ULTRASTRUCTURE OF FERTILIZATION IN A CRYPTOMONAD1

By means of various electron microscopic techniques, the ultrastructure of fusing gametes in a cryptomonad is described for the first time. The isolate used in this study is bisexual, and vegetative cells may act as isogametes. Plasmogamy usually is initiated at the posterior end of one gamete and the mild-ventral region of the other gamete. A posterior, pointed protuberance may be a specialized mating structure which initiates the fusion process. Fusion proceeds toward the anterior end, forming a quadriflagellate cell which becomes spherical and settles to the bottom of the culture flask. The quadriflagellate, spherical cell contains two nuclear-nucleomorph-chloroplast complexes which remain intact throughout karyogamy. During karyogamy the nuclei are positioned close to each other and become lobed on the sides where fusion takes place. At the points where the lobes touch, the nuclear membranes break down and direct karyogamy is initiated. Nuclear fusion continues and eventually a single zygotic nucleus is formed. The zygote nucleus and the two nucleomorphs and chloroplasts become enclosed in a common periplastidial compartment. The nucleomorphs, however, remain apart and do not fuse. Meiosis presumably is zygotic, but the stages of post-karyogamy remain to be elucidated.     

Electron microscopic observations of karyogamy in the fish egg

In the initial step of pronuclear association in fertilized fish eggs, the female and male pronuclei (containing large nucleolus-like bodies) were juxtaposed in the center of the blastodisc and formed nucleoplasmic projections along adjacent surfaces. After contact of the pronuclei, small internuclear bridges joining them were formed by fusion at several regions of the nuclear envelope projections. No specific site of fusion or breakdown of nuclear envelopes was recognized in the pronuclei during karyogamy. In the advanced stage, clumps of condensing chromatin appeared in the nucleoplasm of the newly fused pronuclei. The number and diameter of the internuclear bridges increased gradually by progressive fusion in many regions, finally yielding a spherical zygote nucleus. Following complete formation of the zygote nucleus, the pronuclear envelope began to break down concomitantly with shrinkage of the nucleoplasm, which was highly convoluted around the entire nuclear surface. The nucleoplasm containing chromosomes then mingled with the perinuclear cytoplasm.     

THE FINE STRUCTURE OF PRONUCLEAR DEVELOPMENT AND FUSION IN THE SEA URCHIN, ARBACIA PUNCTULATA

Fertilization events following coalescence of the gamete plasma membranes and culminating in the formation of the zygote nucleus were investigated by light and electron microscopy in the sea urchin, Arbacia punctulata. Shortly after the spermatozoon passes through the fertilization cone, it rotates approximately 180° and comes to rest lateral to its point of entrance. Concomitantly, the nonperforated nuclear envelope of the sperm nucleus undergoes degeneration followed by dispersal of the sperm chromatin and development of the pronuclear envelope. During this reorganization of the sperm nucleus, the sperm aster is formed. The latter is composed of ooplasmic lamellar structures and fasciles of microtubules. The male pronucleus, sperm mitochondrion, and flagellum accompany the sperm aster during its migration. As the pronuclei encounter one another, the surface of the female pronucleus proximal to the advancing male pronucleus becomes highly convoluted. Subsequently, the formation of the zygote nucleus commences with the fusion of the outer and the inner membranes of the pronuclear envelopes, thereby producing a small internuclear bridge and one continuous, perforated zygote nuclear envelope.     

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.     

Double Fertilization in Gnetum gnemon: The Relationship between the Cell Cycle and Sexual Reproduction

Gnetum gnemon, a nonflowering seed plant and member of the Gnetales, expresses a rudimentary pattern of double fertilization that results in the formation of two zygotes per pollen tube. The process of double fertilization in G. gnemon was examined with light and fluorescence microscopy, and the DNA content of various nuclei involved in sexual reproduction was quantified with 4[prime],6-diamidino-2-phenylindole microspectrofluorometry.Male and female gamete nuclei pass through the synthesis phase of the cell cycle and increase their DNA content from 1C to 2C before fertilization. Each of the two zygotes found in association with a pollen tube is diploid and contains the 4C quantity of DNA at inception. Based on these results as well as previous studies of nuclear DNA content in plant sperm, eggs, and zygotes, three fundamental and distinct patterns of gamete karyogamy among seed plants can be circumscribed: (1) G1 karyogamy, in which male and female gametes contain the 1C quantity of DNA throughout karyogamy and the zygote undergoes DNA replication; (2) S-phase karyogamy, in which gamete nuclei initiate fusion at 1C but pass through the S phase of the cell cycle before completely fusing; and (3) G2 karyogamy, in which male and female gamete nuclei pass through the S phase of the cell cycle before the onset of fertilization. Our results show definitively a pattern of G2 karyogamy in G. gnemon.     

Nuclear congression and membrane fusion: two distinct events in the yeast karyogamy pathway

Karyogamy is the process where haploid nuclei fuse to form a diploid nucleus during yeast mating. We devised a novel genetic screen that identified five new karyogamy (KAR) genes and three new cell fusion (FUS) genes. The kar mutants fell into two classes that represent distinct events in the yeast karyogamy pathway. Class I mutations blocked congression of the nuclei due to cytoplasmic microtubule defects. In Class II mutants, nuclear congression proceeded and the membranes of apposed nuclei were closely aligned but unfused. In vitro, Class II mutant membranes were defective in a homotypic ER/nuclear membrane fusion assay. We propose that Class II mutants define components of a novel membrane fusion complex which functions during vegetative growth and is recruited for karyogamy.     

Electron microscopy of sexual reproduction in Nephroselmis olivacea (Prasinophyceae, Chlorophyta)

The electron microscopy of zygote formation and the early stages of zygote germination in Nephroselmis olivacea Stein are presented. Although the gametes differ behaviorally during the early stages of gamete fusion, the alga is isogamous. The minus gamete settled on the substrate, and attached with its left side. The plus gamete swam to the minus gamete, attached ventral to the right side of the minus gamete, while slightly on its left side, and plasmogamy started. No specialized organelle for gamete fusion was seen using either scanning or transmission electron microscopy. Gametic fusion was uniform; the right side of the minus gamete always fused with the ventral, slightly left side of the plus gamete, which suggests the participation of the d‐rootlets of the flagellar apparatus of the two gametes. Body scales were retained throughout the entire sexual process. Before karyogamy, a network of endoplasmic reticulum developed between the nuclei. This position corresponded to the contractile vacuole of the plus gamete. Fusion proceeded as the minus gamete was drawn to the plus gamete and resulted in a hemispherical zygote. Fibrous material appeared on the cell surface, embedding the body scales to form a layer that thickened and contributed to the strong adhesion of the zygote to the substrate. During this stage, karyogamy was completed. A thick zygotic wall composed of two layers, an electron‐dense outer layer and a straticulate electron‐lucent inner layer developed beneath the layer of fibrous material and scales. Zygote germination was induced. After the first meiotic division, the layer of fibrous material and scales ruptured and the inner layer of the zygotic wall thinned, allowing the emergence of two germ cells. They had newly formed scales and two starch grains, but no typical pyrenoid.     

ER membrane protein complex required for nuclear fusion

Diploid cells of the yeast Saccharomyces cerevisiae form after the mating of two haploid cells of the opposite mating type. After fusion of the two plasma membranes of the mating cells, a dinucleated cell forms initially in which the two haploid nuclei then rapidly fuse to form a single diploid nucleus. This latter event, called karyogamy, can be divided into two distinct steps: the microtubule-based movement that causes the two nuclei to become closely juxtaposed and the fusion of the nuclear membranes. For the membrane fusion step, one required component, the ER luminal protein Kar2p (BiP), has been identified. For topological reasons, however, it has been unclear how Kar2p could function in this role. Kar2p is localized to the luminal (i.e., noncytoplasmic) face of the ER membrane, yet nuclear fusion must initiate from the cytosolic side of the outer nuclear membrane or the ER membrane with which it is contiguous. There is both genetic and biochemical evidence that Kar2p interacts with Sec63p, an ER membrane protein containing both luminal and cytosolic domains that is involved in protein translocation across the membrane. We have isolated novel sec63 mutant alleles that display severe karyogamy defects. Disruption of the genes encoding other Sec63p-associated proteins (Sec71p and Sec72p) also results in karyogamy defects. A suppressor mutant (sos1-1) partially corrects the translocation defect but does not alleviate the karyogamy defect. sec61 and sec62 mutant alleles that cause similar or more severe protein translocation defects show no karyogamy defects. Taken together, these results suggest a direct role for Sec63p, Sec71p, and Sec72p in nuclear membrane fusion and argue against the alternative interpretation that the karyogamy defects result as an indirect consequence of the impaired membrane translocation of another component(s) required for the process. We propose that an ER/nuclear membrane protein complex composed of Sec63p, Sec71p, and Sec72p plays a central role in mediating nuclear membrane fusion and requires ER luminally associated Kar2p for its function.     

The kinetics of cytological events during double fertilization in Zea mays L.

By using a clearing method, the process of double fertilization in Zea mays L. (line A 188) was analysed and the precise sequence of events was determined. The period from pollen tube arrival to gamete fusion was relatively short, possibly less than 1 h. The karyogamy was of premitotic type, and the time from the contact of male and female nuclei to the fusion of male and female nucleoli was about 5 h in the egg cell and 3 h in the central cell. In the central cell, the sperm nucleus fused with either one of the polar nuclei or the secondary nucleus, the latter being observed for the first time in maize. The zygote was in the resting period for 13–16 h before division commenced, changing the cell polarity during karyogamy and the resting period. The primary endosperm nucleus divided immediately after karyogamy was completed in the central cell. The embryo sacs with two-celled proembryos contained four to eight endosperm nuclei. The timetable of fertilization events could be a standard for further studies on in vitro fertilization at the cytological and molecular levels.     

Confocal observations of late-acting self-incompatibility in Theobroma cacao L.

Cocoa (Theobroma cacao) has an idiosyncratic form of late-acting self-incompatibility that operates through the non-fusion of incompatible gametes. Here, we used high-resolution confocal microscopy to define fine level changes to the embryo sac of the strongly self-incompatible cocoa genotype SCA 24 in the absence of pollination, and following compatible and incompatible pollination. All sperm nuclei had fused with the female nuclei by 48?h following compatible pollinations. However, following incompatible pollinations, we observed divergence in the behaviour of sperm nuclei following release into the embryo sac. Incomplete sperm nucleus migration occurred in approximately half of the embryo sacs, where the sperm nuclei had so far failed to reach the female gamete nuclei. Sperm nuclei reached but did not fuse with the female gamete nuclei in the residual cases. We argue that the cellular mechanisms governing sperm nucleus migration to the egg nucleus and those controlling subsequent nuclear fusion are likely to differ and should be considered independently. Accordingly, we recommend that future efforts to characterise the genetic basis of LSI in cocoa should take care to differentiate between these two events, both of which contribute to failed karyogamy. Implications of these results for continuing efforts to gain better understanding of the genetic control of LSI in cocoa are discussed.     

NUCLEAR FUSION DEFECTIVE1 encodes the Arabidopsis RPL21M protein and is required for karyogamy during female gametophyte development and fertilization

Karyogamy, or nuclear fusion, is essential for sexual reproduction. In angiosperms, karyogamy occurs three times: twice during double fertilization of the egg cell and the central cell and once during female gametophyte development when the two polar nuclei fuse to form the diploid central cell nucleus. The molecular mechanisms controlling karyogamy are poorly understood. We have identified nine female gametophyte mutants in Arabidopsis (Arabidopsis thaliana), nuclear fusion defective1 (nfd1) to nfd9, that are defective in fusion of the polar nuclei. In the nfd1 to nfd6 mutants, failure of fusion of the polar nuclei is the only defect detected during megagametogenesis. nfd1 is also affected in karyogamy during double fertilization. Using transmission electron microscopy, we showed that nfd1 nuclei fail to undergo fusion of the outer nuclear membranes. nfd1 contains a T-DNA insertion in RPL21M that is predicted to encode the mitochondrial 50S ribosomal subunit L21, and a wild-type copy of this gene rescues the mutant phenotype. Consistent with the predicted function of this gene, an NFD1-green fluorescent protein fusion protein localizes to mitochondria and the NFD1/RPL21M gene is expressed throughout the plant. The nfd3, nfd4, nfd5, and nfd6 mutants also contain T-DNA insertions in genes predicted to encode proteins that localize to mitochondria, suggesting a role for this organelle in nuclear fusion.     

New Observations on Gametogenesis,Fertilization, and Zygote Transformation in Plasmodium gallinaceum1

The ultrastructure of the sexual stages of Plasmodium gallinaceum during gametogenesis, fertilization, and early zygote transformation is described. New observations are made regarding the parasitophorous vacuole (PV) of gametocytes and the process of emergence in male and female gametocytes. Whereas female gametocytes readily disrupted both the PV membrane and host cell plasmalemma during emergence, male gametocytes frequently failed to break down the plasmalemma of the host cell. New observations and hypotheses are presented on the behavior of the male gamete nucleus. Following fertilization, the male nucleus appears to travel through a channel of endoplasmic reticulum in the female gamete before fusing with the female nucleus at a region in which the nuclear envelope is thrown into extensive convoluted folds. Polarization of the zygote nucleus, in association with the appearance of a perinuclear spindle of cytoplasmic microtubules, preceded all other changes in the developing zygote. After nuclear polarization becomes apparent, electron-dense material is deposited beneath the zygote pellicle, and a canopy is formed which eventually extends over the entire apical end of the developing ookinete. As the apical end begins to extend outward, polar rings, micronemes, and subpellicular microtubules become visible in this portion and a “virus-like” inclusion known as a crystalloid is formed in the posterior portion of the zygote. When female gametes are prevented from being fertilized, the cytoplasm at 24 h after gametogenesis is devoid of most of those organelles found in the developing zygote or the mature ookinete. The cell is surrounded only by a single membrane. Although at various points beneath the membrane there are deposits of electron-dense material reminiscent of those deposited in the zygote, no further development of ookinete structures takes place in the unfertilized female gamete.     

Nuclear fusion during yeast mating occurs by a three-step pathway

In Saccharomyces cerevisiae, mating culminates in nuclear fusion to produce a diploid zygote. Two models for nuclear fusion have been proposed: a one-step model in which the outer and inner nuclear membranes and the spindle pole bodies (SPBs) fuse simultaneously and a three-step model in which the three events occur separately. To differentiate between these models, we used electron tomography and time-lapse light microscopy of early stage wild-type zygotes. We observe two distinct SPBs in ~80% of zygotes that contain fused nuclei, whereas we only see fused or partially fused SPBs in zygotes in which the site of nuclear envelope (NE) fusion is already dilated. This demonstrates that SPB fusion occurs after NE fusion. Time-lapse microscopy of zygotes containing fluorescent protein tags that localize to either the NE lumen or the nucleoplasm demonstrates that outer membrane fusion precedes inner membrane fusion. We conclude that nuclear fusion occurs by a three-step pathway.     

KARYOGAMY BLOCK BY HEAT STRESS IN THE FERTILIZATION OF BROWN ALGAE

Karyogamy was inhibited by heat stress in zygotes of Scytosiphon lomentaria (Lyngbye) Link (isogamy), Cutleria cylindrica Okamura (anisogamy), and Fucus distichus subsp. evanescens (C. Agardh) Powell (oogamy). Although high temperatures did not inhibit migration of the male and female nuclei, nuclear envelope fusion was blocked. The ultrastructural stage at which karyogamy was inhibited varied among these species. In S. lomentaria, the outer membranes fused with each other, but the inner membranes did not fuse. Partial fusion of the nuclear envelope occurred in C. cylindrica. In F. distichus, the block of karyogamy at high temperature was incomplete, and nuclear fusion proceeded gradually. The block to karyogamy in S. lomentaria zygotes was reversible, and karyogamy proceeded when zygotes were transferred from 22° to 14° C. Experiments using inhibitors suggested that proteins that might be formed de novo after fertilization do not participate in karyogamy or its inhibition at either 14° or 22° C.     

CYTOLOGICAL BASIS FOR CYTOPLASMIC INHERITANCE IN PSEUDOTSUGA MENZIESII. II. FERTILIZATION AND PROEMBRYO DEVELOPMENT

Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) ovules were used to study male gamete formation, insemination of the egg, and free nuclear and cellular proembryo development. Two male nuclei form as the pollen tube either reaches the megaspore wall or as it enters the archegonial chamber. No cell wall separates them. They are contained within the body-cell cytoplasm. A narrow extension of the pollen tube separates the neck cells and penetrates the ventral canal cell. The pollen tube then releases its contents into the egg cytoplasm. The two male gametes and a cluster of paternal organelles (plastids and mitochondria) migrate within the remains of the body-cell cytoplasm toward the egg nucleus. Microtubules are associated with this complex. The leading male gamete fuses with the egg nucleus. The zygote nucleus undergoes free nuclear division, but the cluster of paternal organelles remains discrete. Free nuclei, paternal and maternal nucleoplasm, maternal perinuclear cytoplasm, and the cluster of paternal organelles migrate en masse to the chalazal end of the archegonium. There, paternal and maternal organelles intermingle to form the neocytoplasm, the nuclei divide, and a 12-cell proembryo is formed. The importance of male nuclei or cells, the perinuclear zone, and large inclusions in cytoplasmic inheritance are discussed in the Pinaceae and in other conifer families. This completes a two-part study to determine the fate of paternal and maternal plastids and mitochondria during gamete formation, fertilization, and proembryo development in Douglas fir.     

Occurrence of Mitochondria in the Nuclei of Tobacco Sperm Cells

Tobacco sperm cells contain intact mitochondria within their nuclei with a frequency of 0.35 [plusmn] 0.13 per cell. These inclusions appear to originate from mitochondria found among chromatids in the highly elongated metaphase plate of the dividing generative cell. These organelles are apparently captured during the reconstitution of the nuclear envelope. Only sperm cells were observed to contain these nuclear mitochondria; generative cells, vegetative pollen cells, transmitting tissue cells, unfertilized egg cells, and central cells lacked them. Nuclear mitochondria were also seen in the nuclei of the egg and central cell after fusion with sperm nuclei, suggesting that nuclear mitochondria are transmitted into the zygote and primary endosperm cells during double fertilization. Organellar inclusions in the sperm nucleus provide a potential mechanism for transmitting organellar DNA into the next generation and could potentially facilitate the transfer of genetic material between the nucleus and other organelles.     

An ultrastructural analysis of mitosis and cytokinesis in the zygote of the sea urchin, Arbacia punctulata

Post-fertilization events leading to the cleavage of the zygote of the sea-urchin, Arbacia punctulata were examined with the light and electron microscopes. Prior to prophase of the first cleavage division, endoplasmic reticulum and annulate lamellae become organized around the zygotic nucleus to produce a crescent-shaped structure which is defined as the streak (Harvey, '56). With the advent of prophase the streak undergoes morphogenic events which lead to the formation of the mitotic asters. During this transition there is a loss of annulate lamellae and a concomitant increase in endoplasmic reticulum. Annulate lamellae are not found as a part of the mitotic apparatus and are not again observed within the embryo until the two cell stage. During telophase, karyomeres are formed which consist of chromosomes delimited by a porous bilaminar envelope. Blastomere nuclei are produced following the fusion of the outer laminae, and subsequently by the fusion of the inner laminae of the envelopes encompassing the karyomeres.     

Cytological events leading to the cleavage of golden hamster zygotes.

Fertilized golden hamster eggs were examined between 6 and 20 hours post-ovulation to determine the events leading to the two-cell stage. Following their migration the pronuclei remain in the central region of the zygote for approximately ten hours. The morphologically, indistinguishable male and female pronuclei remain relatively unchanged during this period, i.e., they do not interdigitate or fuse with one another as described for the zygotes of other organisms. Following this period and at the time of pronuclear breakdown elongate vesicles appear along the nucleoplasmic surface of the pronuclear envelopes. Later the pronuclear envelopes fragment into elongate cisternae; these and the vesicles formed along the inner lamina of the pronuclear envelopes remain closely associated and constitute quadrilaminar structures. The chromosomes which condense prior to and during pronuclear envelope breakdown, migrate to the equatorial plate of the forming cleavage spindle. After cytokinesis the chromosomes in the blastomere nuclei disperse. Increase in the nuclear envelope to accommodate this dispersion may involve the addition of membrane from the quandrilaminar structures.     

NUCLEAR MEMBRANE FUSION IN FERTILIZED LYTECHINUS VARIEGATUS EGGS

Fusion of apposed nuclear envelopes is frequently seen at telophase during postmitotic reorganization of the nucleus, but only rarely at other times in the cell cycle. We attempted to define an experimental system for studying changes in the nuclear envelope related to the cell cycle by varying the time of pronuclear apposition in fertilized Lytechinus variegatus eggs. This approach was based on the assumption that the period from fertilization to metaphase of the first cleavage division corresponds to the period from telophase to metaphase in the generalized cell cycle. The experimental approach used was to block the movement of the pronuclei with Colcemid and then to release this block at varying times after insemination by photochemically inactivating the Colcemid. The results show that apposed pronuclear envelopes can fuse from soon after insemination until the anticipated time of prometaphase. Fusion occurred in about 3 min as scored by light microscopy and this time did not vary significantly with the time after insemination. The potential for nuclear fusion is not restricted to pronuclei alone since diploid nuclei in binucleate cells could be fused using centrifugation in solutions of Colcemid to bring the nuclei into apposition. It is suggested that the potential for nuclear fusion is not necessarily related to the cell cycle and that modification of the nuclear envelope, possibly by association with chromatin or other fibrous material restricts nuclear fusion in most multinucleated cells.