William J. Lennarz

Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction

Abstract Jelly coat, a multicomponent extracellular matrix surrounding the sea urchin egg, induces the acrosome reaction in sperm. The jelly coats of the four species studied, Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus drobachiensis , and Lytechinus variegatus , were found to be very similar in chemical composition. A sialoprotein (approximately 20% of the mass of the jelly coat) and a fucose sulfate polysaccharide (approximately 80%) are the major macromolecular components of the jelly coat. The acrosome reaction inducing capacity resides solely in the fucose sulfate polysaccharide. Induction of the acrosome reaction ranges from highly species specific to nonspecific. Thus, A. punctulata and S. drobachiensis sperm are induced to undergo the acrosome reaction only with their homologous jelly coat, while S. purpuratus sperm react equally well with homologous or L. variegatus jelly coat, but not with A. punctulata jelly coat. L. variegatus sperm seem to be relatively nonspecific in response. Species-specific induction of the acrosome reaction resides solely in the fucose sulfate polysaccharide, suggesting that there must be structural differences in this polysaccharide in the various species. Therefore, in some species, fertilization appears to involve sperm-egg recognition at the level of the jelly coat as well as at the level of sperm-egg receptors.

The Function of Saccharide-Lipids in Synthesis of Glycoproteins

As discussed in Chapter 1, one of the major classes of glycoproteins is that in which the carbohydrate chain is linked to the polypeptide chain by means of an N-glycosidic bond. As shown in Figure 1, this N-glycosidic bond is between an N-acetylglucosaminyl residue and the amido nitrogen of an asparagine residue in the polypeptide chain.

Changes in the organization of the sea urchin egg plasma membrane upon fertilization: Indications from the lateral diffusion rates of lipid-soluble fluorescent dyes

We have used fluorescence photobleaching recovery to measure the diffusion of lipid probes (1,2-acyl-2-(N-4-nitro-benzo-2-oxo-1,3-diazole)aminocaproylphosphatidylcholine (NBD-PC)) and the 3,3′-diacylindocarbocyanine iodides (CndiI) with even acyl chain lengths (n = 10 to 18) in the plasma membranes of the sea urchin egg Strongylocentrotus purpuratus before and after fertilization. At 15°C measured diffusion coefficients, D, ranged from (1 to 5) × 10−9 cm2/sec, diffusing fractions from 36 to 77% for the CndIs. D shows a relative minimum at n = 12 for unfertilized eggs, a relative maximum at n = 14 for fertilized eggs. Thus the effect of fertilization on diffusion coefficient is dependent on probe structure. The fraction of molecules free to diffuse also depends upon acyl chain length; this fraction was smaller in fertilized eggs than in unfertilized eggs for all chain lengths. Our results indicate that different lipid analogs probe different microenvironments and suggest the existence of domains of lipids differing in composition or physical state from the average for the egg plasma membrane.

Jelly coat and induction of the acrosome reaction in echinoid sperm

Abstract In a previous study we established that sperm from four species of echinoids differ in their specificity for induction of the acrosome reaction by heterotypic jelly coat and we presented evidence that there were only small compositional differences in the active component of the jelly coat, a polysaccharide composed of fucose sulfate units. In the current report we present additional studies related to the species specificity of jelly coat with respect to Ca2+ uptake (or exchange), which occurs concomitantly with the acrosome reaction, and activation of phospholipase activity, which appears to occur subsequent to the acrosome reaction. The specificity of jelly coat in inducing these processes is the same as that previously observed in induction of the acrosome reaction. Binding of jelly coat to sperm has been demonstrated, and has been shown to be species specific. This finding raises the possibility that a receptor for jelly coat exists on the surface of the sperm. Finally, based on chemical and physical-chemical studies, evidence is presented that establishes that, despite compositional similarities, the fucose sulfate polysaccharides from the four species of eggs differ in structure.

Characterization of the sperm receptor on the surface of eggs of Strongylocentrotus purpuratus.

Eggs of the sea urchins Strongylocentrotus purpuratus and Arbacia punctulata bind sperm with a high degree of species specificity. By use of an in vitro assay that utilizes bindin (the protein from sperm that mediates sperm-egg binding) egg surface-derived glycoconjugates that function as receptors in this adhesion process have been identified and purified. These glycoconjugates are of extraordinarily high molecular weight and exhibit some properties expected for a proteoglycan. The isolated receptors from both species bind to sperm and inhibit fertilization species specifically. Both receptors contain active carbohydrate-rich fragments that can be liberated by proteolytic digestion. The carbohydrate-rich receptor fragment from S. purpuratus is a very high-molecular-weight (greater than 10(6)), negatively charged glycosaminoglycan-like polymer containing fucose, galactosamine, iduronic acid, and sulfate esters. By contrast, the carbohydrate-rich fragment derived from the A. punctulata receptor is of defined molecular weight (6000) and has no net charge. Incubation of acrosome-reacted sperm with nanomolar amounts of the carbohydrate-rich fragments from either species results in inhibition of fertilization, indicating that these receptor fragments retain sperm binding activity. However, studies utilizing heterologous gametes show that the carbohydrate-rich receptor fragments are not species specific in binding. Thus, it appears that although the carbohydrate chains of the receptor are an adhesive element of the receptor, the intact glycoconjugate is required for species-specific binding.

Distribution of phospholipid-synthesizing enzymes in the wall and membrane subfractions of the envelope of Escherichia coli

The distribution of the enzymes of phospholipid synthesis in cell wall and membrane subfractions of the cell envelope of Escherichia coli, isolated by a procedure involving particle electrophoresis and sucrose gradient density centrifugation, has been studied. The results show that, with the possible exception of CDP-diglyceride:L-serine phosphatidyltransferase all of the enzymes involved in the synthesis of phosphatidic acid, CDP-diglyceride, phosphatidylglycerol, and phosphatidylethanolamine, are localized in the inner cytoplasmic membrane.

Induction of calcium-dependent, localized cortical granule breakdown in sea-urchin eggs by voltage pulsation

A technique that employs a high-voltage pulses to produce pores in cell membranes (Kinosita and Tsong (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1923) has been used to investigate the role of Ca2+ in the early events of activation of sea-urchin eggs. Exposure of eggs to a voltage pulse of 1 kV/cm for 100 microseconds resulted in localized exocytosis of the contents of cortical granules and development of a partial fertilization envelope. This effect was triggered by entrance of Ca2+ through the voltage-induced pores. In a medium containing 100 microM Ca2+ and 45Ca2+ tracer, the voltage-treated eggs admitted 3.6 +/- 0.3 fmol Ca2+/egg within a few seconds. Untreated eggs took up only 1.0 +/- 0.2 fmol/egg after minutes of incubation. Furthermore, depletion of Ca2+ or the presence of EGTA in the external medium prevented elevation of the fertilization envelope by the voltage pulsation. Delay in Ca2+ addition after the voltage pulsation reduced the fraction of eggs that developed partial fertilization envelope. Loss of essential cytoplasmic components during the delay period is judged unlikely, since these eggs were viable, could form partial fertilization envelopes if re-pulsed in the presence of Ca2+, and could develop to normal blastula stage embryos upon fertilization with sperm. Thus, we interpret this effect as due to a resealing of pores; the half-life of pores being 20 s. The elevation of partial fertilization envelopes occurred only at the loci facing the anode, and multiple pulses with mixing resulted in the formation of multiple fertilization envelopes. These envelopes were stable for up to several hours; further propagation (wave spreading) was not observed. The above results indicate that a primary reaction in the sequence of steps in fertilization envelope formation involves Ca2+ to trigger cortical granule breakdown and formation of the fertilization envelope.

The effect of the acrosome reaction on the respiratory activity and fertilizing capacity of echinoid sperm

Abstract We have examined the relationship between the acrosome reaction, sperm respiration, and fertilization using gametes of the sea urchin Strongylocentrotus purpuratus. The results indicate that when sperm are exposed to jelly coat isolated from homologous eggs, the following sequence of events occurs: (1) Sperm undergo the acrosome reaction within 30 sec with little or no loss in their capacity to fertilize eggs; (2) by 60 sec there is a dramatic decrease in fertilizing capacity which stabilizes after 4 or 5 min at a greatly reduced level; (3) by 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration becomes detectable and continues for 8 to 10 min, finally stabilizing at a greatly reduced rate. This decrease in respiration rate is paralleled by a decline in sperm motility. The effects of jelly coat on the acrosome reaction, sperm respiration, and motility are species specific. From these results we conclude that sperm which have undergone the acrosome reaction retain full fertilizing capacity for a very short time. The rapid decline in fertilizing capacity is followed by a decrease in respiration rate and motility.

Studies on the interactions of sperm with the surface of the sea urchin egg.

We have examined the relationship between sperm adhesion and fertilization in the cross species insemination of Arbacia punctulata eggs by Strongylocentrotus purpuratus sperm. As previously reported (Kinsey et al., 1980) the addition of S. purpuratus egg jelly results in induction of the acrosome reaction in sperm and significant numbers of S. purpuratus sperm adhere to A. punctulata eggs. However, in the absence of S. purpuratus egg jelly, S. purpuratus sperm fail to bind to A. punctulata eggs. Although at least 200 S. purpuratus sperm bind to an A. punctulata egg in the presence of S. purpuratus jelly, less than 8% of the eggs are fertilized. The adhesion of S. purpuratus sperm meets the same functional criteria as homologous A. punctulata sperm-egg adhesion. Electron microscopy shows that S. purpuratus sperm that have undergone the acrosome reaction adhere to A. punctulata eggs by their bindin-coated acrosomal process in a manner that is morphologically identical to that observed with homologous A. punctulata sperm. We have also compared the ability of S. purpuratus and A. punctulata sperm to fuse and fertilize with A. punctulata eggs after removal of the vitelline layer. Using high levels of sperm of either species, heterologous as well as homologous fertilization is readily detectable. Under these conditions, where stable binding is not demonstrable, there is no difference in the ability of S. purpuratus and A. punctulata sperm to fertilize A. punctulata eggs. These observations suggest that the failure of S. purpuratus sperm to fertilize A. punctulata eggs under normal conditions may be due to their inability to penetrate the vitelline layer so that they can fuse with the egg plasma membrane. In relation to the possible mechanism of vitelline layer penetration, we have also investigated the mode of action of chymostatin, an inhibitor of chymotrypsin that has been reported to inhibit fertilization of sea urchin eggs (Hoshi et al., 1979). Our findings suggest that the fertilization inhibitory activity of chymostatin is not related to its antichymotrypsin activity. Rather, it appears that this inhibition is due to the induction of an abnormal acrosome reaction in sperm that precludes formation of the acrosome process.

Studies on the specificity of sperm binding in echinoderm fertilization

Abstract Using gametes from the sea urchins Arbacia punctulata and Strongylocentrotus purpuratus , we have evaluated the role of the acrosome reaction and the sperm-egg binding process in the block to interspecific fertilization among echinoids. The results indicate that sperm preinduced to undergo the acrosomal reaction by two different methods still bind to homologous eggs in a species specific manner. These results, taken in conjunction with an earlier study on species specificity of jelly coat induction of the acrosomal reaction (SeGall and Lennarz 1978), indicate that both the acrosome reaction and the sperm binding process contribute to the species specificity of fertilization in S. purpuratus and A. punctulata .