ENZYMO-CYTOCHEMICAL OBSERVATIONS ON THE CORTICAL CHANGE IN THE EGGS OF CYPRINUS CARPIO AND CARASSIUS AURATUS

Acid phosphatase (AcPase) and cholinesterase (ChE) activities were examined by ultracytochemical techniques in the mature unfertilized and the fertilized eggs of Cyprinus carpio and Carassitus auratus , to reveal the differences among three kinds of structures, cortical alveoli, CA- and CB-granules, which discharge their contents on fertilization into the perivitelline space. Deposits of the reaction product for AcPase activity are localized on the plasmalemma of unfertilized eggs, in the cortical alveoli, cytoplasmic matrix, lamellae of the Golgi apparatus and sometimes in multivesicular bodies but not in CA- and CB-granules, mitochondria, rough endoplasmic reticulum or on the plasmalemma of fertilized eggs. Deposits of the reaction product for ChE activity are localized on the inner surface of the plasmalemma, in the cytoplasmic matrix, in mitochondria and on a small number of tubular or cisternal membranes of the endoplasmic reticulum in mature unfertilized eggs, and on the outer surface of the limiting membrane of CB-granules and on membranous structures (possibly Golgi lamellae) associated with their formation, in fertilized eggs. The deposits on the plasmalemma rapidly disappear almost completely, with discharge of the cortical alveoli soon after fertilization, but they are again seen on the inner surface of the plasmalemma when emiocytotic discharge of the CB-granules begins about 10 min after fertilization.     

Immunoelectron Microscopic Demonstration of Cortical Granule Lectins in Coelomic, Unfertilized and Fertilized Eggs of Xenopus laevis

Immunoelectron microscopic studies demonstrated cortical granule lectins (CGLs) in coelomic, unfertilized and fertilized eggs of Xenopus laevis . An antiserum raised against purified cortical granule lectin 1 specifically reacted with the CGLs in immunoblotting and agar diffusion tests. When ultrathin sections were treated with the antiserum and protein A-gold solution, gold particles, indicating antigenic sites, were seen over cortical granules of coelomic and unfertilized eggs, and over the perivitelline space, the vitelline coat and the condensed region of the fertilization layer of fertilized eggs. The pre-fertilization layer immediately adjacent to the outer margin of the vitelline coat in unfertilized eggs was free from gold particles. These observations suggest that released CGLs permeate through the vitelline coat of fertilized eggs and interact with the pre-fertilization layer mainly at the outer margin of the vitelline coat, resulting in formation of the fertilization layer which acts as a block to polyspermy.     

Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi

 Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis) blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively. Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole, whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole. Recieved: 10 June 1996 / Accepted: 12 September 1996     

Fertilization alters the orientation of pigment granule saltations in Arbacia eggs.

Unfertilized eggs of the sea urchin Arbacia punctulata contain pigment granules distributed throughout their cytoplasm. During the first 15 minutes after fertilization, these vesicles move out to the cortex where they become firmly anchored. We have used time-lapse video differential interference microscopy to analyze the motility of these organelles in unfertilized and fertilized Arbacia eggs. Pigment granules exhibit saltatory movement in both unfertilized and fertilized eggs. Quantitation of vesicle saltations before and after fertilization demonstrates that while there is no significant difference in the speed or path-length of vesicle movement, there is a dramatic change in the orientation of these saltations. Saltations in the unfertilized egg are very non-radial and are as likely to be directed toward the cortex as away. In contrast, saltations in the fertilized egg are more radially oriented and more likely to be cortically directed. This transition must reflect underlying changes in the cellular structures necessary for pigment granule saltations. The change in the orientation of pigment granule saltations following fertilization requires both a transient increase in the cytoplasmic concentration of Ca2+ and an elevation of cytoplasmic pH. Similarly, the ability of pigment granules to adhere to the cortex requires both the transient elevation of cytoplasmic Ca2+ and the alkalinization of the cytoplasm. As the reorganization of cortical actin at fertilization is regulated by these ionic fluxes, and both movement and adhesion are sensitive to cytochalasins, we hypothesize that the alterations in directed motility and adhesion reflect underlying changes in the actin cytoskeleton.     

Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.     

Oocyte Maturation and Furrow Formation in an Unfertilized Egg by Fusion with a Fertilized Egg or Blastomeres in the Ascidian, Ascidia sydneiensis divisa

A technique for fusing an ascidian egg with blastomeres using a chemical fusiogen was established and then used to identify cytoplasmic factors that regulate the process of oocyte maturation in ascidian eggs. Unfertilized eggs fused with fertilized eggs or blastomeres in hypotonic artificial sea water containing 20% polyvinyl alcohol within 10 min. After fusion polar bodies were extruded from the unfertilized portion of the fused eggs. Furrows were formed not only in the fertilized portion but also in the unfertilized portion in the fused eggs. No polar body extrusion and furrow formation occur in either portion of fused unfertilized eggs. These results suggest that fertilized eggs and blastomeres contain a factor that induces oocyte maturation. Polar body extrusion and furrow formation were not suppressed in the fertilized portion of fused eggs, suggesting that unfertilized eggs do not contain a factor that inhibits oocyte maturation.     

Reduction in egg hatch after a sublethal dose of chlorfluazuron to larvae of the common cutworm, Spodoptera litura

Abstract.  The effects of a sublethal dose of chlorfluazuron on egg hatching in Spodoptera litura are examined under laboratory conditions. When LD₁₀ of chlorfluazuron is topically applied to newly moulted fifth-instar larvae of either sex, a significant reduction in both the number of eggs laid and subsequent hatching is observed after mating but no significant differences in daily of oviposition are observed when compared with the controls. In addition, examination of the unhatched eggs reveals that the number of unfertilized eggs is greater than those that were fertilized but there are significantly more unfertilized eggs laid by treated insects. Interference by chlorfluazuron, transferred by copulation through sperm fluids or ova, appears improbable. All the fertilized unhatched eggs in the treated crosses die at an earlier stage than those of the controls. In the female-treated crosses, the egg size is significantly reduced compared with the control or male-treated crosses. During mating, the treated-males transferred significantly lower-weight spermatophores into the females. The weight of spermatophores transferred by untreated males is the same to both treated and untreated females. The duration of mating is not affected by chlorfluazuron treatment.     

Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.     

ON PROPAGATION OF CORTICLA FACTOR AND CYTOPLASMIC FACTOR PARTICIPATING IN CLEAVAGE FURROW FORMATION OF THE NEWT'S EGG*

From Cynops pyrrhogaster eggs just after the start of the first cleavage, a fragment of cortical layer with a small entire cleavage furrow was cut out. In the fragment, the cortex had already acquired susceptibility to and the subcortical cytoplasm had already accquired inducibility for furrow formation. The fragment was transplanted to the animal hemisphere of uncleaved fertilized eggs or eggs immediately after the onset of the first cleavage, from which a portion of the host cortex was removed. Observation was made on division of the graft, and on propagation of the cortical susceptibility and the cytoplasmic inducibility of the graft onto the host egg. The transplant divided succesively on the host egg in many cases, but the furrow of the graft never advanced to the surface of the host egg. Neither the cortical factor nor the cytoplasmic factor was transmitted across the graft to the recipient egg.     

Sex allocation in a field population of an autoparasitoid

Autoparasitoid wasps lay fertilized eggs in homopteran nymphs, and these eggs develop into female primary parasitoids. Unfertilized, male-producing eggs are laid in immatures of the wasps' own or another primary parasitoid species; males then develop as secondary or hyperparasitoids. In the population of Encarsia pergandiella studied in Ithaca, NY, fertilized eggs were laid in the nymphs of the whitefly Trialeurodes packardi (primary hosts) and unfertilized eggs were laid almost exclusively in pupal females of their own species (secondary hosts). In the two years the population was studied, secondary hosts were always much less abundant than primary hosts at both sites. However, secondary hosts were parasitized at a significantly greater rate than primary hosts. In a laboratory experiment, the encounter rate of females with primary and secondary hosts was not significantly different. Moreover, there was no evidence from the field that wasps found leaves bearing secondary hosts more frequently than leaves without secondary hosts. Dissections of field-collected females showed them to be mated, and thus capable of laying both unfertilized and fertilized eggs. These results suggest that wasps did not encounter secondary hosts at a greater rate, nor were they constrained to lay unfertilized eggs, but rather secondary hosts were preferred. The oviposition sex ratios were influenced by the proportion of secondary hosts, but were less female-biased than would be predicted from the proportion of secondary hosts alone. The results do not support the predictions of Godray and Waage (1990) for either strictly host-limited autoparasitoids (sex ratio should reflect the proportion of secondary hosts) or for egg-limited autoparasitoids (sex ratio should be equal, and independent of the proportion of secondary hosts).     

Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342

When unfertilized sea urchin eggs are pretreated with the bisbenzimide DNA-specific fluorochrome Hoechst 33342, then washed and fertilized, a single sperm bound to the egg surface becomes intensely fluorescent. The location of the fluorescent sperm on the egg surface coincides exactly with the epicenter of the cortical reaction and the site at which the insemination cone subsequently appears. These observations, coupled with studies of eggs treated with quercetin to prevent fusion, as well as eggs made polyspermic by halothane exposure, indicate that the sperm acquires fluorescence as a consequence of fusion with the fluorochrome preloaded egg. Using a modification of this technique, we have found that cytoplasmic continuity between the sperm and egg is established at 4-8 sec after the onset of the sperm-induced conductance increase in the egg.     

Hypotonic buffer induces meiosis and formation of anucleate cytoplasmic islands in the egg of the two-spotted cricket Gryllus bimaculatus

In insects, egg activation is known to occur in vivo and independently of fertilization, but its mechanisms are poorly understood. To gain understanding of these mechanisms, an attempt was made to activate the egg of Gryllus bimaculatus in vitro. It was found that meiosis resumed and was completed in unfertilized eggs treated with hypotonic buffer. Early developmental processes in activated, unfertilized eggs were investigated and compared with those in fertilized eggs. Mitosis did not progress, resulting in formation of anucleate cytoplasmic islands (pseudoenergids). Development in the activated, unfertilized eggs stopped at this stage and both yolk subdivision and cellularization did not occur. To elucidate the role of the nucleus in the developmental process to the syncytial stage in fertilized eggs, eggs were treated with aphidicolin to inhibit DNA polymerization. It was found that pseudoenergids also formed in these aphidicolin-treated fertilized eggs. These results demonstrate that pseudoenergids can increase in number independently of nuclei, suggesting that the cytoplasm rather than the nucleus plays the primary role in development to the syncytial stage in G. bimaculatus.     

Effects of alpha-latrotoxin on the early developmental events of the sea urchin Paracentrotus lividus: a histochemical study

The effects of the spider toxin alpha-latrotoxin (alpha-LTX) on gametes, zygotes, and early embryos of the sea urchin Paracentrotus lividus have been investigated by in vivo experiments and by histochemical studies of acetylcholinesterase (AChE) activity. Treatment of unfertilized eggs with nanomolar amounts (1 to 0.3 nmol/l) of alpha-LTX neither triggered cortical granule exocytosis, nor prevented the elevation of the fertilization layer by sperms. Instead, fertilized eggs exposed to alpha-LTX showed noticeable alterations in cell surface topography, including the appearance of prominent membrane-limited blebs. Moreover, the zygotes treated with 1 nmol/l alpha-LTX failed to cleave. The histochemical staining of treated zygotes revealed a very strong AChE activity in the cortical region, including blebs. An enzyme reaction was also found in the perivitellin space. Our results suggest the hypothesis that some alpha-LTX receptors may appear after fertilization, supporting the awareness that fertilized eggs display excitable cell features.     

ELECTRON MICROSCOPIC DEMONSTRATION OF A DITHIOTHREITOL-LABILE VITELLINE COAT SURROUNDING THE UNFERTILIZED EGG OF COMANTHUS JAPONICA (ECHINODERMATA: CRINOIDEA)*

In Comanthus, the unfertilized egg is surrounded by a vitelline coat, which is separated from the underlying plasma membrane by a space several hundred Ångstroms wide. By electron microscopy, the vitelline coat is a distinct layer 100 to 150 Å thick, which consists of finely granular material of moderate electron density. Treatment for 3 min in 0.01 M dithiothreitol in sea water buffered to pH 9.2 almost completely removes the vitelline coat and causes the irregularly shaped egg to become spherical. After such DTT-treated eggs have been washed for 2 min in sea water, they cannot be fertilized, but they can undergo a cortical reaction when treated with ionophore A23187. This cortical reaction consists of the exocytosis of cortical granule material directly into the surrounding sea water. By several hours after DTT treatment, most of the eggs, whether exposed to ionophore or not, fragment into spheres of diverse sizes.     

Egg Deposition Behavior in the Haplodiploid Sawfly Athalia rosae ruficornis Jakovlev (Hymenoptera: Symphyta: Tenthredinidae)

The egg deposition behavior of the turnip sawfly, Athalia rosae (Hymenoptera: Symphyta), is described. Both unmated and mated females lay eggs individually inside of fresh young leaves of cruciferous plants. During an oviposition event, females exhibit a distinct pause in abdominal contractions just before the actual egg deposition act. Unmated females show a longer pause (11.31 s on average) than mated females (4.38 s on overall average). By employing an eye color mutation, the sex of the eggs laid by females was ascertained. Females mated once lay mostly fertilized (diploid female) eggs initially but begin to lay a considerable number of unfertilized (haploid male) eggs later in life. The laying of an unfertilized egg is associated with a longer pause (6.98 s on average) than the laying of a fertilized egg (3.76 s on average). These results are in contrast to previous reports on apocritan Hymenoptera, where the presence of a pause or a longer pause during oviposition was associated with the deposition of fertilized eggs rather than unfertilized eggs. The possibility that mated Athalia rosae females control fertilization and its implications for sex allocation strategies are discussed.     

Dominant-negative mutant dynein allows spontaneous centrosome assembly, uncouples chromosome and centrosome cycles

Cleavage cycles commence and chromosome and centrosome cycles proceed in harmony following fertilization of Drosophila eggs and completion of the meiotic divisions. The sperm-introduced centrioles replicate, separate, and while recruit pericentriolar material centrosomes (CS) form. The CS nucleate asters of microtubules (MT). Spindles form following interaction of some astral MT with kinetochores. In unfertilized eggs, chromosomes do not replicate, and CS and MT asters never form, although their components are present in the egg cytoplasm; unknown mechanisms prevent chromosome replication and CS and MT assembly. In unfertilized Laborc(D) eggs, rudimentary CS assemble spontaneously and instantaneously and nucleate small MT asters. In fertilized Laborc(D) eggs, normal CS form and organize normal asters. However, the CS replicate prior to accomplishment of the first mitosis, and spindles with multiple CS develop. In fertilized Laborc(D) eggs, while the chromosome cycles cease, CS cycles proceed as in wild type. Knowing that Laborc(D) is a dominant-negative mutation and encodes the formation of mutant cytoplasmic dynein heavy chain molecules, we show here that cytoplasmic dynein is involved in prevention of CS assembly in unfertilized eggs and establishing harmony between the chromosome and the CS cycles.     

THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions.     

The alphaBbetaC integrin is expressed on the surface of the sea urchin egg and removed at fertilization

Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.     

Reciprocal inheritance of centrosomes in the parthenogenetic hymenopteran Nasonia vitripennis

Background: In the majority of animals, the centrosome-the microtubule-organizing center of the cell-is assembled from components of both the sperm and the egg. How the males of the insect order Hymenoptera acquire centrosomes is a mystery, as they originate from virgin birth.Results: To address this issue, we observed centrosome, spindle and nuclear behavior in real time during early development in the parthenogenetic hymenopteran Nasonia vitripennis. Female meiosis was identical in unfertilized eggs. Centrosomes were assembled before the first mitotic division but were inherited differently in unfertilized and fertilized eggs. In both, large numbers of asters appeared at the cortex of the egg after completion of meiosis. In unfertilized eggs, the asters migrated inwards and two of them became stably associated with the female pronucleus and the remaining cytoplasmic asters rapidly disappeared. In fertilized eggs, the Nasonia sperm brought in paternally derived centrosomes, similar to Drosophila melanogaster. At pronuclear fusion, the diploid zygotic nucleus was associated only with paternally derived centrosomes. None of the cytoplasmic asters associated with the zygotic nucleus and, as in unfertilized eggs, they rapidly degenerated.Conclusions: Selection and migration of the female pronucleus is independent of the sperm and its aster. Unfertilized male eggs inherit maternal centrosomes whereas fertilized female eggs inherit paternal centrosomes. This is the first system described in which centrosomes are reciprocally inherited. The results suggest the existence of a previously undescribed mechanism for regulating centrosome number in the early embryo.     

Intracellular pH shift leads to microtubule assembly and microtubule-mediated motility during sea urchin fertilization: correlations between elevated intracellular pH and microtubule activity and depressed intracellular pH and microtubule disassembly

The regulation of the microtubule-mediated motions within eggs during fertilization was investigated in relation to the shift in intracellular pH (pHi) that occurs during the ionic sequence of egg activation in the sea urchins Lytechinus variegatus and Arbacia punctulata. Microtubule assembly during formation of the sperm aster and mitotic apparatus was detected by anti-tubulin immunofluorescence microscopy, and the microtubule-mediated migrations of the sperm and egg nuclei were studied with time-lapse video differential interference contrast microscopy. Manipulations of intracellular pH were verified by fluorimetric analyses of cytoplasmic fluorescein incorporated as fluorescein diacetate. The ionic sequence of egg activation was manipulated i) to block the pHi shift at fertilization or reduce the pHi of fertilized eggs to unfertilized values, ii) to elevate artificially the pHi of unfertilized eggs to fertilized values, and iii) to elevate artificially or permit the normal pHi shift in fertilized eggs in which the pHi shift at fertilization was previously prevented. Fertilized eggs in which the pHi shift was suppressed did not assemble microtubules or undergo the normal microtubule-mediated motions. In fertilized eggs in which the pHi was reduced to unfertilized levels after the assembly of the sperm aster, no motions were detected. If the intracellular pH was later permitted to rise, normal motile events leading to division and development occurred, delayed by the time during which the pH elevation was blocked. Microtubule-mediated events occurred in eggs in which the intracellular pH was elevated, even in unfertilized eggs in which the pH was artificially increased. These results indicate that the formation and normal functioning of the egg microtubules is initiated, either directly or indirectly, by the shift in intracellular pH that occurs during fertilization.