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Viability of meiotic prophase spermatocytes of rats is facilitated in primary culture of dispersed testicular cells on collagen gel by supplementing epinephrine or norepinephrine: evidence that meiotic prophase spermatocytes complete meiotic divisions in vitro.

Dispersed testicular cells prepared from 14-d-old rats were cultured on type 1 collagen gels using a medium composed of a 1:1 mixture of Ham's F12 medium and Leibovitz's L15 medium (F12-L15 medium) containing 10% (vol/vol) fetal bovine serum. The viability of the spermatogenic cells was facilitated by supplementing a rat adrenal extract into the medium. The effective substance(s) (the survival factor) was purified from acid extracts of adrenals by molecular sieve high performance liquid chromatography and identified as epinephrine and norepinephrine. Both epinephrine and norepinephrine promoted the survival of the spermatogenic cells with a half saturating dose of 10 ng/ml. The spermatogenic cells, which could be cultured for 2 wk on a collagen gel by supplementing with the survival factor (epinephrine or norepinephrine), were subjected to Giemsa staining and to DNA flow cytometry. The following results were obtained: a) The spermatogenic cells from 14-d-old rats did not contain spermiogenic cells (1c-cells). b) During a culture period of 2 to 7 d the ratio of meiotic prophase spermatocytes (4c-cells) to premeiotic cells (2c-cells) increased. On Day 7, more than 90% of the surviving cells were meiotic prophase spermatocytes. c) On Day 10, spermatids (1c-cells) appeared for the first time. The time of the first appearance of spermatids in the culture was consistent with that in vivo. These results suggest that both epinephrine and norepinephrine facilitated the viability of meiotic prophase spermatocytes and that a part of the meiotic prophase spermatocytes completed the meiotic divisions in the testicular cell culture.

Adrenal Cortex↗

An electron microscopic study of mitosis in mouse duodenal crypt cells confirms that the prophasic condensation of chromatin begins during the DNA-synthesizing (S) stage of the cycle.

The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H-thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde-formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography. Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: 1) numerous chromatin "specks" ranging in size from about 5 to 70 nm and averaging 47 nm; 2) a few roughly circular or elongated chromatin "packets" measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20-30 or more per nuclear profile; their average diameter is 128 nm. During mid-prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid- and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleoplasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps. Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro-, meta-, ana-, and telophase. Finally, radioautography 20 min after 3H-thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid-prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid-prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid-prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: 1) a slow, moderate condensation of the chromatin packets during early and mid-prophase and 2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes.

Anaphase↗

Serotonin-induced meiosis reinitiation from the first prophase and from the first metaphase in oocytes of the marine bivalve Hiatella flaccida: respective changes in intracellular Ca2+ and pH.

In the marine bivalve Hiatella flaccida, full-grown oocytes in ovaries are arrested at the first prophase (prophase-I) of meiosis, whereas spawned oocytes have reinitiated meiosis from prophase-I and are again arrested at the first metaphase (metaphase-I). The neurohormone serotonin (5-hydroxytryptamine, 5-HT) was able to trigger meiosis reinitiation both from prophase-I and from metaphase-I. Exposure of prophase-I oocytes to 5-HT caused an increase in intracellular Ca2+ ([Ca2+]i) composed of an initial towering transient and a following lower but sustained elevation. 5-HT-stimulated prophase-I oocytes also showed a gradual rise in intracellular pH (pHi), reaching a plateau level. None of these 5-HT-induced responses was affected by the complete absence of external Ca2+. On the other hand, these responses were suppressed by preinjection of heparin, an antagonist of inositol 1,4,5-trisphosphate-sensitive receptors. Metaphase-I oocytes also exhibited a [Ca2+]i increase in response to 5-HT; the initial [Ca2+]i transient was larger than that in prophase-I oocytes when stimulated with the same 5-HT concentration. Furthermore, after the initial transient, the elevated [Ca2+]i was not sustained but sometimes returned to the prestimulus level and then increased again. Metaphase-I oocytes had higher resting pHi levels than prophase-I oocytes and showed no significant pHi changes after addition of 5-HT. These results suggest that both a [Ca2+]i increase and a pHi rise are responsible for the release from prophase-I arrest, while a [Ca2+]i increase alone is concerned with the release from metaphase-I arrest.

Ammonia↗

Reorganization and condensation of chromatin in mitotic prophase nuclei of Allium cepa.

This paper studies the process and features of chromosome construction in mitotic prophase cells of Allium cepa. The results showed that a prominent reorganization of chromatin occurred during G2--early prophase. The 250-400 nm thick compact chromatin threads in G2 nuclei began to disorganize into about 30, 100 and 220 nm chromatin fibres which constituted the loosely organized chromosome outlines in early prophase before chromosome condensation. In middle prophase, chromosome condensation was characterized by the formation of many condensed regions (aggregates of chromatin), which increased in size (1-1.5 microns) when prophase proceeded. Meanwhile, the chromatin threads that constituted and connected the condensed regions became increasingly thicker (120-250 nm). In late prophase adjacent condensed regions fused to form cylinder-shaped chromosomes. Based on these observations, we come to the conclusion that the construction of prophase chromosomes is a two-step process, that is, the reorganization and condensation of chromatin. In addition, we report the study of silver-stained, DNA- and histone-depleted prophase chromosomes, describe morphological features of the non-histone protein (NHP) residue in early, middle and late prophase chromosomes, and discuss the roles of NHPs in chromosome construction.

Allium↗

Entry into mitosis in vertebrate somatic cells is guarded by a chromosome damage checkpoint that reverses the cell cycle when triggered during early but not late prophase.

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.

Animals↗

Involvement of Chk1 kinase in prophase I arrest of Xenopus oocytes.

Chk1 kinase, a DNA damage/replication G2 checkpoint kinase, has recently been shown to phosphorylate and inhibit Cdc25C, a Cdc2 Tyr-15 phosphatase, thereby directly linking the G2 checkpoint to negative regulation of Cdc2. Immature Xenopus oocytes are arrested naturally at the first meiotic prophase (prophase I) or the late G2 phase, with sustained Cdc2 Tyr-15 phosphorylation. Here we have cloned a Xenopus homolog of Chk1, determined its developmental expression, and examined its possible role in prophase I arrest of oocytes. Xenopus Chk1 protein is expressed at approximately constant levels throughout oocyte maturation and early embryogenesis. Overexpression of wild-type Chk1 in oocytes prevents the release from prophase I arrest by progesterone. Conversely, specific inhibition of endogenous Chk1 either by overexpression of a dominant-negative Chk1 mutant or by injection of a neutralizing anti-Chk1 antibody facilitates prophase I release by progesterone. Moreover, when ectopically expressed in oocytes, a Chk1-nonphosphorylatable Cdc25C mutant alone can induce prophase I release much more efficiently than wild-type Cdc25C; if endogenous Chk1 function is inhibited, however, even wild-type Cdc25C can induce the release very efficiently. These results suggest strongly that Chk1 is involved in physiological prophase I arrest of Xenopus oocytes via the direct phosphorylation and inhibition of Cdc25C. We discuss the possibility that Chk1 might function either as a G2 checkpoint kinase or as an ordinary cell cycle regulator in prophase-I-arrested oocytes.

Amino Acid Sequence↗

Chromosome no. 1 of Crepis capillaris shows defined 3D-shapes in mitotic prophase.

The shape of mitotic prophase chromosomes has been studied in root tip nuclei by confocal microscopy and 3D-image analysis. Crepis capillaris chromosome no. 1 was used as a test object. Chromosome conformation was studied in early, mid- and in late prophase. In mid- and late prophase, individual chromosomes could be distinguished on the basis of their length. Early prophase chromosomes could not be distinguished as individuals. The central axes of prophase chromosomes were traced with an automated computer procedure and then represented as a string of 3D coordinates. This representation facilitated measurement along the chromosome axis of shape parameters such as curvature (amount of bending), torsion (helical winding) and torsion sign (helical handedness). Stretches of early prophase chromosomes showed full helical turns, which could be left- or right-handed. In the later prophase stages curvature and torsion were statistically analysed. Our data on 40 midprophase chromosomes no. 1 show that they are still highly curved, but full helical turns were no longer found. Instead, an overall meandering pattern was observed. In late prophase, one central loop persisted, flanked by two preferential regions of high curvature.

Algorithms↗

Prophase chromosome unique band sequences: definition and utilization.

Extensive experience with the analysis of human prophase chromosomes and studies into the complexity of prophase banding patterns have suggested that at least some prophase chromosomal segments can be accurately identified and characterized independently of the morphology of the chromosome as a whole. The feasibility of identifying and analyzing specified prophase chromosome segments was thus investigated as an alternative approach to prophase chromosome analysis based on whole-chromosome recognition. Through the use of prophase idiograms at the 850-band stage (Francke, 1981) and a systematic comparison system, we have demonstrated that it is possible to divide the 24 human prophase idiograms into a set of 94 unique band sequences, each of which has a banding pattern that is recognizable and distinct from any other nonhomologous chromosome portion. The use of a unique band sequence approach in prophase chromosome analysis is expected to increase efficiency and sensitivity through more effective use of available banding information.

Chromosome Banding↗

Mitotic reversion in prophase of PTK1 cells induced by argon laser microirradiation.

In this paper, we report the effects of laser microirradiation of prophase nucleoli and mitotic chromosomes in cells of female rat kangaroo kidney epithelial cell line PTK1. When the laser power delivered to sample surface was 90-190 mW, irradiation of one of the two nucleoli in the prophase cell did not inhibit the mitotic progress, but resulted in the loss of the irradiated nucleolus in daughter cells. When the laser power was increased to 360-420 mW, either irradiation of the nucleolus or chromosome in midprophase caused a blockage of mitosis at terminal midprophase. The irradiated cells returned morphologically to early prophase. No mitotic reversion occurred in the case of irradiation of chromosomes at late prophase, prometaphase, metaphase, and anaphase. Irradiation of the cytoplasm in prophase cells caused a 50-70 min mitotic delay at prophase. However, the irradiated cells underwent successive mitotic divisions. The mechanism of laser-induced mitotic prophase reversion is discussed.

Animals↗

Observations on pre-prophase bands of microtubules in uniseriate hairs, stomatal complexes of sugar cane, and Cyperus root meristems.

Three aspects of the location and properties of pre-prophase bands of microtubules inplant tissues were examined. (i) Anatomical locations: Pre-prophase bands were found preceding mitosis in the basal meristematic cell of uniseriate hairs in Salvinia auriculata and in intercalary dividing cells in the uniseriate hairs of Tradescantia stamens. Previously they had only been found in 2- or 3-dimensional aggregates of cells. Other new locations were Tradescantia stamen filaments, and periclinal and anticlinal divisions in root and root cap meristems of Cuperus eragrostis. (ii) Prediction of the site of cytokinesis: Developing stomatal complexes of Saccharum officinarum were examined in view of recent reports that guard mother cells in this plant violate the otherwise general rule that the pre-prophase band predicts the line of fusion of the cell plate and the parental wall. The generality of the prediction phenomenon was upheld. (iii) Bisection of pre-prophase band sites: Evidence that the site of the pre-prophase band in the cell cortex is (at least approximately) bisected at cytokinesis was obtained for asymmetrical divisions in Cyperus roots, stomatal complexes of Saccharum, and Salvinia hairs, and symmetrical divisions in Tradescantia stamen hairs and Saccharum guard mother cells. The observations are discussed with particular reference to possible roles of the pre-prophase band site after its microtubules have disappeared at prophase.

Cell Cycle↗

Mutant meiotic chromosome core components in mice can cause apparent sexual dimorphic endpoints at prophase or X-Y defective male-specific sterility.

Genetic modifications causing germ cell death during meiotic prophase in the mouse frequently have sexually dimorphic phenotypes where oocytes reach more advanced stages than spermatocytes. To determine to what extent these dimorphisms are due to differences in male versus female meiotic prophase development, we compared meiotic chromosome events in the two sexes in both wild-type and mutant mice. We report the abundance and time course of appearance of structural and recombination-related proteins of fetal oocyte nuclei. Oocytes at successive days post coitus show rapid, synchronous meiotic prophase development compared with the continuous spermatocyte development in adult testis. Consequently, a genetic defect requiring 2-3 days from the onset of prophase to reach arrest registers pachytene as the developmental endpoint in oocytes. Pachytene spermatocytes, on the other hand, which normally accumulate during days 4-10 after the onset of prophase, will be rare, giving the appearance of an earlier endpoint than in oocytes. We conclude that these different logistics create apparent sexually dimorphic endpoints. For more pronounced sexual dimorphisms, we examined meiotic prophase of mice with genetic modifications of meiotic chromosome core components that cause male but not female sterility. The correlations between male sterility and alterations in the organization of the sex chromosome cores and X-Y chromatin may indicate that impaired signals from the XY domain (XY chromosome cores, chromatin, dense body and sex body) may interfere with the progression of the spermatocyte through prophase. Oocytes, in the absence of the X-Y pair, do not suffer such defects.

Animals↗

Mapping G-bands on human prophase chromosomes.

OHNUKI's method for demonstrating coils in human metaphase chromosomes also reveals a fine G-band pattern on prophase chromosomes of sufficient clarity to justify an attempt at mapping. Maps are provided for each chromosome to show the maximum number of prophase bands observed, and an intermediate stage in chromosome contraction, tracing the pathways of apparent band fusion as the cell progresses to metaphase, is presented. The prophase bands on many chromosomes tend to occur in distinct groups, the members of which ultimately merge to give the dark G-bands of metaphase chromosomes. Every G-band of the standard metaphase chromosomes. Every G-band of the standard metaphase pattern is compounded from two or more prophase bands. In at least contracted prophase chromosomes examined, some bands are seen which have no obvious metaphase counterpart. There are marked similarities between banded prophases and the chromoomere pattern seen at meiotic prophase. However, since chromosome contraction is a dynamic process, agreement between maps will be expected only for corresponding degrees of chromosome contraction.

Cells, Cultured↗

Cytoskeletal asymmetry in Zea mays subsidiary cell mother cells: a monopolar prophase microtubule half-spindle anchors the nucleus to its polar position.

Double labeling of microtubules and actin filaments revealed that in prophase subsidiary mother cells of Zea mays a monopolar prophase microtubule "half-spindle" is formed, which lines the nuclear hemisphere distal to the inducing guard mother cell. The nuclear hemisphere proximal to the guard mother cell is lined by an F-actin cap, consisting of a cortical F-actin patch and actin filaments originating from it. The microtubules of the "half-spindle" decline from the nuclear surface and terminate to the preprophase microtubule band. After disintegration of the latter, a bipolar metaphase spindle is organized. The polar F-actin cap persists during mitosis and early cytokinesis, extending to the chromosomes and the subsidiary cell daughter nucleus. In oryzalin treated subsidiary mother cells the prophase nuclei move away from the polar site. Cytochalasin B and latrunculin-B block the polar migration of subsidiary mother cell nuclei, but do not affect those already settled to the polar position. The prophase nuclei of latrunculin-B treated subsidiary mother cells are globally surrounded by microtubules, while the division plane of latrunculin-B treated subsidiary mother cells is misaligned. The prophase nuclei of brick 1 mutant Zea mays subsidiary mother cells without F-actin patch are also globally surrounded by microtubules. The presented data show that the prophase microtubule "half-spindle"-preprophase band complex anchors the subsidiary mother cell nucleus to the polar cell site, while the polar F-actin cap stabilizes the one metaphase spindle pole proximal to the inducing guard mother cell.

Bridged Bicyclo Compounds, Heterocyclic↗

Plk1 promotes nuclear translocation of human Cdc25C during prophase.

The nuclear accumulation of active M-phase promoting factor (MPF) during prophase is thought to be essential for coordinating M-phase events in vertebrate cells. The protein phosphatase Cdc25C, an activator of MPF, enters the nucleus to keep MPF active in the nucleus during prophase. However, the molecular mechanisms that control nuclear translocation of Cdc25C during prophase are unknown. We show that phosphorylation of a serine residue (Ser198) in a nuclear export signal sequence of human Cdc25C occurs during prophase and promotes nuclear localization of Cdc25C. We also show that Polo-like kinase 1 (Plk1) is responsible for this phosphorylation and that constitutively active Plk1 promotes nuclear localization of Cdc25C. Remarkably, a mutant Cdc25C in which Ser198 is replaced by alanine remains in the cytoplasm when wild-type Cdc25C accumulates in the nucleus during prophase. These results suggest that Plk1 phosphorylates Cdc25C on Ser198 and regulates nuclear translocation of Cdc25C during prophase.

Cell Cycle Proteins↗

Evolution of the meiotic prophase and of the chromosome pairing process during human fetal ovarian development.

BACKGROUND: Studies on human oocytes in prophase I are limited due to the difficulty in obtaining the sample. However, a complete study of meiotic prophase evolution and the homologue pairing process is necessary to try to understand the implication of oogenesis in the origin of human aneuploidy. METHODS: A complete analysis of meiotic prophase progression comprising the long developmental time period during which meiotic prophase takes place, based on the analysis of a total of 8603 oocytes in prophase I from 15 different cases is presented. The pairing process of chromosomes 13 and 18 is also described. RESULTS: The findings significantly relate for the first time the evolution of meiotic prophase to fetal development. Although for both chromosomes 13 and 18 a high pairing efficiency is found, pairing failure at the pachytene stage has been observed in 0.1% of oocytes. However, errors at the diplotene stage are substantially increased, suggesting that complete, premature disjunction of the homologues commonly occurs. Moreover, pre-meiotic errors are also described. CONCLUSIONS: Our findings show that homologous chromosomes pair very efficiently, but the high frequency of complete, premature homologue separation found at diplotene suggests that mechanisms other than the pairing process could be more likely to lead to the high aneuploidy rate observed in human oocytes.

Aneuploidy↗

High resolution ordering of DNA markers by multi-color fluorescent in situ hybridization of prophase chromosomes.

To improve resolution for physical ordering of adjacent DNA loci, prophase chromosomes were used for multi-color fluorescent in situ hybridization (FISH). The prophase chromosomes were prepared from cultured lymphocytes by a thymidine synchronization, bromodeoxyuridine release technique and then treating the synchronized cultures with topoisomerase II inhibitors ICRF154 or ICRF193. Almost all mitotic figures exhibited highly elongated prophase chromosomes without significant reduction of the mitotic index. Using multi-color FISH with these prophase chromosomes, we were able to distinguish signals for loci separated by as little as 50 kb, and determine their orientation. Furthermore, using this prophase ordering system, we confirmed the linear order and defined the orientation of seven cosmid markers within a 360-kb region surrounding D10S102, a locus that is closely linked to the disease locus in families segregating an allele causing multiple endocrine neoplasia IIA (MEN2A). This prophase FISH system, by rapidly and precisely providing the linear order of loci that are very close, can expedite construction of fine cytogenetic maps and contribute to positional-cloning studies in which the precise ordering of DNA loci in a target region is critical.

Cells, Cultured↗

Nucleolar and perichromosomal RNA synthesis during meiotic prophase in the mouse testis.

The transcriptional activity during meiotic prophase in the mouse testis is studied with light microscopy and high-resolution autoradiographic techniques using [(3)H]uridine as a labeled precursor. In the present study, two types of RNA synthesis are detected during meiotic prophase: an extranucleolar RNA synthesis of perichromosomal localization and a nucleolar RNA synthetic activity. In some of the autosomes and close to the basal knobs, the activity of the nucleolar organizers is evidenced by the incorporation of [(3)H]uridine into nucleolar masses from zygotene on and at earlier labeling times. The evolution of nucleoli and the formation of a nucleolus attached to the sex pair are described during the different meiotic stages. Perichromosomal labeling, from leptotene on, reaches a maximum during middle pachytene and falls progressively to a low level at longer incorporation times. Sertoli's cell, the most active RNA synthetic cell in the seminiferous epithelium, rises to a maximum of labeling and drops at earlier times compared with the meiotic prophase cells. The condensed sex chromosomes show some scattered silver grains especially at middle pachytene. The axial chromosome cores and synaptonemal complexes are devoid of silver grains during the meiotic prophase. The observations suggest that a control mechanism operates during meiotic prophase to regulate transcriptional activity in the sex chromosomes and to provide differential RNA synthesis in autosomal bivalents at various stages of prophase and within certain segments of the chromosomes.

Animals↗