FERTILIZATION IN OEDOGONIUM. III. KARYOGAMY

Karyogamy is described in Oedogonium cardiacum from ultrastructural studies. Close proximity of the two gamete nuclei in the fusion cell is established by plasmogamy, whereas karyogamy appears to be initiated by multiple contacts formed between the outer membranes of the adjoining nuclear envelopes. Blebs of endoplasmic reticulum (ER) originate from the outer membrane of each nuclear envelope; these ER blebs presumably contact and fuse with the outer membrane of the nuclear envelope of the opposing nucleus. This is followed by the fusion of the inner membranes of the opposing nuclear envelopes, thereby resulting in a series of small connective bridges between the two gamete nuclei. It is estimated that in this manner 30–50 bridges are formed, perhaps many more. Several of these bridges enlarge relative to the others; one presumably becomes the major connection between the fusing nuclei. As it continues to enlarge, any organelles positioned between the fusing nuclei are pushed aside. There is also evidence, particularly in later stages of karyogamy, that the smaller connective bridges fuse to form larger ones. Temporary cytoplasmic channels often result at the juncture of fusion. In other instances, isolated inclusions of cytoplasm may be delimited by remnants of nuclear envelope deep within the developing zygote nucleus; these inclusions disappear with subsequent development. Throughout karyogamy the contribution of the male gamete nucleus is readily recognized by the characteristic appearance of its highly condensed chromatin. Ultimately, however, this distinction is lost and the content of the mature zygote nucleus assumes a more uniform appearance very similar to that of an egg nucleus. The complete process of fertilization in Oedogonium may occur within 15 min of mixing the spermatozoids with eggs.     

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.     

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.     

Karyogamy after Electrofusion of Single Egg and Sperm Cell Protoplasts from Maize: Cytological Evidence and Time Course

In maize, in vitro fusion of isolated male gametes with isolated egg cell protoplasts can be induced by electric pulses. Until now, karyogamy has not been demonstrated. In this study, we cytologically examined fusion products fixed at different times after electrofusion with phase contrast microscopy and transmission electron microscopy. We obtained a precise timetable from 23 samples studied during the first 3 hr. The sperm nucleus was integrated within the egg cell protoplast, migrated toward the egg cell nucleus, and fused with it within 1 hr, as demonstrated by ultrastructural observations, three-dimensional reconstructions of nuclei, and subsequent nuclear volume estimates. Fusion of nuclei occurred before zygotic mitosis, as is the case in vivo. These findings demonstrate karyogamy during in vitro fertilization of maize.     

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.     

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.     

Cytological Events during Zygote Formation of the Fern Ceratopteris thalictroides

The cytological events, including nuclear fusion, digestion of male organelles and rebuilding of the plasmalemma and cell wall, during zygote formation of the fern Ceratopteris thalictroides (L.) Brongn. are described based on the observations of transmission electron microscopy. When the spermatozoid enters the egg and contacts the cytoplasm, the male chromatin relaxes continually. The microtubular ribbon (MTr) is separated from the male nucleus and then an envelope reappears around the male nucleus. During nuclear fusion, the egg nucleus becomes highly irregular and extends some nuclear protrusions. It is proposed that the protrusions fuse with the male nucleus actively. After nuclear fusion the irregular zygotic nucleus contracts gradually. It becomes spherical before the zygote divides. The male chromatin is identifiable as fibrous structure in the zygotic nucleus in the beginning, but it gradually becomes diffused completely. The male organelles, including the MTr, multilayered structure, flagella and the male mitochondria are finally digested in the zygotic cytoplasm. Finally a new plasmalemma and cell wall are formed outside the protoplast. The organelles in the zygote are rearranged, which produces a horizontal polarity zygote. The zygote divides with an oblique-vertical cell plate facing the apical notch of the gametophyte.     

Gamete fusion site on the egg cell and autonomous establishment of cell polarity in the zygote

Gamete fusion activates the egg in animals and plants, and the gamete fusion site on the zygote might provide a possible cue for zygotic development and/or embryonic patterning. In angiosperms, a zygote generally divides into a two-celled proembryo consisting of an apical and a basal cell with different cell fates. This is a putative step in the formation of the apical-basal axis of the proembryo. We observed the positional relationship between the gamete fusion site and the division plane formed by zygotic cleavage using an in vitro fertilization system with rice gametes. There was no relationship between the gamete fusion site and the division plane leading to the two-celled proembryo. Thus, the gamete fusion site on the rice zygote does not appear to function as a determinant for positioning the zygote division plane, and the zygote apparently possesses autonomous potential to establish cell polarity along the apical-basal axis for its first cleavage.Key words: asymmetric division, egg cell, fertilization, gamete fusion, rice, sperm cell, two-celled proembryo, zygote     

FERTILIZATION IN OEDOGONIUM. I. PLASMOGAMY12

Events prior to, during, and immediately following plasmogamy have been investigated in Oedogonium cardiacum using combined techniques of light and electron microscopy. Maturation of the oogonium involves the formation of an oogonial pore and the differentiation of the single egg from the larger oogonial protoplast from which it is formed. The fine structure of the sperm cell at the time of plasmogamy is described as well as the nature of its entrance into the oogonium. Cinematographic films were used to analyze the movements of the spermatozoids prior to plasmogamy and, similarly, 26 complete acts of gametic fusion were recorded and analyzed. Prior to plasmogamy the flagella-bearing anterior extremity of the spermatozoid typically becomes elongate and is thereafter capable of flexible movements and rapid changes in shape which appear more or less independent of the rest of the cell. The sperm cell always makes initial contact with the egg surface by means of this agile, proboscis-like, anterior end. Contact results through a combination of thrusting movements of the entire sperm cell and rapid, lateral sweeping movements of its flagellated anterior extremity against the egg surface. Gametic fusion is initiated with violent, vibrational movements of the sperm cell accompanied by loss of its flagella. Apparent fusion of the gamete membranes unites their protoplasts by a narrow cytoplasmic bridge which gradually increases in size as the sperm cell cytoplasm flows into the egg. An average time of 30.5 sec was required for complete fusion as determined from 25 typical sequences of plasmogamy recorded cinematographically. Fusion occurs even more rapidly when diploid oogonia are substituted for daploid oogonia. The entire sperm cell, with the exception of the flagella, fuses with the egg during plasmogamy. The dissimilar gamete nuclei are clearly distinguished ultrastructurally in the binucleate fusion cell. Concentrations of sperm cell mitochondria and remains of the flagellar apparatus (but no flagella) are readily recognized in the fusion cell. The fate of these and other cytoplasmic constituents of the sperm cell is discussed. Immediately after plasmogamy, and prior to karyogamy, a thin, finely fibrous layer is formed us an investment exterior to the fusion cell. Karyogamy follows shortly after plasmogamy, and both events may take place within 15 min after mixing eggs and spermatozoids.     

FERTILIZATION IN PLUMBAGO ZEYLANICA: GAMETIC FUSION AND FATE OF THE MALE CYTOPLASM

Developmental phases surrounding the processes of gametic delivery and fusion were examined ultrastructurally in the reduced megagametophyte of Plumbago zeylanica, which lacks synergids. Gametic delivery occurs at the end of pollen tube growth and results in deposition of two male gametes, a vegetative nucleus, and a limited amount of pollen cytoplasm between the egg and central cell. Discharge of these materials from the tube is accompanied by loss of inner and outer pollen tube plasma membranes, loss of sperm-associated cell wall components, and disruption of the formerly continuous cell wall between the egg and central cell. The dispersion of egg cell wall components directly exposes female reproductive cell membranes to the unfused male gametes and pollen tube without disrupting gametic cell plasma membranes. Presence of unfused sperms within the female gametophyte appears to be a transitory phenomenon, lasting less than 5 min at the end of over 8½ hr of pollen tube growth. At the time of gametic deposition, plasma membranes of unfused sperm cells become directly appressed to plasma membranes of both the egg and central cell. Gametic fusion is initiated by a single fusion event between membranes of participating male and female cells, which is rapidly followed by subsequent, secondary fusion events between the same two cells at different locations along their surface. Gametic fusion results in the transmission of male gamete nuclei with co-transmission of nearly the entire sperm cytoplasmic volume and organellar complement, and it is possible to identify heritable male cytoplasmic organelles within both the incipient zygote and endosperm. Paternally originating plastids may be distinguished from maternal plastids by differences in morphology and staining characteristics, whereas paternal mitochondria may be distinguished from maternal mitochondria by populational differences in mitochondrial size which are statistically significant. Such observations further indicate that transmitted paternal mitochondria seem to remain viable, as judged by their ultrastructural appearance, and are transmitted exclusively by sperm cytoplasm rather than discharged pollen cytoplasm. The presence of anucleate, membrane-bounded cytoplasmic bodies between the egg and central cell are identifiable on the basis of their enclosed organelles and indicate that fragmentation of a small amount of the sperm cytoplasm associated with the vegetative nucleus commonly occurs. The presence and identification of sperm cytoplasmic organelles and associated membranes within female reproductive cells following gametic transmission represents strong evidence in support of the cellular basis of nuclear and cytoplasmic transmission during sexual reproduction in Plumbago.     

Inheritance pattern of chloroplast DNA is correlated with gamete types based on sex-specific arrangement of the cell fusion site in Caulerpa (Ulvophyceae, Chlorophyta)

Using field emission scanning electron microscopy (FE‐SEM) and fluorescence microscopy, the respective relationships between the arrangement of the gamete cell‐fusion site and the inheritance pattern of chloroplast DNA (cp‐DNA) were studied for Caulerpa brachypus Harvey, C. okamurae Weber‐van Bosse, C. racemosa (Forsskål) J. Agardh var. laete‐virens (Montagne) Weber‐van Bosse, and C. serrulata (Forsskål) J. Agardh var. serrulata f. lata (Weber‐van Bosse) Tseng. The eyespot of the biflagellate gamete was visualized using FE‐SEM. The female gamete, but not the male, has one eyespot on the cell body posterior. In most mating pairs, the female gamete is fused at the anterior left side of the eyespot and the male gamete at a cell surface that is perpendicular to the plane of the flagellar beat when both gametes are mixed. Then, the inheritance pattern of cp‐DNA was observed using fluorescence microscopy after staining with 4′6‐diamidino‐2‐phenylindole. Male and female gametes have one cell nucleus and one chloroplast each. Chloroplasts of the female gamete usually contain 1–11 spherical or rod‐shaped nucleoids. In contrast, nucleoids are not usually detected in the male gamete’s chloroplast. After mixing male and female gametes, the male gamete without nucleoids and female gametes with nucleoids are always associated at the lateral side and become planozygotes. Such a correlation between the arrangement of the cell fusion site and the inheritance pattern of cp‐DNA was found in another member of Caulerpales, Bryopsis maxima Okamura. These results suggest the possibility that the arrangement of the cell fusion site in the gamete is not determined randomly regardless of sex, but is rather correlated with specific mating types. The relation of these results to those for Chlamydomonas is discussed.     

EVIDENCE FOR SEXUAL REPRODUCTION IN THE MARINE DINOFLAGELLATES, PYROCYSTIS NOCTILUCA AND PYROCYSTIS LUNULA (DINOPHYTA)

Evidence for sexual reproduction was observed in two oceanic dinoflagellate species, Pyrocystis noctiluca Murray ex Haeckel and Pyrocystis lunula (Schütt) Schütt. Observations suggest that cells underwent fertilization as opposed to cell division because of the following: first, fusing cells had a conspicuous pore (fusion pore) connecting the two gametes; dividing cells lacked this feature. In culture, about 0.1% of P. noctiluca cells had a fusion pore, which serves as a possible site for gamete recognition on the cell wall. Second, we document a temporal progression of plasmogamy and karyogamy. Fusion events in both species were observed at the beginning of the day, whereas division stages were most apparent at the end of the day.     

Studies on Metamastophora (Corallinaceae,Rhodophyta). I. M. flabellata (Sonder) Setchell: Morphology and anatomy

A morphological-anatomical study of Australian populations of Metamastophora flabellata (Sonder) Setchell, the type species of Metamastophora (Corallinaceae, Rhodophyta), has revealed that the primarily erect or ascending non-geniculate thallus possesses a dorsi-ventral organization of tissues. All conceptacles are uniporate and arise dorsally. Two distinct vegetative meristems occur: an apical primary meristem from which hypothallial cells are produced basipetally and a sub-epithallial secondary meristem which generates perithallial cells basipetally and secondary epithallial cells acropetally. Primary epithallial cells arise from divisions of subapical hypothallial cells. In younger parts, tissues are produced only dorsal to the hypothallium; in veins and stipes, tissue production occurs both dorsal and ventral to the hypothallium. Mature tetrasporic conceptacles contain peripheral tetrasporangia with zonately divided contents and a central sterile columella. Gametic conceptacles produce fertile tissue across the entire conceptacle chamber floor. After fertilization, the zygotic nucleus or a derivative is transferred (presumably) to an auxiliary cell through cells of the carpogonial branch; no tubular transfer siphon develops. Mature fusion cells are composed of the amalgamated supporting cells of carpogonial branches and are initiated from a single supporting cell which functions as an auxiliary cell. Unbranched 3–4 celled gonimoblast filaments arise from the fusion cell, do not become connected to other cells, and produce terminal carposporangia. Results from this study have led to a redefinition of hypothallium and perithallium in relation to meristems rather than substrate. In addition, carposporophyte ontogeny in the Corallinaceae is considered in terms of the presumed mode of transfer of the zygotic nucleus to the fusion cell, the extent of fusion cell development, and gonimoblast filament production in relation to auxiliary cells and fusion cells.     

The changes in ultrastructure during fertilization of the colourless flagellate Polytoma papillatum with special reference to the configural changes of their mitochondria

Changes in the morphology of the mitochondrial inventory (= chondriome), the nucleus and the flagellar apparatus during the generative (sexual) life cycle of Polytoma papillatum were examined by means of the serial sectioning technique. At the onset of copulation gametes do not differ obviously from interphase cells of the vegetative (asexual) life cycle, in that, both primarily contain one basket-shaped mitochondrion. The quadriflagellate and binucleate zygote exhibits a chondriome which consists of one large highly reticulated basket at the periphery of the zygote and 33 smaller mitochondrial units. Therefore, the basket clearly results from fusion of the two gamete chondriomes. The smaller mitochondrial fragments are either spherical to ovoid or elongated and poorly branched; they tend to occupy more central regions of the zygote. After karyogamy the mitochondrial basket disintegrates into several fragments of various shapes and sizes. Most of the mitochondrial fragments are located at the periphery. At the onset of karyogamy the nuclei and the flagellar apparatuses do not differ significantly from those of the gametes and vegetative interphase cells. The diploid nucleus, however, is characterized by: 1. many spherical bodies (diameter: ca. 200 to 600 nm) which are found both in the nucleoplasm and in the nucleolus. The major part of these bodies consists of material whose ultrastructure resembles that of the "pars fibrosa" in the nucleolus; 2. three deep invaginations of the nuclear membrane, two of which extend to the nucleolus; 3. an increase of nucleoplasm-filled cavities in the nucleolar "pars granulosa". The four flagella are considerably shortened; the basal bodies bound to the flagella have lost their striated connection and the roots have nearly completely disappeared. The results are compared with those obtained from investigations in Chlamydomonas; their significance in extranuclear genetics and in the systematics of Volvocales is discussed.     

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.     

Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae

We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid cell fusion in budding yeast may forgo cell cycle checkpoints present in vegetative mitosis.     

Eyespot behavior during the fertilization of gametes in Ulva arasakii Chihara (Ulvophyceae, Chlorophyta)

Behavior of the eyespots during the fertilization of Ulva arasakii Chihara was studied using field emission scanning electron microscopy (FE‐SEM). FE‐SEM enabled the visualization of the eyespot of biflagellate male and female gametes. The smaller male gamete has one protruded smaller (1.3 ± 0.15 μm× 1.0 ± 0.29 μm) eyespot and the larger female gamete has a larger (1.6 ± 0.2 μm× 1.1 ± 0.13 μm) one on a posterior position of the cell. The cell membrane over the eyespot region is relatively smooth compared to other parts of the cell body and exhibits hexagonal arranged lipid globules. Because the size of the cell and the morphology of the eyespot are different between male and female gametes, we could follow the fate of the eyespots during the fertilization. The initial cytoplasmic contact and fusion of the gametes takes place at their anterior end, slightly posterior to the flagellar base. The morphology of the fusing gametes followed two clearly distinguishable patterns. About half the gamete pairs lie side‐by‐side with their longitudinal axes nearly parallel, while the rest are oriented anti‐parallel to each other. In all cases, the larger female gamete fused along the same side as the eyespot, while the smaller male gamete fused along the side away from its eyespot. As fusion proceeds, the gamete pair is transformed into the quadriflagellate planozygote, in which the eyespots are positioned side‐by‐side on the region of cell fusion. These observations indicated that the opposite positioning of the eyespot relative to the cell fusion site in male and female gametes is important for the proper arrangement of the eyespots in the planozygote. The significance of this feature in advanced green algae is briefly discussed.     

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.