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.     

Microvilli in sea urchin eggs : Differences in their formation and type

The elongation of microvilli, which normally occurs upon fertilization in sea urchin eggs, was also observed in unfertilized eggs treated with the enzyme, papain. Cortical granule exocytosis, which is thought to be the source of membrane used in microvillar elongation, does not occur in the papain-treated eggs. It appears, therefore, that there is more than one way in which the egg plasma membrane can increase very quickly and to a great extent. In addition, the kinds of microvilli formed in the two instances appear to be different. Previous work with reaggregating sea urchin cells is also cited to support the suggestion that microvilli can form in different ways and are of different types.     

Activation of amino acid uptake at fertilization in the sea urchin egg. Requirement for proton compartmentalization during cytosolic alkalosis

The comparative importance of the release of intracellular ionic calcium, Na+/H+ exchange and cytosolic alkalosis as activator signals was studied on the development of amino acid uptake at fertilization in sea urchin eggs. We show that, once stimulated, the rate of valine uptake is greatly dependent upon intracellular pH. Suppression of the Na+/H+ exchange at the time of activation, by applying ionophore (A23187) in sodium-free artificial sea water (ONaASW), inhibits the development of valine influx. This cannot be restored by a further (30 min later) alkalosis by transferring eggs into sea water. Suppressing the alkalosis in the presence of Na+/H+ exchange at fertilization by simultaneous addition of acid into sea water results in activation of the amino acid carrier which exhibits an increased rate of transport as soon as the eggs are replaced in sea water at pH 8.0. The absence of alkalosis in eggs activated in ONaASW can be counterbalanced either by adding NH4Cl 10 mM or by transfer into ASW at pH 9.0 at activation. Ammonia-treated eggs absorbed amino acid as controls, whereas eggs in sea water at pH 9.0 failed to develop a valine uptake system, suggesting that ammonia can completely replace the effect of Na+/H+ exchange. Furthermore, addition of NH4Cl immediately before fertilization conceals the Na+/H+ exchange but stimulates valine uptake as in controls. These data suggest that: the occurrence of the intracellular calcium increase alone is not sufficient for the develpment of the amino acid transport system; cell alkalinization at fertilization derives from the cytoplasmic membrane-located Na+/H+ exchange and an inward movement of protons into a cortical acidic compartment, which is discussed.     

Changes in the topography of the sea urchin egg after fertilization

Changes in the topography of the sea urchin egg after fertilization were studied by scanning and transmission electron microscopy. Strongylocentrotus purpuratus eggs were treated with dithiothreitol to modify the vitelline layer and to prevent formation of a fertilization membrane. Dithiothreitol treatment caused the microvilli to become more irregular in shape, length, and diameter than those of untreated eggs. The microvilli were similarly modified by trypsin treatment. This effect did not appear to be due to disruption of cytoskeletal elements beneath the plasma membrane, for neither colchicine nor cytochalasin B altered microvillar morphology. Thus, it appears that the vitelline layer may act in the maintenance of surface form of unfertilized eggs. Since dithiothreitol-treated eggs did not elevate a fertilization membrane, scanning electron microscopy could be used to directly observe modifications in the egg plasma membrane after fertilization. The wave of cortical granule exocytosis initiated at the point of attachment of the fertilizing sperm was characterized by the appearance of pits that subsequently opened, releasing the cortical granule contents and leaving depressions upon the egg surface. The perigranular membranes inserted during exocytosis were seen as smooth patches between the microvillous patches remaining from the original egg surface. This produced a mosaic surface with more than double the amount of membrane of unfertilized eggs. The mosaic surface subsequently reorganized to accommodate the inserted membrane material by elongation of microvilli. Blebs and membranous whorls present before reorganization suggested the existence of an unstable intermediate state of plasma membrane reorganization. Exocytosis and mosaic membrane formation were not blocked by colchicine or cytochalasin B, but microvillar elongation was blocked by cytochalasin B treatment.     

Ca2+ influx and stimulation of protein synthesis in sea urchin eggs

Acid release, Ca2+ influx and stimulation of protein synthesis were investigated with sea urchin eggs submitted to an excess of KCl, to NH4Cl, and to a combination of both. KCl, though unable to promote any acid release, triggers a large 45Ca uptake by eggs and slightly stimulates protein synthesis, provided that external Ca2+ is present. NH4Cl, which induces an intracellular pH increase, triggers a late and small 45Ca uptake but highly stimulates protein synthesis. The combined use of NH4Cl + KCl allows a large 45Ca uptake to occur but the level of protein synthesis is similar to that obtained with NH4Cl alone and is identical whether external Ca2+ is present or not. In contrast to previous works, our results show that the large stimulation of protein synthesis triggered by an intracellular pH increase, as after NH4Cl activation, cannot be enhanced by a Ca2+ influx. This suggests that the Ca2+ influx occurring after fertilization has only a minimal effect on the overall stimulation of protein synthesis.     

Analysis of sea urchin egg cortical transformation in the absence of cortical granule exocytosis

A burst of endocytosis accompanying microvillar elongation follows cortical granule exocytosis in normal sea urchin development. By 5 min postfertilization the burst is over and a lower level of endocytosis ensues (constitutive phase). To determine whether microvillar elongation and initiation of endocytosis are necessary concommitants of cortical granule exocytosis we utilized Chase's (1967, Ph.D. thesis, University of Washington, Seattle) high-hydrostatic pressure technique to block the latter and then examined developing eggs for endocytosis and microvillar elongation. To accomplish this, eggs were fertilized, after which hydrostatic pressure was quickly raised to 6000-7000 psi at the start of cortical granule exocytosis and maintained for 5 min. Only the cortical granules immediately surrounding the sperm penetration site were secreted (about 3% or less of the egg's total number of cortical granules). Blockage of major cortical granule exocytosis had the following consequences on surface events during first division: (1) The endocytosis burst normally associated with cortical granule exocytosis was effectively eliminated as was early microvillar elongation and elevation. Both occurred to a limited extent around the sperm penetration site which resulted in a highly localized surface transformation. (2) By 20 min after fertilization endocytosis began over the rest of the egg surface in the absence of any further cortical granule exocytosis. (3) Subsequently, during a 30-min period starting midway between fertilization and first cleavage microvilli more than doubled in length and endocytosis levels increased severalfold. These events brought about a complete surface transformation similar to that which normally occurs in early development but in the absence of cortical granule exocytosis. By first cleavage surfaces and cortices of high-pressure-treated and control eggs were nearly indistinguishable except for the presence of cortical granules in cortices of the former. Pressure-treated eggs cleaved normally and developed to larval forms overnight. The period of late surface transformation in high-pressure-treated Strongylocentrotus purpuratus eggs corresponds in timing and some of its characteristics to second phase microvillar elongation observed in normal development in this species and also in S. droebachiensis development. These observations suggest, therefore, that microvillar elongation and endocytosis are necessary membrane remodelling events which must occur for normal development even in the absence of membrane addition from the cortical granules.     

The relation between intracellular pH and rate of protein synthesis in sea urchin eggs and the existence of a pH-independent event triggered by ammonia

The relation between rate of protein synthesis and intracellular pH (pHi) was investigated in the eggs of the sea urchin Strongylocentrotus purpuratus. Increasing external pH (pHo) resulted in raising pHi of eggs and also in increased rate of protein synthesis. Similarly, at constant pHo, adding various concentrations of NH4Cl to eggs caused graded increases of both pHi and protein synthesis. Using various concentrations of NH4Cl at a low pHo and incubating eggs at high pHo, we compared protein synthesis under similar pHi conditions and this revealed that at least half the increased protein synthesis stimulated by NH4Cl is independent of induced rise of pHi, as also seems to be chromosome condensation which was never observed in eggs incubated at high pHoS. The additional pH-independent event triggered by NH4Cl does not appear related to elevated free Ca2+, since protein synthesis and chromosome condensation do not require external Ca2+ and no increases of free Ca2+ sufficient to activate the Ca2+-calmodulin-mediated enzyme NAD kinase occurred. Monensin disrupts intravesicular pH gradients but does not stimulate protein synthesis, indicating that this local effect, also promoted by NH4Cl, is not involved in ammonia-induced increase of protein synthesis. Using two other amines which have low pKa values, benzocaine and tricaine, we observed 2-fold increases in protein synthesis rates, even though pHi was lowered. While the exact nature of the pH-independent event(s) triggered by NH4Cl, and possibly by other amines, remains unidentified, its possible involvement in normal mitosis is stressed.     

Spatiotemporal relationships among early events of fertilization in sea urchin eggs revealed by multiview microscopy.

Four early events of egg fertilization, changes in intracellular calcium concentration and intracellular pH, reorientation of the surface membrane, and the elevation of the fertilization envelope, were imaged in real time and in pairs in single sea urchin eggs. The paired imaging allowed the correlation of the four events spatially and temporally. Three of them propagated as waves starting at the sperm entry site. The earliest was the calcium wave, visualized with fluorescent indicator dyes. After a delay of 10 s there followed a large decrease in the fluorescence polarization of membrane-bound dyes, which we interpret as arising from membrane reorientation as a result of cortical granule exocytosis and microvillar elongation. With a further delay of 15 s the fertilization envelope was seen to rise in transmitted light. All three waves propagated with similar velocities of approximately 10 microns/s, supporting the view that calcium triggers the latter two events. The fluorescence polarization changed in two steps with a clear pause of 10-20 s in between. The second step, which also propagated as wave, reflects either further elongation of microvilli or straightening of irregular microvilli. This second step was abolished by cytochalasin B and was coincident with an increase in cytoplasmic pH, suggesting that pH-induced actin reorganization may play a role. The cytoplasmic alkalinization, imaged with a fluorescent probe, was quite different from the other events in that it took place homogeneously throughout the egg and slowly (over 100 s). Apparently, the alkalinization is not on a direct downstream pathway of calcium origin. An opposing possibility, that the alkalinization may in fact be triggered by the traveling calcium wave, is also discussed.     

Changes in intracellular acidic compartments in sea urchin eggs after activation

Acridine orange (AO) was used as a vital probe for looking at acidic intracellular compartments in sea urchin eggs. This weak base is concentrated by acidic compartments, shifting its fluorescence from green to red due to the formation of dye aggregates. Fertilization or parthenogenetic activation with ionophore A23187 resulted in the appearance of orange fluorescent granules of sizes ranging from 1 to 2 microns at the cortical region of the egg. In one species of sea urchin (Lytechinus pictus), these granules migrate inward before cell division and associate with the forming mitotic apparatus. Treatments that discharge the transmembrane pH gradient (NH4Cl, nigericin, monensin, and acidic external pH) eliminate the orange fluorescence, indicating they are acidic compartments. Spectrofluorimetric measurements showed a decrease in monomer fluorescence accompanying egg activation which is reversible by similar treatments as seen with the fluorescence microscopic observations. Stratified eggs which were subsequently fertilized had acidic granules concentrated at the centripetal pole. This allowed the electron microscopic identification of the granules and showed they are present in the unfertilized egg, although not able to concentrate the AO. Activation of eggs in the absence of Na+ prevented the cytoplasmic alkalinization and also inhibited the appearance of acidic granules. The results indicate that the internal pH rises after egg activation triggers the acidification of these granules. Their possible functions, as in intracellular pH regulation, are discussed.     

Uptake and release of calcium by isolated egg cortices of the sea urchin Paracentrotus lividus

Isolated cortices from sea urchin eggs accumulate calcium in a non-mitochondrial store at a rate dependent on ATP and enhanced by oxalate. Calcium efflux is increased by ionomycin and by A23187 which has been used as a parthenogenetic agent, but not by weak base (NH4Cl). Accumulated calcium is partially released by 1,4,5 inositol triphosphate.     

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.     

Internal pH can regulate Ca2+ uptake and the acrosome reaction in sea urchin sperm

The egg jelly-induced acrosome reaction of sea urchin sperm is accompanied by intracellular alkalinization and Ca2+ entry. We have previously shown that in the absence of egg jelly, NH4Cl, which increases intracellular pH (pHi), induces Ca2+ uptake and the acrosome reaction in sperm of the sea urchin, Strongylocentrotus purpuratus. Here we show that at a constant concentration of NH4Cl (20 mM) in seawater, sperm react less as external pH is lowered from the normal 8 to 7.25. The pH dependence of the NH4Cl response is not very sensitive to temperatures between 12 and 17 degrees C. NH4Cl (15-50 mM) stimulates Ca2+ uptake and acrosome reactions in sperm suspended in Na+-free seawater, a condition known to inhibit the inductive effect of jelly. Jelly does not further stimulate Ca2+ uptake of sperm preincubated in NH4Cl, indicating that once the permeability to Ca2+ is increased by raising the pHi, the jelly has no further effect. We have used the membrane potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide to follow the membrane potential change that occurs when NH4Cl is added. Depolarization (25 mV) is associated with the acrosome reaction when either the natural inducer, egg jelly, or NH4Cl is added to sperm. Response to both inducers is inhibited under conditions known to abolish the acrosome reaction, i.e., low-pH seawater and nisoldipine. These results indicate that the NH4Cl-induced depolarization that accompanies the reaction is probably due to the opening of channels that allow Ca2+ to enter the cell and not to the depolarization by NH4+ ions. High-K+ seawater, which depolarizes sperm, and tetraethylammonium, a K+ channel blocker, inhibit the jelly-induced depolarization and the acrosome reaction, but do not inhibit NH4Cl-induced changes. It has already been shown that nigericin promotes Ca2+ entry and the acrosome reaction in sea urchin sperm. We found that the action of this ionophore depends on the pH of normal seawater. In the absence of external Na+ (replaced by choline), nigericin does not induce the reaction and does not stimulate Ca2+ uptake.     

Separation of microvilli from Tubifex eggs upon activation: Its inhibition by concanavalin A hinders ooplasmic segregation and cleavage

The surface of mature eggs of the freshwater oligochaete Tubifex exhibits numerous microvilli. Upon activation, microvilli become narrower at their base and separated from the ooplasmic surface. Here it is shown that concanavalin A (Con A) reversibly inhibits the separation of microvilli from activated Tubifex eggs. The Con A-treated eggs undergo meioses and mitoses at a normal rate. Microvilli on these eggs change their length in a meiotic cycle-dependent manner; their core bundles of microfilaments elongate significantly during the second meiosis. The Con A-treated eggs fail to complete polar body formation, ooplasmic segregation and cleavages. Treatment with Con A of eggs that have accomplished microvillar separation does not exert any inhibitory effect on their development. Succinyl-Con A, a dimeric derivative of Con A, does not prevent microvillar separation, suggesting that the tetravalent form of Con A is essential for Con A to exert its inhibitory effect on microvillar separation.     

Microfilament association of ASGP-2, the concanavalin A-binding glycoprotein of the cell-surface sialomucin complex of 13,762 rat mammary ascites tumor cells

Microfilament-associated proteins and membrane-microfilament interactions are being investigated in microvilli isolated from 13,762 rat mammary ascites tumor cells. "Phalloidin shift" analyses on velocity sedimentation gradients of Triton X-100 extracts of [3H]-glucosamine-labeled microvilli identified a 120-kDa cell-surface glycoprotein associated with the microvillar microfilament core. The identification was verified by concanavalin A (Con A) blots of one- and two-dimensional (2D) electrophoresis gels of sedimented microfilament cores. By 2D-electrophoresis and lectin analyses the 120-kDa protein appeared to be a fraction of ASGP-2, the major Con A-binding glycoprotein of the sialomucin complex of the 13,762 cells. This identity was confirmed by immunoblot analyses using immunoblot-purified anti-ASGP-2 from anti-membrane serum prepared against microvillar membranes. Proteolysis of the microvilli with subtilisin or trypsin resulted in an increase in the amount of ASGP-2 associated with the microfilament cores. An increase was also observed with sialidase treatment of the microvilli, suggesting that negative charges, probably present on the highly sialated sialomucin ASGP-1 of the ASGP-1/ASGP-2 sialomucin complex, reduce ASGP-2 association with the microfilament core. Proteolysis of isolated microvillar membranes, which contain actin but not microfilaments, also increased the association of ASGP-2 with a Triton-insoluble, actin-containing membrane fraction. Purified ASGP-2 does not bind to microfilaments in sedimentation assays. Since the Triton-insoluble membrane residue is enriched in an actin-containing transmembrane complex, which contains a different glycoprotein, we suggest that the ASGP-2 is binding indirectly via this complex to the microfilament core in the intact microvilli.     

Microfilaments during sea urchin fertilization: Fluorescence detection with rhodaminyl phalloidin

Rhodaminyl-labeled phalloidin is used to demonstrate the distribution of microfilaments during fertilization and early development in eggs of the sea urchins Arbacia punctulata and Lytechinus variegatus. The surface of unfertilized eggs have numerous punctate fluorescence sites at which rhodaminyl phalloidin binds, indicating the presence of actin oligomers or polymers. During fertilization this punctate pattern of fluorescence begins to change. Within thirty seconds of insemination, the fertilization cone is first detectable with this technique as an erect structure on the surface of the egg. The fertilization cone grows to a maximum size by 8–9 minutes, and is resorbed by 16 minutes after insemination. The surface of the fertilized egg displays numerous fluorescent fibers by 10 minutes after insemination rather than the punctate fluorescence observed in unfertilized eggs, indicative of the burst of microfilament assembly resulting in microvillar elongation. The elongated microfilaments persist through cytokinesis. Staining is also detected throughout the cortices of unfertilized, fertilized, and cleaving eggs. Cytochalasin E (10 μM, 30 min) prevents microfilament elongation and cytokinesis and reduces the cortical staining intensity after fertilization. At cleavage, contractile rings, appearing as narrow equatorial bundles of fibers, have been detected in Lytechinus variegatus as transient structures.     

Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions.     

Membrane-microfilament interactions in ascites tumor cell microvilli. Identification and isolation of a large microfilament-associated membrane glycoprotein complex

[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with ascites tumor cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.     

Effect of calyculin A on the surface structure of unfertilized sea urchin eggs

Calyculin A, a potent inhibitor of type 1 and type 2A protein phosphatases, induces contractile ring formation when applied to unfertilized sea urchin eggs [Tosuji et al., 1992: Proc. Natl. Acad. Sci. USA 89:10613-10617]. We report here the elongation of microvilli in the unfertilized eggs exposed to calyculin A. The elongated microvilli and associated sperm-egg binding sites (egg receptor for sperm) then became concentrated into a constriction site corresponding to the cleavage furrow. The egg receptor for sperm was also in close connection to the microfilaments. Okadaic acid is another known inhibitor of protein phosphatase type-1 and type-2A. Its effect, however, is about a hundredfold feebler for type-1 phosphatase than type-2A. Even after treatment with okadaic acid, no change was observed, suggesting that these morphological changes were induced by calyculin A solely though its inhibitory effect on the type-1 protein phosphatase.     

Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.     

The Role of Intracellular pH in Fertilization of Sand Dollar Eggs Analyzed by Microinjection Method

The change in intracellular pH (pH_i) upon fertilization and the effects of changing the pH_i by microinjection of pH buffers were investigated in the eggs of the sand dollar, Clypeaster japonicus. The pH_i was determined by the tint of a pH indicator, phenol red, microinjected into eggs. The pH_i ranged from 6.5 to 6.75 in unfertilized eggs and it rose by 0.4 to 0.5 unit within 3 min upon fertilization. The elevated pH_i ranging from 7.0 to 7.25 was maintained at least until the first cleavage. As reported in eggs of other species of sea urchin (1–4), development of fertilized eggs which had been transferred to Na-free sea water immediately after insemination was arrested and the pH_i did not rise remaining at the level of unfertilized eggs. Development was initiated in eggs arrested in Na-free sea water when the pH_i was elevated up to the level of fertilized eggs, i.e. 7.0 to 7.25, by microinjecting 1 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-KOH buffer at pH 8.0. By microinjection of pH 7.5 buffer, some eggs started development though none of them underwent cleavage. By microinjection of pH 7.0 or pH 6.5 buffer, development was not initiated. The initiation of development depended on the pH value of microinjected pH buffer, and in consequence, on the final pH_i. The elongation of microvilli which had been arrested in eggs in Na-free sea water was also induced by microinjection of pH 8.0 or 7.5 buffer.