DNA methylation and demethylation events during meiotic prophase in the mouse testis.

The genes encoding three different mammalian testis-specific nuclear chromatin proteins, mouse transition protein 1, mouse protamine 1, and mouse protamine 2, all of which are expressed postmeiotically, are marked by methylation early during spermatogenesis in the mouse. Analysis of DNA from the testes of prepubertal mice and isolated testicular cells revealed that transition protein 1 became progressively less methylated during spermatogenesis, while the two protamines became progressively more methylated; in contrast, the methylation of beta-actin, a gene expressed throughout spermatogenesis, did not change. These findings provide evidence that both de novo methylation and demethylation events are occurring after the completion of DNA replication, during meiotic prophase in the mouse testis.     

Chromatin dynamics in the male meiotic prophase

During the male meiotic prophase in mouse and man, pairing and recombination of homologous chromosomes is accompanied by changes in chromatin structure. In this review, the dynamics of assembly and disassembly of the chromatin-associated complexes that mediate sister chromatid cohesion (cohesin) and maintain chromosome pairing (the synaptonemal complex) are described. Special features of the meiotic S phase are discussed, and also the dynamics of several key players that act together after the S phase at sites of meiotic double-strand break DNA repair. Current knowledge on histone modifications that occur during the male meiotic prophase is discussed, with special attention for the inactive chromatin of the X and Y chromosomes that constitutes the sex body. Finally, it is discussed that in the future, it will be possible to view the true chromatin dynamics during male meiosis in time, in living cells, through analysis of fluorescent-tagged proteins expressed in transgenic mice, using advanced fluorescent microscopy techniques.     

Silver-stained structures in mammalian meiotic prophase

Silver staining of mammalian spermatocytes revealed, in light microscopy, synaptonemal complex and structures within the sex vesicle. It is feasible to follow the chromosome pairing phenomenon from zygotene to pachytene by examining the behavior of synaptonemal complexes. Nucleolus organizer regions take heavy silver stain in pachytene but are no longer detectable in later stages of meiosis.     

Chromosome behaviour during meiotic prophase in the solanaceae

The molecular structure of chromatin during dogfish spermiogenesis was examined by electron microscopy after the dispersion of nuclei at low ionic strength. In early and late stages of differentiation (round and elongating spermatids), chromatin is globular, although basic nuclear proteins are different from those present in somatic nuclei. Three protein fractions are complexed with DNA in sperm nuclei. These fractions appear at the end of differentiation (elongated spermatids), subsequently undergoing a modification of their solubilization properties; only one protein fraction remains acid-soluble. Dispersed chromatin from sperm nuclei again shows a beads-on-a-string configuration both in the presence of the three specific sperm proteins and when the acid soluble fraction is extracted. Variations of the mean diameter of chromatin subunits during spermiogenesis appear rather limited compared to extensive modifications of chromatin superstructures.     

Chromatin organization during meiotic prophase ofBombyx mori

Chromatin organization during the early stages of male meiotic prophase inBombyx mori was investigated by electron microscopy. The analysis of nuclei prepared by the Miller spreading procedure, suggests that chromatin fibers which are 200–300 Å in diameter undergo an orderly folding coincident with the formation of the synaptonemal complex. In very early stages the chromatin is released in linear arrays typical of interphase chromatin material. With time loops containing 5–25 of B conformation DNA, initially visualized at the periphery of early meiotic prophase nuclei, aggregate into discrete foci. These foci coalesce to form the longitudinal axis of the chromosome in conjunction with the initial appearance of the axial elements of the synaptonemal complex. At pachytene, the loops are evenly distributed along the length of the chromosome and extend radially so that in well spread preparations the chromosome has a brush-like appearance. Throughout this period nascent RNP-fibers were visualized along some of the loops.     

Histone synthesis during meiotic prophase in Lilium

Anthers of Lilium candidum L. were cultivated on artificial media containing labelled amino acids. Histones were isolated from meiocytes and fractionated by the use of polyacrylamide gel electrophoresis (PAGE). Total histone synthesis was found not to terminate at the end of premeiotic interphase but to continue until at least zygotene. However, the rate of synthesis was reduced during prophase I compared to interphase. Separate fractions were synthesized asynchronously during the period from late interphase to zygotene. Tissue specific histone of meiosis (FM) was synthesized during late interphase and leptotene.Dedicated to Professor A. A. Prokofieva-Belgovskaia on the occasion of the seventieth anniversary of her birthday.     

Checkpoint mechanisms: the puppet masters of meiotic prophase

The coordinated execution of cell cycle processes during meiosis is essential for the production of viable gametes and fertility. Coordination is particularly important during meiotic prophase, when nuclei undergo a dramatic reorganization that requires the precise choreography of chromosome movements, pairing interactions and DNA double-strand break (DSB) repair. Analysis of the underlying regulatory mechanisms has revealed crucial and widespread roles for DNA-damage checkpoint proteins, not only in cell cycle surveillance, but also in controlling many processes uniquely characteristic of meiosis. The resulting regulatory network uses checkpoint machinery to provide an integral coordinating mechanism during every meiotic division and enables cells to safely maintain an error-prone event such as DSB formation as an essential part of the meiotic program.     

The structure of chromatin at meiotic prophase I

The qualitative and quantitative changes in molecular chromatin structures during the meiotic prophase I were studied. The following patterns were discovered: (1) unlike somatic cells, the syntheses of total histone and DNA and its integration into the chromatin occur independently and asynchronously: DNA replication is completed by the interphase, whereas the synthesis of histone and its integration into the chromatin continue to late meiotic prophase I, and (2) individual histone fractions are synthesized and integrated into the chromatin during meiotic prophase independently and asynchronously. Chromatin hydrolysis with nucleases DNI, STN, and SI demonstrated considerable differences in the hydrolysis products obtained at different stages of the meiotic prophase I; presumably, this reflects the differences between the structures of initial chromatin at different stages of the meiotic prophase I.     

The structure of chromatin at meiotic prophase I

The qualitative and quantitative changes in molecular chromatin structures during the meiotic prophase I were studied. The following patterns were discovered: (1) unlike somatic cells, the syntheses of total histone and DNA and its integration into the chromatin occur independently and asynchronously: DNA replication is completed by the interphase, whereas the synthesis of histone and its integration into the chromatin continue to late meiotic prophase I, and (2) individual histone fractions are synthesized and integrated into the chromatin during meiotic prophase independently and asynchronously. Chromatin hydrolysis with nucleases DNI, STN, and SI demonstrated considerable differences in the hydrolysis products obtained at different stages of the meiotic prophase I; presumably, this reflects the differences between the structures of initial chromatin at different stages of the meiotic prophase I.     

Colchicine action at meiotic prophase revealed by SC-spreading

In the present paper earlier findings on the interference of colchicine with presynaptic alignment and synaptonemal complex formation in Allium ursinum are corroborated and several other direct or indirect consequences of colchicine treatment, like deformations and decomposition of lateral elements and a high incidence of non-homologous associations are reported. Mechanisms of meiotic alignment and pairing are discussed with respect to their susceptibility to the known cytological and biochemical activities of colchicine.     

Structural organization of the meiotic prophase chromatin in the rat testis

Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4. The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin. However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions. Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels. While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones. A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles. Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA.     

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).     

Robertsonian chromosomes and the nuclear architecture of mouse meiotic prophase spermatocytes

Background

The nuclear architecture of meiotic prophase spermatocytes is based on higher-order patterns of spatial associations among chromosomal domains from different bivalents. The meiotic nuclear architecture depends on the chromosome characteristics and consequently is prone to modification by chromosomal rearrangements. In this work, we consider Mus domesticus spermatocytes with diploid chromosome number 2n = 40, all telocentric, and investigate a possible modification of the ancestral nuclear architecture due to the emergence of derived Rb chromosomes, which may be present in the homozygous or heterozygous condition.

Results

In the 2n = 40 spermatocyte nuclei random associations mediated by pericentromeric heterochromatin among the 19 telocentric bivalents ocurr at the nuclear periphery. The observed frequency of associations among them, made distinguishable by specific probes and FISH, seems to be the same for pairs that may or may not form Rb chromosomes. In the homozygote Rb 2n = 24 spermatocytes, associations also mediated by pericentromeric heterochromatin occur mainly between the three telocentric or the eight metacentric bivalents themselves. In heterozygote Rb 2n = 32 spermatocytes all heterochromatin is localized at the nuclear periphery, yet associations are mainly observed among the three telocentric bivalents and between the asynaptic axes of the trivalents.

Conclusions

The Rb chromosomes pose sharp restrictions for interactions in the 2n = 24 and 2n = 32 spermatocytes, as compared to the ample possibilities for interactions between bivalents in the 2n = 40 spermatocytes. Undoubtedly the emergence of Rb chromosomes changes the ancestral nuclear architecture of 2n = 40 spermatocytes since they establish new types of interactions among chromosomal domains, particularly through centromeric and heterochromatic regions at the nuclear periphery among telocentric and at the nuclear center among Rb metacentric ones.     

The diffuse diplotene stage of meiotic prophase in Neurospora

The prophase stages of meiosis in Neurospora crassa are re-examined following McClintock (1945) and Singleton (1953). A diffuse chromosome stage occurring between pachynema and diakinesis is described. It is proposed that the diffuse stage does not necessarily represent a condition of intense gene activity in the sense of directing the metabolic activity of the ascus.Supported by U. S. Public Health Service grants AI-01462 and GM-14263.