Sea Urchin Egg Cortical Granules Research Articles

An analysis of the lamellar structure of sea urchin egg cortical granules using X-ray scattering

Cortical granules (CGs) are secretory vesicles associated with egg and oocyte plasma membranes that undergo exocytosis at fertilisation. In the sea urchin Strongylocentrotus purpuratus, the internal organisation of these CGs exhibits a lamellar-type morphology. The different lamellar layers correspond to proteoglycans, structural proteins and enzymes required for fertilisation envelope assembly and modification of the post-fertilisation egg surface. We have studied the lamellar structure of CGs using X-ray scattering and reveal the contrast density variation of the lamellae in the native state. The structure of functionally competent CGs in situ differs significantly from that determined by electron microscopic studies. We observed a strong periodicity of the lamellar structure of 280 Å as opposed to the 590 Å repeat observed previously. Fusion of the CGs produced a loss of the lamellar repeat and the development of a broad peak corresponding to a 20 Å periodicity that may be indicative of the molecular packing in the resulting hydrated gel structure.

Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium.

We have investigated the consequences of having multiple fusion complexes on exocytotic granules, and have identified a new principle for interpreting the calcium dependence of calcium-triggered exocytosis. Strikingly different physiological responses to calcium are expected when active fusion complexes are distributed between granules in a deterministic or probabilistic manner. We have modeled these differences, and compared them with the calcium dependence of sea urchin egg cortical granule exocytosis. From the calcium dependence of cortical granule exocytosis, and from the exposure time and concentration dependence of N-ethylmaleimide inhibition, we determined that cortical granules do have spare active fusion complexes that are randomly distributed as a Poisson process among the population of granules. At high calcium concentrations, docking sites have on average nine active fusion complexes.

Using Caged Calcium to Study Sea Urchin Egg Cortical Granule Exocytosis in Vitro

We present a simple method for releasing calcium from the photolabile chelator DM-nitrophen in a microscope chamber and following the calcium-triggered exocytosis of sea urchin egg cortical granules. With this method, calcium can be introduced at different rates and the biological response can be monitored under these conditions. We find that this "uncaging" of calcium, but not magnesium, barium, or strontium (up to 500 μM), from DM-nitrophen triggers exocytosis. The timing and identity of which granules first fuse with the plasma membrane appear to be stochastic, although there does seem to be some subsequent positive cooperativity in the fusion of neighboring granules. Calcium-triggered exocytosis is a threshold phenomenon, and this threshold appears to be independent of the rate of calcium uncaging. Mild treatment with N-ethylmaleimide shifts this threshold so that higher concentrations of calcium are required for exocytosis and decreases the apparent rate of exocytosis despite the fact that all of the granules remain fusion competent, albeit with higher concentrations of calcium.

Immunocytochemical evidence suggesting heterogeneity in the population of sea urchin egg cortical granules

Unfertilized eggs of many species of animals contain cortical granules, which are specialized secretory granules that upon fertilization release their contents from the egg. The unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, contain cortical granules that all display an identical and elaborate internal morphology. It has been assumed that they all contain identical components. In this report we present immunocytochemical data which indicate that the cortical granule population of S. purpuratus eggs is heterogeneous. Two monoclonal antibodies are shown to react to the spiral lamellae region of approximately 20% of the cortical granules, implying that the contents of the reactive granules differ from the contents of the majority of the population. An egg protein of greater than 320 kDa is recognized by the antibody. These antibodies also stain a 130-kDa protein expressed on the surface of primary mesenchyme cells in later development. Both antibodies recognize a post-translational modification of this protein. This suggests that an antigenically similar epitope is present both on the 130-kDa primary mesenchyme cell-specific protein and in the cortical granules. To determine if the primary mesenchyme and cortical granule proteins are related, a fusion protein antibody specific for a region of the 130-kDa protein was used to stain unfertilized eggs. This antibody did not stain cortical granules. Thus, 20% of the cortical granules contain a molecule that has an epitope antigenically similar to the post-translational modification recognized in primary mesenchyme cells by the monoclonal antibodies.

Sea urchin egg‐cortical granule protease has arginyl proteolytic specificity

AbstractSea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse‐phase high‐performance liquid chromatography (RP‐HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83–88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP‐HPLC analysis with a spectrophotometric Nα‐benzoyl‐L‐arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin‐like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.

SPECIFICITY IN THE INDUCTION OF POLYSPERMY IN SEA URCHIN EGGS BY SOYBEAN TRYPSIN INHIBITOR

Sea urchin egg cortical granules contain a soybean trypsin inhibitor (SBTI)-sensitive protease that is activated and secreted upon fertilization. Results obtained in several laboratories have implicated this activity in the cortical reaction, elevation of the fertilization envelope, and the establishment of the block to polyspermy. These conclusions are supported in part by studies using a variety of trypsin inhibitors. This interpretation has recently been challenged, however, and past results with soybean trypsin inhibitor (SBTI) and other agents were attributed to a non-specific protein effect (Dunham et al., 1982). We have re-examined this question by studying the effects of native and inactivated SBTI on purified egg protease and the promotion of polyspermy. Protease was purified from unfertilized eggs by SBTI-affinity chromatography in the presence of benzamidine-HCl to minimize autodegradation. Inactivation of SBTI by acid or alkali treatment greatly diminished its ability to inhibit the egg protease or to cause polyspermy. We also observed a correlation between the relative potencies of five serine protease inhibitors and their ability to promote polyspermy. We conclude that induction of polyspermy by SBTI and other protease inhibitors is indeed due to inhibition of the egg protease.

Sea urchin egg cortical granule exocytosis is followed by a burst of membrane retrieval via uptake into coated vesicles

Using improved fixation procedures we have found that extensive endocytotic activity is turned on at fertilization in eggs of three species of sea urchins. Beginning after completion of cortical granule exocytosis and after exocytotic pits have completely smoothed over, the entire activated egg surface engages in a limited period of extensive removal of membrane via uptake into coated vesicles. This "burst phase" lasts about 3-5 min after which the number of invaginating coated vesicles decreases rapidly. At the end of this burst phase all the patches of cortical granule membranes have disappeared, and the egg surface is left uniformly covered by microvilli. For the remainder of the first cell cycle coated pits continue to form at a slower but steady rate. Endocytotic activity continues past the time of first cleavage. There is distinct overlap in onset and duration of the burst phase of endocytosis with the period of medium acidification during normal development. However, activation of eggs in choline sea water, which inhibits acid secretion, results in an endocytic burst whose timing and duration are similar to those in normal eggs. The endocytic burst is, therefore, independent of cytoplasmic alkalinization. These results suggest, in accord with the two-step model of egg activation (D. Epel, R. A. Steinhardt, and R. A. Humphreys, 1974; Dev. Biol. 40, 245-255; D. Epel, 1978, Curr. Top. Dev. Biol. 12, 185-246) that initiation of endocytosis is most likely a Ca2+-dependent event.

Isolation and characterization of sea urchin egg cortical granules.

A method has been developed to isolate cortical granules (CG) free in suspension. It involves the mechanical disruption of the CG from CG lawns (CGL; Dev. Biol. 43:62-74, 1975) and concentration of the CG by low speed centrifugation. The isolated CG are intact and are a relatively pure population as judged by electron microscopy. Granule integrity is confirmed by the fact that isolated intact CG are radioiodinated to only 0.05% of the specific activity of hypotonically lysed CG. Purity of the CG preparation is assessed by the enrichment (four- to sevenfold) of CG marker enzymes and the absence or low activity of plasma membrane, mitochondrial, cytoplasmic, and yolk platelet marker enzyme activities. CG isolated from 125I-surface-labeled eggs have a very low specific radioactivity, demonstrating that CG contamination by the plasma membrane-vitelline layer (PM-VL) is minimal. CG yield is approximately 1% of the starting egg protein. The CG isolation method is simple and rapid, 4 mg of CG protein being obtained in 1 h. Isolated CG and PM-VL display distinct electrophoretic patterns on SDS gels. Actin is localized to the PM-VL, and all bands present in the CGL are accounted for in the CG and PM-VL. Calmodulin is associated with the CGL, CG, and PM-VL fractions, but is not specifically enriched in these fractions as compared with whole egg homogenates. This method of isolating intact CG from unfertilized sea urchin eggs may be useful for exploring the mechanism of Ca2+-mediated CG exocytosis.

Novel procedures for collection of sea urchin egg cortical granule exudate: Partial characterization and evidence for postsecretion processing

AbstractWe have developed two procedures to collect total cortical granule exudate in a soluble form from eggs of the sea urchin Strongylocentrotus purpuratus. Egg suspensions were either treated with dithiothreitol to disrupt the vitelline envelope or divalent cations were removed postinsemination to prevent the normal vitelline‐to‐fertilization envelope transition. Rapid acidification of the insemination mixture (dithiothreitol‐treated eggs) to pH 6.0 prevented precipitation of the paracrystalline protein fraction described by Bryan [1970a]. Exudate was partitioned into three fractions. The pH 8.0‐insoluble fraction appeared to be identical to the paracrystalline protein fraction. The pH 8.0‐soluble fraction was separated into pH 4.0‐soluble and‐insoluble fractions. Analysis for peroxidase and protease activities showed that peroxidase activity was localized in all three fractions whereas protease activity was restricted to the pH 4.0 insoluble fraction as reported [Carroll and Epel, 1975]. A minimum of six major proteins were detected on native polyacrylamide gels of total exudate. Under reducing and denaturing conditions, 12 polypeptides ranging from 19,000 to 165,000 in molecular weight were detected in total exudate; six polypeptides were recovered in the pH 8.0‐insoluble fraction. To test the hypothesis that protease and peroxidase activities process cortical granule proteins after secretion, we inseminated eggs in solutions containing peroxidase and protease inhibitors. The paracrystalline protein fraction crystallized slowly from insemination mixtures containing both inhibitors compared to controls and there were dramatic differences in exudate electrophoretic patterns. We suggest that cortical granule protease and peroxidase activities process the exudate so that the paracrystalline protein fraction rapidly crystallizes during normal fertilization.

The effect of local anesthetics and ammonia on cortical granule-plasma membrane attachment in the sea urchin egg

The normal attachment of sea urchin egg cortical granules to the overlying plasma membrane can be disrupted by urethane, the tertiary amines procaine and tetracaine, and ammonia. Following such treatment, and contrary to the normal situation in mature eggs, the cortical granules can be dislodged from the egg cortex by centrifugal force. In some experiments, procaine treatment alone can disrupt cortical granule attachment enough to prevent exocytosis at fertilization. In such eggs, cortical granules can then be seen in the blastomeres. These observations suggest that a special attachment exists between the cortical granules and the plasma membrane and that if this attachment is sufficiently disrupted, cortical granule exocytosis is prevented. A new technique for the isolation of cortical granules has been developed based on the urethane sensitivity of their attachment to the plasma membrane.

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

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.

Calcium activation of esteroproteolytic activity obtained from sea urchin egg cortical granules

Calcium activation of esteroproteolytic activity obtained from sea urchin egg cortical granules