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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

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

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

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

Prophasing of interphase nuclei and induction of nuclear envelopes around metaphase chromosomes in HeLa and Chinese hamster homo- and heterokaryons.

Fusing human HeLa metaphase cells with HeLa interphase cells resulted within 30 min in either of two phenomena in the resultant binucleate cell: either prophasing of the interphase nucleus or formation of a normal-appearing nuclear envelope around the metaphase chromosomes. The frequency of either occurrence was strongly dependent on environmental pH. At pH's of 6.6-8.0, prophasing predominated; at pH 8.5 nuclear envelope formation predominated. Additionally, the frequencies of the two events in multinucleate cells depended on the metaphase/interphase ratio. When the ratio was 0.33 nuclear envelope formation predominated; when it was 2.0 prophasing predominated. In their general features, the results with fused HeLa cells resembled those reported earlier with fused Chinese hamster Don cells. However, the results provided an indication that between pH 6.6 and 8.0 the HeLa metaphase cells possessed a much greater capacity than the Don metaphase cells to induce prophasing. Fusion of Don metaphase cells with HeLa interphase cells or of Don interphase cells with HeLa metaphase cells at pH 8.0 resulted in nuclear envelope formation or prophasing in each kind of heterokaryon. As in the homokaryons, the frequencies of the two events in the heterokaryons depended on the metaphase/interphase ratio. The statistics of prophasing and nuclear envelope formation in the homo- and heterokaryon populations were consistent with the notion that disruption or formation of the nuclear envelope depends on the balance attained between disruptive and formative processes.

Cell Division

Some comments regarding chromosome pulverization (premature chromosome condensation or PCC, prophasing).

Premature chromosome pulverization (PCC) or prophasing is a much misunderstood cytological entity. It must be separated from chromosome damage caused by a number of chemical, physical and biological agents. Prophasing is observed in fused cells in which one of the constituent cells must be in metaphase and another in interphase. The morphology of the "pulverized" interphase nucleus will depend on the phase of the cell cycle in which the interphase cell was in when exposed to a substance present in the cytoplasm of the metaphase cell leading to "prophasing". Prophasing is a normal cellular phenomenon occurring prematurely or under abnormal conditions (fusion of cells) and its demonstration in human cells or tumors may be indicative of the presence of a virus (or its products) which leads to cell fusion, but does not play a role in prophasing.

Animals

Dose-response relationships and R.B.E. values of dominant lethals induced by X-rays and 1.5 MeV neutrons in prophase-1 oocytes and in mature sperm of the two-spotted spider mite Tetranychus urticae Koch (acari, tetranychidae).

Mature sperm and prophase-1 oocytes of Tetranychus urticae Koch were irradiated with 250-kVp X-rays or 1.5 MeV fast neutrons. The X-ray doses ranged from 0.5 to 24.0 krad, and those of the fast neutrons from 0.1 to 16.0 krad. The genetic endpoint measured was lethality, expressed in the stages from egg to adulthood in the F1 progeny. The frequency of recessive lethals in female germ cells was estimated by comparing survival of fertilized versus unfertilized F1 eggs, after irradiation with the same dosage. X-Rays induce dominant lethals in prophase-1 oocytes by the action of both single hits on single targets and multiple hits on multiple targets. 1.5-MeV neutrons induce these effects predominantly by the action of multiple tracks on multiple targets. Dominant lethals were induced in mature sperm by X-rays and by fast neutrons by the action of both single hits on single targets and multiple hits on multiple targets. Both for prophase-1 oocytes and for mature sperm the low R.B.E. value corresponded with the relatively large multiple-target component of induction of dominant lethals by fast neutrons. The nature of dominant lethality in relation to the kinetochore organization of the chromosome is discussed. A non-linear trend in the dose--effect relationship was observed for both X-rays and fast neutrons for the estimated frequency of recessive lethals induced in prophase-1 oocytes. X-Rays were more effective than neutrons in inducing recessive lethals in prophase-1 oocytes at doses lower than 3 krad.

Animals

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.

Animals

Role of calcium in regulating intracellular pH following the stepwise release of the metabolic blocks at first-meiotic prophase and second-meiotic metaphase in amphibian oocytes.

31P-NMR has been used to monitor changes in intracellular pH following the sequential release of the block at first-meiotic prophase by hormones and the block at second-meiotic metaphase by fertilization in Rana eggs and oocytes. The broad phosphoprotein signal was eliminated by a combination of spin-echo and deconvolution techniques. pHi was determined from the pH-dependent separation of intracellular Pi and phosphocreatine resonances. Agents that release the prophase block (progesterone, insulin, D-600, La3+) increased pHi from 7.38 to 7.7-7.8 within 1-3 h. Noninducers such as 17 beta-estradiol were without effect. By second-metaphase arrest (ovulated, unfertilized) the pHi had fallen to 7.1-7.2. pHi underwent a transient increase to about 7.7 within the first 30 min at fertilization, with a slow 0.1-0.2 pH unit oscillation during early cleavage. The progesterone-induced elevation of intracellular pH is not blocked by amiloride and occurs in Na+-free medium. A transient rise in pHi occurs when the prophase-arrested oocyte is transferred to Ca2+-free medium or when ionophore A23187 is added to the Ca2+-containing medium. Agents that inhibit the resumption of the first meiotic division either block the rise in pHi (procaine, PMSF) or shorten the time-course of the rise in pHi (ionophore A23187). Conditions that elevate intracellular Ca2+ levels and/or increase Ca2+ exchange produce an increase in pHi, whereas those conditions that decrease intracellular Ca2+ levels and/or exchange produce a fall in pHi within 1 h. The time-course of the increase in pHi both following release of the prophase block and at fertilization coincide with a fall in intracellular cAMP and release of surface and/or intracellular Ca2+. These results suggest that: (1) pHi is a function of cytosolic free Ca2+ levels and/or Ca2+ exchange across the oocyte plasma membrane, and (2) meiotic agonists (progesterone, insulin, D-600) and mitogens (sperm, ionophore A23187) modulate intracellular and/or membrane Ca2+ with the resulting changes in pHi and cAMP and resumption of the meiotic divisions.

Animals

Fertilization and in vitro development of cryopreserved human prophase I oocytes.

OBJECTIVE: To determine the potential for in vitro maturation, fertilization, and cleavage after cryopreservation of immature, prophase I human oocytes. DESIGN: Immature oocytes obtained in excess of the number required by the patient were randomized and cryopreserved at the prophase I stage or cultured as control. After thawing and maturation in vitro, test and control oocytes were inseminated with husband's sperm and evaluated for fertilization and cleavage in vitro. SETTING: In vitro fertilization program. PATIENTS: Consenting patients undergoing controlled ovarian hyperstimulation for the purposes of IVF. MAIN OUTCOME MEASURES: Rates of maturation to metaphase II, fertilization, and cleavage were compared between control and cryopreserved oocytes. RESULTS: Upon thaw, 58.5% (72/123) of prophase I oocytes were viable. Control oocytes demonstrated a 74.8% (98/131) maturation rate to metaphase II, a 56.5% (52/92) fertilization rate, and an 11.5% (6/52) blastocyst rate. Cryopreserved oocytes showed a 83.3% (60/72) rate of maturation, a 57.7% (30/52) fertilization rate, and a 3.3% (1/30) blastocyst rate. No significant differences were noted between any of these parameters. CONCLUSIONS: These results demonstrate that prophase I oocytes from stimulated IVF cycles are able to survive cryopreservation and resume meiosis to achieve full nuclear maturation post-thaw. In addition, cryopreserved oocytes retain the same capacity for fertilization and development as control oocytes.

Adult

A novel mitotic spindle pole component that originates from the cytoplasm during prophase.

Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the 115/110-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.

Autoantibodies