Influence of centriole behavior on the first spindle formation in zygotes of the brown alga Fucus distichus (Fucales, Phaeophyceae)

The influence of centrioles, derived from the sperm flagellar basal bodies, and the centrosomal material (MTOCs) on spindle formation in the brown alga Fucus distichus (oogamous) was studied by immunofluorescence microscopy using anti-centrin and anti-beta-tubulin antibodies. In contrast to a bipolar spindle, which is formed after normal fertilization, a multipolar spindle was formed in polyspermic zygote. The number of mitotic poles in polyspermic zygotes was double the number of sperm involved in fertilization. As an anti-centrin staining spot (centrioles) was located at these poles, the multipolar spindles in polyspermic zygotes were produced by the supplementary centrioles. When anucleate egg fragments were fertilized, chromosome condensation and mitosis did not occur in the sperm nucleus. Two anti-centrin staining spots could be detected, microtubules (MTs) radiated from nearby, but the mitotic spindle was never produced. When a single sperm fertilized multinucleate eggs (polygyny), abnormal spindles were also observed. In addition to two mitotic poles containing anti-centrin staining spots, extra mitotic poles without anti-centrin staining spots were also formed, and as a result multipolar spindles were formed. When karyogamy was blocked with colchicine, it became clear that the egg nucleus proceeded independently into mitosis accompanying chromosome condensation. A monoastral spindle could be frequently observed, and in rare cases a barrel-shaped spindle was formed. However, when a sperm nucleus was located near an egg nucleus, the two anti-centrin staining spots shifted to the egg nucleus from the sperm nucleus. In this case, a normal spindle was formed, the egg chromosomes arranged at the equator, and the associated MTs elongated from one pole of the egg spindle toward the sperm chromosomes which were scattered. From these results, it became clear that paternal centrioles derived from the sperm have a crucial role in spindle formation in the brown algae, such as they do during animal fertilization. However, paternal centrioles were not adequate for the functional centrosome during spindle formation. We speculated that centrosomal materials from the egg cytoplasm aggregate around the sperm centrioles and are needed for centrosomal activation.     

IMMUNOFLUORESCENCE MICROSCOPY OF FERTILIZATION AND PARTHENOGENESIS IN LAMINARIA ANGUSTATA (PHAEOPHYTA)1

Normal fertilization and parthenogenesis of unfertilized eggs were observed in Laminaria angustata Kjellman by indirect immunofluorescence microscopy using a tubulin antibody. Sperm aster formation did not occur at plasmogamy. The centrosome of the egg gradually disappeared. Shortly after karyogamy, one centrosome reappeared near the zygote nucleus. During mitosis, the centrosome replicated and the daughter centrosomes migrated to opposite poles. The mitotic spindle was formed by microtubules that elongated from both poles. After the first cell division, each of the daughter cells received one centrosome that persisted throughout the development of the sporophyte. During parthenogenetic development, abnormal mono-, tri-, and multi-polar spindles were formed. These abnormal spindles caused abnormal nuclear and cytoplasmic division. Thus, cells were produced with 1) no nuclei, 2) multiple nuclei, 3) irregular numbers of chromosomes, and/or 4) no centrosomes. This is one of the reasons for the abortion and abnormal morphogenesis during parthenogenesis. Ultrastructural observations showed that, although cells of some parthogenetic sporophytes have centrioles, cells of almost all abnormally shaped parthenogenetic sporophytes lack centrioles. These results suggest that centrioles are required for normal centrosomal functions in Laminaria. Although centrioles are inherited paternally, some centrosomal material appears to be present or produced de novo in unfertilized eggs.     

Selective disappearance of maternal centrioles after fertilization in the anisogamous brown alga Cutleria cylindrica (Cutleriales, Phaeophyceae): Paternal inheritance of centrioles is universal in the brown algae

The behavior of centrioles in zygotes and female gametes developing parthenogenetically in the anisogamous brown alga Cutieria cyiindrica Okamura was studied using electron and immunofluorescence microscopy. Two pairs of centrioles, detected using anti-centrin antibody, were observed in the vicinity of the male and female nuclei, respectively, just after plasmogamy. The fluorescence intensity of one of the two centrin foci became weak 6 h after plasmogamy and finally disappeared. It was impossible to determine whether the male- or female-derived centrioles disappeared in zygotes, because there was nothing to detect morphological differences between the two centrioles. However, a prominent anti-centrin staining focus was located at the condensed male nucleus in zygotes in which karyogamy had not occurred yet. As a result, it was considered that the maternally inherited centrioles had selectively disappeared during development in C. cylindrica. The paternal inheritance of centrioles in zygotes was also confirmed by electron microscopy. Considering previous observations from oogamous and isogamous species of brown algae, we concluded that the paternal inheriance of centrioles could be universal in the brown algae.     

PREMATURE CHROMOSOME CONDENSATION OF THE KARYOGAMY-BLOCKED SPERM PRONUCLEUS IN THE FERTILIZATION OF FUCUS DISTICHUS (FUCALES,PHAEOPHYCEAE)1

Mitosis of egg and sperm pronuclei of Fucus distichus subsp. evanescens (C. Agardh)Powell was examined by fluorescence and electron microscopy when migration of the sperm pronucleus and, as a result, karyogamy were blocked by colchicine treatment after plasmogamy. Chromosome condensation was obsewed in both pronuclei Microspectrophotometric studies after staining the nuclei with mithramycin A clearly showed that DNA synthesis ocurred in the egg pronucleus but not in the sperm pronucleus. This means that chromosomes condensed prematurely in the sperm pronucleus (premature chromosome condensation). In some cases, the egg chromosomes became arranged on a metaphase plate, whereas the sperm chromosomes lay scattered near the egg pronucleus. Immuno fluorescence microscopy using anti-β-tubulin antibody confirmed that a normal spindle was formed at the egg pronucleus. A pair of centrioles existed at the two poles of this spindle. The sperm nuclear membrane disappeared, and microtubules radiated to the sperm chromosomes from one pole of the egg spindle.     

TRANSIENT LOCALIZATION OF γ-TUBULIN AROUND THE CENTRIOLES IN THE NUCLEAR DIVISION OF BOERGESENIA FORBESII (SIPHONOCLADALES, CHLOROPHYTA)

Mitosis in Boergesenia forbesii (Harvey) Feldman was studied by immunofluorescence microscopy using anti-β–tubulin, anti-γ–tubulin, and anti-centrin antibodies. In the interphase nucleus, one, two, or rarely three anti-centrin staining spots were located around the nucleus, indicating the existence of centrioles. Microtubules (MTs) elongated randomly from the circumference of the nuclear envelope, but distinct microtubule organizing centers could not be observed. In prophase, MTs located around the interphase nuclei became fragmented and eventually disappeared. Instead, numerous MTs elongated along the nuclear envelope from the discrete anti-centrin staining spots. Anti-centrin staining spots duplicated and migrated to the two mitotic poles. γ–Tubulin was not detected at the centrioles during interphase but began to localize there from prophase onward. The mitotic spindle in B. forbesii was a typical closed type, the nuclear envelope remaining intact during nuclear division. From late prophase, accompanying the chromosome condensation, spindle MTs could be observed within the nuclear envelope. A bipolar mitotic spindle was formed at metaphase, when the most intense staining of γ-tubulin around the centrioles could also be seen. Both spindle MT poles were formed inside the nuclear envelope, independent of the position of the centrioles outside. In early anaphase, MTs between separating daughter chromosomes were not detected. Afterward, characteristic interzonal spindle MTs developed and separated both sets of the daughter chromosomes. From late anaphase to telophase, γ-tubulin could not be detected around the centrioles and MT radiation from the centrioles became diminished at both poles. γ-Tubulin was not detected at the ends of the interzonal spindle fibers. When MTs were depolymerized with amiprophos methyl during mitosis, γ-tubulin localization around the centrioles was clearly confirmed. Moreover, an influx of tubulin molecules into the nucleus for the mitotic spindle occurred at chromosome condensation in mitosis.     

The first spindle formation in brown algal zygotes

Regulation of the first spindle formation in brown algal zygotes was described. It is well known that there are three types of sexual reproduction in brown algae; isogamy, anisogamy and oogamy. Paternal inheritance of centrioles can be observed in all these cases, similar to animal fertilization. In isogamy and anisogamy, female centrioles (= flagellar basal bodies) selectively disappear and male centrioles remain after fertilization. In a typical oogamy (e.g. fucoid members), liberated egg does not have centrioles, and sperm centrioles are introduced in zygote. Participation of sperm centrioles to the spindle formation in zygotes was also described using Fucus distichus as a model system. Sperm centrioles function as a part of centrosome, namely microtubule organizing center, in zygote. Therefore, they have a crucial role in the spindle formation. Observations on the spindle formation in polygyny and karyogamy-blocked zygotes strongly suggest that egg nucleus can form a mitotic spindle by itself without centrosome, even though the resulting spindles are of abnormal shapes. %     

Fertilization-induced changes in the microtubular architecture of the maize egg cell and zygote—an immunocytochemical approach adapted to single cells

By using single cell micromanipulation techniques, we developed an immunocytochemical procedure to examine subcellular protein localization in isolated and cultured cells. Localization of microtubules was examined in isolated single egg cells and developing zygotes of maize with anti--tubulin antibodies. In egg cells, a few cortical microtubules were detected but well organized microtubules were rarely observed. In contrast, distinct cortical microtubules and strands of cytoplasmic microtubules radiating from the nucleus to the cell periphery were observed in developing zygotes. Solely cortical microtubules were observed in zygotes up to 7 h after in vitro fertilization. After this time, radiating microtubules additionally appeared, and persisted during zygote development. These results indicate early and pronounced fertilization-induced changes in microtubular organization in the fertilized egg cell of maize.     

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.     

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.     

Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation

Centrioles organize microtubules in two ways: either microtubules elongate from the centriole cylinder itself, forming a flagellum or a cilium ("template elongation"), or pericentriolar material assembles and nucleates a microtubule aster ("astral nucleation"). During spermatogenesis in most species, a motile flagellum elongates from one of the sperm centrioles, whereas after fertilization a large aster of microtubules forms around the sperm centrioles in the egg cytoplasm. Using Xenopus egg extracts we have developed an in vitro system to study this change in microtubule-organizing activity. An aster of microtubules forms around the centrioles of permeabilized frog sperm in egg extracts, but not in pure tubulin. However, when the sperm heads are incubated in the egg extract in the presence of nocodazole, they are able to nucleate a microtubule aster after isolation and incubation with pure calf brain tubulin. This provides a two-step assay that distinguishes between centrosome assembly and subsequent microtubule nucleation. We have studied several centrosomal antigens during centrosome assembly. The CTR2611 antigen is present in the sperm head in the peri-centriolar region. gamma-tubulin and certain phosphorylated epitopes appear in the centrosome only after incubation in the egg extract. gamma-tubulin is recruited from the egg extract and associated with electron-dense patches dispersed in a wide area around the centrioles. Immunodepletion of gamma-tubulin and associated molecules from the egg extract before sperm head incubation prevents the change in microtubule-organizing activity of the sperm heads. This suggests that gamma-tubulin and/or associated molecules play a key role in centrosome formation and activity.     

Kinesin-1 prevents capture of the oocyte meiotic spindle by the sperm aster

Centrioles are lost during oogenesis and inherited from the sperm at fertilization. In the zygote, the centrioles recruit pericentriolar proteins from the egg to form a mature centrosome that nucleates a sperm aster. The sperm aster then captures the female pronucleus to join the maternal and paternal genomes. Because fertilization occurs before completion of female meiosis, some mechanism must prevent capture of the meiotic spindle by the sperm aster. Here we show that in wild-type Caenorhabditis elegans zygotes, maternal pericentriolar proteins are not recruited to the sperm centrioles until after completion of meiosis. Depletion of kinesin-1 heavy chain or its binding partner resulted in premature centrosome maturation during meiosis and growth of a sperm aster that could capture the oocyte meiotic spindle. Kinesin prevents recruitment of pericentriolar proteins by coating the sperm DNA and centrioles and thus prevents triploidy by a nonmotor mechanism.     

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.     

The orientation of primary cilia during the wound response in 3Y1 cells

Summary— The behavior of the primary cilia of 3Y1 cells in the interphase was investigated by indirect immunofluorescence microscopy and transmission electron microscopy, using an antibody for tubulin. At 4.5 h after scraping a part of a confluent cell sheet, the primary cilia of cells facing the wound were located predominantly forward of the nucleus on the wounded side, and were oriented in the direction of the leading lamellae. Cytoplasmic microtubules (MTs), emanating from around the base of the cilia, were well developed in the leading lamellae on the wounded side. On the other hand, in the cells of an unperturbed area away from the wounded edge, the primary cilia remained randomly distributed near the nucleus. The position and a certain well-defined orientation of a pair of centrioles seem to play an important role for the development of cytoplasmic MTs, and consequently the orientation of the centrioles is controlled by the primary cilia.     

Physiological polyspermy: Selection of a sperm nucleus for the development of diploid genomes in amphibians

The union between a sperm and an egg nucleus in egg fertilization is necessary to mix genetic materials to create a new diploid genome for the next generation. In most animals, only one sperm is incorporated into the egg (monospermy), but several animals exhibit physiological polyspermy in which several sperms enter the egg during normal fertilization. However, only one sperm nucleus forms the zygote nucleus with the egg nucleus, even in a polyspermic egg. The cellular and molecular mechanisms involved in the selection of sperm nuclei in the egg cytoplasm have been well investigated in urodele amphibians. The principal sperm nucleus develops a larger sperm aster and contacts the egg nucleus to form a zygote nucleus, whereas other accessory sperm nuclei are unable to approach the egg nucleus. The diploid zygote nucleus induces cleavage and participates in embryonic development, whereas the accessory sperm nuclei undergo pyknosis and degenerate. We propose several models to account for the mechanisms of the selection of one sperm nucleus and the degeneration of accessory sperm nuclei. The roles of physiological polyspermy in animal reproduction are discussed by comparison with other polyspermic species.     

The cytology of the gametes and fertilization of Allomyces macrogynus

The gametes and the process of fertilization were examined by light and electron microscopy in the lower eukaryote Allomyces macrogynus. Differences in gamete morphology included the overall larger size and the presence of a larger nuclear apparatus, along with the association of a side-body complex and many more mitochondria in the female gamete. In this species of Allomyces, fertilization was initiated by contact and fusion of specialized regions of the gamete plasma membranes resulting in a binucleate fusion cell surrounded by plasma membrane contributed by both partners. Following plasmogamy, nuclear fusion was initiated by multiple nuclear membrane contacts between adjacent outer membranes. Following inner membrane fusion, small nucleoplasmic bridges were observed which presumably fused with one another and resulted in a single bridge which widened, forming the mature diploid nucleus. After karyogamy, fusion of the nuclear caps did not always occur and zygotes with and without fused caps were observed. Coalescence of the nucleoli completed the events of fertilization, forming a zygote with a single nuclear apparatus (sometimes with two caps) and two flagella. These observations are discussed in relation to fertilization mechanisms and compared to fertilization in other organisms.     

The first two cleavages in Tubifex involve distinct mechanisms to generate asymmetry in mitotic apparatus

We have investigated factors which determine inequality of the first two cleavages in Tubifex hattai. A mitotic spindle for the first cleavage, which is located at the center of the egg, possesses an aster at one pole, but not at the other pole. Inequality of the first cleavage is determined by the asymmetric organization of the spindle poles, rather than by the spindle position in the egg. A centrosome which appears as a dot stained with an anti--tubulin antibody is found at one pole (at the center of the aster) of the spindle, but not at the other pole. This centrosome appears to be maternal in origin. In contrast to the first cleavage, the poles of the second cleavage spindle are not different from each other either in their ability to form asters or in -tubulin distribution. As a result of an interaction of one of the spindle poles with the cell cortex, however, an asymmetric spindle is formed in the cell CD, giving rise to unequal division in this cell. Thus, factors generating asymmetry in spindle organization are intrinsic to the mitotic spindle in the first cleavage, but not in the second cleavage.     

Destruction of maternal centrioles during fertilization of the brown alga, Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae)

In brown algal fertilization, a pair of centrioles is derived from the male gamete, irrespective of the sexual reproduction pattern, i.e., isogamy, anisogamy, or oogamy. In this study, the manner in which the maternal centriole structure is destroyed in early zygotes of the isogamous brown alga Scytosiphon lomentaria was examined by electron microscopy. At fertilization, the zygote had two pairs of centrioles (flagellar basal bodies) derived from motile male and female gametes, and there was no morphological difference between the two pairs. The flagellar basal plate and the axonemal microtubules were still connected with the distal end of centrioles. Ultrastructural observations showed that the integrity of maternal-derived centrioles began to degenerate even in the 1-h-old zygote. At that time, the cylinder of triplet microtubules of the maternal centrioles became shorter from the distal end, and a section passing through the centrioles indicated that a part of the nine triplets of microtubules changed into doublet or singlet microtubules by degeneration of B and/or C tubules. In 2-h-old zygote, there was no trace of maternal centrioles ultrastructurally, and only the paternal centrioles remained. Further, reduction of centrin accompanying destruction of the maternal centrioles was examined in immunofluorescence microscopy. Centrin localized at the paternal and the maternal centrioles had the same fluorescence intensity in the early zygotes. At 4-6 h after fertilization, two spots indicating centrin localization showed different fluorescence intensity. Later, the weaker spot disappeared completely. These results showed that there is a difference in time between the destruction of the centriolar cylinders and the reduction of centrin molecules around them.     

Tubulin and actin topology during zygote formation of Saccharomyces cerevisiae

The topology of tubulin and actin during mating of Saccharomyces cerevisiae was analysed by fluorescence microscopy with the monoclonal anti-tubulin antibody Tu01 and rhodamine-labelled phalloidin. Preconjugatory cells displayed an asymmetric distribution of the microtubule and actin cytoskeleton and an overall polarization of the cells preceding cell fusion. Prior to karyogamy, the haploid spindle pole bodies were associated with abundant cytoplasmic microtubules. Budding zygotes revealed the same tubulin and actin patterns as vegetative cells. Treatment of the mating mixture with the microtubule inhibitor nocodazole (10 micrograms ml-1) did not prevent polarization and fusion of haploids, zygote formation and emergence of the first zygotic bud. In marked contrast, the migration of the nucleus in preconjugatory cells as well as nuclear migration and fusion within the zygotes was unequivocally blocked by the action of the drug. It is suggested that the problem of the morphogenesis of mating should be approached by considering interactions at the cell periphery.     

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.