants even after direct exposure to the musk has ended. These findings also point to the need to determine whether other environmental chemicals have similar long-term effects on these transporters. The results are relevant to human health because they raise the possibility that exposure to common xenobiotics and pharmaceuticals could cause similar long-term inhibition of these transporters and lead to increased exposure to normally excluded toxicants.

Methods for removal of the vitelline membrane of sea urchin eggs: I. Use of dithiothreitol (Cleland Reagent)☆

Abstract A simple procedure is described for removing the vitelline membrane of sea urchin eggs. The method is based on the observation that brief incubation of unfertilized sea urchin eggs in 5–10 mM dithiothreitol (DTT or Cleland Reagent) at pH 9.2 effectively removes this membrane. Membrane removal and normal development have been obtained with the eggs of the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus . That DTT removes membranes, combined with other observations and experiments, indicates that the structural integrity of the vitelline membrane results in part from disulfide linkages between neighboring protein molecules.

Isolation and biological activity of the proteases released by sea urchin eggs following fertilization

Abstract The protease activity released from sea urchin egg cortical granules into the surrounding seawater at fertilization is involved in vitelline layer elevation and the block to polyspermy. The cortical granule protease components were isolated by isoelectric precipitation and affinity chromatography on p -aminobenzamidine-Sepharose columns. Elution profiles from affinity columns suggested heterogeneity of the proteases, and polyacrylamide-gel electrofocusing of affinity-purified preparations established the presence of two proteins. Dramatically different biological activities were resolved by affinity chromatography. Early-eluting fractions of low specific activity delaminated the vitelline layer from the egg plasma membrane; this activity is termed vitelline delaminase. Late-eluting fractions of high specific activity modified the egg vitelline layer surface such that sperm could not bind or fertilize them; this activity is referred to as sperm receptor hydrolase. The biological activities of the sea urchin proteases are apparently the result of limited action on the vitelline layer, unlike bovine trypsin which simply digests the vitelline layer. The cortical granule proteases lost biological specificity when stored at 0°C at pH 8.0. Esterase activity increased, and the preparation acquired the ability to digest the vitelline layer. Increase of the esterase activity in protease preparations was prevented by storage at low pH. The molecular weight of both enzymes was estimated by sucrose gradient centrifugation to be 47,000, whereas multiple components with molecular weights between 10 5 and 10 6 were demonstrated by gel filtration.