Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10^?5 to 10^?3M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10^?3M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.     

A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization

A trypsin-like proteinase was localized within a single subcellular compartment of unfertilized Strongylocentrotus purpuratus eggs, the cortical granules. Homogenates of eggs were fractionated by rate-zonal centrifugation. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1, 3-glucanase) in the sucrose density gradient. A bimodal distribution pattern was obtained for aryl esterase activity (substrate: β-naphthyl acetate), with one peak in the microsomal and the other in the cortical granule fractions. The cortical granule enzyme was characterized as a trypsin-like proteinase, since it also hydrolyzed another typical tryptic substrate α-N-benzoyl-l-arginine ethyl ester and was completely inactivated by soybean trypsin inhibitor (SBTI). The aryl esterase activity in the microsomal fractions was not inhibited by SBTI, while 50% of the total aryl esterase activity in the original egg homogenate was inactivated by SBTI. The identity of the enzyme(s) responsible for the aryl esterase activity associated with the microsomal particles is unknown at present.The cortical granule proteinase functions in the elevation of the fertilization membrane and establishment of the block to polyspermy at fertilization. Arbacia punctulata eggs inseminated in the presence of trypsin inhibitors, SBTI or tosyl lysine chloromethyl ketone (TLCK), failed to elevate normal fertilization membranes and became heavily polyspermic.On the basis of these results and observations made by other investigators with a wide variety of biological systems, it is proposed that trypsin-like proteinases function in the discharge of secretory granules from all types of cells.     

An electrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis

In 27% DeBoer's saline (DBS), which yields maximum fertility rates, Xenopus eggs fertilized in vitro are monospermic, regardless of sperm concentration. One block to polyspermy (the “slow” block), described previously, occurs at the fertilization envelope that is elevated in response to the cortical reaction. This paper describes properties of an earlier, “fast” block at the plasma membrane and evaluates the functional significance of the two blocks at physiological sperm concentrations in natural mating conditions. Unfertilized eggs have a resting membrane potential of ?19 mV in 27% DBS. Fertilization triggers a rapid depolarization to +8 mV (the fertilization potential, FP); the potential remains positive for ca. 15 min. Activation of eggs with the ionophore, A23187, produces a slower but similar depolarization (the activation potential, AP). As in other amphibian eggs, the FP appears to result from a net efflux of Cl^?, since the peak of the FP (or the AP in ionophore-activated eggs) decreases as the concentration of chloride salts in the medium is increased. In 67% DBS no FP or AP is observed; eggs fertilized in 67% DBS become polyspermic and average 2 sperm entry sites per egg. In the 5–37 mM range, I^? and Br^?, but not F^?, are more effective than Cl^? in producing polyspermy. In 20 mM NaI the plasma membrane hyperpolarizes in response to sperm or ionophore; 100% levels of polyspermy and an average of 14 sperm entry sites per egg are observed. NaI does not inhibit or retard elevation of the fertilization envelope; the cortical reaction and fertilization envelope are normal in transmission electron micrographs. In 67% DBS, which also inhibits the fast block, the slow block was estimated to become functional 6–8 min after insemination. Eggs fertilized by natural mating in 20 mM NaI exhibit polyspermy levels of 50–90% and average 5 sperm entry sites per egg. Since eggs become polyspermic when fertilized by natural mating under conditions that inhibit the fast, but not the slow, block to polyspermy, we conclude that the fast block is essential to the prevention of polyspermy at the sperm concentrations normally encountered by the egg.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.     

Ionophore-induced efflux of sodium and potassium ions from sea urchin eggs

The efflux of K⁺ and Na⁺ from sea urchin eggs during Ca²⁺ ionophore A23187-induced parthenogenesis was studied in a K⁺ and Na⁺-free artificial seawater using extracellular ion-specific electrodes. We have probed this model system with monovalent cation-specific ionophores to determine if they affect K⁺ efflux in the unfertilized egg and whether any changes in ionophore sensitivity are observed during egg activation. In 500 mM choline chloride, 10 mM CaCl₂, 50 mM MgCl₂, 10 mM Tris-Cl pH 8.0, A23187 induced a rapid efflux of K⁺ and Na⁺ from the eggs after a short lag time (10–15 seconds). After the burst, the rate of K⁺ efflux remained higher than the pre-activation rate, but was lower than during the burst phase, while the rate of Na⁺ efflux became nearly zero. Monovalent cation-specific ionophores (valinomycin, gramicidin and nigericin) had no effect on K⁺ efflux from the unfertilized eggs in our model system. However, once the egg was activated by A23187, each of the above ionophores caused a prolongation of the burst phase for many minutes. These results show that the unfertilized egg plasma membrane (using our artificial conditions) is not susceptible to the monovalent cation-specific antibiotics and suggest that either the inserted cortical granule membrane or the developing fertilization envelope interacts with these ionophores to cause the change in rate-limiting step for K⁺ efflux observed egg activation.     

An ultrastructural examination of polyspermy induced by soybean trypsin inhibitor in the sea urchin Arbacia punctulata

Fine structural studies have been conducted to determine the role of the trypsinlike proteinase during fertilization which is localized within the cortical granules of sea urchin eggs. Eggs were treated with soybean trypsin inhibitor (SBTI), inseminated, and prepared for microscopic examination at various intervals during fertilization. In the presence of SBTI the breakdown of the cortical granules is delayed and the elevation of the vitelline layer is incomplete. Although all the cortical granules in SBTI-treated eggs are dehisced in a manner similar to controls, the rate at which their breakdown is propagated is reduced. Consequently, dehiscence of all the cortical granules in SBTI-treated specimens requires approximately 3 min to complete, vs. 1 min for controls. In addition the fertilization membrane has a blistered appearance owing to its attachment, at multiple loci, to the surface of the zygote. During the early stages of fertilization many of the treated eggs are monospermic, later all are polyspermic. Supernumerary sperm enter treated eggs (zygotes) up to 10 min after the initiation of the cortical granule reaction, apparently at those sites where the fertilization membrane has failed to separate from the zygote's surface.     

Reaction of sperm with egg-derived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin

Recent evidence suggests roles for egg derived hydrogen peroxide (H₂O₂) and ovoperoxidase (secreted by cortical granules) in both fertilization envelope hardening and the block to polyspermy in sea urchins. Strongylocentrotus purpuratus eggs were found to release H₂O₂ during the cortical reaction at fertilization. Treatment of sperm with equivalent concentrations of H₂O₂ resulted in a rapid loss of sperm fertilizing ability. Attempts were made to induce polyspermy by utilizing ovoperoxidase inhibitors at concentrations known to inhibit fertilization envelope hardening. Eggs fertilized in phenylhydrazine became polyspermic, while 3-amino-1,2,4-triazole-treated eggs did not. These data suggested that a sperm peroxidase might be involved in preventing polyspermy. This hypothesis was tested by the addition of phenylhydrazine or 3-amino-1,2,4-trizaole to H₂O₂-treated sperm. Phenylhydrazine acted to protect sperm fertility from H₂O₂, while 3-amino-1,2,4-triazole increased the adverse effect of H₂O₂. Simultaneous addition of both inhibitors to sperm incubated in H₂O₂ gave an intermediate value of sperm fertility. These data indicate that (1) H₂O₂ generated by sea urchin eggs during the cortical reaction at fertilization is used for two separate processes, fertilization envelope hardening and the prevention of polyspermy; (2) ovoperoxidase is probably not involved in preventing polyspermy; and (3) egg-derived H₂O₂ reacts directly with sperm enzymes to prevent polyspermy. The phenylhydrazine-sensitive enzyme in the sperm is probably a peroxidase that acts to inactivate sperm, while the 3-amino-1,2,4-triazolesensitive enzyme is probably a catalase which protects sperm from H₂O₂. This hypothesis is consistent with model experiments on horseradish peroxidase and bovine liver catalase.     

Polyspermy block in jellyfish eggs: Collaborative controls by Ca and MAPK

Jellyfish eggs neither undergo apparent cortical reaction nor show any significant change in the membrane potential at fertilization, but nevertheless show monospermy. Utilizing the perfectly transparent eggs of the hydrozoan jellyfish Cytaeis uchidae, here we show that the polyspermy block is accomplished via a novel mechanism: a collaboration between Ca²⁺ and mitogen-activated protein kinase (MAPK). In Cytaeis, adhesion of a sperm to the animal pole surface of an egg was immediately followed by sperm–egg fusion and initiation of an intracellular Ca²⁺ rise from this site. The elevated Ca²⁺ levels lasted for several minutes following the sperm–egg fusion. The Ca²⁺ rise proved to be necessary and sufficient for a polyspermy block, as inhibiting a Ca²⁺ rise with EGTA promoted polyspermy, and conversely, triggering a Ca²⁺ rise by inositol 1,4,5-trisphosphate (IP₃) or excess K⁺ immediately abolished the egg’s capacity for sperm–egg fusion. A Ca²⁺ rise at fertilization or by artificial stimulations evoked dephosphorylation of MAPK in eggs. The eggs in which phosphorylated MAPK was maintained by injection of mRNA for MAPK kinase kinase (Mos), like intact eggs, exhibited a Ca²⁺ rise at fertilization or by IP₃ injection, and shut down the subsequent sperm–egg fusion. However, the Mos-expressing eggs became capable of accepting sperm following the arrest of Ca²⁺ rise. In contrast, addition of inhibitors of MAPK kinase (MEK) to unfertilized eggs caused MAPK dephosphorylation without elevating Ca²⁺ levels, and prevented sperm–egg fusion. Rephosphorylation of MAPK by injecting Mos mRNA after fertilization recovered sperm attraction, which is known to be another MAPK-dependent event, but did not permit subsequent sperm–egg fusion. Thus, it is possible that MAPK dephosphorylation irreversibly blocks sperm–egg fusion and reversibly suppresses sperm attraction. Collectively, our data suggest that both the fast and late mechanisms dependent on Ca²⁺ and MAPK, respectively, ensure a polyspermy block in jellyfish eggs.     

Mechanistic studies of the plasma membrane block to polyspermy in mouse eggs

The mechanisms responsible for the plasma membrane associated block to polyspermy in mouse eggs were studied. Reinsemination experiments using zona-free eggs indicated that, after fertilization, the egg plasma membrane is altered such that sperm binding to the egg plasma membrane is blocked, except in the region of the second polar body. Activation of the egg with either ethanol or strontium chloride did not result in a block to polyspermic penetration, as artificially activated eggs displayed identical penetration levels as to nonactivated control eggs. The penetrability of activated eggs was not altered by the presence or absence of the zona pellucida during activation. Lectin staining for egg cortical granule material indicated that activation did cause cortical granule exocytosis; however, activated eggs remained penetrable. These data support the following conclusions: (1) an alteration in the ability of the egg plasma membrane to allow sperm adherence accounts for the block to polyspermy; (2) establishment of the plasma membrane block to polyspermy is sperm dependent, since artificial egg activation does not result in a block response; (3) the contents of the egg's cortical granules do not play a role in the establishment of the plasmalemma block response. © 1993 Wiley-Liss, Inc.     

Studies of the kinetics and ionic requirements for the phosphorylation of ribosomal protein S6 after fertilization of Arbacia punctulata eggs

Fertilization of the eggs of the sea urchin Arbacia punctulata is followed by the phosphorylation of ribosomal protein S6. The increase in phosphorylation starts at the same time that protein synthesis begins to increase, and leads to the appearance of mono-, di-, and triphosphorylated S6 derivatives. Essentially all the S6 is phosphorylated by first cleavage. This phosphorylation requires the occurrence of both the normal Ca²⁺ transient and the consequent Na⁺H⁺ exchange. Protein synthesis can be partially activated by an increase in intracellular pH brought about by weak bases, but this neither causes S6 phosphorylation, nor the inactivation of the specific S6 phosphatase present in unfertilized Arbacia eggs.     

Growth in high-K+ medium induces Friend cell differentiation.

Friend erythroleukemic cells can be induced to differentiate by growth in high-K⁺ medium. Growth of Friend cells in medium containing 60–90 mM K⁺ and 90-60 mM Na⁺ (keeping the osmotic pressure constant) induced differentiation as measured by iron-59 incorporation into heme, accumulation of globin mRNA, the appearance of benzidine-positive cells, and the expression of erythrocyte membrane antigens. In addition, these “high-K⁺, low-Na⁺” conditions were synergistic with low doses of dimethylsulfoxide (DMSO) for the induction of erythroid differentiation. Not all Friend cell clones examined could be induced to differentiate in high-K⁺, low-Na⁺ medium alone, but the synergism between DMSO and high-K⁺, low-Na⁺ was observed in all cases.     

Voltage response to fertilization and polyspermy in sea urchin eggs and oocytes

Successful collision rates of sperm with eggs and oocytes of the sea urchins Psammechinus microtuberculatus and Paracentrotus lividus have been studied using an electrophysiological method. A monospermic response in eggs consists of a 1- to 2-mV step depolarization of the egg plasma membrane accompanied by an increase in voltage noise. The step precedes the main positive-going depolarization by approximately 13 sec at room temperature. If other successful collisions occur during this 13-sec period (indicated by additional steps), the egg is polyspermic. It is shown by direct observation that each step depolarization signifies the entry of a single sperm. No evidence for an electrically mediated fast block was found. The average rate of successful sperm-egg encounters increases with sperm density, although individual steps appear to occur randomly. Step depolarizations also occur in oocytes, however, they usually decay after several seconds and are not followed by a large, positive-going depolarization. The rate of occurrence of such steps increases with sperm density over the range 10⁵ to 10⁹ sperm/ml. The original evidence of Rothschild and Swann for a fast partial block is compared with a model of polyspermy suggested by our experiments. Reasonable agreement between our method of counting successful collisions (in oocytes and eggs) and the method used by Rothschild and Swann (for eggs) was obtained for sperm densities below 10⁶/ml. The results diverge for higher sperm densities, our method giving higher values. A test for the hypothesis of a fast partial block to polyspermy is suggested, using our method of counting successful sperm-egg collisions.     

The polyspermy block in eggs of Urechis caupo: Evidence for a “rapid” block

Experiments have established the presence and time course of a polyspermy block in eggs of the echiuroid worm Urechis caupo. At various times after an initial insemination the eggs were reinseminated with concentrated sperm suspensions and the polyspermy determined. The block is rapid; partial protection against polyspermy is evident within a few seconds after insemination of the eggs. Although there are other rapid responses approximately correlated in time with the beginning of the block and possibly related to it, I also consider the possibility that a relatively ‘late’ event might account for the apparent ‘rapid’ block in these eggs.     

Abnormal fertilization is responsible for reduced fecundity following thiram-induced ovulatory delay in the rat

Brief exposure to some pesticides, applied during a sensitive window for the neural regulation of ovulation, will block the preovulatory surge of LH and, thus, delay ovulation. Previously, we have shown that a single i.p. injection of 50 mg/kg of thiram, a dithiocarbamate fungicide that decreases norepinephrine synthesis, on proestrus (1300 h) suppresses the LH surge and delays ovulation for 24 h without altering the number of oocytes released. However, when bred, the treated dams had a decreased litter size and increased postimplantation loss. We hypothesized that the reduced litter size in thiram-delayed rats was a consequence of altered oocyte function arising from intrafollicular oocyte aging. To test this hypothesis, we examined delayed oocytes, zygotes, and 2-cell embryos for evidence of fertilization and polyspermy. In addition, we used confocal laser-scanning microscopy to evaluate and characterize cortical granule localization in oocytes and release in zygotes, because the cortical granule response is a major factor in the normal block to polyspermy. Our results demonstrate that a thiram-induced, 24-h delay in ovulation alters the fertilizability of the released oocyte. Although no apparent morphological differences were observed in the unfertilized mature oocytes released following the thiram-induced delay, the changes observed following breeding include a significant decrease in the percentage of fertilized oocytes, a significant increase in polyspermic zygotes (21%), and a 10-fold increase in the number of supernumerary sperm in the perivitelline space. Importantly, all the polyspermic zygotes exhibited an abnormal pattern of cortical granule exudate, suggestive of a relationship between abnormal cortical reaction and the polyspermy in the delayed zygotes. Because polyspermy is associated with polyploidy, abnormal development, and early embryonic death, the observed polyspermy could explain the abnormal development and decreased litter size that we observed previously following thiram-delayed ovulation.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : II. STIMULATION OF CELLULAR OXIDATION AND REVERSIBLE INHIBITION OF CELL DIVISION BY DIHALO AND TRIHALOPHENOLS

The dihalo and trihalophenols, and phenols containing both halo and nitro substituents in the same molecule, produce, in fertilized eggs of Arbacia punctulata, a rise in rate of oxygen consumption and a reversible block to cell division. To define the conditions which affect the degree of this activity, the following factors have been varied: the arrangement of substituents in the molecule, the concentration of reagent, and the time after fertilization at which the reagent is added. The stimulation of oxygen consumption and reversible block to cell division produced by the dihalophenols are qualitatively the same as those previously produced in fertilized Arbacia eggs by certain dinitrophenols. To yield optimum respiratory effect and maximum division block, it usually requires a higher concentration of dihalo than of the corresponding dinitrophenol. For example, with fertilized Arbacia eggs at 20°C. 2,4-dinitrophenol, in optimum concentration of 3 x 10^–5 molar, raises oxygen consumption to 292 per cent of normal (4). The corresponding values for two dihalo analogues are: 2,4-dichlorophenol, 10^–4 molar and 236 per cent; 2,4-dibromophenol, 6 x 10^–5 molar and 282 per cent. The halophenols differ from the nitrophenols in two interesting respects: (a) The monohalophenols produce little or no oxidative stimulation or division block in fertilized Arbacia eggs; p-nitrophenol is very active in both respects. (b) The symmetrical trihalophenols have an appreciable ability to stimulate oxygen consumption and block division; symmetrical trinitrophenol is inactive in both respects (4). The increases in oxygen consumption produced in fertilized Arbacia eggs by 2,4-dichloro and 2,4-dinitrophenol are larger than the percentage increases given by methylene blue and o-cresol indophenol under the same experimental conditions. The dihalo and dinitrophenols produce a reversible block to the cell division of fertilized marine eggs. The oxidation-reduction indicators, in contrast to the dihalo and dinitrophenols, block cell division irreversibly and fertilized eggs of Arbacia do not recover from optimum respiratory stimulating concentrations of these oxidation-reduction dyes. The present experiments with halophenols are in harmony with and lend considerable support to the hypothesis (4) that nitro and similarly substituted phenols derive their biological activity from the presence and properties of the phenolic OH group, as modified by proper substitution in the phenolic benzene ring.     

A sodium-dependent, fast block to polyspermy occurs in eggs of fucoid algae

More than 70% of Pelvetia fastigiata eggs and about 15% of Fucus distichus eggs become polyspermic when fertilized at natural sperm concentrations in a low-sodium (2.5 mM Na+, 450 mM N-methyl glucamine) artificial seawater. Natural levels of polyspermy are 1-3% for both species. Polyspermic eggs germinate and respond to photopolarization, but do not develop beyond an abnormal, "stumpy," four-cell stage. They die within 1-1.5 weeks. The sodium-dependent block is a fast block, and it is replaced by a second block (probably cell wall formation) no later than 9 min (Pelvetia) after eggs are shed. The sodium-dependent block in Pelvetia is very efficient; when external sodium is raised to only 47.5 mM, the level of polyspermy drops to about 25%. These results are compared with data on marine invertebrates in the context of factors such as the sperm/egg concentration at fertilization and natural, osmotic (salinity) stress.     

Voltage Noise Changes during Monospermic and Polyspermic Fertilization of Mature Eggs of the Anuran, Rana temporaria

The electrical response of mature anuran eggs to the fertilizing sperm consists of a rapid depolarization and a decrease in resistance of the plasma membrane (fertilization potential) and serves as a fast block to polyspermy. We report here that the fertilization potential, previously thought to be the earliest electrical response of the egg, is preceded in Rana temporaria by changes in voltage noise. Voltage noise was recorded after insemination and compared in monospermic and NaI-induced polyspermic eggs. Fertilization potential in monospermic eggs arised at 1 min 45 sec to 2 min 15 sec after insemination, and that in NaI-induced polyspermic eggs did at 3 min to 3 min 30 sec after insemination. However, the increase in voltage noise was detected at the similar time (1–2 min 30 sec) after insemination in both the eggs. The duration of voltage noise increase before the fertilization potential was larger in polyspermic eggs (50–105 sec) than in monospermic eggs (10–40 sec). Polyspermic fertilization in Rana temporaria induced by NaI was checked by visualizing multiple sperm entry sites with the scanning microscope. The process of sperm entry and the development of the fertilization body are similar to those occurring with monospermic fertilization; furthermore all supernumerary sperm fuse only with the animal hemisphere of the egg. Although the physiological basis of the changes in voltage noise is unclear, these alterations appear to be the earliest electrical response to sperm yet reported.     

Localization of cortical granule constituents before and after exocytosis in the hamster egg

Electrical activation of the hamster egg was used to study cortical granule constituents before and after exocytosis. The activated hamster eggs underwent cortical granule decondensation just prior to and at the time of exocytosis. Some of the cortical granules of aged, unactivated eggs underwent similar changes. FITC- and gold-conjugated Lens culinaris agglutinin (LCA) bound intensely to the surfaces of activated but not unactivated eggs. This labelling was associated with the microvilli. Permeabilized eggs exhibited discrete cortical labelling before activation, with a subsequent decrease following the cortical reaction. Gold-conjugated LCA specifically bound to cortical granules when incubated with thin sections. FITC-soybean trypsin inhibitor (SBTI) bound in discrete foci in the cortex of unactivated eggs. Following activation, cortical labelling by SBTI decreased. Aprotinin and benzamidine hydrochloride inhibited FITC-SBTI from binding to the egg cortex. Gold-avidin localization of biotin-SBTI in the electron microscope demonstrated that condensed cortical granules did not bind SBTI but decondensed or exocytosing granules did. This suggests that a cortical granule protease is exposed just prior to exocytosis. Activated eggs exhibited dramatic decreases in the number of hamster sperm penetrating the cytoplasm, suggesting that a plasma membrane block to polyspermy is temporally related to cortical granule exocytosis.     

Sodium and potassium balance of captive meadow voles (Microtus pennsylvanicus) fed laboratory chow and vegetation diets

• 1.1. Mineral balance was studied in meadow voles (Microtus pennsylvanicus) maintained in the laboratory.
• 2.2. Urine and fecal Na⁺ contents of voles on low-Na⁺ diets were comparable to those reported for other herbivore species, but urine and fecal K levels were higher.
• 3.3. Voles approached Na⁺ balance (input = output) on diets with Na⁺ content as low as 56 ppm.
• 4.4. There was not a clearcut hypertrophy of the adrenal-gland zona glomerulosa in voles maintained on low-Na⁺ diets.
• 5.5. Plasma K content and bone water content were higher in voles maintained on high-Na ⁺ vegetation diets, suggesting expansion of extracellular fluid volume.

     

U.V. Irradiation Inhibits The Electrical Block to Polyspermy in Echinoderms*

Oocytes of the sea urchin Sphaerechinus granularis and the startish Marthasterias glacialis have been submitted to U.V. irradiation before fertilization. This treatment significantly increased the incidence and severity of polyspermy in the sea urchin and was also found effective on starfish oocytes. These were found more resistant to damage than sea urchin eggs and U.V. irradiation did not affect either their response to the hormone l-methyladenine or the rate of elevation of the fertilization envelope, which assures the late and definitive block to polyspermy. Electrophysiological measurements performed on M. glacialis oocytes definitively demonstrate that U.V. irradiation completely inactivates voltage-dependent sodium channels, without altering the other main conductances, Cl^?, K⁺ or Ca²⁺. After such a treatment, the relative permeability of the membrane to Na⁺ as compared to K⁺ shifted from 0.019±0.003 to 0.003±0.002 and only the calcium component of the action potentials could be observed. However, a fertilization potential, preceded by small sperm induced steps, is still present in these conditions, although its peak and plateau level are greatly reduced. These new findings are discussed, which confirm the electrical nature of the fast block to polyspermy but question about the specificity of those sperm-gated channels which are supposed to trigger the fertilization potential.