Pre-fertilization ovule development inCapsella: ultrastructure and ultracytochemical localization of acid phosphatase in the meiocyte

Summary Pre-meiotic and prophase I ovules ofCapsella bursa-pastoris (L.) Medic.(monosporic,Polygonum type of gametophyte development) were fixed routinely or incubated in a modified Gomori medium containing -glycerophosphate as a substrate. Prior to the beginning of meiosis the potential meiocyte is ultrastructurally similar to the other cells of the nucellus and is distinguished only by its size and position. At the initiation of prophase I dramatic ultrastructural and ultracytochemical changes take place in the female meiocyte. These include the sudden appearance of cytoplasmic structures composed of single and multiple concentric cisternae, distinctive changes in plastids and mitochondria, and the blebbing of 0.3 m double-membraned vesicles from the nuclear envelope. The concentric cisternae encapsulate portions of cytoplasm containing ribosomes, plastids, mitochondria, ER fragments and vesicles. Both single and multiple concentric cisternae localize high levels of acid phosphatase and function as autophagic vesicles (AVs) that sequester ribosomes and organelles for destruction during meiosis. Plastids stop dividing and become more spherical during prophase I. Some plastids localize acid phosphatase and many show continuities between the outer membrane and the plastid envelope and acid phosphatase-rich RER cisternae. Mitochondria appear as dense, contracted spheres or rods. Some mitochondria localize acid phosphatase but they do not show membrane confluencies with the ER. Some of the plastids and mitochondria that are segregated into the functional megaspore at meiosis II are destroyed but others apparantly survive meiosis and give rise to the plastid and mitochondrial populations of the young gametophyte (Schulz andJensen, unpublished). The lateral and end walls of the meiocyte show patches of intense aniline blue fluorescence and the chalazal end wall of the cell is perforated with large numbers of plasmodesmata.Research supported by NSF Grant PCM-79-11018. The authors gratefully acknowledge the valuable assistance of David Lee Ivans in this project.     

The activity of the nucleolus organising region and the origin of cytoplasmic nucleoloids in meiocytes ofLilium

Summary The origin of the nucleolus-like bodies (nucleoloids) released into the cytoplasm during the meiotic divisions in pollen mother cells ofLilium has been traced. Chains of accessory nucleoli are formed at the nucleolus organising regions (NOR) of the nucleolar chromosomes during pachytene and diplotene while the parent-cell nucleolus is undergoing dissolution. Autoradiography using³H-uridine as a tracer shows that this involves the resumption of RNA synthesis at the NOR, although no new synthesis is associated with the parent-cell nucleolus. The accessory nucleoli are released from the NOR to become distributed throughout the nucleus in late prophase; there is no evidence that they contain DNA. In division phases, their material is probably held at the chromosome surfaces as part of the metaphase sheath. After the divisions, globuli are re-formed, and these eventually appear as the nucleoloids after detachment into the cytoplasm. It seems improbable that a gene amplification phase is associated with accessory nucleolus or nucleoloid formation. Evidence from a wide range of species suggests that the production of cytoplasmic nucleoloids during microsporogenesis is a general phenomenon among angiosperms, probably linked with the rapid build-up of ribosome numbers which follows upon the period of elimination in the meiotic prophase.     

Behavior of silver stainable material during mitosis inVicia faba

To reveal the behavior of silver stainable material localized mainly in the nucleoli and nucleolar organizing regions (NORs), the somatic cells ofVicia faba were investigated by silver staining throughout the mitotic cell cycle. Nucleoli of interphase and early prophase nuclei were darkly stained. From late prophase to anaphase the secondary constrictions were discriminated as silver stained NORs and many silver grains appeared throughout the cytoplasm. At late prophase the NOR condensed at the same rate as the chromosome arm. Small spherical bodies and two new nucleoli appeared in telophase nuclei and at the same time the cytoplasmic grains disappeared. On the basis of the above observations on the silver stainable material during each mitotic phase, the behavior of silver stainable material is interpreted.     

Meiosis in Mesostoma ehrenbergii ehrenbergii (Turbellaria,Rhabdocoela)

Female meiosis in Mesostoma ehrenbergii ehrenbergii is achiasmate. Electron microscope serial section reconstructions of nuclei from the germarium region of the ovary have shown that synaptonemal complex (SC) is present during the early prophase stages. The greatest amounts present were in two nuclei containing 389 and 401 respectively. The reconstructions showed that discontinuities in the SC existed along the lengths of the bivalents in these two nuclei which were taken to represent the maximally paired stage, i.e., pachytene. — Reconstructions of post pachytene nuclei showed that SC was absent from the bivalents and therefore retention of SC until metaphase I is not the mechanism used by this achiasmate species to ensure homologous chromosome segregation. An alternative mechanism for this function is proposed based on observations made on the later stages of meiosis in maturing oocytes.     

Ultrastructural changes in the cell wall,nucleus and cytoplasm of pollen mother cells during meiotic prophase I inLycopersicon esculentum (Mill.)

Summary Throughout the premeiotic to late prophase I stages of meiosis in the anthers of tomato (Lycopersicon esculentum) extensive changes occurred in the ultrastructure of pollen mother cells (PMCs). During early prophase, the wall of each PMC developed a layered appearance and was broadened both by the widening of the middle lamella as well as by intensive deposition of microfibrils in the wall. By late prophase, however, the microfibrils adjacent to the plasmalemma dissipated. At the same time, callose was deposited between the wall and the plasmalemma. The nucleus of the PMCs also underwent changes. During early prophase, the nucleolus consisted of a linear series of three segments, with a separation of the granular and fibrillar portions. By late prophase, the nucleoli were less distinct as the nucleus was highly vacuolate. Mitochondria were initially simple with lightly stained matrix and few cristae but, during the course of prophase, they acquired a more densely-stained matrix with dilated cristae. Plastids remained relatively undifferentiated and, at late prophase, many were convoluted in appearance and constricted at intervals indicating their division. Cytoplasmic connections between adjacent PMCs were broad enough to permit the passage of organelles and were retained through to metaphase I. These cytological and wall changes appear to be a prerequisite for the subsequent development of microspores.Abbreviations PMC pollen mother cell - NOR nucleolus organizing region     

Nucleolar structures in chromosome and SC preparations from human oocytes at first meiotic prophase

Summary We describe a comparative study of the behavior of nucleolar structures and their relationship with nucleolar chromosomes and synaptonemal complexes at first meiotic prophase of human oocytes in an attempt to elucidate the nature of this cellular organization and to learn more about maternal nondisjunction. The number of main nucleoli varies along the different stages of prophase I and is usually low. It shows an increase from leptotene to pachytene and a decrease from pachytene to diplotene related to a decrease and an increase of main nucleoli volume, respectively. The methodology employed has enabled us to analyze in detail dark bodies, round bodies, dense bodies, and main nucleoli in chromosome or synaptonemal complex spreads. The relationship between nucleolar chromosomes or synaptonemal complexes and the nucleoli implies the existence, in a very reduced space, of chromosomal regions that contain homologous sequences and that are often unpaired. This situation may facilitate the production of heterologous pairing and chromosomal exchanges between nonhomologous chromosomes and finally result in aneuploidy. THus, the situation explained above together with the differences between the oocyte and spermatocyte NOR cycles could be one of the reasons for the higher incidence of aneuploidies of maternal origin at meiosis I.     

Polar cytoplasmic evaginations in dividing spermatocytes of the firebug,Pyrrhocoris apterus (Pyrrhocoridae,Hemiptera)

Summary The present fine structure and anti-tubulin immunofluorescence study deals with evaginations from the cell surface in metaphase I spermatocytes of the firebugPyrrhocoris apterus (Pyrrhocoridae, Hemiptera). The surface of spermatogonia and prophase spermatocytes was smooth throughout. Only in metaphase I and anaphase I, cytoplasmic threads projected from polar portions of the spermatocytes. In contrast, equatorial portions of these cells possessed a smooth surface. By mid to late telophase, the evaginations were no longer detectable in spermatocytes. Three ideas are at hand to explain the development of polar cytoplasmic evaginations. First, they could represent a membrane reserve used up during spindle elongation in telophase of meiosis. In order to test this idea, spindle structure was analyzed in meiosis I using simultaneously antibodies to -tubulin and -tubulin. -Tubulin represents a tubulin isoform prevalent in centrosomes. The observations showed that spindle elongation was not very prominent in meiosis of the bug. Although it cannot be ruled out, the formation of a polar membrane reserve prior to spindle elongation is not a likely explanation for the evaginations from the cell surface. Second, the development of surface extensions could bring about increased exchange of metabolites during a particularly active stage of meiosis. Third, the polar evaginations could be an inadvertent product of the aster microtubules protruding towards the plasma membrane.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole.2 HCl - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N-tetraacetic acid - FITC fluorescein-isothiocyanate - PBS phosphate-buffered saline - PIPES piperazine-N,N bis (2-ethane sulfonic acid) - MT microtubule - MTOC microtubule-organizing centre     

Unusual prophase structures and multiple nucleoli in male meiosis of Drosophila species of the virilis group

With silver nitrate (Ag-NOR) staining, unusual fibrillar structures, apparently coupled to the nucleolus, were found is several species of the D. virilis group. In D. littoralis, beaded strings appear in connection with these structures, whereas the late prophase is characterized by the appearance of multiple nucleoli in the nucleoplasm. In D. virilis, the nucleus has a prominent pointed protrusion in the region of the nucleolus and often a fibril protrudes from this point. Small nucleoli are budding from the nucleolus during prophase. The multiple nucleoli at late prophase are smaller and fewer. A nucleolar body with black spots appears at prometaphase and persists through metaphase and anaphase. In D. lummei, the nucleolus becomes surrounded by fibrils, which are released into the nucleoplasm and on which multiple nucleoli are synthesized.These phenomena are similar to the events described in oocyte meiosis of many animals, where rDNA amplification, coupled to the synthesis of multiple nucleoli in late prophase, has been established.     

Ultrastructure of meiosis in the hollyhock rust fungus,Puccinia malvacearum I. Prophase I — Prometaphase I

Summary A thoroughly documented account of the ultrastructure of the meiotic spindle pole body (SPB) cycle in a rust (Basidiomycota, Uredinales) is presented for the first time. The three-dimensional structure of the SPB and spindle during meiosis in the hollyhock rust fungusPuccinia malvacearum is analyzed from serial sections of preselected stages. This paper covers prophase I to prometaphase I. At late prophase I, the nucleolus disperses and does not reappear until the end of meiosis. The SPB at late prophase I consists of two, 4-layered discs, 0.8–1.0 m in diameter, connected by a middle piece (MP). The SPB is associated with a differentiated region of the nuclear envelope and nucleoplasm. At late diplotene to diakinesis, each disc generates a half spindle as it inserts into an otherwise intact nuclear envelope. The MP connecting the interdigitating half spindles elongates and eventually splits transversely during subsequent spindle elongation. Each half MP, which is attached to a SPB disc, becomes inserted in a sheath-like extension of the nuclear envelope. The intranuclear late prometaphase I spindle always becomes oriented perpendicularly to the longitudinal axis and sagittal plane of the metabasidium. There are 200–290 spindle microtubules (MTs) at each SPB at late prometaphase. The nonkinetochore MTs form a coherent central spindle around which the kinetochore MTs and bivalents are spread. A metaphase plate is absent. The results are compared with SPB behavior and spindle structure in early meiosis of other basidiomycetes and ascomycetes.     

The F-actin distribution during microsporogenesis in Magnolia soulangeana Soul. (Magnoliaceae)

Summary F-actin distribution during male meiosis in Magnolia soulangeana was studied by means of fluorescence microscopy following staining with rhodaminephalloidin. Actin filaments were observed to persist during all of the developmental stages of meiosis. Four main types of configurations were recognized: (1) peripheral filaments underlying the plasma membrane (cortical network); (2) filaments dispersed throughout the inner cytoplasm (central cytoplasmic network); (3) filaments associated with the meiotic spindles; (4) filaments associated with the phragmoplasts. The cortical and central cytoplasmic filaments exhibited different behaviours. Whereas the cortical network remained present in an apparently unchanged form during all of the meiotic stages, the central cytoplasmic filaments, although they never completely disappeared, were reduced and concentrated around the nucleus at the end of prophase. At metaphase, fluorescent spindles consisting of filament bundles running from pole to pole or being interrupted at the equatorial zone could be seen. At the end of both the first and second division of meiosis, fluorescent bands of filaments (disks) appeared at the level of the cell division planes (equatorial regions) where cleavage furrows were constituted. These cleavage furrows did not form when floral buds were cultivated in a cytochalasin-containing medium. Our results show that during microsporogenesis in M. soulangeana the actin filaments constitute a highly complex and dynamic system that is involved in particular in cytoplasm cleavage of the meiocytes.     

Microtubule distribution in dv, a maize meiotic mutant defective in the prophase to metaphase transition

Microsporogenesis in Zea mays, the meiotic reduction of diploid sporocytes to haploid microspores, proceeds through a well-defined developmental sequence. The ability to generate mutants that affect the process makes this an ideal system for elucidating the role of the cytoskeleton during plant development. We have used immunofluorescence microscopy to compare microtubule distribution in wild-type and mutant microsporocytes. During normal meiosis the distribution of microtubules follows a specific temporal and spatial pattern that reflects the polar nature of microspore formation. Perinuclear microtubule staining increases and the nucleus elongates in the future spindle axis during late prophase I. Metaphase I spindles with highly focused poles align along the long axis of the anther locule. Cytokinesis occurs perpendicular to the spindle axis. The second division axis shifts 90 degrees with respect to the first division plane, thereby yielding an isobilateral tetrad of microspores. Microtubule distribution patterns during meiosis suggest that a nuclear envelope-associated microtubule organizing center (MTOC) controls the organization of cytoplasmic microtubules and contributes to spindle formation. The meiotic mutant dv is defective in the transition from a prophase microtubule array to a metaphase spindle. Instead of converging to form focused poles, the metaphase spindle poles remain diffuse as in prometaphase. This defect correlates with several abnormalities in subsequent developmental events including the formation of multinucleate daughter cells, multiple microspindles during meiosis II, multiple phragmoplasts, polyads of microspores, and cytoplasmic microtubule foci. These results suggest that dv is a mutation that affects MTOC organization.     

Microtubular configurations during meiosis and megasporogenesis in Gasteria verrucosa and Chamaenerion angustifolium

Summary In Gasteria and Chamaenerion, microtubular configurations were visualized immunocytochemically during meiosis and megasporogenesis in order to study their relationship to cell development, meiotic divisions and selection of the functional megaspore. In Chamaenerion, the intensity of the fluorescence found in megaspores was weaker than that found in Gasteria. Both plants exhibited concentrations of microtubules around the meiocyte nuclei during pachytene-diplotene. Preprophase bands were not observed. In Chamaenerion, cytoplasmic microtubules radiating from meiocyte nuclei were found at late prophase, the dyad stage and in the functional megaspore; in Gasteria, they were observed only at the dyad stage and in the functional megaspore. During the second meiotic division of Gasteria, dividing cells and their nuclei exhibited differences in volumes. Also, the two microtubular spindles of the dyad cells had different widths. Fluorescence indicating the presence of the cytoskeleton diminished during maturation of the large functional megaspores in both plants, whereas in the three degenerating smaller megaspores, fluorescence intensity persisted. Our conclusion is that only an indirect relationship exists between the organization of the microtubular cytoskeleton and selection of the functional megaspore.     

Sequential FISH analysis with rDNA genes and Ag-NOR banding in the lady beetle Olla v-nigrum (Coleoptera: Coccinellidae)

We have characterized the meiosis of Olla v-nigrum by standard analysis, performed a NOR study using NOR banding, FISH of rDNA genes and sequential FISH/AgNOR analysis, and adapted the FISH methodology to Coccinellidae. The chromosome number determined at metaphase I was n = 9 + Xyp. At zygotene it was possible to identify the sex vesicle which presented a deeply stained heteropycnotic block. Chromosome X is much larger than the y and the two combine, forming a "parachute" in metaphase I. FISH analysis using a probe of rDNA genes 18S, 28S and 5.8S of D. melanogaster was used to map the genes in the sex vesicle. The NOR band showed high gene activity in this region. These results were confirmed using sequential FISH/Ag NOR analysis. The data obtained for Olla v-nigrum agree with the classical hypothesis raised to explain the type of sex chromosome association in a parachute format (Xyp) as being due to the presence of nucleolar material. The chromosome number and parachute configuration during metaphase I in this species agree with the basic karyotype of most Coleopterans. The major adaptation of the FISH method was the simultaneous denaturation and hybridization that permitted preservation of chromosome morphology, an essential factor when the chromosomes are small.     

Noncanonical meiosis in the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> as a model for studying the molecular bases of the homologous chromosome synapsis,crossing over,and segregation

The nematode C. elegans is a classic study object of developmental biology and genetics, which is particularly suitable for studying the molecular bases of meiosis. Developing meiocytes are located in the threadlike gonads of C. elegans in linear gradient order of the stages of meiosis, which facilitates studying the order of intracellular events during meiosis. C. elegans has polycentric chromosomes. This causes a special order of events during meiosis, and as a consequence, meiosis in C. elegance differs from canonical meiosis of most eukaryotes. In the meiotic prophase I, all chromosomes carry single protein “pairing centers.” They are responsible for joining homologous chromosomes in pairs. This initiates the formation of synaptonemal complexes (SCs). Programmed double-stranded DNA breaks appear after initiation of the SC assembly, and they give rise to meiotic recombination. The initiation of meiotic recombination after the chromosome pairing distinguishes the C. elegans meiotic pattern from those in the absolute majority of eukaryotes studied. C. elegans has strict crossing over interference, which allows for the formation of one chiasma per bivalent. In the late prophase I, the polycentric centromeres are remodeled, one of the chromosome ends acquires a cuplike kinetochore, and during two meiotic divisions, chromosomes behave as monocentric. The study of meiosis in C. elegans allows for separate investigation of synapsis and recombination of homologous chromosomes and provides material for studying the evolution of meiosis.     

Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse (Equus caballus)

Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag-NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staining. The NOR foci were observed to be variable in number, size, and shape, but were usually located centrally and appeared as one or two nucleolus-like structures in the spermatozoa head. Three distinctive FISH signals identified the NOR-bearing chromosome pairs during the synaptic cell stage of meiosis I. At diakinesis/metaphase I, as well as different stages of meiosis II, FISH signals clearly depicted the NOR-bearing sister chromatids. The synaptonemal complexes of primary spermatocytes consistently showed three rDNA foci following FISH, but variably demonstrated two or three Ag-NOR bodies following silver staining. We propose rDNA loss and gain during unequal crossing-over events could be both a direct and indirect cause of variation in equine NOR foci. Additionally, our cytogenetic analysis did not confirm the presence of a fourth pair of NORs-bearing chromosomes in the horse, which is contrary to previously mitotic published data.     

Meiosis readiness in Lilium longiflorum “Croft”

It is proposed that anthers of Lilium longiflorum Croft approaching the end of premeiotic mitosis reach a state described as meiosis readiness after which cells in premeiotic prophase are unable to complete a mitotic division but despiralize to interphase and enter a meiotic division. Many of the laggard premeiotic cells begin despiralization before reaching an extremely contracted state of late prophase. Premeiotic despiralization is not, therefore, attributed to a deficiency in metaphase but to an inability of these cells to complete prophase. Premeiotic despiralization appears to be preceded by a slowing-down of prophase development. There is variation among anthers and anther regions in the onset of prophase retardation and meiosis readiness. It is suggested that meiosis readiness depends upon a gradual accumulation of meiosis-inducing substances in the cytoplasm of the premeiotic cells. It has not been determined whether the cells that undergo premeiotic despiralization give rise to the giant microsporocytes with shattered chromosomes observed at late prophase of meiosis.     

Inverted meiosis and its place in the evolution of sexual reproduction pathways

Inverted meiosis is observed in plants (Cyperaceae and Juncaceae) and insects (Coccoidea, Aphididae) with holocentric chromosomes, the centromeres of which occupy from 70 to 90% of the metaphase chromosome length. In the first meiotic division (meiosis I), chiasmata are formed and rodlike bivalents orient equationally, and in anaphase I, sister chromatids segregate to the poles; the diploid chromosome number is maintained. Non-sister chromatids of homologous chromosomes remain in contact during interkinesis and prophase II and segregate in anaphase II, forming haploid chromosome sets. The segregation of sister chromatids in meiosis I was demonstrated by example of three plant species that were heterozygous for chromosomal rearrangements. In these species, sister chromatids, marked with rearrangement, segregated in anaphase I. Using fluorescent antibodies, it was demonstrated that meiotic recombination enzymes Spo11 and Rad5l, typical of canonical meiosis, functioned at the meiotic prophase I of pollen mother cells of Luzula elegance and Rhynchospora pubera. Moreover, antibodies to synaptonemal complexes proteins ASY1 and ZYP1 were visualized as filamentous structures, pointing to probable formation of synaptonemal complexes. In L. elegance, chiasmata are formed by means of chromatin threads containing satellite DNA. According to the hypothesis of the author of this review, equational division of sister chromatids at meiosis I in the organisms with inverted meiosis can be explained by the absence of specific meiotic proteins (shugoshins). These proteins are able to protect cohesins of holocentric centromeres from hydrolysis by separases at meiosis I, as occurs in the organisms with monocentric chromosomes and canonical meiosis. The basic type of inverted meiosis was described in Coccoidea and Aphididae males. In their females, the variants of parthenogenesis were also observed. Until now, the methods of molecular cytogenetics were not applied for the analysis of inverted meiosis in Coccoidea and Aphididae. Evolutionary, inverted meiosis is thought to have appeared secondarily as an adaptation of the molecular mechanisms of canonical meiosis to chromosome holocentrism.