SEXUAL REPRODUCTION IN EPHEDRA NEVADENSIS (EPHEDRACEAE): FURTHER EVIDENCE OF DOUBLE FERTILIZATION IN A NONFLOWERING SEED PLANT

Since the initial discovery of double fertilization in angiosperms in 1898, a number of reports of double fertilization-like events in the genus Ephedra have appeared. Until recently, convincing documentation of double fertilization in Ephedra had not been presented. In Ephedra nevadensis, following entry of a single binucleate sperm cell into the egg cell, one sperm nucleus migrates in a chalazal direction to fuse with the egg nucleus. Contemporaneous with this first fertilization event, the ventral canal nucleus regularly migrates from its initially apical position within the egg cell to a more central position within the egg cytoplasm, where it fuses with a second sperm nucleus. Based on quantitative microspectrofluorometric analysis, occasional supernumerary nuclei within the egg cell (derived by migration through pores in the cell walls between jacket cells and the central cell or egg cell) can be ruled out as participating in the second fertilization event. The evolutionary establishment of double fertilization in Ephedra (or its ancestors) was dependent on a number of specific developmental preconditions: 1) persistence of the ventral canal nucleus (which is degenerate in many groups of nonflowering seed plants) through the time of normal fertilization; 2) regular displacement of the ventral canal nucleus from its initially apical position within the egg cell to a position within the egg cytoplasm where fusion of the egg nucleus with the first sperm nucleus earlier occurred; 3) acquisition of egg-like features by the ventral canal nucleus that allow it to attract and fuse with a sperm nucleus; and 4) consistent entry of a second sperm nucleus into the archegonial cavity to participate in a second fertilization event. Although it cannot be determined definitively whether double fertilization in Ephedra is evolutionarily homologous with double fertilization in flowering plants, comparative evidence is consistent with the hypothesis that double fertilization arose in a common ancestor of the Gnetales and angiosperms.     

Division of the generative nucleus in cultured pollen tubes of the Bromeliaceae

Pollen germination, division of the generative nucleus and position of the generative nucleus in the pollen tube during in vitro germination were examined for six bromeliad cultivars. The influence of mixed amino acids (casein hydrolysate) and individual amino acids (Arg, Asn, Asp, Glu, Gly, Met, Phe, Orn, Tyr) were tested. Aechmea fasciata and A. chantinii pollen tubes showed more generative nuclear division in cultured pollen tubes than the other four cultivars tested. Casein hydrolysate did not stimulate generative nuclear division. In general arginine (1 mM) improved division of the Aechmea generative nucleus and to a lesser extent this of Vriesea `Christiane', Guzmania lingulata and Tillandsia cyanea. A concentration of 2 mM arginine reduced pollen tube growth of Aechmea. The vegetative nucleus was ahead of the generative nucleus in approximately 50% of the pollen tubes of all cultivars studied. In about 25% of the pollen tubes, the generative nucleus was ahead and in ±25% pollen tubes the vegetative and generative nuclei were joined together. The distance between the two generative nuclei and the distance from the generative nuclei to the pollen tube tip differed significantly for Aechmea fasciata and A. chantinii. The influence of different amino acids for Aechmea fasciata and A. chantinii varied with respect to pollen germination and generative nuclear division. Arg and Met improved nuclear division of both Aechmea cultivars. Pollen germination and sperm cell production were not linked. This information is important to ameliorate in vitro pollination methods used to overcome fertilization barriers in Bromeliaceae and other higher plants.     

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.     

Autoradiographs of Pollen Tube Nuclei with Calcium-45

Autoradiography with Ca⁴⁵ has been used to obtain information about the relation between calcium and chromosomes. Labelled pollen from the Easter lily, Lilium longiflorum, was allowed to develop into pollen tubes between 5 and 6 cm. long in the styles of non-radioactive flowers. All of the nuclei, namely the tube nucleus and the two sperm nuclei, retain Ca⁴⁵ after this period of growth and development. Since the two sperm nuclei have formed during this interval by the mitotic division of the generative nucleus and growth of the tube has occurred under the influence of the tube nucleus, it is inferred that the calcium was bound in a stable nuclear component, the chromosomes.     

Prematurely condensed human sperm chromosomes after in vitro fertilization (IVF)

Summary During an in vitro fertilization (IVF) program 122 inseminated eggs showing polar body extrusion, but neither formation of pronuclei nor cell cleavage were analysed cytogenetically. Nine of these eggs showed prematurely condensed sperm chromosomes of the G₁-phase (G₁-PCC) besides the haploid set of maternal metaphase II chromosomes. This phenomenon can be explained by the permanent arrest of the oocytes at metaphase II after sperm penetration and hence the continuing presence of cytoplasmic chromosome condensing factors which lead to the induction of PCC in the sperm nucleus. The overall frequency of this aberrant type of fertilization was calculated to be in the order of 3–4% of all in vitro fertilized eggs.     

The organization of microtubules during generative-cell division inConvallaria majalis

Summary The organization of the microtubule cytoskeleton in the generative cell ofConvallaria majalis has been studied during migration of the cell through the pollen tube and its division into the two sperm cells. Analysis by conventional or confocal laser scanning microscopy after tubulin staining was used to investigate changes of the microtubule cytoskeleton during generative-cell migration and division in the pollen tube. Staining of DNA with 4,6-diamidino-2-phenylindole was used to correlate the rearrangement of microtubules with nuclear division during sperm cell formation. Before pollen germination the generative cell is spindle-shaped, with microtubules organized in bundles and distributed in the cell cortex to form a basketlike structure beneath the generative-cell plasma membrane. During generative-cell migration through the pollen tube, the organization of the microtubule bundles changes following nuclear division. A typical metaphase plate is not usually formed. The generative-cell division is characterized by the extension of microtubules concomitant with a significant cell elongation. After karyokinesis, microtubule bundles reorganize to form a phragmoplast between the two sperm nuclei. The microtubule organization during generative-cell division inConvallaria majalis shows some similarities but also differences to that in other members of the Liliaceae.Abbreviations CLSM confocal laser scanning microscopy - EM electron microscopy - GC generative cell - GN generative nucleus - MT microtubule - SC sperm cell - SN sperm nucleus - VN vegetative nucleus     

The developmental fate of angiosperm pollen is associated with a preferential decrease in the level of histone H1 in the vegetative nucleus

In angiosperm pollen, the vegetative cell is assumed to function as a gametophytic cell in pollen germination and growth of the pollen tube. The chromatin in the nucleus of the vegetative cell gradually disperses after microspore mitosis, whereas the chromatin in the nucleus of the other generative cell remains highly condensed during the formation of two sperm nuclei. In order to explain the difference in chromatin condensation between the vegetative and generative nuclei, we analyzed the histone composition of each nucleus in Lilium longiflorum Thunb. and Tulipa gesneriana immunocytochemically, using specific antisera raised against histones H1 and H2B of Lilium. We found that the level of histone H1 decreased gradually only in the vegetative nucleus during the development of pollen within anthers and that the vegetative nucleus in mature pollen after anther dehiscence contained little histone H1. By contrast, the vegetative nucleus contained the same amount or more of histone H2B than the generative nucleus. The preferential decrease in the level of histone H1 occurred in anomalous pollen with one nucleus (uninucleate pollen) or with two similar nuclei (equally divided pollen), which had been induced by treatment with colchicine. The nuclei in the anomalous pollen resembled vegetative nuclei in terms of structure and staining properties. The anomalous pollen was able to germinate and extend a pollen tube. From these results, it is suggested that the preferential decrease in level of histone H1 in pollen nuclei is essential for development of the male gametophytic cell through large-scale expression of genes that include pollen-specific genes, which results in pollen germination and growth of the pollen tube. Received: 9 May 1998 / Accepted: 4 June 1998     

Ordered arrangement and rearrangement of chromosomes during spermatogenesis in two species of planarians (Plathelminthes)

The pattern of distribution of telomeric DNA (TTAGGG), 28S rDNA, and 5S rDNA has been studied using fluorescence in situ hybridization (FISH) and primed in situ labelling during spermatogenesis and sperm formation in the filiform spermatozoa of two species of planarians, Dendrocoelum lacteum and Polycelis tenuis (Turbellaria, Plathelminthes). In both species, the positions of FISH signals found with each probe sequence are constant from cell to cell in the nuclei of mature sperm. Chromosome regions containing 5S and 28S rDNA genes are gathered in distinct bundles of spiral form. In early spermatids with roundish nuclei, the sites of a given sequence on different chromosomes remain separate. Centromeres (marked by 5S rDNA) gather into a single cluster in the central region of the slightly elongated sperm nucleus. During spermatid maturation, this cluster migrates to the distal pole of the nucleus. In Polycelis, telomeric sites gather into three distinct clusters at both ends and in the middle of the moderately elongated nucleus. These clusters retain their relative positions as the spermatid matures. All the chromosome ends bearing 28S rDNA gather only into the proximal cluster. Our data suggest that structures in the nucleus selectively recognise chromosome regions containing specific DNA sequences, which helps these regions to find their regular places in the mature sperm nucleus and causes clustering of the sites of these sequences located on different chromosomes. This hypothesis is supported by observations on elongated sperm of other animals in which a correlation exists between ordered arrangement of chromosomes in the mature sperm nucleus and clustering of sites of the same sequence from different chromosomes during spermiogenesis. Received: 15 December 1997; in revised form: 24 March 1998 / Accepted: 14 April 1998     

Sperm dimorphism in terms of nuclear shape and microtubule accumulation in Cyrtanthus mackenii

Pollen tubes of Cyrtanthus mackenii, a species with bicellular pollen, were cultured in vitro to investigate nuclear phase changes during generative cell division and male germ unit (MGU) formation, using flow cytometric analysis. Results revealed that sperm cells were formed after 12 h of culture. During sperm maturation, the nuclei of sperm cells were not associated with the vegetative nucleus (unassociated sperm cells; Sua) and became longer than those of sperm cells associated with the vegetative nucleus (Svn). These findings indicate that the pair of sperm cells in the C. mackenii MGU is dimorphic in terms of nuclear shape. Dimorphism coincides with anti-α-tubulin antibody immunofluorescence, which was higher in the Sua than in Svn. Following treatment with oryzalin, triggering microtubule depolymerization, differences between nuclear shapes in the two sperm nuclei disappeared, suggesting that microtubule accumulation between sperm cells in the MGU correlates with differences in the nuclear shape.     

Fine structure of sperm cells in pollen grains ofBeta

Summary The structure of sperm cells in mature trinucleate pollen grains ofBeta vulgaris L. was studied with the electron microscope. The ellipsoidal sperm cell nuclei and cytoplasm are products of mitosis and cytokinesis of the ellipsoidal generative cell. Each sperm cell is separated from the vegetative cytoplasm by two contiguous membranes which enclose its cytoplasm and nucleus. Microtubules present in the sperm cell cytoplasm may be responsible for sperm cell motility.Approved as Journal paper Nr. 846, Utah Agricultural Experiment Station, Logan, Utah.     

Brassica napus pollen development during generative cell and sperm cell formation

Summary Brassica napus pollen development during the formation of the generative cell and sperm cells is analysed with light and electron microscopy. The generative cell is formed as a small lenticular cell attached to the intine, as a result of the unequal first mitosis. After detaching itself from the intine, the generative cell becomes spherical, and its wall morphology changes. Simultaneously, the vegetative nucleus enlarges, becomes euchromatic and forms a large nucleolus. In addition, the cytoplasm of the vegetative cell develops a complex ultrastructure that is characterized by an extensive RER organized in stacks, numerous dictyosomes and Golgi vesicles and a large quantity of lipid bodies. Microbodies, which are present at the mature stage, are not yet formed. The generative cell undergoes an equal division which results in two spindle-shaped sperm cells. This cell division occurs through the concerted action of cell constriction and cell plate formation. The two sperm cells remain enveloped within one continuous vegetative plasma membrane. One sperm cell becomes anchored onto the vegetative nucleus by a long extension enclosed within a deep invagination of the vegetative nucleus. Plastid inheritance appears to be strictly maternal since the sperm cells do not contain plastids; plastids are excluded from the generative cell even in the first mitosis.     

Lebendbeobachtungen der Chromosomen-Aufregulation bei der Gallmücke Heteropeza pygmaea (Cecidomyiidae,Diptera)

In male-determined, paedogenetically developing eggs of Heteropeza pygmaea a restitutive fertilization takes place after meiosis. Two small nuclei of maternal origin (somatic nuclei) and the egg nucleus migrate to the center of the egg chamber. Their chromosomes then form the metaphase plate of the primary cleavage nucleus. The in vitro observations and the analysis of photomicrographs and time lapse films revealed that the metaphase stage can be reached in three different ways: 1. The egg nucleus and the two somatic nuclei form one common spindle. 2. The egg nucleus forms a spindle and the two somatic nuclei together form another one. The two spindles then fuse in late prometaphase and form a single spindle. 3. The egg nucleus alone forms a spindle. The chromosomes of the somatic nuclei migrate to the equator of this spindle. This variation in the restitutive fertilization is explained by an increasing asynchrony between the development of the egg nucleus and the slower somatic nuclei from the first to the third type.     

Sperm Identification in Maize by Fluorescence in Situ Hybridization

The two sperm cells of common origin within the pollen tube of flowering plants are each involved in a fertilization event. It has long been recognized that preferential fusion of one sperm with the egg can occur in B chromosome-containing lines of maize. If the second pollen mitosis begins with a single B chromosome, nondisjunction will result in one sperm possessing two B chromosomes and the other containing no B chromosomes. The B chromosome-containing sperm most often fertilizes the egg, whereas the sperm nucleus with no B chromosomes fuses with the polar nuclei. Despite the obvious advantages of being able to recognize and then track, separate, and analyze one sperm type from the other, it has not been possible because of the lack of sufficient detectable differences between the two types of sperms. In this study, we used a B chromosome-specific DNA sequence (pZmBs) and in situ hybridization to identify and track the B chromosome-containing sperm cell within mature pollen and pollen tubes. Our results are consistent with conclusions from previous genetic studies related to B chromosome behavior during pollen formation. Within pollen tubes, the position in which the B chromosome-containing sperm travels (leading or trailing) in relation to the sperm cell lacking B chromosomes appears to be random.     

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in <Emphasis Type="Italic">Alstroemeria aurea</Emphasis>

This study aimed to analyze male gamete behavior from mature pollen to pollen tube growth in the bicellular pollen species Alstroemeria aurea. For mature pollen, pollen protoplasts were examined using flow cytometry. The protoplasts showed two peaks of DNA content at 1C and 1.90C. Flow cytometry at different developmental stages of pollen tubes cultured in vitro revealed changes in the nuclear phase at 9 and 18 h after culture. Sperm cell formation occurred at 6–9 h after culture, indicating that the first change was due to the division of the generative cells into sperm cells. After sperm cell formation, the number of vegetative nucleus associations with sperm cells showed a tendency to increase. This association was suggested as the male germ unit (MGU). When sperm cells, vegetative nuclei, and partial MGUs were collected separately from pollen tubes cultured for 18 h and analyzed using a flow cytometer, the sperm cells and vegetative nuclei contained 1C DNA, while the DNA content of partial MGUs was counted as 2C. Therefore, the second change in the nuclear phase, which results in an increase in 2C nuclei, is possibly related to the formation of MGUs.     

Paternal cytoplasmic transmission in Chinese pine (Pinus tabulaeformis)

Summary. In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles – plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine. Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, People’s Republic of China.     

Some events of mitosis and cytokinesis in the generative cell of Ornithogalum virens L.

The organization of the microtubule (Mt) cytoskeleton during mitosis and cytokinesis of the generative cell (GC) in Ornithogalum virens L. (bicellular pollen type, chromosome number, n = 3) from prophase to telophase/sperm formation was investigated by localization of -tubulin immunofluorescence using a conventional fluorescence microscope and a confocal laser scanning microscope. Chromosomes were visualized with DNA-binding fluorochrome dyes (ethidium bromide and 46-diamino-2-phenyl-indole). The GC of O. virens is characterized by G2/M transition within the pollen grain and not in the pollen tube as occurs in the majority of species with bicellular pollen. It was found that prophase in the GC starts before anthesis and prometaphase takes place after 10 min of pollen germination. The prophase Mts are organized into three prominent bundles, located near the generative nucleus. The number of these Mt bundles is the same as the number of GC chromosomes, a relation which has not previously been considered in other species. The most evident feature in the prophase/ prometaphase transition of O. virens GC is a direct rapid rearrangement of Mt bundles into a network which appears to interact with kinetochores and form a typical prometaphase Mt organization. The metaphase chromosomes are arranged into a conventional equatorial plate, and not in tandem as is thought to be characteristic of GC metaphase. The metaphase spindle consists of kinetochore fibres and a few interzonal fibres which form dispersed poles. Anaphase is characterized by a significant elongation of the mitotic spindle concomitant with the extension of the distance between the opposite poles. At anaphase the diffuse poles converge. Cytokinesis is realized by cell plate formation in the equatorial plane of the GC. The phragmoplast Mts between two future sperm nuclei appear after Mts of the mitotic spindle have disappeared.Abbreviations DAPI 46-diamino-2-phenyl-indole - GC generative cell - GN generative nucleus - Mt microtubule This research was made possible in part due to TEMPUS Programme and Global Network for Cell and Molecular Biology UNESCO grants to Magorzata Bana. The experimental part of the work was done in Siena University. M. Banas is very grateful to Prof. Mauro Cresti and his group for scientific interest, offering the excellent laboratory facilities, and kind reception.     

The organization of the cytoskeleton in the generative cell and sperms ofHyacinthus orientalis

Summary The microtubular cytoskeleton of the generative cell (GC) ofHyacinthus orientalis has been studied until the formation of the sperm cells (SCs). Immunofluorescence procedures in combination with confocal laser scanning microscopy (CLSM) has enabled the visualization of the organization of the microtubular cytoskeleton. Chemical fixation and freeze-fixation electron microscopy have been used to investigate the cytoskeleton and the ultrastructural organization of the GC and SCs. During pollen activation the GC is spindle-shaped. Microtubules (MTs) are organized as bundles and distributed in proximity of the GC plasmamembrane, forming a basket-like structure. Following migration through the pollen tube, the basket-like structure becomes more intertwined. During the nuclear division the MTs are involved in the segregation of the chromosomes and kinetochores are clearly discernible. Association with organelles is also observed. The chromosomes of the GC remain condensed until they separate in two sperm nuclei. The pre-prophase band was never observed. At the end of the GC division the microtubular network reorganizes in the two SCs.Abbreviations CLSM confocal laser scanning microscopy - DAPI 46-diamidino-2-phenyl-indole - F-S freeze-substitution - GC generative cell - MT microtubule - PBS phosphate buffered saline - R-F rapid freeze-fixation - SC sperm cell - TBS tris buffered saline - VN vegetative nucleus     

The male germ unit of Rhododendron: quantitative cytology,three-dimensional reconstruction,isolation and detection using fluorescent probes

Summary The sperm cells of Rhododendron laetum and R. macgregoriae differentiate within the pollen tube about 24 h after germination in vitro. Threedimensional reconstruction shows that the sperm cells are paired together, and both have extensions that link with the tube nucleus, forming a male germ unit. Quantitative analysis shows that the sperm cells in each pair differ significantly in surface area, but not in cell volume nor in numbers of mitochondria or plastids. When isolated from pollen tubes by osmotic shock, the sperm cells became ellipsoidal and surrounded by their own plasma membrane, while a proportion remained in pairs linked by the inner tube plasma membrane. Both generative and sperm cells are visualized in pollen tube preparations by immunofluorescence with anti-tubulin and anti-actin monoclonal antibodies (MAbs) combined with H33258 fluorescence of the nuclei. Video-image processing shows the presence of an axial microtubule cage in the generative cells, and some microtubules are present in the cytoplasmic extensions that clasp the tube nucleus. Following sperm cell division, the extensive phragmoplast between the sperm nuclei is partitioned by the plasma membranes.     

Origin of facultative heterochromatin in the endosperm ofGagea lutea (Liliaceae)

Summary Facultative heterochromatin occurs not only in certain animals in connection with sex determination but also in members of at least one plant genus,Gagea (Liliaceae s. str.), but here in the course of embryo sac development, fertilization, and endosperm formation. The present contribution intends to provide undebatable photographic and cytometric evidence, previously not available, for the events in the course of which three whole genomes in the pentaploid endosperm nuclei ofGagea lutea become heterochroma-tinized. In this plant, embryo sac formation usually follows the Fritillaria type, i.e., the embryo sac is tetrasporic, and a 1 + 3 position of the spore nuclei is followed by a mitosis in which the three chalazal spindles fuse and two triploid nuclei are formed. A triploid chalazal polar nucleus is derived from one of these, which contributes to the pentaploid endosperm. These nuclei in the chalazal part of the embryo sac show stronger condensation compared with the micropylar ones. The pycnosis of the triploid polar nucleus is maintained and even enhanced during endosperm proliferation, while the micropylar polar nucleus and the sperm nucleus maintain their euchromatic condition. The origin of the heterochromatic masses in the endosperm nuclei from the three chalazal genomes of the central cell is unambiguously evident from the distribution of heterochromatic chromosomes in the first endosperm mitosis and the following interphase. DNA content measurements confirm a 3 2 relationship of heterochromatic and euchromatic chromosome sets, which is usually maintained up to the cellularized endosperm. Pycnotic nuclei in the chalazal part of megagametophytes are characteristic of several embryo sac types, but only forGagea spp. it is documented that such nuclei can take part in fertilization and endosperm formation.Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday