Nucleolar structure during the meiotic prophase in Allium cepa anthers

Summary Naturally segregated nucleoli have been observed during the prophase in meiocytes of Allium cepa anthers. Under the light microscope the nucleolus is seen to consist of two clearly differentiated regions: a central core, which is strongly argyrophilic (ochre or dark brown) and slightly basophilic, surrounded by a basophilic peripheral region, which shows a low degree of argyrophilia. Under the electron microscope the central region appears as consisting of fibrillar elements (pars fibrosa), while the peripheral region proved to consist mainly of granules about 150 Å in diameter (pars granulosa).When the pachytene nucleolus of Allium cepa is stained with basic fuchsin, a small circular area appears intensely stained, which gradually grows larger as the pachytene proceeds. This characteristic structure, eventually reaching a size of 0.5–1.5 is regularly to be observed with a central vacuole. Under the electron microscope this area appears as a circular structure of high electron density, which corresponds in shape and size with the area revealed by the light microscope.The relationship between this new structure, which we have called globulus with other nucleolar structures is discussed.This work was partially supported by a grant from the Fondo de Ayuda a la Investigación, 1968, Spain. We wish to thank most especially Dr. M. C. Risueño, M.I. Rodriguez-García and J. M. Sogo, of the Cell Structures Section, (Department of Cytology) for their efficient collaboration. We also wish to thank M. C. Partearroyo and A. Partearroyo for technical assistance. One of the authors (J.C.S.) has a Research Training Fellowship awarded by the International Agency for Research on Cancer (World Health Organization).     

Nucleolar structure and synthetic activity during meiotic prophase and spermiogenesis in the rat

The ultrastructure of nucleoli was examined in developing rat spermatocytes and spermatids, with the help of serial sections. In addition, the radioautographic reaction of nucleoli as examined in rats sacrificed 1 hr after intratesticular injection of 3H(5')-uridine and taken as an index of the rate of synthesis of ribosomal RNA (rRNA). Primary spermatocytes from preleptotene to zygotene have small nucleoli typically composed of fibrillar centers, a fibrillar component, and a granular component, within which are narrow interstitial spaces. During early and mid-pachytene, nucleoli enlarge to about nine times their initial size, with the fibrillar and granular components forming an extensive network of cords--a nucleolonema--within which are wide interstitial spaces. Meanwhile, there appear structures identical to the granular component but distinct from nucleoli; they are referred to as extranucleolar granular elements. Finally, from late pachytene to the first maturation division, nucleoli undergo condensation, as shown by contraction of fibrillar centers into small clumps, while fibrillar and granular components condense and segregate from each other, with a gradual decrease in interstitial spaces. In secondary spermatocytes, nucleoli are compact and rather small, while in young spermatids they are also compact and even smaller. Nucleoli disappear in elongating spermatids. In 3H-uridine radioautographs, nucleolar label is weak in young primary spermatocytes, increases progressively during early pachytene, is strong by the end of mid pachytene, but gradually decreases during late pachytene up to the first maturation division. In secondary spermatocytes and spermatids, there is no significant nucleolar label. In conclusion, rRNA synthesis by nucleoli is low in young spermatocytes. During pachytene, while nucleoli enlarge and form a lacy nucleolonema, rRNA synthesis increases gradually to a high level by the end of mid pachytene. However, during the condensation and segregation of nucleolar components occurring from late pachytene onward, the synthesis gradually decreases and disappears. The small, compact spermatids arising from the second maturation division do not synthesize rRNA.     

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.     

Regulation of the meiotic cell cycle in oocytes

The mitotic and meiotic cell cycle share many regulators, but there are also important differences between the two processes. The meiotic maturation of Xenopus oocytes has proved useful for understanding the regulation of Cdc2-cyclin-B, a key activator of G2/M progression. New insights have been made recently into the signalling mechanisms that induce G2-arrested oocytes to resume and complete the meiotic cell cycle.     

Involvement of ecdysone in the control of meiotic reinitiation in oocytes of Locusta migratoria (Insecta,orthoptera)

Following their biosynthesis in the follicle cells of vitellogenic ovaries, large amounts of ecdysteroids pass into the oocytes where they accumulate and persist during ovulation and egg-laying. The present paper shows that free ecdysone is unevenly distributed in the oocytes exhibiting the highest concentrations in the region of the posterior pole where the final sequences of nuclear maturation, including germinal vesicle breakdown (GVBD), occur. A correlative study indicates that the concentrations of free ecdysone in this region are particularly high (10 to 20 μM) during two periods of meiotic reinitiation observed in the oocytes: reinitiation I, leading from prophase I to metaphase I with GVBD; and reinitiation II, from metaphase I to the end of meiosis. In vitro incubations of oocytes in meiotic arrest (prophase I) in the presence of exogenous ecdysone demonstrate that complete reinitiation (including GVBD) can be triggered in a dose-dependent manner by this hormone.     

Nucleolar cycle and localization of NORs in early embryos of Parascaris univalens

During early embryogenesis of the nematode Parascaris univalens (2n=2) the processes of chromatin diminution and segregation of the germ and somatic cell lineages take place simultaneously. In this study we analyzed the nucleolar cycle in early embryos, both in germinal and somatic blastomeres, by means of silver staining and antibodies against the nucleolar protein fibrillarin. We observed an identical nucleolar cycle in both types of blastomeres, hence, the chromatin diminution process has no effect on the nucleolar cycle of somatic blastomeres. We report the existence of outstanding differences between this cycle and those previously reported during early embryogenesis of other species. There is a true nucleolar cycle in early embryos that shows a peculiar nucleolar disorganization at prophase, and a preferential localization of prenucleolar bodies only on the euchromatic regions during nucleologenesis. Moreover, fibrillarin does not form a perichromosomal sheath in metaphase or anaphase holocentric chromosomes, probably owing to their special centromeric organization. The number and location of nucleolus organizer regions (NORs) in the chromosomal complement have been determined using silver impregnation, chromomycin A₃/distamycin A staining, and fluorescent in situ hybridization using an rDNA probe. There are only two NORs, one per chromosome, and these are lost in blastomeres after chromatin diminution. Moreover, the constant presence of two nucleoli in somatic blastomeres suggests that NORs are not affected during the fragmentation of euchromatic regions when this process occurs.     

Protein kinase cascades in meiotic and mitotic cell cycle control

Eukaryotic cell cycle progression during meiosis and mitosis is extensively regulated by reversible protein phosphorylation. Many cell surface receptors for mitogens are ligand-stimulated protein-tyrosine kinases that control the activation of a network of cytoplasmic and nuclear protein-serine (threonine) kinases. Over 30 plasma membrane associated protein-tyrosine kinases are encoded by proto-oncogenes, i.e., genes that have the potential to facilitate cancer when disregulated. Proteins such as ribosomal protein S6, microtubule-associated protein-2, myelin basic protein, and casein have been used to detect intracellular protein-serine (threonine) kinases that are activated further downstream in growth factor signalling transduction cascades. Genetic analysis of yeast cell division control (cdc) mutants has revealed another 20 or so protein-serine (threonine) kinases. One of these, specified by the cdc-2 gene in Schizosaccharomyces pombe, has homologs that are stimulated during M phase in maturing sea star and frog oocytes and mammalian somatic cells. Furthermore, during meiotic maturation in these echinoderm and amphibian oocytes, this is followed by activation of many of the same protein-serine (threonine) kinases that are stimulated when quiescent mammalian somatic cells are prompted with mitogens to traverse from G0 to G1 phase. These findings imply that a similar protein kinase cascade may oversee progression at multiple points in the cell cycle.     

Ecdysteroids and meiotic reinitiation in Palaemon serratus (Crustacea Decapoda Natantia) and in Locusta migratoria (Insecta orthoptera). A comparative study

Summary

Meiotic reinitiation has been studied in Locusta migratoria and Palaemon serratus in relation to the titre of free ecdysteroids present in the maturing oocyte. In both species meiotic reinitiation is characterized by two meiotic arrests, in prophase I and in metaphase I, and the first meiotic resumption which leads to germinal vesicle breakdown (GVBD) is correlated with increasing titres of ecdysteroids in the oocyte. Meiotic reinitiation has been successfully triggered in the oocytes of both species by incubation with physiological doses of ecdysteroids.     

Formation of chiasmata in the synchronous meiotic cycle of Lilium longiflorum

The results of treatment of meiotic cells of Lilium longiflorum by cycloheximide at the time of chiasma initiation and formation suggest that in the sequential phases of nuclear development, complete chiasma suppression resulting in achiasmate cells is attained prior to a quantitative reduction of chiasma frequency. Reduction of chiasma frequency and complete suppression are believed to be based on two different mechanisms.     

Recombination nodules and chiasma localization in two orthoptera

The distribution of recombination nodules (RNs) is reported from observations on two-dimensional spreads of Locusta migratoria and Chloealtis conspersa spermatocytes; C. conspersa is a known example of a species with terminally localized chiasmata, while L. migratoria has nonspecific positioning of chiasmata. Measurements of the distances from 102 RNs to the ends of the synaptonemal complexes (SCs) on which they were found show the RNs to be near-terminally localized in C. conspersa and to occur along the lengths of the SCs in L. migratoria. Thus, the localization of RNs appears to reflect the localization of chiasmata. These observations are interpreted as support for the proposed recombinant function of RNs.     

Checkpoint kinase 1 is essential for meiotic cell cycle regulation in mouse oocytes

Checkpoint kinase 1 (Chk1) plays key roles in all currently defined cell cycle checkpoints, but its functions in mouse oocyte meiosis remain unclear. In this study, we report the expression, localization and functions of Chk1 in mouse oocyte meiosis. Chk1 was expressed from germinal vesicle (GV) to metaphase II (MII) stages and localized to the spindle from pro-metaphase I (pro-MI) to MII stages in mouse oocytes. Chk1 depletion facilitated the G₂/M transition while Chk1 overexpression inhibited the G₂/M transition as indicated by germinal vesicle breakdown (GVBD), through regulation of Cdh1 and Cyclin B1. Chk1 depletion did not affect meiotic cell cycle progression after GVBD, but its overexpression after GVBD activated the spindle assembly checkpoint and prevented homologous chromosome segregation, thus arresting oocytes at pro-MI or metaphase I (MI) stages. These results suggest that Chk1 is indispensable for prophase I arrest and functions in G₂/M checkpoint regulation in meiotic oocytes. Moreover, Chk1 overexpression affects meiotic spindle assembly checkpoint regulation and thus chromosome segregation.     

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.