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.     

CHEMICAL ANALYSIS OF TOAD EGG-JELLY IN RELATION TO ITS 'SPERM-CAPACITATING' ACTIVITY

In an attempt to characterize a factor in anuran egg-jelly that is essential for fertilization, dejellied, non-fertilizable eggs of the toad, Bufo bufo , were inseminated in the following jelly preparations: jelly solubilized by KCN followed by dialysis (Dialyzed jelly: DJ), jelly solubilized by ultraviolet irradiation (UVJ), a diffusible factor released from jelly coat into deionized water (DF), the dialyzable fraction of DF (DFD), and the non-dialyzable fraction of DF (DFR). It was found that all the preparations except DFR are active in supporting the fertilization of dejellied eggs. DFD is thermo-stable, and characterized by a rise in pH accompanying increase in concentration. DF obtained from Rana japonica also capacitated the fertilization of dejellied Bufo eggs.
Chemical analyses indicated that DJ, UVJ, DF and DFR contain various amounts of fucose, hexoses, hexosamines, and proteins. Sialic acid was present in DJ and UVJ, but not in DF. In DFD, only hexoses and proteins were detectable to a measurable degree. A salient feature of the paper chromatographic analyses was the predominance in DFD of an unspecified reducing sugar which was found in common in all the preparations with fertilization-supporting activity. Gel-filtration in combination with bioassay for fertilization led to the isolation of the active substance, which had a molecular weight of less than 500, and was characterized by a basic nature and the presence of a reducing sugar.
The possible importance in fertilization of this small molecular weight jelly component is stressed, together with the suggestion that the component represents some terminal group of the jelly macromolecule in either diffusible or non-diffusible form.     

Mechanism of univalent antisperm antibody inhibition of fertilization in the sea urchin, Arbacia punctulata

Univalent antisperm antibodies (IFab) markedly inhibited the fertilizing capacity of sperm when tested on intact, dejellied, and "demembranated" Arbacia punctulata eggs. Sperm motility and egg jelly penetration were not affected by IFab. Antifertilizin was excluded as the essential sperm antigen involved in the fertilization-inhibiting action. Sperm pretreated with IFab did not bind to the surfaces of either dejellied or demembranated eggs, whereas control globulin (CFab) and seawater-pretreated sperm bound to such eggs in high numbers. Electron microscopy showed that IFab-treated sperm failed to undergo the acrosome reaction. This excluded "bindin" as the essential antigen. Inhibition of fertilization by IFab was reversed or bypassed by artificial induction of the acrosome reaction with ionophore A23187. It is concluded that univalent antisperm antibody treatment inhibits the fertilizing capacity of sperm by preventing a sperm-egg interaction that results in the acrosome reaction; consequently, attachment of the sperm to the egg is prevented.     

Egg water from the amphibian Bufo arenarum induces capacitation-like changes in homologous spermatozoa

Mammalian sperm acquire fertilizing capacity after residing in the female tract, where physiological changes named capacitation take place. In animals with external fertilization as amphibians, gamete interactions are first established between sperm and molecules of the egg jelly coat released into the medium. Since dejellied oocytes are not normally fertilized, the aim of this study was to determine if the jelly coat of the toad Bufo arenarum promotes a “capacitating” activity on homologous sperm. We found that sperm incubation in diffusible substances of the jelly coat (egg water) for 90-180 s is sufficient to render sperm transiently capable of fertilizing dejellied oocytes. The fertilizing state was correlated with an increase of protein tyrosine phosphorylation and a decrease of sperm cholesterol content. Inhibition of either the increase in tyrosine phosphorylation or cholesterol efflux affected the acquisition of fertilizing capacity. Phosphorylation and fertilization could be promoted with NaHCO₃ and also by addition of beta cyclodextrin. Moreover, sperm could gain the ability to fertilize dejellied oocytes in the presence of these compounds. These data indicate that sperm should undergo a series of molecular changes to gain fertilizing capacity; these changes are reminiscent of mammalian sperm capacitation and take place before the acrosome reaction.     

THE EFFECT OF SOLUBLE EGG JELLY ON THE FERTILIZABILITY OF ACID-DEJELLIED SEA URCHIN EGGS*

Acid-dejellied Lytechinus pictus eggs bind few sperm and show decreased fertilizability. Addition of solubilized egg jelly increases sperm binding and fertilizability, presumably by increasing the frequency of the acrosome reaction. However, dejellied Strongylocentrotus purpuratus bind more sperm and show increased fertilizability in the complete absence of soluble egg jelly. Addition of soluble egg jelly greatly decreases fertilizability in S. purpuratus. Such species differences may be the basis for the controversy between Lillie and Tyler on the one hand, who believed that egg jelly increased egg fertilizability; while Loeb and Hagström on the other hand, believed jelly had no effect on, or actually decreased egg fertilizability. ¹²⁵I-labeling of dejellied S. purpuratus egg surfaces and immunofluorescent studies show that egg jelly persists on the surfaces of acid-dejellied eggs. Egg jelly appears to be a non-removable component of the vitelline layer of this species.     

THE FERTILIZING CAPACITY OF SEA URCHIN SPERM RAPIDLY DECREASES AFTER INDUCTION OF THE ACROSOME REACTION*

When immotile, flagella-less sperm were added to acid-dejellied eggs of Strongylocentrotus purpuratus 11% of the eggs fertilized. Addition of soluble egg jelly increased the percentage fertilization to 90.5. Over 50% of the sperm exposed to egg jelly had undergone the acrosome reaction compared to only 3–5% in the absence of jelly. Egg jelly was added to flagella-less sperm to induce the acrosome reaction and dejellied eggs added at various times thereafter. The fertilizing capacity of the sperm decreased with first order kinetics with 50% loss by 23 sec after induction of the acrosome reaction. Intact, motile sperm bind to formaldehyde-fixed eggs with maximum binding occurring 40 sec after sperm addition. After 40 sec the sperm begin to detach from the fixed eggs and by 240 sec none remain attached. Sperm detachment from fixed eggs and loss of fertilizing capacity after the acrosome reaction show a close temporal correlation.     

Toad egg-jelly as a source of divalent cations essential for fertilization

Dejellied uterine eggs of the toad Bufo bufo japonicus are not fertilizable in 1/20 De Boer's solution (1/20 DB), but are fertilized when inseminated in a uv-solubilized jelly (UVJ) or the dialyzate of UVJ (UVJD). The present study was carried out to define this fertilization-supporting activity of egg-jelly. Dejellied eggs were fertilized in a high frequency when inseminated in a medium containing the ashes obtained by heating UVJD at 600 degrees C for 16 hr. Similarly, a reconstituted salt solution (RSS), which mimics the ionic composition of UVJD, supported a high rate of fertilization. To be effective in fertilization, however, RSS had to be present at the time of insemination. Analyses of individual salts revealed that dejellied eggs are successfully fertilized in CaCl2 and/or MgCl2 at 1-5 mM, only slightly in KCl at 10 mM, but not at all in NaCl at any of the concentrations tested. The activity of UVJD was lost reversibly when divalent cations were chelated by EDTA. The fertilization of dejellied eggs is therefore possible in a medium without any organic components of egg-jelly, provided that 2-5 mM Ca2+ or Mg2+ is present. Sperm were motile in media containing cations below 20-25 mM, regardless of the ionic composition. The egg-jelly possessed cations in a concentration of about 130 mM, but most ions were lost from intact jelly on immersion of eggs in water for 2-3 min, accompanied by the acquisition of fertilizability by sperm. Examination of the behavior of salts on dialysis or gel-filtration of jelly molecules revealed that the jelly retains Ca2+ and Mg2+, and possibly K+ as well, but not Na+ and Cl-. We propose that toad egg-jelly plays a function in fertilization by retaining Ca2+ and/or Mg2+ around each egg at the level necessary for successful sperm entrance into the egg.     

Gamete interaction in the white sturgeon Acipenser transmontanus: a morphological and physiological review

Synopsis Sturgeon gametes differ from those of most fish in that the sperm possess acrosomes that undergo exocytosis and filament formation while the eggs possess numerous micropyles. Acipenser transmontanus eggs are encased by multilayered envelopes that consist of outer adhesive jelly coats and three structured layers interior to the jelly. The glycoprotein jelly layer only becomes adhesive upon exposure to freshwater. The layer interior to the jelly, layer 3, is the other carbohydrate-containing component of the egg envelope. This layer consists of a water-insoluble glycoprotein that, upon freshwater exposure, is hydrolyzed by a trypsinlike protease to yield a water-soluble, lower molecular weight carbohydrate-containing component. This component can be identified in the surrounding medium when unfertilized eggs are incubated in freshwater. This egg water component elicits acrosome reactions only in homologous sperm. The A. transmontanus sperm acrosome reaction is a Ca⁺⁺ and/or Mg⁺⁺ dependent event that includes the formation of a 10 μ long fertilization filament. A. transmontanus fertilization can occur at low sperm per egg ratios; however, crossfertilization of A. transmontanus eggs with lake sturgeon, A. fluvescens, sperm results in a very low number of fertilized eggs, even at high sperm per egg ratios. The morphological, physiological, and biochemical phenomenon reviewed in this paper are related to the environment in which they occur. Also, the possible role of the acrosome and the presence of numerous micropyles are discussed.     

Fertilization of investment-free Xenopus eggs

The vitelline envelope of unfertilized Xenopus egg can be removed manually after treating the dejellied eggs for 10 min with 20% (w/v) sucrose in F-1 saline. Fertilization occurred in 52% of the eggs denuded in this way when UV-solubilized jelly was added to the sperm-egg mixture; without the jelly the level of fertilization was only 6%. Fertilization did not occur synchronously in the denuded eggs; the average delay between insemination and fertilization was 19 +/- 18 min.     

The vitelline envelope to fertilization envelope conversion in eggs of Xenopus laevis

Fertilization of the Xenopus laevis egg causes the conversion of the vitelline envelope to the fertilization envelope, a change reflected in the loss of sperm penetrability of the egg and the appearance of an electron-dense layer on the outer aspect of the fertilization envelope. As seen by one-dimensional gel electrophoresis, two components with molecular weights of 69,000 and 64,000 in the vitelline envelope were converted to 66,000 and 61,000 in the fertilization envelope. By two-dimensional gel electrophoresis, the components in the 69,000 and 64,000 molecular weight regions of the vitelline envelope were seen to shift to more basic isoelectric points upon conversion to the fertilization envelope. Peptide mapping by limited proteolysis suggested that the 69,000 and 64,000 molecular weight components shared the same polypeptide chains but the smaller glycoprotein lacked a carbohydrate side chain found on the larger species. Similar sites on each glycoprotein were affected when the vitelline envelope was converted to the fertilization envelope. No N-terminal amino acids could be identified on the envelope components, indicating that these glycoproteins have blocked N-termini. Ionophore A23187-activation of jellied eggs (but not dejellied eggs) caused the molecular weight changes in the absence of sperm. Thus, factors from the jelly and the cortical granules but not from sperm apparently are involved in the processing of the 69,000 and 64,000 molecular weight components.     

Difference of fertilizing capacity between testicular sperm and vas deferens sperm in Cynops pyrrhogaster

The fertilizing capacity was compared between testicular and vas deferens sperm in Cynops pyrrhogaster. The testicular sperm was not capable of fertilizing jelly eggs. In contrast, the vas deferens sperm was already capable of fertilizing the newt jelly eggs. There was no inhibitory factor for fertilizing jelly eggs in the testis. These results suggest that the testicular sperm is immature as to the fertilizing capacity. The testicular sperm gained the fertilizing capacity for the jelly eggs by treatment with Holtfreter's solution or 1/20 strength Holtfreter's solution. The treatment may promote the step of maturation to achieve the fertilizing capacity. The treated testicular sperm did not fertilize dejellied eggs, although vas deferens sperm fertilized dejellied eggs. Therefore, the maturation state of the treated testicular sperm is different from that of vas deferens sperm. Newt sperm may be matured within the vas deferens, as the newt does not have an organ like the mammalian epididymis.     

The jelly envelopes and fertilization of eggs of the newt, Notophthalmus viridescens

Fertilization in Notophthalmus viridescens is internal and involves passage of the sperm through five layers of egg jelly (J5-J1, from outermost to innermost), each of which is secreted by a discrete region of the oviduct. Polyspermy is normal. Passage of the sperm through the jelly and into the egg was studied by a technique of artificial insemination similar to natural insemination, in that undiluted fluid from the vas deferens was applied directly to eggs with various layers of jelly present, followed by flooding with water three to five minutes later. In general, successful fertilization increased as the number of jelly layers increased; jellyless coelomic eggs were not fertilizable. Sperm passage through the jelly and into the egg usually occurs within one to three minutes. Upon hydration of the jelly, barriers to sperm penetration develop in layers J5 and J3. Changes in the egg jelly thus seem to be involved in the restriction of polyspermy to a low level.     

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.     

Cortex reorganization of <Emphasis Type="Italic">Xenopus laevis</Emphasis>eggs in strong static magnetic fields

Observations of magnetic field effects on biological systems have often been contradictory. For amphibian eggs, a review of the available literature suggests that part of the discrepancies might be resolved by considering a previously neglected parameter for morphological alterations induced by magnetic fields – the jelly layers that normally surround the egg and are often removed in laboratory studies for easier cell handling. To experimentally test this hypothesis, we observed the morphology of fertilizable Xenopus laevis eggs with and without jelly coat that were subjected to static magnetic fields of up to 9.4 T for different periods of time. A complex reorganization of cortical pigmentation was found in dejellied eggs as a function of the magnetic field and the field exposure time. Initial pigment rearrangements could be observed at about 0.5 T, and less than 3 T are required for the effects to fully develop within two hours. No effect was observed when the jelly layers of the eggs were left intact. These results suggest that the action of magnetic fields might involve cortical pigments or associated cytoskeletal structures normally held in place by the jelly layers and that the presence of the jelly layer should indeed be included in further studies of magnetic field effects in this system.     

Fertilization of the eggs ofCynops pyrrhogaster (japanese newt) after immersion in water

Summary Cynops pyrrhogaster oviducal eggs with and without jelly envelopes (jelly egg and dejellied egg respectively) were immersed in water, and then inseminated artificially. After 1 h of immersion in water, more than half the dejellied eggs were fertilized and developed, but no jelly eggs developed. The rapid decrease in the ability of jelly eggs to be fertilized after immersion in water is not due to a deficiency in the egg. Our results make it clear that hydrated jelly envelopes prevent the eggs from fertilizing. The ability of the egg to be fertilized decreases for a long time in water and this decrease proceeds more slowly in De Boer's solution or Holtfreter's balanced salt solution than in water.     

Egg envelope conversion following fertilization in Bufo japonicus

The envelope of the Bufo japonicus egg becomes impenetrable to sperm following fertilization. Electrophoretic analysis of envelopes showed that two glycoprotein components with apparent molecular weights of 65,000 and 61,000 were hydrolyzed during fertilization to 62,000 and 58,000, respectively. These two envelope components were structurally related as shown by peptide mapping and deglycosylation studies. Hardening of the envelope following egg activation was also observed, as detected by an increase in the envelope melting temperature. The involvement of proteolytic activities in the envelope hydrolysis and hardening reactions was demonstrated using protease inhibitors, and was verified for the hydrolysis reaction by observing a loss of mass in deglycosylated envelope components obtained before and after fertilization. A low ionic strength medium (less than 50 mM) was required for both the hardening and hydrolysis reactions. Envelopes from eggs activated in a high ionic strength medium were resistant to lysin from sperm, indicating that neither hydrolysis nor hardening was necessary to block lysin activity on the envelope. Both envelope hydrolysis and hardening could be effected in the absence of sperm (i.e., when eggs were activated by electric shock) and after egg jelly had been removed, indicating that neither sperm nor jelly factors were required for the envelope modifications. In addition, when eggs were activated in the presence of NH4Cl to suppress cortical granule exocytosis, envelope hardening and hydrolysis were still observed, indicating that a cortical granule-derived factor may not be involved.     

Fertilization of newt coelomic eggs in the absence of jelly envelope material

Summary Fertilization ofCynops pyrrhogaster (Japanese newt) coelomic eggs was studied in the absence of jelly envelope material or synthetic high polymer. An undiluted sperm fluid from the vas deferens fertilized coelomic eggs in the absence of the jelly envelope material or synthetic high polymer. The fertilized eggs developed beyond gastrulae and formed tail bud embryos. These results indicate that the fertilization process does not depend upon the presence of jelly envelope material or synthetic high polymer and that the sperms within the vas deferens are already capable of fertilizing the eggs inC. pyrrhogaster. The sperm suspension in Holtfreter's balanced salt solution (H-sperm) fertilized the coelomic eggs without the jelly envelope material or synthetic high polymer. These eggs had been suspended in Holtfreter's balanced salt solution (H) or in 1/20 strength H (1/20 H) prior to insemination (H-eggs or 1/20 H-eggs). In contrast, the sperm suspension in 1/20 H (1/20 H-sperm) did not fertilize 1/20 H-eggs, but dit H-eggs. In the latter case, H surrounding the eggs may affect sperms, allowing them to be fertilized. The 1/20 H-sperms regained their ability to fertilize 1/20 H-eggs on re-exposure to H. The 1/20 H-sperm also fertilized jelly eggs. The results of the dejellied egg experiment showed the same pattern. These results indicate that the sperms within the vas deferens lose their capacity to fertilize 1/20 H-eggs on exposure to low ionic strength solution (1/20 H); this capacity is restored by exposure to high ionic strength solution (H) or to jelly envelope.     

Egg jelly components responsible for histone degradation and acrosome reaction in the starfish, Asterina pectinifera.

In the starfish, Asterina pectinifera, egg jelly induces the degradation of sperm histones as well as the acrosome reaction. We have isolated histone degradation-inducing components from the egg jelly. The histone degradation and the acrosome reaction are induced by a co-operative action of ARIS, which is an extremely large, sulfated glycoprotein with diffusible substance(s) in the jelly. Co-ARIS I, a steroidal saponin of the jelly, is effective to induce both reactions in the presence of ARIS.     

Acrosome Reaction-Inducing Substance Purified from the Egg Jelly Inhibits the Jelly-Induced Acrosome Reaction in Starfish: An Apparent Contradiction

Previous studies indicated that two components of the egg jelly are required for induction of the acrosome reaction in starfish: a sulfated glycoprotein called acrosome reaction-inducing substance (ARIS) and a diffusible organic substance(s) called Co-ARIS. In the present study the sites of action of ARIS and Co-ARIS and their temporal relationships were examined. When sperm had been treated for a few minutes with ARIS, or a crude preparation of Co-ARIS (Fraction M₈), or inadequate amounts of jelly, or sufficient jelly in low Ca²⁺ sea water, they did not undergo the acrosome reaction when the deficiencies were corrected. Moreover, they became nonresponsive to the jelly. Pronase digest of ARIS (P-ARIS) but not of Fraction M₈ retained this capacity. A steroidal saponin purified as Co-ARIS did not have this capacity. This suggests the presence of a third jelly component, probably an oligopeptide(s), participating in induction of the acrosome reaction. Activation of Ca²⁺ -uptake seems to be at least one, if not the only, action site of ARIS and Co-ARIS, because ARIS, P-ARIS, and Fraction M₈ inhibited jelly-induced Ca²⁺ -uptake by sperm, and because the calcium ionophore A23187 by-passed the blockage by these components of the jelly-induced acrosome reaction.     

Fertilization selection on egg and jelly-coat size in the sand dollar Dendraster excentricus

Organisms with external fertilization are often sperm limited, and in echinoids, larger eggs have a higher probability of fertilization than smaller eggs. This difference is thought to be a result of the more frequent sperm-egg collisions experienced by larger targets. Here we report how two components of egg target size, the egg cell and jelly coat, contributed to fertilization success in a selection experiment. We used a cross-sectional analysis of correlated characters to estimate the selection gradients on egg and jelly-coat size in five replicate male pairs of the sand dollar Dendraster excentricus. Results indicated that eggs with larger cells and jelly coats were preferentially fertilized under sperm limitation in the laboratory. The selection gradients were an average of 922% steeper for egg than for jelly-coat size. The standardized selection gradients for egg and jelly-coat size were similar. Our results suggest that fertilization selection can act on both egg-cell and jelly-coat size but that an increase in egg-cell volume is much more likely to increase fertilization success than an equal change in jelly-coat volume. The strengths of the selection gradients were inversely related to the correlation of egg traits across replicate egg clutches. This result suggests the importance of replication in studies of selection of correlated characters.