Fertilization acid release in Urechis eggs: I. The nature of the acid and the dependence of acid release and egg activation on external pH

The amount of fertilization acid produced by eggs of Urechis caupo, monitored by automatically back-titrating egg suspensions with base, depends linearly on the pH of the seawater. Above pH 7.0, at which no acid is released (Paul, M., Dev. Biol.43, 299–312, 1975), acid release increased approximately 0.34 pmole/egg/0.1 pH unit. Activation (germinal vesicle breakdown) depended on the amount of acid release in natural seawater; it did not occur if eggs released <1.5 pmole acid/egg. When fertilization acid is released into HCO^?₃-free seawater and the pH permitted to decrease, the supernatant can be tested for the presence of a volatile acid, such as CO₂, by bubbling with N₂ and comparing the increase in pH as volatile acid is driven off with experiments in which HCl or CO₂ is substituted for fertilization acid. An increase in pH of <0.2 pH units occurred on N₂ bubbling when fertilization acid or HCl was used to acidify HCO^?₃-free seawater compared to an increase of >0.5 pH units when CO₂ was used. Therefore, most, if not all, of Urechis fertilization acid is not volatile, and since Paul (1975) showed that it is not a nonvolatile weak acid, it must be H⁺.     

A volatile inhibitor immobilizes sea urchin sperm in semen by depressing the intracellular pH

Sea urchin spermatozoa are normally immotile in semen, but motility can be initiated by increasing gas flow over the semen--for example, by blowing N2 gas over a thin layer of semen. This result indicates that sperm motility is not O2 limited and suggests that seminal fluid contains a volatile inhibitor of motility which is responsible for the paralysis of sperm in semen. This inhibitor might be carbon dioxide, which reversibly immobilizes sperm. 31P-NMR measurements of pH show that the sperm intracellular pH (pHi) increases by 0.36 pH unit upon dilution of semen into seawater. Since previous studies have shown that this magnitude of pH increase is sufficient to trigger sperm motility, we suggest that the volatile inhibitor is inhibiting sperm motility in semen by depressing the pHi. A simple hypothesis that explains these observations is that the volatile motility inhibitor is CO2, which could acidify pHi as a diffusable weak acid. In this regard, sperm diluted into seawater release acid, and this acid release is related to the pHi increase and motility initiation. In fact, nearly half of the acid released by sperm upon dilution is volatile and may therefore be due to CO2 efflux. Most of the acid, however, cannot be attributed to CO2 release because it is not volatile. Thus, when sperm are diluted into seawater, they raise their pHi by releasing CO2 and protons from the cytoplasm into the surrounding seawater.     

Fertilization acid of sea urchin eggs: Evidence that it is H+, not CO2

The report of R. J. Gillies, M. P. Rosenberg, and D. W. Deamer (1981, J. Cell. Phys., 108, 115–122) that sea urchin fertilization acid is anaerobically produced CO₂, was reinvestigated by inseminating Strongylocentrotus purpuratus eggs in HCO^?₃-free seawater, then bubbling the seawater with N₂ to remove volatile acid. Fertilization acid production occurred in HCO^?₃-free seawater and with N₂-bubbling, the pH rose 0.28 ± 0.08 unit, significantly less than the rise of 0.63 ± 0.14 unit during N₂-bubbling of HCO^?₃-free seawater that had been acidified with CO₂ and similar to the rise of 0.18 ± 0.07 unit when acidification was with HCl. We conclude that most, if not all, of the sea urchin fertilization acid is nonvolatile and thus is not CO₂; since it is not a weak acid, it must be H⁺.     

Isoelectric precipitation of soybean protein using carbon dioxide as a volatile acid

A novel process is presented for the isoelectric precipitation of soy protein, using carbon dioxide as a volatile acid. By contacting a soy meal extract with pressurized carbon dioxide, the solution pH was decreased to the isoelectric region of the soy proteins. Complete precipitation of the precipitable soy proteins could be achieved for protein concentrations up to 40 g/l at pressures less than 50 bar. Isoelectric precipitation with a volatile acid enabled accurate control of the solution pH by pressure and eliminated the local pH overshoot, usual in conventional precipitation techniques. The advantage of the improved precipitation control was reflected by the morphology of the precipitate particles. Protein aggregates formed by CO₂ were perfectly spherical whereas protein precipitated by sulfuric acid had an irregular morphology. The influence of process variables to control particle size is discussed.     

Calcium-mediated mitochondrial movement in ascidian sperm during fertilization.

Sperm of the solitary ascidian Ascidia ceratodes shed their mitochondria on contact with the chorion. The mitochondrion forms a sphere which slides down the sperm tail ultimately to be released in about 2 min. During this process the sperm emits acid into the surrounding seawater, lowering the pH. Raising the external pH to 9.5 stimulates the shedding process in the absence of eggs. This requires Ca²⁺ but not Na⁺. Removal of Na⁺ from the medium also induces the reaction in the presence of Ca²⁺. Sperm freshly diluted in pH 9.5 seawater require 2.5 min for 50% fertilization, while the same sperm 13 min later require only 1 min, suggesting that mitochondrial shedding is rate limiting in fertilization. Acid release can be induced by diluting the sperm in Na⁺-free seawater containing Ca²⁺ or by the ionophores A23187 or Nigericin. The necessity of Ca²⁺ for both acid release and the morphological changes suggests that an exchange of Ca²⁺ for H⁺ may be occurring which triggers the shedding process.     

Calcium influx following fertilization of Urechis caupo eggs.

Measurements of ⁴⁵Ca flux into and out of Urechis eggs indicate that, during the first 10 min after insemination, the eggs take up 0.24 pmole of Ca/egg. Total egg Ca measured by atomic absorption (AA) spectroscopy increased by 0.23 pmole of Ca/egg (0.56, 0.79, and 0.76 pmole of Ca/egg for unfertilized, 10-min fertilized, and 60-min fertilized eggs, respectively). Thus, the total change in egg Ca is accounted for by the influx even though the rate of efflux, measured as a release of ⁴⁵Ca from preloaded eggs, increases to twice the unfertilized rate by 15 min. The fertilization influx follows saturation kinetics (K_a = 1.3 mM). It is competitively inhibited by procaine, but is not inhibited by dinitrophenol, mersalyl acid, or ruthenium red. Ten percent of the total Ca influx has occurred by 10 sec, and it is, therefore, the most rapid response to fertilization yet known in these eggs. The influx is also observed in eggs partially activated by insemination in pH 7 seawater (SW); the other fertilization responses, except sperm penetration, do not occur in pH 7 SW. Although Ca influx alone is insufficient to activate the eggs, it may be a prerequisite for cytoplasmic activation and development, inducing other secondary responses which are prevented by low external pH.     

Intracellular pH of sea urchin eggs measured by the dimethyloxazolidinedione (DMO) method

Intracellular pH (pH1) of sea urchin eggs and embryos was determined using DMO (5,5-dimethyl-2,4-oxazolidinedione). By this method, the pH1 of Lytechinus pictus eggs increased after fertilization from 6.86 to 7.27, and this higher pHi was maintained thereafter, as has been previously observed with pH microelectrodes. The same general result was obtained with the eggs of Strongylocentrotus purpuratus, in contrast to previous estimates of the pH of egg homogenates from this species, which had indicated a rise and then fall of pHi after fertilization. pHi did not significantly change during early cell divisions. Studies of treatments that alter pHi confirmed that ammonia alkalizes and acetate acidifies the cells. The regulation of pHi by embryos in the acidic seawater is impaired if sodium is absent, whereas unfertilized eggs can regulate pHi in acidic, sodium-free seawater.     

Sodium-potassium exchange in sea urchin egg. II. Ionic events stimulating the Na+-K+ pump activity at fertilization

The preceding paper (Ciapa et al., 1984) provided biochemical and kinetic characterization of the Na+-K+ exchange in Paracentrotus lividus eggs. The present work is a study of the ionic events involved in the stimulation of the Na+-K+ transporter after fertilization. Fertilization in low Na+-external medium containing amiloride (0.1 mM) suppresses the stimulation of the net efflux of H+ and 86Rb uptake. Activation of eggs with the ionophore A23187 leads to stimulation of both Na+-H+ exchange and ouabain-sensitive 86Rb influx. When eggs were activated with A23187 in artificial seawater, 86Rb uptake and 24Na influx showed similar saturable kinetics with respect to the external Na+. A23187 treatment of eggs in Na+-free artificial seawater did not stimulate the Na+-K+ exchange until 10 mEq Na+ was added. Activation of eggs by NH4Cl (5 mM) stimulated 86Rb influx and Na+ exit; both fluxes were ouabain sensitive. Monensin increased cell Na+ of unfertilized eggs without any significant increase in intracellular pH: a condition in which 86Rb influx was not markedly stimulated. Addition of 10 mEq Na+ to unfertilized eggs in Na+-free artificial seawater stimulated 86Rb uptake but to a lower extent that did 10 mEq Na+ plus sperm. It is concluded that (1) the stimulation of the Na+-K+ pump at fertilization has an absolute requirement for the Na+-H+ exchange; (2) the alkalinization of eggs resulting from the acid efflux is a prerequisite for the enhancement of the Na+-K+ pump; (3) the amount of Na+ entering eggs at fertilization determines the intensity of the Na+-K+ exchange; (4) early events of fertilization such as exocytosis and calcium release which may be involved in the stimulation of the Na+-K+ pump must necessarily be coupled to cell alkalinization.     

Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg

Experiments have been carried out to test the proposal that the pH increase at fertilization in sea urchin eggs promotes microvillar elongation. Results presented herein show that microvillar elongation and microfilament formation occurred when sea urchin eggs were incubated in sodium-free seawater containing the calcium ionophore A23187, a treatment which initiates activation, i.e., induces a transient increase in intracellular free calcium, but prevents subsequent cytoplasmic alkalinization. Within elongated microvilli and cortices of these eggs, microfilaments were arranged in a loose meshwork. However, if the pH of the egg cytoplasm was increased experimentally, microfilament bundles appeared within individual microvilli. These findings suggest that: (1) microvillar elongation and microfilament formation in the sea urchin egg at fertilization may occur when cytoplasmic alkalinization is inhibited, and (2) formation of the microvillus bundle of microfilaments at egg activation is pH sensitive. Additionally, if the cytoplasmic pH of unfertilized eggs was experimentally elevated by NH₄Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.     

CO2-induced fertilization impairment in Strongylocentrotus droebachiensis collected in the Arctic

Fertilization depends on distribution and aggregation patterns of sea urchins which influence gamete contact time and may potentially enhance their vulnerability to ocean acidification. In this study, we conducted fertilization experiments to assess the effects of selected pH scenarios on fertilization success of Strongylocentrotus droebachiensis, from Spitsbergen, Arctic. Acidification was achieved by aerating seawater with different CO₂ partial pressures to represent pre-industrial and present conditions (measured ~180–425 µatm) and future acidification scenarios (~550–800, ~1,300, ~2,000 µatm). Fertilization success was defined as the proportion of successful/unsuccessful fertilizations per treatment; eggs were classified according to features of their fertilization envelope (FE), hyaline layer (HL) and achievement of cellular division. The diagnostic findings of specific pathological aberrations were described in detail. We additionally measured intracellular pH changes in unfertilized eggs exposed for 1 h to selected acidification treatments using BCECF/AM. We conclude that (a) acidified conditions increase the proportion of eggs that failed fertilization, (b) acidification may increase the risk of polyspermy due to failures in the FE formation supported by the occasional observation of multiple sperms in the perivitelline space and (c) irregular formation of the embryo may arise due to impaired formation of the HL. The decrease in fertilization success could be also related to the observed changes in intracellular pH at pCO₂ ~ 1,000 μatm or higher.     

Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle.     

Release of acid and changes in light-scattering properties following fertilization of Urechis caupo eggs.

The release of a fertilization acid, monitored by measuring the pH of egg suspensions, begins within 10 sec of insemination of Urechis caupo eggs. This is 4 min before the vitelline layer begins to elevate and is apparently unrelated to that process. The eggs of two molluscs, Mytilus californianus and Acmaea incessa, do not form a fertilization acid. The acid of Urechis eggs is not accompanied by release of “fertilization” carbohydrate, sulfate, or a nonvolatile weak acid into the seawater. The light-scattering properties of Urechis eggs change during the first 10 min after insemination. A decrease in light scattering begins by 10 sec and is complete by 1 min (Phase I). This is followed by a further decrease (3–6 min, Phase II) and an increase (6–10 min, Phase III). In striking contrast to an overtly similar situation in sea urchin eggs (fertilization acid and coincident light-scattering decrease), the release of acid and the initial light-scattering change are not the result of cortical granule discharge, and the acid, at least, is not related to the changes in shape or surface area which the eggs undergo. The processes underlying these rapid events are not yet known.     

Ocean acidification alters sperm responses to egg-derived chemicals in a broadcast spawning mussel

The continued emissions of anthropogenic carbon dioxide are causing progressive ocean acidification (OA). While deleterious effects of OA on biological systems are well documented in the growth of calcifying organisms, lesser studied impacts of OA include potential effects on gamete interactions that determine fertilization, which are likely to influence the many marine species that spawn gametes externally. Here, we explore the effects of OA on the signalling mechanisms that enable sperm to track egg-derived chemicals (sperm chemotaxis). We focus on the mussel Mytilus galloprovincialis, where sperm chemotaxis enables eggs to bias fertilization in favour of genetically compatible males. Using an experimental design based on the North Carolina II factorial breeding design, we test whether the experimental manipulation of seawater pH (comparing ambient conditions to predicted end-of-century scenarios) alters patterns of differential sperm chemotaxis. While we find no evidence that male–female gametic compatibility is impacted by OA, we do find that individual males exhibit consistent variation in how their sperm perform in lowered pH levels. This finding of individual variability in the capacity of ejaculates to respond to chemoattractants under acidified conditions suggests that climate change will exert considerable pressure on male genotypes that can withstand an increasingly hostile fertilization environment.     

Artificial Insemination 'Regulated by EDTA' in the Monoecious Brown Alga Fucus evanescens

As part of an attempt to control the fertilization of a monoeciousbrown alga Fucusevanescens, the effects of EDTA on the releaseand fertilization of gametes were studied. When receptacleswere treated for liberation of gametes by soaking in plain seawater,egg-packets and spermatophores were discharged from oogoniaand antheridia, respectively, with the gametes retained withintheir packing envelopes. After a while, the envelopes were disruptedand the gametes were released. Thirty min after the soaking,over 99% of eggs were fertilized. However, when receptacleswere soaked in seawater that contained 0.5 mg m1^–1 Na₂.EDTAat 4°C, the release and fertilization of gametes were preventedafter egg-packets and spermatophores had been released fromthe receptacles. Release of gametes from such egg-packets andspermatophores occurred rapidly when the medium was dilutedwith an excess of plain seawater. The chelating agent affectedthe disruption of egg-packets and spermatophores, but it didnot affect the subsequent fertilization and development of thefertilized eggs. On the basis of these results, normal unfertilizedeggs and sperm were isolated separately by filtration and centrifugationof the released packing envelopes in the presence of 0.5 mgml^–1 Na₂EDTA at 4°C. Artificial insemination usingthe isolated gametes was successful. ³ Present address: Akan Board of Education, Akan, 085-02 Japan.     

Acid release,cytoplasmic alkalinization,and chromosome condensation during sea urchin fertilization and amine-lnduced parthenogenesis

We have studied the relationship between acid release, cytoplasmic alkalinization, and the extent of chromosome condensation during parthenogenetic activation of sea urchin eggs. The relative rate of acid release in Strongylocentrotus purpuratus eggs was determined from pH measurements of egg suspensions. Acid release in inseminated eggs began after a lag of 0.4 min and the relative rate increased 108-fold, declined, and release was essentially complete by 8-min postinsemination. An average of 3.8 ± 0.23 × 10^?12moles H⁺ cell^? was released as determined by backtitration with NaOH. Acid release characteristics of eggs parthenogenetically activated with either NH₄C1, methylamine ethylamine, n-propylamine, n-butylamine, or benzylamine were qualitatively similar. There was no detectable lag peroid and the increase in relative rate of acid release was directly proportional to the carbon number of the amine used, eg, from 8.3-fold methylamine to 470-fold with benzylamine. The total equivalents of acid released ranged from 0.50–8.2 × 10^?12 moles H⁺·cell^? in direct proportion to the concentration of amine used. The degree fo cytoplasmic alkalinization induced as a function of methylamine and benzylamine concentration was determined by pH measurements fo egg homogenates; egg cultures were also prepared for microscopic examination of chromosome condensation. None of the eggs had condensed chromosomes at 0.5-mM methylamine whereas a cytoplasmic alkalinization of 0.6 pH units was observed. Increased methylamine levels up to 10mM resulted in chromiosome condensation in only 20% of the eggs. A similar result was found with benzylamine. We conclude that acid release and cytoplasmic alkalinization during chemical parthenogenesis are insufficient to mimic sperm induction of chromiosome condensation and suggest that an additional factor(s) is required for chromosome condensation by low concentration of amines.     

Activation of sea urchin eggs by inositol phosphates is independent of external calcium.

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.     

Hardening of the sea urchin fertilization envelope by peroxidase-catalyzed phenolic coupling of tyrosines

Within minutes after its elevation from the egg surface, the sea urchin fertilization envelope (FE) becomes "hardened" by a reaction that renders it resistant to agents that solubilize, denature or degrade most proteins. Peroxidase activity is released into the surrounding seawater from Stronglyocentrotus purpuratus eggs during fertilization. Evidence from several sources indicate that the catalytic action of the peroxidase is responsible for hardening the FE through the phenolic coupling of tyrosyl residues of the FE proteins. First, the peroxidase is localized within the hardened FE and within the crystalline FE precursor material released from egg cortical granules during the fertilization reaction. Second, a direct correlation is established between the effectiveness of compounds in inhibiting the cortical granule peroxidase (CGP) and their effectiveness in inhibiting hardening of the FE. Third, the CGP catalyzes the cross-linking of tyrosines in solution, a reaction known to be catalyzed by horseradish peroxidase (HRP). Fourth, acid hydrolysates of hardened FEs contain cross-linked tyrosines that are identified by comparing their chromatographic ultraviolet absorption and fluorescent characteristics to those known for cross-linked tyrosines formed by HRP. Finally, when eggs are fertilized in the presence of 125I, the CGP heavily labels proteins of the FE and of the crystalline FE precursor material released with the enzyme from the cortical granules. The iodide label reflects the localization of the CGP and may reflect the sites of peroxidase-generated tyrosyl phenyl radicals involved in the tyrosine coupling reaction. Maximal iodide labeling occurs during the first 5 min period following fertilization, corresponding to the period of FE hardening.     

Formation of Short-Chain Fatty Acids from H2 and CO2 by a Mixed Culture of Bacteria

The biological utilization of CO₂ and H₂ for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37°C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Glycolipid linkage of a polyspermy blocking glycosidase to the ascidian egg surface.

Ascidian eggs release N-acetylglucosaminidase rapidly into the seawater following fertilization. This glycosidase is detected seconds after fertilization, and histochemical tests suggest the cell surface as the prefertilization storage site (Lambert, C. C. (1989). Development 105, 415-420). Living eggs of Ascidia ceratodes, A. callosa, and A. paratropa all cleave a fluorogenic substrate in seawater. Following cell surface biotinylation and activation of the eggs, enzyme activity binds to streptavidin further substantiating the cell surface localization. The released glycosidase has a molecular weight of 180 kDa by size exclusion chromatography and exhibits bands at 62 and 70 kDa by SDS-PAGE, suggesting a possibly multimeric enzyme. The enzyme is released by a glycophosphatidylinositol-specific phospholipase C and HNO2 deamination, both of which are specific indicators of linkage to the cell surface via phosphatidylinositol. The enzyme from unfertilized eggs is quite hydrophobic in Triton X-114 phase partition experiments but becomes hydrophyllic after release by activation or deamination. All of these observations are consistent with the glycosidase being anchored to the cell surface via a GPI anchor that is cleaved at fertilization to yield the soluble form of the enzyme which helps protect the egg against polyspermy. We discuss the possible role of a cell surface PLC in this release.     

Fertilization acid release in Urechis eggs. II. The stoichiometry of Na+ uptake and H+ release

We tested the hypothesis that Na+ uptake and H+ release at fertilization of Urechis eggs might occur via a Na+:H+ exchange. Previous studies have shown that (1) Na+ uptake is proportional to the number of entering sperm in seawater with or without lowered Na+ and (2) H+ release is proportional to external pH. Therefore, to determine if Na+ uptake and H+ release are always proportional, we determined the effect of polyspermy on H+ release in natural and low Na+ seawater and the effect of external pH on Na+ uptake and release. Na+ uptake and H+ release do not covary in a manner consistent with a Na+:H+ exchange. H+ release under most conditions was manner consistent with a Na+:H+ exchange. H+ release under most conditions was independent of the number of sperm/egg and in low Na+ seawater was at most 53 +/- 16% of that in natural seawater. In contrast, Na+ uptake in low Na+ seawater can be more than in natural seawater (Jaffe et al., J. Gen. Physiol. 73, 469-492, 1979). In natural seawater Na+ uptake exceeded H+ release; at pH 7 Na+ uptake was 2 pmol/egg, but there was no H+ release. Since Na+ release did not increase at fertilization at pH 7, neither Na+:Na+ nor Na+:H+ exchange could account for the Na+ uptake. An alternate hypothesis is suggested: Na+ uptake is primarily via the channels responsible for the fertilization potential, while H+ release is by another route that is affected by the membrane potential during the fertilization potential.