Experimental separation of pronuclei in fertilized sea urchin eggs: chromosomes do not organize a spindle in the absence of centrosomes

We tested the ability of chromosomes in a mitotic cytoplasm to organize a bipolar spindle in the absence of centrosomes. Sea urchin eggs were treated with 5 X 10(-6) colcemid for 7-9 min before fertilization to block future microtubule assembly. Fertilization events were normal except that a sperm aster was not formed and the pronuclei remained up to 70 microns apart. After nuclear envelope breakdown, individual eggs were irradiated with 366-nm light to inactivate photochemically the colcemid. A functional haploid bipolar spindle was immediately assembled in association with the male chromosomes. In contrast to the male pronucleus, the female pronucleus in most of these eggs remained as a small nonbirefringent hyaline area throughout mitosis. High-voltage electron microscopy of serial semithick sections from individual eggs, previously followed in vivo, revealed that the female chromosomes were randomly distributed within the remnants of the nuclear envelope. No microtubules were found in these pronuclear areas even though the chromosomes were well-condensed and had prominent kinetochores with well-developed coronas. In the remaining eggs, a weakly birefringent monaster was assembled in the female pronuclear area. These observations demonstrate that chromosomes in a mitotic cytoplasm cannot organize a bipolar spindle in the absence of a spindle pole or even in the presence of a monaster. In fact, chromosomes do not even assemble kinetochore microtubules in the absence of a spindle pole, and kinetochore microtubules form only on kinetochores facing the pole when a monaster is present. This study also provides direct experimental proof for the longstanding paradigm that the sperm provides the centrosomes used in the development of the sea urchin zygote.     

Anti-tubulin immunofluorescence microscopy of microtubules present during the pronuclear movements of sea urchin fertilization

Anti-tubulin immunofluorescence microscopy is used here to demonstrate the configurations of the microtubule-containing structures which participate in the pronuclear movements of sea urchin fertilization. This technique shows that the egg is devoid of microtubules until after the fertilizing sperm is fully incorporated. All the microtubules which appear during the course of fertilization are organized around the base of the sperm head and the sperm aster thus formed behaves in a way that could account for the characteristic motions of the male and female pronuclei as documented by time-lapse video microscopy. Extension of astral microtubules appears to be responsible for the slow (ca. 2.5 μm min^?1) movement of the sperm aster into the cytoplasm of the egg; the rapid (ca. 15 μm min^?1) migration of the female pronucleus to the sperm aster seems to depend on connection of the female pronucleus to microtubules of the sperm aster. Continued extension of astral microtubules after the pronuclei are brought into conjunction can account for the centripetal motion of the paired (or fused) pronuclei and for the positioning of the zygote nucleus in the center of the egg. The behavior of astral microtubules during these motions suggests that they are capable of transmitting both pushing and pulling forces. All the pronuclear movements, and the assembly of detectable microtubules, are sensitive to the microtubule inhibitors griseofulvin and colchicine. Because of this sensitivity, and since all the observable microtubules within the egg during fertilization arise at the sperm aster, it is concluded that the pronuclear movements of fertilization result from the actions of the sperm aster. The pronuclear movements of sea urchin fertilization represent a simple but striking example of microtubule-mediated motility.     

Motility and centrosomal organization during sea urchin and mouse fertilization

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.     

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.     

Microtubule distribution during fertilization in the rabbit.

The distribution of microtubules was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti-alpha-tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronuclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.     

Maternal gamma (gamma)-tubulin is involved in microtubule reorganization during bovine fertilization and parthenogenesis

In this study, gamma-tubulin distribution was determined chronologically in conjunction with microtubule dynamics during bovine fertilization and parthenogenesis. In unfertilized bovine oocytes, gamma-tubulin was identified in the cytoplasm, mainly in the cortex and concentrated in the meiotic spindle. Following sperm penetration, gamma-tubulin in the cytoplasm was recruited by a sperm component. During pronuclear apposition, gamma-tubulin was localized as spots at the spindle poles. gamma-tubulin spots were observed in blastomeres of embryos cleaved in vitro. Following electrical stimulation, gamma-tubulin and microtubule matrix were noted in oocyte cortex. In the late pronuclear stage, considerably less gamma-tubulin and microtubules were detected in the cytoplasm. At the mitotic metaphase of parthenotes, gamma-tubulin was recruited to the condensed chromatin and concentrated in the spindle. The gamma-tubulin spots were not detected until the 8-cell stage of parthenotes. This suggests that maternal gamma-tubulin is recruited by a sperm component to reconstitute the zygotic centrosome. In the absence of sperm components, the cell cycle-related assembly of gamma-tubulin organizes microtubule nucleation for positioning the pronucleus and spindle protein of mitotic metaphase during the first cell cycle of bovine parthenotes.     

Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm

Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.     

Nuclear envelope breakdown is under nuclear not cytoplasmic control in sea urchin zygotes

Nuclear envelope breakdown (NEB) and entry into mitosis are though to be driven by the activation of the p34cdc2-cyclin B kinase complex or mitosis promoting factor (MPF). Checkpoint control mechanisms that monitor essential preparatory events for mitosis, such as DNA replication, are thought to prevent entry into mitosis by downregulating MPF activation until these events are completed. Thus, we were surprised to find that when pronuclear fusion in sea urchin zygotes is blocked with Colcemid, the female pronucleus consistently breaks down before the male pronucleus. This is not due to regional differences in the time of MPF activation, because pronuclei touching each other break down asynchronously to the same extent. To test whether NEB is controlled at the nuclear or cytoplasmic level, we activated the checkpoint for the completion of DNA synthesis separately in female and male pronuclei by treating either eggs or sperm before fertilization with psoralen to covalently cross-link base-paired strands of DNA. When only the maternal DNA is cross-linked, the male pronucleus breaks down first. When the sperm DNA is cross-linked, male pronuclear breakdown is substantially delayed relative to female pronuclear breakdown and sometimes does not occur. Inactivation of the Colcemid after female NEB in such zygotes with touching pronuclei yields a functional spindle composed of maternal chromosomes and paternal centrosomes. The intact male pronucleus remains located at one aster throughout mitosis. In other experiments, when psoralen-treated sperm nuclei, over 90% of the zygote nuclei do not break down for at least 2 h after the controls even though H1 histone kinase activity gradually rises close to, or higher than, control mitotic levels. The same is true for normal zygotes treated with aphidicolin to block DNA synthesis. From these results, we conclude that NEB in sea urchin zygotes is controlled at the nuclear, not cytoplasmic, level, and that mitotic levels of cytoplasmic MPF activity are not sufficient to drive NEB for a nucleus that is under checkpoint control. Our results also demonstrate that the checkpoint for the completion of DNA synthesis inhibits NEB by acting primarily within the nucleus, not by downregulating the activity of cytoplasmic MPF.     

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.     

The movements and fusion of the pronuclei at fertilization of the sea urchin Lytechinus variegatus: Time-lapse video microscopy

The penetration of the sperm into the egg, and the movements of the male and female pronuclei were followed from sperm attachment through pronuclear fusion, using time-lapse video microscopy of gametes and zygotes of the sea urchin Lytechinus variegatus (23° C). The pronuclei move in four stages: I. Sperm Entry Phase, following sperm-egg fusion and a rapid radiating surface contraction (5.9 ± 1.3 μm/second) when egg microvilli engulf the sperm head, midpiece, and tail to form the fertilization cone and the sperm tail beats in the egg cytoplasm; II. Formation of the Sperm Aster, which pushes the male pronucleus centripetally at a rate of 4.9 ± 1.7 μm/minute starting 4.4 ± 0.5 minutes after sperm-egg fusion, as the male pronucleus undergoes chromatin decondensation; III. Movement of the Female Pronucleus, the greatest and fastest of the pronuclear motions at a rate of 14.6 ± 3.5 μm/minute at 6.8 ± 1.2 minute after sperm-egg fusion, which establishes the contact between the pronuclei; and IV. Centration of the Pronuclei to the egg center at a rate of 2.6 ± 0.9 μm/minute by 14.1 ± 2.6 minutes after sperm-egg fusion. Pronuclear fusion typically occurs after stage IV and proceeds rapidly starting 14.7 ± 3.6 minutes after sperm-egg fusion with the male pronucleus coalescing into the female pronucleus at a rate of 14.2 ± 2.6 μm/minute.     

Derivation of the membrane comprising the male pronuclear envelope in inseminated sea urchin eggs

Investigations were conducted in an effort to determine the origin of the membrane comprising the male pronuclear envelope of inseminated sea urchin eggs. The events of fertilization in zygotes treated with 200 μg/ml of puromycin are not impaired even though incorporation of [³H]leucine is inhibited up to 80% when compared to control specimens. Developing male pronuclei in zygotes treated with puromycin form nuclear envelopes structurally similar to and within the same period as controls. In puromycin-treated and untreated zygotes morphologically recognizable portions of the sperm nuclear envelope are incorporated into the structure of the male pronuclear envelope. Pronuclear development was also examined in inseminated ova where most of the endoplasmic reticulum (ER) was confined to a specific area of the zygote. Eggs were centrifuged in order to stratify their organelles into specific layers (stratified eggs); with further centrifugation stratified eggs are bisected to form nucleate (rich in ER) and nonnucleate halves (containing little ER). Observations of inseminated stratified eggs and nucleate and nonnucleate halves demonstrate an inverse relation between the amount of ER present in the vicinity of a reorganizing sperm nucleus and the time it takes to form the male pronuclear envelope. Computation of the maximum quantity of membrane in the male pronucleus that may be derived from the sperm nuclear envelope is approximately 15%. These investigations suggest that a major portion of the male pronuclear envelope is derived from endoplasmic reticulum within the egg and only a small portion (up to 15%) originates from the sperm nuclear envelope.     

Nascent protein requirement for completion of meiotic maturation and pronuclear development: examination of fertilized and A-23187-activated surf clam (Spisula solidissima) eggs

The involvement of newly synthesized proteins and calcium in meiotic processes, sperm nuclear transformations, and pronuclear development was examined in emetine-treated, fertilized, and A-23187-activated Spisula eggs by observing changes in the morphogenesis of the maternal and paternal chromatin. Emetine treatment (50 micrograms/ml) initiated 30 min before fertilization or A-23187 activation inhibited incorporation of [3H]leucine into TCA-precipitable material and blocked second polar body formation. Sperm incorporation and the initial enlargement of the sperm nucleus were unaffected; however, the dramatic enlargement and transformation of the sperm nucleus into a male pronucleus, which normally follow polar body formation, were delayed 10 to 20 min. Unlike the situation in untreated, control eggs, male pronuclear development took place while the maternally derived chromosomes remained condensed. It was not until approximately 20 min after the normal period of pronuclear development that the maternal chromosomes dispersed and formed a female pronucleus in emetine-treated, fertilized eggs. Formation of pronuclei, however, was unaffected in both emetine-treated, A-23187-activated eggs and fertilized eggs incubated with A-23187. These observations indicate that germinal vesicle breakdown, first polar body formation, and initial transformations of the sperm nucleus are independent of newly synthesized proteins. Inhibition of second polar body formation and the delay in pronuclear development brought about by emetine, as well as the appearance of silver grains over pronuclei in autoradiographs of control eggs incubated with [3H]leucine demonstrate that nascent proteins are involved with the completion of meiotic maturation and the development of male and female pronuclei. The ability of A-23187 to override the inhibitory effects of emetine on pronuclear development suggests that both nascent protein and calcium signals are involved in regulating the status of the maternal and paternal chromatin during pronuclear development.     

Remodeling the sperm nucleus into a male pronucleus at fertilization

After fertilization, the dormant sperm nucleus undergoes morphological and biochemical transformations leading to the development of a functional nucleus, the male pronucleus. We have investigated the formation of the male pronucleus in a cell-free system consisting of permeabilized sea urchin sperm nuclei incubated in fertilized sea urchin egg extract containing membrane vesicles. The first sperm nuclear alteration in vitro is the disassembly of the sperm nuclear lamina as a result of lamin phosphorylation mediated by egg protein kinase C. The conical sperm nucleus decondenses into a spherical pronucleus in an ATP-dependent manner. The new nuclear envelope (NE) forms by ATP-dependent binding of vesicles to chromatin and GTP-dependent fusion of vesicles to each other. Three cytoplasmic membrane vesicle fractions with distinct biochemical, chromatin-binding and fusion properties, are required for pronuclear envelope assembly. Binding of each fraction to chromatin requires two detergent-resistant lipophilic structures at each pole of the sperm nucleus, which are incorporated into the NE by membrane fusion. Targeting of the bulk of NE vesicles to chromatin is mediated by a lamin B receptor (LBR)-like integral membrane protein. The last step of male pronuclear formation involves nuclear swelling. Nuclear swelling is associated with import of soluble lamin B into the nucleus and growth of the nuclear envelope by fusion of additional vesicles. In the nucleus, lamin B associates with LBR, which apparently tethers the NE to the lamina. Thus male pronuclear envelope assembly in vitro involves a highly ordered series of events. These events are similar to those characterizing the remodeling of somatic and embryonic nuclei transplanted into oocytes. The relationship between sperm nuclear remodeling at fertilization and nuclear remodeling after nuclear transplantation is discussed.     

Sequential transformations of human sperm nucleus in human egg

In-vitro insemination of human zona-free oocytes prepared from oocytes that failed to fertilize in an in-vitro fertilization programme was used as an experimental model to study the time course and morphological events during the development of sperm nuclei into male pronuclei. At 30 min after insemination, 22 eggs were cultured in a CO2 incubator for further 3.5 h and 17 eggs were placed individually between a slide and coverslip for randomly repeated microscopical observations in a controlled environment for at least 3.5 h. Simultaneous arrest of maternal meiosis and sperm nuclear development occurred in 36.4% (8/22) eggs cultured in the CO2 incubator and 47.1% (8/17) of those cultured between a slide and coverslip. Sequential transformation of the human sperm nucleus in human eggs was studied in 6 eggs that showed continuous development of sperm nuclei into male pronuclei during at least 3.5 h after insemination. The early sperm nuclear development in human egg ooplasm can be divided into three phases: the sperm nucleus first decondenses (phase 1) then partly recondenses (phase 2) before expanding again to form an early male pronucleus (phase 3). The prepronuclear stages (phases 1 and 2) took about 60 min each and the pronuclear formation (phase 3) began between 120 and 170 min after insemination. Early pronuclear formation was associated with the occurrence of dense outline material, probably a precursor of the future pronuclear membrane, around the recondensed nucleus in re-expansion (phase 3). Between 30 and 60 min after the beginning of phase 3, numerous (greater than 20) dense grains, considered as nucleolar precursors, were clearly visible inside the growing male pronucleus. Moreover, we have examined sperm nuclear changes in some eggs in which the progression of late meiosis was abnormal. Meiotic arrest of maternal chromatin was always associated with arrest of sperm head development. In 75% (6/8) of the eggs arrested in the metaphase II stages and in 87.5% (7/8) of the eggs arrested in late anaphase II, sperm nuclear development was stopped at the decondensed and recondensed stages, respectively. We have always observed male pronuclei when a maternal pronucleus was present in the egg. These observations suggested that maternal chromatin and sperm nuclear development are probably regulated by common factor(s).     

Microtubule and centrosome distribution during sheep fertilization

The distribution of microtubules and centrosomes was studied during sheep fertilization by electron and immunofluorescence microscopy. Tubulin and centrosomal material was identified with monoclonal anti-alpha-tubulin and MPM-2 antibodies, respectively. In ovulated eggs, microtubules were exclusively found in the meiotic spindle and centrosomal material at each of its poles. At fertilization, sperm centrosomes were incorporated into the egg and organized the sperm astral microtubules. During pronuclear development and migration, the sperm aster increased in size; microtubules of the sperm aster extended from the male pronucleus to the egg center and towards the female pronucleus. The position of the sperm aster during pronuclear migration suggests that it plays a role in this process. When the pronuclei were in apposition in the egg center, a dense array of microtubules and the centrosomal material were present between the two pronuclei. The proximal centriole of the sperm was identified by electron microscopy, between the apposed pronuclei. The centrosomal material extending around the centriole and the sperm neck and proximal mid-piece, apparently contained several foci from which microtubules radiated. These data suggest that in sheep unlike in mice, centrosomal material originating from the sperm is involved in the fertilization events.     

Microtubules in ascidian eggs during meiosis, fertilization, and mitosis

The sequential changes in the distribution of microtubules during germinal vesicle breakdown (GVBD), fertilization, and mitosis were investigated with antitubulin indirect immunofluorescence microscopy in several species of ascidian eggs (Molgula occidentalis, Ciona savignyi, and Halocynthia roretzi). These alterations in microtubule patterns were also correlated with observed cytoplasmic movements. A cytoplasmic latticework of microtubules was observed throughout meiosis. The unfertilized egg of M. occidentalis had a small meiotic spindle with wide poles; the poles became focused after egg activation. The other two species had more typical meiotic spindles before fertilization. At fertilization, a sperm aster first appeared near the cortex close to the vegetal pole. It enlarged into an unusual asymmetric aster associated with the egg cortex. The sperm aster rapidly grew after the formation of the second polar body, and it was displaced as far as the equatorial region, corresponding to the site of the myoplasmic crescent, the posterior half of the egg. The female pronucleus migrated to the male pronucleus at the center of the sperm aster. The microtubule latticework and the sperm aster disappeared towards the end of first interphase with only a small bipolar structure remaining until first mitosis. At mitosis the asters enlarged tremendously, while the mitotic spindle remained remarkably small. The two daughter nuclei remained near the site of cleavage even after division was complete. These results document the changes in microtubule patterns during maturation in Ascidian oocytes, demonstrate that the sperm contributes the active centrosome at fertilization, and reveal the presence of a mitotic apparatus at first division which has an unusually small spindle and huge asters.     

Effects of motility inhibitors during sea urchin fertilization : Microfilament inhibitors prevent sperm incorporation and restructuring of fertilized egg cortex, whereas microtubule inhibitors prevent pronuclear migrations

The sensitivity of specific stages of fertilization to microfilament inhibitors (cytochalasins B (CB), D (CD), and E (CE) and phalloidin) and to inhibitors of microtubule assembly (colcemid (CMD), colchicine (CLC), griseofulvin (GSF), maytansine (MAY), nocodazole (NCD), podophyllotoxin (PDP), and vinblastine (VB)) was investigated using differential interference contrast, time-lapse video microscopy of the sea urchin Lytechinus variegatus. Cytochalasins (CDCE>CB) will prevent sperm incorporation if added prior to or simultaneous with insemination. Sperm-egg fusion and the cortical reaction appear normal, but then the subsequent elevation of the fertilization coat lifts and eventually detaches the ‘fertilizing’ sperm from the egg plasma membrane. When the cytochalasins are added after fusion, the forming fertilization cone is rapidly resorbed, and the lateral displacement of the sperm along the egg cortex is terminated; the pronuclear migrations and mitoses occur normally though cytokinesis is never observed. Cytochalasin treatment before or within 2 min of insemination results in the development of aberrant egg cortices, whereas cytochalasin treatments after 2 min post-fusion have little effect. Phalloidin results in large and long-lasting fertilization cones and a retardation of the rate of sperm incorporation. Eggs exposed to any of the microtubule inhibitors 15 min prior to insemination will incorporate the spermatozoon, though the formation of the sperm aster and the accompanying pronuclear migrations are prevented. Interestingly, the final stage of sperm incorporation involving a lateral displacement of the sperm along the egg cortex is greater (27.1 vs 12.4 μm in controls) and faster (5.4 vs 3.5 μm/min in controls) in microtubule-inhibited eggs. GSF and VB, which readily permeate fertilized eggs, will prevent the formation of the sperm aster if added 3 min after sperm-egg fusion, they will prevent the migration of the female pronucleus if added 5 or 7 min after sperm-egg fusion, pronuclear centration if added 10 min post-fusion, and syngamy if added 12 min post-fusion. CLC- or CMD- treated eggs will develop normally if these drugs are photochemically inactivated with 366 nm light within 4 min post-fusion, arguing that sperm incorporation is completely independent of assembling microtubules. These results indicate that microfilament inhibitors will prevent sperm incorporation and the restructuring of the fertilized egg cortex, and that microtubule inhibitors will prevent the formation and functioning of the sperm aster during the pronuclear migrations; an interplay between cortical microfilaments and cytoplasmic microtubules appears required for the successful completion of fertilization.     

Fertilization and the cytoskeleton in the mouse

The behaviour and roles of the microtubule network and the microfilaments following fertilization in the mouse oocyte are described. The microtubule network is organized by multiple microtubule organizing centres (MTOCs) and these play a major role in establishing spindle structure and pronuclear movement following fertilization; in contrast to sea urchin and frog eggs, the sperm centriole plays little part in organization of the post-fertilization spindle. The microfilaments are required for spindle rotation, polar body formation, certain changes in the egg cortex, and also for pronuclear movement. Influences of the chromosomes on microtubule and microfilament organisation are also discussed.     

Alkaline pH favors microtubule self-assembly in surf clam, Spisula solidissima, oocyte extracts

Microtubule assembly in surf clam oocytes is dependent upon events that occur during fertilization. Prior to fertilization there are few, if any microtubules, but within minutes after fertilization microtubules assemble to form the meiotic apparatus. This study demonstrates that the assembly of microtubules after fertilization may be dependent on the fertilization-induced pH change of the cytoplasm. Since the magnitude of the intracellular pH (pHi) change in Spisula oocytes has not been determined, surf clam microtubule assembly was examined at pH values that reflect the pHi change that occurs during sea urchin fertilization. The results indicate that microtubule assembly in crude oocyte extracts is favored at alkaline pH. In contrast, purified surf clam tubulin assembles to a greater extent at pH 6.6 than at pH 7.2. These results reveal that the tubulin in unfertilized oocytes can assemble into microtubules at pH 6.6 but that they are prevented from doing so by pH-dependent cytoplasmic regulatory factors in the oocyte.