Egg Jelly Research Articles

The Role of Jelly Coats in Sperm-Egg Encounters, Fertilization Success, and Selection on Egg Size in Broadcast Spawners

Sperm limitation may be an important selective force influencing gamete traits such as egg size. The relatively inexpensive extracellular structures surrounding many marine invertebrate eggs might serve to enhance collision rates without the added cost of increasing the egg cell. However, despite decades of research, the effects of extracellular structures on fertilization have not been conclusively documented. Here, using the sea urchin Lytechinus variegatus, we remove jelly coats from eggs, and we quantify sperm collisions to eggs with jelly coats, eggs without jelly coats, and inert plastic beads. We also quantify fertilization success in both egg treatment groups. We find that sperm‐egg collision rates increase as a function of sperm concentration and target size and that sperm are not chemotactically attracted to eggs nor to jelly coats in this species. In fertilization assays, the presence of the jelly coat is correlated with a significant but smaller‐than‐expected improvement in fertilization success. A pair of optimality models predict that, despite the large difference in the energetic value of egg contents and jelly material, the presence of the jelly coat does not diminish selection for larger egg cell size when sperm are limiting.

EVOLUTION OF EGG TARGET SIZE: AN ANALYSIS OF SELECTION ON CORRELATED CHARACTERS

In broadcast-spawning marine organisms, chronic sperm limitation should select for traits that improve chances of sperm-egg contact. One mechanism may involve increasing the size of the physical or chemical target for sperm. However, models of fertilization kinetics predict that increasing egg size can reduce net zygote production due to an associated decline in fecundity. An alternate method for increasing physical target size is through addition of energetically inexpensive external structures, such as the jelly coats typical of eggs in species from several phyla. In selection experiments on eggs of the echinoid Dendraster excentricus, in which sperm was used as the agent of selection, eggs with larger overall targets were favored in fertilization. Actual shifts in target size following selection matched quantitative predictions of a model that assumed fertilization was proportional to target size. Jelly volume and ovum volume, two characters that contribute to target size, were correlated both within and among females. A cross-sectional analysis of selection partitioned the independent effects of these characters on fertilization success and showed that they experience similar direct selection pressures. Coupled with data on relative organic costs of the two materials, these results suggest that, under conditions where fertilization is limited by egg target size, selection should favor investment in low-cost accessory structures and may have a relatively weak effect on the evolution of ovum size.Corresponding Editor: D. Wheeler

The vasculopathy of autosomal dominant polycystic kidney disease: Insights from animal models

A 37-year-old white man with autosomal dominant polycystic kidney disease (ADPKD) was admitted to Boston's Beth Israel Hospital with sudden severe headache of two-hour duration. He was unconscious on admission. Emergency angiography revealed a ruptured 15 mm cerebral aneurysm. Emergency surgery to clip the aneurysm was unsuccessful, and the patient died 36 hours after admission. His serum creatinine was 3.2 mg/dL on admission; the rest of his laboratory values were within normal limits. A request for an autopsy was denied. At age 20, the patient and his then 15-year-old sister had abdominal ultrasound examinations that revealed bilateral renal cysts, consistent with the diagnosis of ADPKD. At age 32, the patient had a sudden, relatively severe, acute myocardial infarction (MI). Cardiac catheterization revealed three-vessel disease, and he subsequently underwent a triple coronary artery bypass operation. Throughout his medical course, the patient's blood pressure was in the 120/80 to 140/80 mm Hg range while he was taking lisinopril, 10 mg/day, which was started after his MI. The patient's mother has ADPKD and end-stage renal disease, and she is on hemodialysis. She recently underwent unsuccessful renal transplantation. There is no known family history of cerebral aneurysms. However, the mother had two cerebrovascular accidents and recovered from both. The patient's father had an MI at age 32, but he lived to age 64. A paternal uncle also died at an early age of an MI. The patient's younger sister was later screened for cerebral aneurysms and the test was negative. The patient's two young children have not been screened for ADPKD.

Maternal energy investment in eggs and jelly coats surrounding eggs of the echinoid Arbacia punctulata

In free-spawning marine invertebrates, the amount of maternal energy that is invested in each egg has profound implications for all life-history stages of the offspring. The eggs of echinoids are freely spawned into the water and are surrounded by several structurally complex extracellular layers. These extracellular layers, or jelly coats, do not contribute energy to embryonic development but must impose an energy cost on the production of each egg. The investment of maternal energy reserves in the jelly coats of echinoid eggs may have important implications for the number of eggs that can be produced (i.e., fecundity) and the amount of energy that can be invested in each egg. We estimated the degree to which maternal energy is invested in the jelly coats surrounding eggs of the echinoid Arbacia punctulata. Estimates were derived from measurements of the amount of energy contained in the combined eggs and jelly coats, and in the eggs alone. The amount of energy contained in A. punctulata eggs ranged from 2.70 to 5.53 x 10(-4) J egg(-1). The amount of energy contained in the jelly coats ranged from 0.13 to 0.48 x 10(-4) J jelly coat(-1). The mean concentration of energy in the eggs was 2.15 mm(-3) and 0.29 J mm(-3) in the jelly coats. These results indicate that between 3% and 11% (mean = 7%) of the total energy invested in each A. punctulata egg is partitioned to the jelly coat alone. A significant positive relationship was found between the volumes of the jelly coats and the amount of energy they contained. Based on this relationship and an analysis of differences in the size of jelly coats between echinoid species, we suggest that the degree to which energy is invested in jelly coats may vary among echinoid species and is therefore likely to be an important life-history characteristic of these organisms.

Fertilization in Callochiton castaneus (Mollusca).

A fine-structural study of fertilization in Callochiton castaneus has revealed that the mechanism of sperm penetration into the egg is intermediate between the primitive condition found in members of the order Lepidopleurida and the more derived condition found in the Chitonida. C. castaneus sperm have the long needlelike nuclear filament and reduced acrosome that characterizes all Chitonida, but they have retained several plesiomorphic features such as an unspecialized mid-piece and a lack of flagellar reinforcement. As in some Lepidopleurida but unlike any Chitonida, the egg hull in this species comprises a thick, smooth jelly coat permeated by pores that permit sperm rapid access to the vitelline layer. The jelly coat is delicate and quickly dissolves when a sperm concentrate is used, suggesting that excess acrosomal enzymes may be responsible. Once the sperm have penetrated the vitelline layer, the long nuclear filament bridges the gap to cups in the egg membrane. However, once the fertilization membrane is raised, the perivitelline space exceeds the length of the nuclear filament, preventing other sperm from penetrating the egg. A fertilization cone forms around the nuclear filament of the penetrating sperm, but it does not appear to engulf the body of the sperm. Rather, the nuclear chromatin is injected into the egg as a long thread. The remaining sperm organelles are apparently abandoned on the egg surface. If this is the case, it would be a significant departure from fertilization in other molluscs and many other metazoans, in which sperm organelles, such as centrioles and mitochondria, enter the egg. New sperm and egg characters, as well as significant differences in fertilization, indicate that Callochitonidae are basal to all other members of the order Chitonida and may warrant separation as the sister taxon to the suborders Chitonina and Acanthochitonina.

Alginate Coating of Xenopus laevis Embryos

Xenopus laevis eggs were coated, immediately after squeeze-stripping and fertilization, with a thin layer ( approximately 50 microm) of film based on one of three different types of alginates which varied in their mannuronic/guluronic acid ratio. The alginate was cross-linked with either Ca or Ba ions at three different concentrations. The developmental, survival, and hatching of these embryos and the swelling of their natural jelly coats or hydrocolloid coatings were studied over 7 days, while embryos were maintained in flowing aerated water at a ratio of 85 mL per embryo or at a very diminished ratio of 1.6 mL of sterile or nonsterile MMR solution per embryo. All experiments were conducted in triplicate at 20+/-1 degrees C. Oxygen was monitored continuously. Mineral content was determined in the alginate-jelly coat and within the embryos over time. The coating conferred major advantages when the ratio between the embryos and the surrounding medium was at a minimum under nonsterile conditions, perhaps as a result of the film's resistance to diffusion. In the studied systems, the coating seemed to postpone embryo hatching to a more developed stage. In addition, the coating served as a barrier to microbial contamination and thus improved survival prospects.

Subcellular Localization of Ca-ATPase and Calcium in Bufo arenarum Oviducts.

The localization of calcium and Ca−ATPase activity in Bufo arenarum oviducts was investigated by ultrahistochemical techniques during the sexual cycle. In the preovulatory period prominent calcium deposits were detected in the secretory granules of epithelial and glandular cells as well as in close proximity to the plasma membranes. Furthermore, intense Ca−ATPase activity was found outlining the plasma and secretory granule membranes. Regional differences along the oviduct were observed. Immediately after ovulation the above−mentioned cells exhibited a marked reduction of calcium deposits and Ca−ATPase activity, but during hibernation this situation progressively reverted. These results demonstrate that the most prominent calcium deposits can be detected in the cellular structures that, through different secretion mechanisms, release their products towards the oviductal lumen to form the structural network of the jelly coats. The variations in calcium contents in the oviduct and the animal cycle changes in serum calcium levels are closely related with the fluctuations of estradiol serum levels during the sexual cycle, suggesting hormonal regulation.

Acrosome reaction in starfish: signal molecules in the jelly coat and their receptors

Animal eggs are generally encased in one or more extra-cellular coats that protect the egg from biological, chemical and mechanical hazards. These coats contain some essential molecules for sperm to fertilise an appropriate egg, such as the specific ligand for sperm binding and the specific signal for induction of the acrosome reaction. In starfish, the outermost egg coat is a relatively thick gelatinous layer called the jelly coat. When starfish sperm encounter the jelly coat of homologous eggs, they undergo the acrosome reaction within a second or less (Dale et al., 1981; Ikadai & Hoshi, 1981; Sase et al., 1995). We have thus searched the jelly coat for the signal molecule(s) that triggers the acrosome reaction in the starfish, Asterias amurensis. It is known that three components in the jelly coat, namely acrosome reaction-inducing substance (ARIS), Co-ARIS and asterosap, act in concert on homologous spermatozoa to elicit the acrosome reaction immediately and efficiently (Hoshi et al., 1994,1999).ARIS alone induces the acrosome reaction only in high calcium or high pH seawater. In normal seawater, besides ARIS, either Co-ARIS or asterosap is required for the induction. Without ARIS, no combination of Co-ARIS and asterosap can induce the acrosome reaction in normal, high calcium or high pH seawater. A mixture of ARIS and Co-ARIS increases the intracellular Ca2+ level, whereas asterosap increases the intra-cellular pH (Matsui et al., 1986a, b; Nishigaki et al., 1996). These events are prerequisites for the induction of the acrosome reaction. Indeed, the triad of ARIS, CoARIS and asterosap provides the best conditions for the induction of the acrosome reaction in normal sea-water (Hoshi et al., 1994, 1999).

There is more than just sugar in the recipe for jelly

An egg goes through a series of mysterious processes as it readies for encountering sperm. In frogs, as the egg passes through the oviduct, it is coated with a series of layers – rich glycoconjugates and proteins – that comprise the jelly coat of the egg. The structure and importance of the egg jelly has long been known – in Xenopus, it comprises three distinct layers, and removal of the jelly renders the egg unfertilizable. Recently, two laboratories have begun characterizing the jelly factors that are required for fertilization.As a first step towards identifying the jelly molecules involved in fertilization, Mozingo and Hedrick1xDistribution of lectin binding sites in Xenopus laevis egg jelly. Mozingo, N.M. and Hedrick, J.L. Dev. Biol. 1999; 210: 428–439CrossRef | PubMed | Scopus (9)See all References1 provided a detailed description of the distribution of the sugar moieties comprising much of the structural components of Xenopus egg jelly. The differences in sugar distribution and abundance in each jelly sublayer was determined by lectin binding. Preincubating eggs with some lectins inhibited sperm penetration of the jelly, suggesting that the sugar moieties might be involved in sperm binding.By contrast, Olson and Chandler2xXenopus laevis egg jelly contains small proteins that are essential to fertilization. Olson, J.H. and Chandler, D.E. Dev. Biol. 1999; 210: 401–410CrossRef | PubMed | Scopus (27)See all References2 report that proteins that can diffuse from the jelly are both necessary and sufficient for restoring sperm binding to jellyless eggs. Significantly, neither the structural components nor a range of charged polymers could restore fertilization to jellyless eggs. The diffusible factor must be present at the time of fertilization as pretreating jellyless eggs with the diffusible fraction and then removing it prior to sperm addition did not promote fertilization. The fertilization-restoration activity is probably a protein as it is destroyed by heat and protease treatment and is small – 60% of the activity is able to pass through a filter possessing a 50 kDa cut off. Although candidates for this diffusible factor have not been reported, once identified, it will be important to understand the relationships between the protein and glycoconjugate ingredients of the egg jelly in order to reconcile the interesting differences between these two studies.

Distribution of Lectin Binding Sites in Xenopus laevis Egg Jelly

Eggs from the anuran Xenopus laevis are surrounded by a thick jelly coat that is required during fertilization. The jelly coat contains three morphologically distinct layers, designated J1, J2, and J3. We examined the lectin binding properties of the individual jelly coat layers as a step in identifying jelly glycoproteins that may be essential in fertilization. The reactivity of 31 lectins with isolated jelly coat layers was examined with enzyme-linked lectin-assays (ELLAs). Using ELLA we found that most of the lectins tested showed some reactivity to all three jelly layers; however, two lectins showed jelly layer selectivity. The lectin Maackia amurensis (MAA) reacted only with J1 and J2, while the lectin Trichosanthes kirilowii (TKA) reacted only with J2 and J3. Some lectins were localized in the jelly coat using confocal microscopy, which revealed substantial heterogeneity in lectin binding site distribution among and within jelly coat layers. Wheat germ agglutinin (WGA) bound only to the outermost region of J3 and produced a thin, but very intense, band of fluorescence at the J1/J2 interface while the remainder of J2 stained lightly. The lectin MAA produced an intense fluorescence-staining pattern only at the J1/J2 interface. Several lectins were also tested for the ability to inhibit fertilization. WGA, MAA, and concanavalin A significantly inhibited fertilization and WGA was found to block fertilization by preventing sperm from penetrating the jelly. Using Western blotting, we identified high-molecular-weight components in J1 and J2 that may be important in fertilization.

Xenopus laevis Egg Jelly Contains Small Proteins That Are Essential to Fertilization

The eggs of Xenopus laevis are surrounded by investment layers of egg jelly that interact with the sperm immediately prior to fertilization. Components of these egg jelly layers are necessary for the fertilization of the egg by incoming sperm. Eggs which are stripped of their jelly layers are refractile to fertilization by sperm, but the addition of solubilized jelly promotes fertilization. We have shown previously that the egg jelly layers are composed of a fibrous network of glycoconjugates which loosely hold smaller diffusible components. Extracts of these diffusible components were prepared by incubation of freshly ovulated eggs in high-salt buffers for 12 h at 4°C. This diffusible component extract, when incubated with sperm, promoted the sperm's ability to fertilize dejellied eggs in a dose-dependent manner. In contrast, the high-molecular-weight “structural” glycoconjugates of jelly that remain after extraction of the diffusible components did not increase fertilization efficiency of dejellied eggs nor did nonspecific proteins, carbohydrate polymers, or organic polymers. The diffusible components, analyzed by SDS–PAGE, consisted of a mixture of proteins from 4 to 180 kDa. The protein responsible for fertilization rescue appeared to be <50 kDa and appeared to self-aggregate or to bind to larger proteins. This protein component was required during sperm binding to the egg, its action required an intact egg vitelline envelope, and its action was independent of large soluble polymers such as Ficoll.

Influence of creep phenomenon and surface roughness on the adhesion of Xenopus laevis eggs to different substrates

The adhesion of fertilized and nonfertilized Xenopus laevis eggs to various surfaces differing in texture, roughness, and chemical composition was examined. Techniques regularly used in the fields of coating and adhesives were adopted. The stresses for the disconnection of eggs from different surfaces were evaluated by a custom-made apparatus, which disconnects the eggs by applying a regulated water pressure. The strength of adhesion depends, among others, on the roughness of the surfaces. A commercial roughness tester evaluated this parameter. It was observed that the rougher the surface, the better the adhesion between the egg and the surface. Maximal adhesion between the eggs and rough surfaces was detected 1 h after the first contact. For smooth surfaces (hydrogels), such a maximum was recognized only after 24 h had elapsed. In addition to the character of the surfaces, the creep of the jelly coat with time significantly influenced the strength of adhesion. The creep of the jelly coat was also dependent on the degree of swelling. The swelling of an egg's jelly coat during the first 8.5 h of the process was monitored by weight. Major changes in weight were observed during the first 100 min. After longer periods, the weight reached an asymptotic level. Incorporating polyvinyl pyrrolidone (PVP) into the medium eliminates the adhesion and stickiness of Xenopus eggs in the laboratory. Tensile bond stress and peel techniques were used to estimate the egg's adhesion to several surfaces. These tests revealed that the first contact between the egg and substrate resulted in a stronger adhesion than when the egg was disconnected and re-glued (re-adhered) to the same surface. Adhesion to natural surfaces (pebbles and Nimephea leaves) was better than to synthetic smooth surfaces. The shorter the distance between the female toad and any surface, the better the adhesion of the eggs.

A Sperm Chemoattractant Is Released from Xenopus Egg Jelly during Spawning

Although the jelly layers surrounding amphibian eggs are known to be essential to fertilization, no biological activities have been reported for jelly macromolecules in any vertebrate. Here we provide evidence that the jelly surrounding Xenopus laevis eggs releases a small diffusible protein into the surrounding media that serve as sperm chemoattractant. Using video microscopy we find that Xenopus sperm will preferentially turn toward and contact a glass capillary filled with egg jelly extract. In experiments using a two-chamber bioassay device we find that egg jelly extracts are capable of stimulating sperm extract. In experiments using a two-chamber bioassay device we find that egg jelly extracts are capable of stimulating sperm movement across a membrane barrier sixfold over controls. This activity is not observed in materials unrelated to egg jelly and the response of sperm to egg jelly extract is clearly chemotactic rather than chemokinetic. A concentration gradient of the extract is absolutely necessary, and the chemotactic activity of the extract exhibits a biphasic dose dependence similar to that of chemotactic agents in other systems. We have been able to characterize the factor as being a heat-stable protein about 10 kDa in size. This study, therefore, provides the first clear evidence for a diffusible sperm chemoattractant in a nonhuman vertebrate, as well as the first demonstration of a physiological role for egg jelly macromolecules in Xenopus fertilization.

Acrosome reaction inactivation in sea urchin sperm

Acrosome reaction inactivation (ARI) is a process that renders sperm irreversibly refractory to the egg jelly (the natural inducer of the acrosome reaction, AR). This process triggered by the egg jelly, is associated with an increase in [Ca 2+] i. However, we show here that a rise in [Ca 2+] i alone is not sufficient to induce ARI, since artificially increasing [Ca 2+] i with either an ionophore or rising external pH, does not trigger ARI. Contrary to the AR which strictly requires Ca 2+, ARI can be triggered almost equally well by Sr 2+. On the other hand, Mn 2+ inhibits ARI and, as we showed earlier, does not affect AR. These observations indicate that the mechanisms involved in ARI differ from those leading to AR. In addition, we report here that high external pH (a non-physiological inducer of AR) triggers the AR in previously inactivated sperm by opening the same Ca 2+ channels activated by the egg jelly. Considering that the opening of Ca 2+ channels is one of the earliest responses triggered by the egg jelly and that ARI requires the egg jelly receptor to be activated, we have concluded that ARI involves the uncoupling between the egg jelly receptor and Ca 2+ channels. Furthermore, intracellular pH (pH i) determinations, in the presence or absence of ionomycin to substitute for the uncoupled Ca 2+ channels, indicate that pH i regulation is also impaired in inactivated sperm. In conclusion, ARI is a manifestation of the uncoupling of the egg jelly receptor from the different ion transport systems required for the acrosome reaction.

Structural analysis of oligosaccharide-alditols released by reductive beta-elimination from the jelly coats of the anuran Bufo arenarum.

Amphibian egg jelly coats are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they are transported toward the cloaca. Mucin type glycoproteins are the major constituents of the egg jelly coats. In this study, the O-linked carbohydrate chains of the jelly coat surrounding the eggs of Bufo arenarum were released by alkaline borohydride treatment. Fractionation of the mixture of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography, ion-exchange chromatography and high-performance liquid chromatography using an amino-bonded silica column, afforded 11 fractions. The primary structures of these O-glycans were determined by one-dimensional and two-dimensional 1H-NMR spectroscopy in conjunction with matrix-assisted laser-desorption-ionization--time-of-flight mass spectrometry. 11 oligosaccharide structures, possessing a core consisting of Galbeta1-->3GalNAc-ol with or without branching through a GlcNAc residue linked beta1-->6 to the GalNAc residue (core type 2 or core type 1, respectively) are described. These oligosaccharide-alditols with these types of cores have been identified previously in mammalian mucins or in oviducal amphibian jellies. These glycans contain blood group determinants such as H, A or Cad antigens.

Structural analysis of a new series of oligosaccharide-alditols released by reductive beta-elimination from oviducal mucins of Rana utricularia.

Egg jelly coats from Rana utricularia are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they pass along the different oviducal portions. In this study, carbohydrate chains of the jelly coat surrounding the eggs of R. utricularia were released by alkali/borohydride treatment. Fractionation of O-linked oligosaccharide-alditols was achieved by a combination of chromatographic techniques comprising anion-exchange chromatography, gel-permeation chromatography and HPLC on a silica column bonded with aminopropyl groups. Structural characterization was performed by one- and two-dimensional 1H-NMR spectroscopy in combination with matrix-assisted laser-desorption ionization-time of flight MS and methylation analysis. Ten oligosaccharide structures possessing a core consisting of Galbeta(1-->3)GalNAc-ol with or without branching through a GlcNAc residue linked beta(1-->6) to the GalNAc residue (core type 2 or core type 1 respectively) are described. The most representative carbohydrate sequences are: GlcNAc(beta1-3)[Fuc(alpha1-4)]GlcNAc, GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)GlcNAc(beta1-3)GlcNAc and Gal(beta1-3)GlcNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)GlcNAc. The carbohydrate chains isolated from R. utricularia are quite different from those found in other amphibian species, in which the presence of species-specific material has been characterized. Since the jellies surrounding amphibian eggs are involved in egg-sperm interactions, these structural investigations can provide biochemical support for investigation of the fertilization process.

Structural analysis of oligosaccharide-alditols released by reductive β-elimination from oviducal mucins of Rana dalmatina

The O-linked oligosaccharides of the jelly coat surrounding the eggs of Rana dalmatina were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by consecutive normal-phase high-performance liquid chromatography on a silica-based alkylamine column. The structures of the oligosaccharide-alditols were determined by 400-MHz 1H-NMR spectroscopy in combination with matrix assisted laser desorption ionization–time of flight analysis. The five structures were identified range in size from trisaccharides to hexasaccharides, possessing a core consisting of Gal( β1–3)GalNAc-ol (core type 1). Novel oligosaccharide-alditols are: The carbohydrate chains isolated from Rana dalmatina are different from those found in other amphibian species, in which the presence of species-specific material has been characterized. Since the role of carbohydrates appears more and more apparent during the fertilization process, the biodiversity of the O-linked oligosaccharides could support such a biological role.

The Fucose Sulfate Polymer of Egg Jelly Binds to Sperm REJ and Is the Inducer of the Sea Urchin Sperm Acrosome Reaction

The sea urchin sperm cell is an advantageous model for studying ligand-mediated exocytosis. Sperm can be obtained in vast quantities and induced to undergo exocytosis of the acrosomal vesicle with great synchrony. During sea urchin fertilization, egg jelly (EJ) triggers the sperm acrosome reaction (AR) which is required for sperm binding and fusion with the egg. Uncertainty exists as to the exact biochemical nature of the AR inducer. The following study was performed in an attempt to clarify the nature of the inducer. EJ from individual females (Strongylocentrotus purpuratus) was analyzed on SDS–PAGE gels. Each female had a unique composition of EJ macromolecules, but all females possessed the previously described fucose sulfate polymer (FSP). Two electrophoretic isotypes of FSP were discovered; 87% of females had only one isotype and 13% had both. Both FSP isotypes bound to the REJ protein (receptor for egg jelly) purified from sperm. The two FSP isotypes had almost equal potency in inducing the AR. EJ was fractionated by DEAE chromatography in 6 M urea/4% β-mercaptoethanol. All AR-inducing activity coeluted with FSP. FSP, purified by trypsin digestion followed by dialysis, was twice as active as the non-trypsin-digested control at inducing the AR. EJ was digested with proteinase K, boiled in detergent and β-mercaptoethanol, and subjected to sucrose density gradient sedimentation. The FSP and AR activity had superimposable sedimentation patterns. Purified FSP had no associated peptide component. Sperm from individual males differed in the concentration dependency of purified FSP to induce the AR. The data indicate that the 138/82 kDa EJ glycoproteins, previously thought to act as AR inducers, do not appear to be involved in triggering the AR. The data are consistent with the hypothesis that FSP is the only inducer of the AR of this sea urchin species.

Spermiogenesis and Sperm Structure in Relation to Early Events of Fertilization in the Limpet Tectura testudinalis (Muller, 1776).

Spermiogenesis and fertilization in the limpet Tectura testudinalis (Mollusca: Archaeogastropoda: Patelloidea) were examined using scanning and transmission electron microscopy, as well as light microscopy. Spermiogenesis was similar to that described for other Lottiidae (Patelloidea), but some key differences were noted. We observed that the acrosomal vesicle forms on the plasma membrane from several Golgi-derived proacrosomal vesicles during the early spermatid. Evidence from anti-actin antibody staining of a related species, T. scutum, indicates that the acrosome probably contains G-actin in three different locations, but there are no preformed actin filaments. The flagellum of mature sperm tapers to a thin, filamentous end-piece. The mature egg of T. testudinalis is enclosed by a thin vitelline layer and a thick jelly coat bound by follicle cells. Where a mature sperm encounters the intact jelly coat, its acrosome tip may elongate, up to twice its original length. The acrosome reaction is not induced, but by this mechanism the acrosome bridges the jelly coat and extends down far enough to contact the vitelline layer. During the elongation of the acrosome, microfilaments are formed inside the tip. As well, new membrane required for this process is supplied by elimination of any slack in the plasma membrane and also perhaps by spontaneous formation of vesicles inside the acrosome. Once the tip of the sperm contacts and binds with the vitelline layer it undergoes the acrosome reaction, in which the plasma and acrosomal membranes fuse and roll back in a manner typical of scaphopods and some polychaetes. Sperm also may bind directly with the vitelline layer when the jelly coat is absent. Furthermore, the acrosome tip may undergo spontaneous fusion with a naked egg microvillus that formerly connected with a follicle cell process. Thus, the sperm either individually or collectively are equipped to deal with any one of several potential routes to successful fertilization of the egg. Current theories of gastropod phylogeny place the limpets as basal to the Gastropoda, but new evidence presented here supports the idea that they are a divergent group, with some unique innovations in sperm design.

Autosomal dominant polycystic kidney disease: A genetic perspective

Summary: Important advances in understanding the common genetic disease, autosomal dominant polycystic kidney disease (ADPKD), have been made in last 3 years through the identification and characterization of the major genes, PKDI and PKD2. the PKDI and PDK2 genes map to the chromosomal regions, 16p13.3 (within a complex reiterated area) and 4q21‐23, respectively, and encode transcripts of over 14kb (PKDI) and 5 kb (PKD2). the predicted PKD1 protein, polycystin, is 4302/3 aa with a calculated molecular mass of approximately 460 kDa. Polycystin has a large extracellular region containing motifs involved in protein‐protein and protein‐carbohydrate interactions, and is attached to the membrane with multiple transmembrane regions. PKD2 is a small integral membrane protein (110 kD) with cytoplasmic N and C termini, 6 transmembrane domains and a region of homology with polycystin. Similarity of PKD2 to voltage‐gated ion channels and an area of homology between polycystin and a receptor for egg jelly protein, which is thought to regulate ion channels, suggests a role for the ADPKD proteins in ion transport. the PKD1 and PKD2 genes are expressed in most tissues and polycystin has been localised to tubular epithelial structures in the fetal and adult kidney. A Western product of approximately 400 kDa has been detected in membrane fractions of renal tissue and strong polycystin expression has been noted in PKD1 cystic epithelia. the mutations detected at PKD1 and PKD2 suggest that these germline changes are inactivating. A specific syndrome due to a deletion of PKD1 and the adjacent TSC2 gene is associated with early onset polycystic kidney disease and tuberous sclerosis. Recent evidence of somatic mutation in cystic epithelia suggests that PKD1 is recessive at the cellular level, although this appears a odds with the continued polycystin expression noted in these cells. Further studies to determine the precise mutational mechanism and the normal role of the ADPKD proteins is required.