Ability of in vitro maturing bovine oocytes to transform sperm nuclei to metaphase chromosomes.

Bovine oocytes at the germinal vesicle stage were inseminated in Brackett & Oliphant's medium with bovine serum albumin, caffeine and heparin. Eight hours after insemination, oocytes were transferred into tissue culture medium-199 containing 10% fetal calf serum and cultured for 5-40 h at 39 degrees C in 5% CO2 in air. The proportions of unpenetrated and penetrated oocytes reaching metaphase II increased as the time of examination increased, reaching 70 and 65% 40 h after transfer, respectively. When oocytes were penetrated by more than four spermatozoa, meiotic maturation was greatly retarded. Sperm nuclei were decondensed in most (81%) penetrated oocytes 5 h after transfer. The decondensed sperm nuclei were recondensed and then transformed to metaphase chromosomes which were morphologically compacted at first but became slightly dispersed later. The formation of the metaphase chromosomes was observed in 86% of penetrated oocytes examined 40 h after transfer, and occurred in all metaphase II oocytes at that time. In oocytes penetrated by more than nine spermatozoa, no such transformation of sperm nuclei was observed. Well-developed male and female pro-nuclei were observed in only three (6%) of 51 oocytes penetrated 40 h after transfer.     

Transformation of sperm nuclei to metaphase chromosomes in the cytoplasm of maturing oocytes of the mouse

Zona-free oocytes of the mouse were inseminated at prometaphase I or metaphase I of meiotic maturation in vitro, and the behavior of the sperm nuclei within the oocyte cytoplasm was examined. If the oocytes were penetrated by up to three sperm, maturation continued during subsequent incubation and became arrested at metaphase II. Meanwhile, each sperm nucleus underwent the following changes. First, the chromatin became slightly dispersed. By 6 h after insemination, this dispersed chromatin had become coalesced into a small mass, from which short chromosomal arms later became projected. Between 12 and 18 h after insemination, each mass of chromatin became resolved into 20 discrete metaphase chromosomes. In contrast, if oocytes were penetrated by four to six sperm, oocyte meiosis was arrested at metaphase I, and each sperm nucleus was transformed into a small mass of chromatin rather than into metaphase chromosomes. If oocytes were penetrated by more than six sperm, the maternal chromosomes became either decondensed or pycnotic, and the sperm nuclei were transformed into larger masses of chromatin. As control experiments, immature and fully mature metaphase II oocytes were inseminated. In the immature oocytes, which were kept immature by exposure to dibutyryl cyclic AMP, no morphological changes in the sperm nucleus were observed. On the other hand, in the fully mature oocytes, which were activated by sperm penetration, the sperm nucleus was transformed into the male pronucleus. Therefore, the cytoplasm of the maturing oocyte develops an activity that can transform the highly condensed chromatin of the sperm into metaphase chromosomes. However, the capacity of an oocyte is limited, such that it can transform a maximum of three sperm nuclei into metaphase chromosomes. Furthermore, the presence of more than six sperm causes a loss of the ability of the oocyte to maintain the maternal chromosomes in a metaphase state.     

Neutron diffraction of chromatin in interphase nuclei and metaphase chromosomes

We have used neutron diffraction to study chromatin structure in interphase nuclei and metaphase chromosomes as a function of decreasing ion concentration. Aliquots of a suspension of rat liver nuclei prepared in a polyamine-free buffer were washed in buffers of 1/3, 1/6 and 1/12 if the original concentration of monovalent and divalent cations (40 mM KCl; 20 mM NaCl; 1.2 mM MgCl2). After the first dilution step (1/1 to 1/3), only small changes occurred in the diffraction pattern. They can be interpreted by a loosening of the original structure, i.e. by the formation of isolated buffer-filled spaces with an overall size of the order of 35-45 nm. Drastic changes in the diffraction pattern were observed, however, when the nuclei were washed in the more diluted buffers (1/6 and 1/12). The profiles of the distances distribution functions indicate the formation of supranucleosomal particles with an overall diameter of 40-50 nm. The compact chromatin structure disassembled directly into these fundamental structural units. Structural transformations in the Chinese hamster ovary metaphase chromosomes were induced by diminishing the Ca2+ ion concentration of the buffer from originally 3.0 mM to 0.3 mM and/or by increasing the pH value of the buffer from originally 7.0 up to 8.0. The neutron diffraction patterns remained essentially unchanged during these treatments, i.e. the decondensation of the chromosomes as observed in the light microscope is not accompanied by disassembly at the ultrastructural level between 2 nm and 150 nm.     

Ultrastructure of the centrometric regions of mouse chromosomes in metaphase chromosomes and interphase nuclei]

The chromatin ultrastructure was studied in the centromeric region of mitotic chromosomes and in interphase nuclei of mouse cells after differential staining on C-band. A new method is suggested to study centromeric region of chromosomes treated by the Giemsa banding technique. Fibers of chromosomes appeared to be packed denser in the centromeric regions of mitotic chromosomes than in arms. The disposition of chromatin fibers in the centromeric chromocentres of interphase nuclei is the same as in the centromeric regions of mitotic chromosomes.     

Comparative studies on the structural organization of membrane-depleted nuclei and metaphase chromosomes

Interphase membrane-depleted nuclei and metaphase chromosomes were prepared in parallel with a nonionic detergent lysis procedure at low ionic strength. By flow microfluorometry we showed for the first time that cell lysates contain all stages of the cell cycle in the same proportions as the starting cell population. Morphologically intact membrane-depleted nuclei and metaphase chromosomes were isolated as non-aggregated structures on sucrose gradients. When analysed in the electron microscope, membrane-depleted nuclei that had been treated with 2M NaCl appeared as residual structures containing the pore complex-lamina layer attached to a halo of DNA filaments. In contrast, no distinct high salt-resistant structure was found with metaphase chromosomes. They formed a highly fragile network which disintegrated easily into small complexes connected with DNA filaments. High salt-resistant DNA-protein complexes were purified by Metrizamide density gradient centrifugation. The main difference in the protein composition of interphase and metaphase residual complexes was the presence in interphase of a protein triplet in the 60–75 kilodalton molecular weight range and its absence in metaphase. This protein triplet most likely corresponds to the lamins A, B, and C of the nuclear lamina. The combined results suggest that the main difference in the structural organization of interphase nuclei and metaphase chromosomes is the presence or absence of the pore complex-lamina layer.     

Inhibition by dibutyryl cyclic AMP of the transition to metaphase of mouse oocyte nuclei and its reversal by cell fusion to metaphase oocytes

Mouse oocytes at metaphase I of meiotic maturation were treated with puromycin, which caused the condensed chromosomes to become decondensed to form an interphase nucleus. The chromosomes returned to a metaphase state 6.3 hr after the oocytes were transferred to puromycin-free medium [H. J. Clarke and Y. Masui (1983) Dev. Biol. 97, 291-301]. In contrast, the chromosomes of the puromycin-treated oocytes remained decondensed within the nucleus if dibutyryl cyclic AMP (dbcAMP) was included in the puromycin-free medium. This implies that dbcAMP inhibited the development of conditions in the oocytes that were required for the transition to metaphase. The chromosomes of puromycin-treated oocytes that were incubated for 7.5 hr in dbcAMP-containing medium returned to metaphase just 1.9 hr after transfer to dbcAMP-free medium. Therefore, the protein synthesis-dependent process that is required for the transition to metaphase could occur in the presence of dbcAMP. Fusion to metaphase II oocytes, or to puromycin-treated oocytes that had returned to metaphase, rapidly induced transition of the nuclei of dbcAMP-inhibited oocytes to metaphase, despite the presence of the inhibitor. These results suggest that the transition of nuclei to metaphase can be induced by a cytoplasmic factor that is present in metaphase oocytes, and that dbcAMP inhibits the development of this factor.     

Regulation of meiotic metaphase by a cytoplasmic maturation-promoting factor during mouse oocyte maturation

During mouse oocyte maturation the regulation of the activity of a cytoplasmic maturation-promoting factor (MPF) was examined. The mouse MPF activity was determined based on its ability to induce maturation in immature starfish oocytes after microinjection with the cytoplasm from mouse oocytes. MPF appeared initially at germinal vesicle breakdown (GVBD), and its activity fluctuated in exact correspondence with meiotic cycles, reaching a peak at each metaphase and almost disappearing at the time of emission of the first polar body. Cycloheximide affected neither the initial MPF appearance nor GVBD. Thereafter, however, in the presence of cycloheximide the meiotic spindle was not formed and MPF disappeared, although the chromosomes remained condensed. After removing cycloheximide, MPF reappeared and was followed by the first metaphase and subsequently by polar body emission. Finally the meiotic cycle progressed to the second metaphase. Thus, for the appearance of MPF, there is a critical period shortly before the first metaphase, after which protein synthesis is required. In the presence of either cytochalasin D or colcemid, MPF activity remained at elevated levels. Addition of cycloheximide to such cytochalasin-treated oocytes, in which the meiotic cycle was arrested at the first metaphase, caused the MPF levels to decrease and was followed by movement of chromosomes to both poles where they decondensed and two nucleus-like structures were formed. Thus, the disappearance of MPF may initiate the metaphase-anaphase transition. Furthermore, detailed cytological examination revealed that chromosomes in cytochalasin-treated oocytes were monovalent while those treated only with cycloheximide were divalent, suggesting that dissociation of the synapsis is a prerequisite for chromosome decondensation after the disappearance of MPF. In all these respects, MPF seems to be a metaphase-promoting factor rather than just a maturation-promoting factor.     

Fluorescence in situ hybridization to Y chromosomes in decondensed human sperm nuclei

Human sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome-specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.     

The morphological relationship between mitochondria and cytoplasmic membranes of the follicular oocyte in domestic pig

Summary An ultrastructural study of the mature follicular oocytes in domestic pig demonstrate a morphological relationship between the mitochondria and the cytoplasmic membranes immediately surrounding the yolk globules of the cells. Frequently, the cytoplasmic membranes are observed to be in close proximity of the mitochondria or are found to be continuous with the outer mitochondrial membrane. Sometimes the cytoplasmic membranes are found to display the formation of one or more oval loops of different diameter located at their presumed ends or free in the nearby cytoplasm. The significance of these observations is discussed in the light of the available informations, which suggest that the cytomembrane system in certain phases of development may take part in the formation of mitochondria.This work was supported by the Agricultural Research Council of Norway.     

In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.     

The arrangement of chromosomes in nuclei of sperm from plethodontid salamanders

Plethodontid salamanders have n = 13 or 14 large metacentric or sub-metacentric chromosomes. Sperm nuclei from Plethodon cinereus measure 72×1 m. The nucleoprotein of spermatids is at first finely granular. In elongate spermatids it clumps into larger granules, which then fuse to form the compact nucleoprotein of the mature sperm. The nuclei of mature sperm are negatively birefringent with respect to their length. — ³H RNA complementary to high-density satellite DNA of centromeric heterochromatin in P. cinereus has been hybridized in-situ to spermatids and sperm, and its site of binding to these cells has been examined by autoradiography. Labelling of round spermatid nuclei is localized in a single patch. Elongate spermatid nuclei are labelled only over the rear quarter of the nucleus. Label over the nuclei of mature sperm is localized in a region extending 10–20 m forwards from the rear of the nucleus. — In P. cinereus the ribosomal genes are located near the centromere on the short arm of chromosome 7. ³H ribosomal RNA hybridizes to a single patch in round spermatid nuclei. Elongate spermatid nuclei show label over a short segment of the rear half of the nucleus. In spermatids nearing maturity the labelled region is never more than 20 m long. — These results indicate that in P. cinereus each chromosome is arranged in a U formation with its centromere at the base of the sperm nucleus, and its arms extended forwards along the length of the nucleus. — Among plethodontids, increase in C value and corresponding increase in chromosome size is accompanied by increase in the length rather than the width of the sperm nucleus. — ³H ribosomal RNA hybridizes to a short segment in spermatid and sperm nuclei from Xenopus and Triturus. In these animals, the position of the labelled segment varies from sperm to sperm.     

Responses of mammalian metaphase chromosomes to endonuclease digestion

Digestion of fixed metaphase chromosomes by endonucleases (micrococcal nuclease and DNase II) under optimal digestion conditions followed by Giemsa staining produces sharp banding patterns identical to G-bands. In ³H-thymidine labeled, synchronized metaphase cells of the chinese hamster (CHO line), the band induction is accompanied by the removal of DNA. The single strand specific nuclease S1 and DNase I do not produce such banding patterns.     

The similarity of DNA sequences remaining bound to scaffold upon nuclease treatment of interphase nuclei and metaphase chromosomes.

The fragments of DNA attached to protein skeleton of interphase nuclei or metaphase chromosomes were obtained. Both the method involving restriction endonuclease treatment/1,2/and a novel procedure based on mild staphylococcal nuclease digestion were used. In the latter case, DNA fragments remaining bound to nuclei or chromosomes are not enriched in satellite but only in abundant middle repetitive DNA. The shorter the fragments of attached DNA, the higher the content of middle repetitive DNA in the fraction. It has a slightly higher density in a CsCl gradient comparing to the main DNA. The yield of attached DNA, its distribution in a CsCl density gradient, and its renaturation properties are essentially the same for interphase and metaphase chromosomes. The average size of DNA loops was found to be equal to approximately 60 kb for both metaphase chromosomes and interphase nuclei. The conclusion has been drawn that the bulk of attachment sites of DNP fibrils to axial chromosomal structures remains unchanged during the cell cycle.     

Low angle x-ray diffraction studies of chromatin structure in vivo and in isolated nuclei and metaphase chromosomes

Diffraction of x-rays from living cells, isolated nuclei, and metaphase chromosomes gives rise to several major low angle reflections characteristic of a highly conserved pattern of nucleosome packing within the chromatin fibers. We answer three questions about the x-ray data: Which reflections are characteristic of chromosomes in vivo? How can these reflections be preserved in vitro? What chromosome structures give rise to the reflections? Our consistent observation of diffraction peaks at 11.0, 6.0, 3.8, 2.7 and 2.1 nm from a variety of living cells, isolated nuclei, and metaphase chromosomes establishes these periodicities as characteristic of eukaryotic chromosomes in vivo. In addition, a 30-40- nm peak is observed from all somatic cells that have substantial amounts of condensed chromatin, and a weak 18-nm reflection is observed from nucleated erythrocytes. These observations provide a standard for judging the structural integrity of isolated nuclei, chromosomes, and chromatin, and thus resolve long standing controversy about the “tru” nature of chromosome diffraction. All of the reflection seen in vivo can be preserved in vitro provided that the proper ionic conditions are maintained. Our results show clearly that the 30-40-nm maximum is a packing reflection. The packing we observe in vivo is directly correlated to the side-by-side arrangement of 20- 30-nm fibers observed in thin sections of fixed and dehydrated cells and isolated chromosomes. This confirms that such packing is present in living cells and is not merely an artifact of electron microscopy. As expected, the packing reflection is shifted to longer spacings when the fibers are spread apart by reducing the concentration of divalent cations in vitro. Because the 18-, 11.0-, 6.0-, 3.8-, 2.7-, and 2.1-nm reflections are not affected by the decondensation caused by removal of divalent cations, these periodicities must reflect the internal structure of the chromaticn fibers.     

In situ hybridization to somatic metaphase chromosomes of potato

Summary An in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a ladderpattern in genomic Southern's of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.     

Architecture of metaphase chromosomes and chromosome scaffolds

We have developed procedures for depositing intact mitotic chromosomes and isolated residual scaffolds on electron microscope grids at controlled and reproducible levels of compaction. The chromosomes were isolated using a recently developed aqueous method. Our study has addressed two different aspects of chromosome structure. First, we present a method for improved visualization of radial chromatin loops in undisrupted mitotic chromosomes. Second, we have visualized a nonhistone protein residual scaffold isolated from nuclease-digested chromosomes under conditions of low salt protein extraction. These scaffolds, which have an extremely simple protein composition, are the size of chromosomes, are fibrous in nature, and are found to retain differentiated regions that appear to derive from the kinetochores and the chromatid axis. When our standard preparation conditions were used, the scaffold appearance was found to be very reproducible. If the ionic conditions were varied, however, the scaffold appearance underwent dramatic changes. In the presence of millimolar concentrations of Mg++ or high concentrations of NaCl, the fibrous scaffold protein network was observed to undergo a lateral aggregation or assembly into a coarse meshlike structure. The alteration of scaffold structure was apparently reversible. This observation is consistent with a model in which the scaffolding network plays a dynamic role in chromosome condensation at mitosis.     

Structure of and homology between pachytene and somatic metaphase chromosomes of the tomato

The pachytene and somatic metaphase chromosomes of tomato are structurally differentiated into proximal chromatic and distal achromatic parts. The pachytene chromosomes have very clear and characteristic chromosome markers, with the help of which all 12 bivalents can be clearly identified. Based on the size, the arm ratio, the ratio of chromatic parts and the presence and size of achromatic parts, all 12 pairs of somatic chromosomes can also be identified, and each pair be homologised with the corresponding pachytene bivalent. A comparison of the lengths of chromatic and achromatic parts of pachytene chromosomes with the chromatic and achromatic parts of the corresponding somatic chromosomes indicate, that, on an average, the chromatic parts are contracted by a factor of 4 to 5, whereas the achromatic parts are contracted by a factor of 30. The heteropycnosis near the centromere in tomato chromosomes therefore is not a special characteristic of meiotic chromosomes, but present in somatic metaphase chromosomes also.This study was part of a project resulting from a contract between the AssociationEuratom-I.T.A.L. and the Agricultural University of Wageningen.     

Banding and spiralization of human metaphase chromosomes

Summary A new technique is described which produces spiralization of human metaphase chromosomes. The important feature is heat followed by trypsin treatment. By varying conditions, it is possible to produce bands, spirals and intermediate stages. This provides a new approach to the understanding of banding and chromosome structure.