Chromosome aberrations in metaphase II-oocytes. Stage sensitivity in the mouse oogenesis to amethopterin and cyclophosphamide

The stage sensitivity in oogenesis of C₃H mice was investigated by transplacental treatment of embryonic oogonia and oocytes at meiotic prophase I. After birth the stages of early and late dictyotene as well as the preovulatory and ovulatory phases were treated. Chromosome analysis was performed in unfertilized metaphase II-oocytes after induced ovulation [pregnant mare's serum (PMS) and human chorionic gonadotrophin (HCG)]. As test compounds both the folic acid antagonist amethopterin (M) and the alkylating agent cyclophosphamide (C) were used.Embryonic oogonia as well as the preovulatory phase of oogenesis proved to be most sensitive for the induction of chromosomal aberrations. The investigation with graded doses during the preovulatory stage demonstrated the dose-dependent frequency of the induced types of chromosomal abnormalities.The high sensitivity of these stages where chromosome segregation takes place, e.g. oogonia, preovulatory stage, seems to be related to an additional induction of aneuploidies.     

Induced chromosomal aberrations in pronuclei, 2-cell stages and morulae of mice

C₃H female mice, 10–12 weeks old, were treated in the pre-ovulatory phase of oogenesis or in the first mitotic interphase after fertilization with amethopterin (M) at 200 mg/kg body weight. The females were mated with untreated males of the same age group and strain and were checked hourly for vaginal plugs. At different times after fertilization the oviducts of the females of experiment and control were flushed out and the cleavage stages obtained were examined cytologically. Chromosome analysis was performed in the pronucleus stage, in the 2-cell stage and in morulae. Separate analyses of maternal and paternal chromosomes of the pronucleus stage can show whether the spontaneous or induced aberrations are derived from oogenesis or spermatogenesis. After treatment the frequency of numerical and structural aberrations increased compared with the matched control. During the early prenatal development at least 2 steps seem to be important for the elimination of chromosomal aberrations: 1, from the unfertilized mII oocyte to the pronucleus stage; and 2, from early morulae to the registration of dominant lethal mutations.     

Analysis of the mechanism(s) of metaphase I-arrest in strain LT mouse oocytes: delay in the acquisition of competence to undergo the metaphase I/anaphase transition.

Fully grown oocytes of most laboratory mice progress without interruption from the germinal vesicle (GV) stage to metaphase II, where meiosis is arrested until fertilization. In contrast, many oocytes of strain LT mice arrest precociously at metaphase I and often undergo subsequent spontaneous parthenogenetic activation. Cytostatic factor (CSF), which prevents the degradation of cyclin B and maintains high maturation-promoting factor (MPF) activity, is required for maintenance of metaphase I-arrest in LT oocytes, similar to its requirement for maintaining metaphase II-arrest in normal oocytes. However, CSF does not instigate metaphase I-arrest since a temporary metaphase I-arrest occurs in MOS-null LT oocytes. This paper addresses the mechanism(s) that may instigate metaphase I-arrest and tests the hypothesis that there may be one or more defects in LT oocytes that delay their acquisition of competence to trigger the cascade of processes that normally drive entry into and progression through anaphase I. To test this hypothesis, MPF activity was artificially abrogated by treating oocytes with a general protein kinase inhibitor, 6-DMAP, at various times during the progression of meiosis I. This allowed a comparison of the time at which LT and normal oocytes become competent to undergo the metaphase I/anaphase transition even if oocytes were arrested at metaphase I when 6-DMAP-treatment was begun. There were no differences between LT and control oocytes in the kinetics of MPF suppression by 6-DMAP. However, it was found that LT oocytes do not acquire competence to undergo the metaphase I/anaphase transition in response to 6-DMAP until 50-60 min after normal oocytes. A similar delay was observed in strain CX8-4 oocytes, which also have a high incidence of metaphase I-arrest, but not in strain CX8-11 oocytes, which exhibit a low incidence of metaphase I-arrest. MOS-null LT oocytes also exhibit a delay in acquisition of competence to undergo the metaphase I/anaphase transition. Thus, a delay in competence to undergo the metaphase I/anaphase transition in response to 6-DMAP-treatment correlates with metaphase I-arrest. It is therefore hypothesized that the observed delay in acquisition of competence to enter anaphase I may instigate the sustained metaphase I-arrest in LT oocytes by allowing CSF activity to rise to a level that prevents cyclin B degradation and maintains high MPF activity before anaphase can be initiated by normal triggering mechanisms.     

Implication of microtubules and microfilaments in the response of the ovarian adenylate cyclase-cyclic AMP system to gonadotropins and prostaglandin E2.

Addition of anti-actin serum or cytochalasin B (3 μg/ml) to the medium abolished the stimulatory effect of LH and of choleragen, and inhibited the action of FSH, but not of PGE₂, on cyclic AMP production in cultured rat Graafian follicles. Colchicine and anti-sera to BSA, tubulin or smooth-muscle myosin, as well as anti-actin serum absorbed with actin, had no effect on the follicular response to LH, but anti-tubulin serum and colchicine inhibited the response to FSH and PGE₂. The inhibitory effect of cytochalasin B on LH-action was fully reversed 24 h after transfer of the follicles to drug-free medium. Neither anti-actin serum nor cytochalasin B had any effect on the binding of ¹²⁵I-hCG by the follicular cell membrane. The results suggest that microfilaments, but not microtubules, are intimately involved in the process of LH- and choleragen-stimulated ovarian adenylate cyclase activity. By contrast, the action of PGE₂ is dependent on microtubule assembly, while the action of FSH seems to depend on both these components of the cytoskeleton.     

Assignment of genes encoding metallothioneins I and II to Chinese hamster chromosome 3: evidence for the role of chromosome rearrangement in gene amplification.

Cadmium resistant (Cdr) variants with coordinately amplified metallothionein I and II (MTI and MTII) genes have been derived from both Chinese hamster ovary and near-euploid Chinese hamster cell lines. Cytogenetic analyses of Cdr variants consistently revealed breakage and rearrangement involving chromosome 3p. In situ hybridization with a Chinese hamster MT-encoding cDNA probe localized amplified MT gene sequences near the translocation breakpoint involving chromosome 3p. These observations suggested that both functionally related, isometallothionein loci are linked on Chinese hamster chromosome 3. Southern blot analyses of DNAs isolated from a panel of Chinese hamster X mouse somatic cell hybrids which segregate hamster chromosomes confirmed that both MTI and MTII are located on chromosome 3. We speculate that rearrangement of chromosome 3p could be causally involved with the amplification of MT genes in Cdr hamster cell lines.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphonidae). I. Ovary structure and previtellogenic growth of oocytes

Glossiphonia heteroclita has paired ovaries whose shape and dimensions change as oogenesis proceeds: during early previtellogenesis they are small and club-shaped, whereas during vitellogenesis they broaden and elongate considerably. During early oogenesis (previtellogenesis), each ovary is composed of an outer envelope (ovisac) that surrounds the ovary cavity and is filled with hemocoelomic fluid, in which a single and very convoluted ovary cord is bathed. The ovary cord consists of germline cells, including nurse cells and young oocytes surrounded by a layer of elongated follicle cells. Additionally, follicle cells with long cytoplasmic projections occur inside the ovary cord, where they separate germ cells from each other. The ovary cord contains thousands of nurse cells. Each nurse cell has one intercellular bridge, connecting it to a central anucleate cytoplasmic mass, the cytophore (rachis); it in turn is connected by one intercellular bridge with each growing oocyte. Numerous mitochondria, RER cisternae, ribosomes, and Golgi complexes are transported from the nurse cells, via the intercellular bridge and cytophore, to the growing oocytes. Oogenesis in G. heteroclita is synchronous with all oocytes in the ovary in the same stage of oogenesis. The youngest observed oocytes are slightly larger than nurse cells, and usually occupy the periphery of the ovary cord. As previtellogenesis proceeds, the oocytes gather a vast amount of cell organelles and become more voluminous. As a result, in late previtellogenesis the oocytes gradually protrude into the ovary cavity. Simultaneously with oocyte growth, the follicle cells differentiate into two subpopulations. The morphology of the follicle cells surrounding the nurse cells and penetrating the ovary cord does not change, whereas those enveloping the growing oocytes become more voluminous. Their plasma membrane invaginates deeply, forming numerous broad vesicles that eventually seem to form channels or conducts through which the hemocoelomic fluid can easily access the growing oocytes.     

Comparison of DNA-repair synthesis,chromosome aberrations and induction of micronuclei in cultured human fibroblast,Chinese hamster ovary and central mudminnow (Umbra limi) cells exposed to chemical mutagens

In mammalian cells it has previously been observed that low DNA-repair activity is correlated wtih high chromosome-aberration frequency. Since fish cells typically express comparatively low amounts of DNA repair, the chromosome aberration test holds potential as a sensitive fish genotoxicity assay. A comparison of in vitro DNA-repairm activity showed HF > CHO > Ul-H = Ul-F following exposure to MNNG and 4NQO. Although peak chromosome-aberration frequency varied CHO > Ul-H > HF, at comparable mutagen concentrations the relationship was Ul-H > HF > CHO following 4NQO exposure and Ul-H > HF = CHO after MNNG exposure. Analyzing for chromosome aberrations at high mutagen concentrations was not possible due t mitotic inhibition/toxicity which varied according to the mutagen and cell line. Micronuclei frequency varied CHO > Ul-H > HF = Ul-F. In CHO and Ul-H, a 10–15 fold increase over the controls compares with only a 2–3 fold increase for HF and Ul-F. These differences are likely related, in part, to the cell-division rate of each line and the coincident repair of the damaged DNA. Reasons for the lack of negative correlation between DNA repair and chromosomal damage in fish cells are discussed.     

Quantitative aspects of the formation and loss of DNA interstrand crosslinks in Chinese hamster cells following treatment with cis-diamminedichloroplatinum(II) (cisplatin). II. Comparison of results from alkaline elution, DNA renaturation and DNA sedimentation studies

The formation of interstrand crosslinks in the DNA of cis-diamminedichloroplatinum(II) (cisplatin)-treated Chinese hamster cells has been demonstrated by three independent, highly sensitive techniques: namely those of alkaline elution, renaturation of crosslinked DNA and alkaline sucrose gradient velocity sedimentation. Simultaneous measurement of DNA break frequency by the first two methods and a computer model combined with the third enabled the calculation of crosslink frequency after application of low doses of cisplatin. Good agreement was found between the values obtained by the three methods used here, and also with those obtained by direct measurement of the amount of crosslinked hybrid DNA in density-labelled cells treated with higher doses of cis-platin (Roberts, J.J. and Friedlos, F. (1981) Biochim. Biophys, Acta 655, 146--151). When measured by all three methods described here, crosslinking was found to increase during several hours after treatment and then to decrease with a half-life of between 12 and 24 h. For low initial levels of crosslinking, this was largely attributed to an excision repair process, since the formation of breaks in DNA was only minimal.     

Direct gene dosage determination in patients with unbalanced chromosomal aberrations using cloned DNA sequences. Application to the regional assignment of the gene for alpha 2(I) procollagen (COLIA2).

We describe a new method of direct gene dosage determination in patients with unbalanced chromosomal aberrations using cloned DNA sequences: the intensity of the signal obtained by hybridization of the radioactive probe to the corresponding DNA fragments can be compared with the intensity of the DNA fragments that hybridize with a nonsyntenic probe used as an internal control. This has been demonstrated by densitometer tracing of the autoradiogram, using an X-specific DNA sequence, beta globin and alpha 2(I) collagen probes, in normal men and women, in one patient trisomic for 11p, and in one patient trisomic for segment 7q21 leads to 7qter. The ratio men/women for the X-specific sequence (DXS) was close to the expected value 0.5, while the ratio trisomy 11/normal control and trisomy 7/normal control were close to 1.5 for beta globin (HBB) and alpha 2(I) collagen (COLIA2), respectively. The gene coding for COLIA2 can therefore be assigned to 7q21 leads to 7qter. This method should also apply to noncoding sequences: the increasing number of cloned DNA segments that have already been assigned to a specific chromosome represent a new tool for prenatal and premorbid diagnosis of unbalanced chromosomal aberrations.     

The induction of chromosomal aberrations and SCEs by visible light in combination with dyes. II. Cell cycle dependence, and the effect of hydroxyl radical scavengers during light exposure in cultures of Chinese hamster ovary cells sensitized with acridine orange

Chinese hamster ovary (CHO) cells were synchronized by mitotic shake-off, treated with the fluorochrome acridine orange (AO; 0.5 micrograms/ml), washed free of excess dye and subsequently exposed to visible light (2 X 40 W/8 Wm-2). The light exposure was performed on cells in the G1, G1/S, S or G2 phase of the cell cycle. AO + light induced high frequencies of aberration in the S phase and even higher in the G1 phase. The aberrations observed were all of the chromatid type. The chromosome-type aberrations (dicentrics, rings) obtained when cells in the G1 phase were exposed to X-rays were not found after corresponding treatments with AO + light. With the exception of an increased frequency of gaps, no chromosomal aberrations were induced in G2-phase cells. Sister-chromatid exchanges were efficiently produced by the photodynamic system in the G1, G1/S and S phase of the cell cycle. In other experiments, AO-treated unsynchronized CHO cells were exposed to light in the presence of the hydroxyl radical scavengers mannitol (100 mM) and 5-dimethyl thiourea (100 mM). In parallel experiments these scavengers were found to reduce markedly the chromosome breaking effects by X-rays but had no influence on the photodynamic induction of chromosomal alterations. The results presented show that the visible light-induced chromosomal alterations in CHO cells sensitized with the fluorochrome AO are obtained by an S-dependent mechanism. Furthermore, the results indicate that the hydroxyl free radical does not play a major role in the production of chromosomal alterations by AO + light.     

The in vivo induction of sister chromatid exchanges in the bone marrow of the Chinese hamster. II.N-nitrosodiethylamine (DEN) and n-isopropyl-alpha-(2-methyl-hydrazino)-p-toluamide (Natulan), two carcinogenic compounds with specific mutagenicity problems.

N-Nitrosodiethylamine (DEN) and N-isopropyl-alpha-(2-methylhyadrazino)-p-toluamide (Natulan) were examined with the in vivo SCE method of Vogel and Bauknecht. Only Natulan showed a positive effect with a significant increase of induced SCE between 10 and 25 mg/kg b.w. The six-point curve of the dose effect was of the plateau type. With DEN only a slight increase with high doses could be obtained, which was not significant when 50 or 100 cells were counted. Compared with the results of other tests published, Natulan gives positive results preferentially with in vivo mammalian tests but not with microorganisms. On the other hand, DEN is inactive in vivo on the chromosomal level, but preferentially induces point mutations at the molecular level in microorganisms and Drosophila. It is recommended to include in a battery of true mutagenicity tests also cytogenetic tests (in vivo SCE test and micronucleus test).     

Analysis of the pigment stoichiometry of pigment-protein complexes from barley (Hordeum vulgare). The xanthophyll cycle intermediates occur mainly in the light-harvesting complexes of photosystem I and photosystem II.

The carotenoid zeaxanthin has been implicated in a nonradiative dissipation of excess excitation energy. To determine its site of action, we have examined the location of zeaxanthin within the thylakoid membrane components. Five pigment-protein complexes were isolated with little loss of pigments: photosystem I (PSI); core complex (CC) I, the core of PSI; CC II, the core of photosystem II (PSII); light-harvesting complex (LHC) IIb, a trimer of the major light-harvesting protein of PSII; and LHC IIa, c, and d, a complex of the monomeric minor light-harvesting proteins of PSII. Zeaxanthin was found predominantly in the LHC complexes. Lesser amounts were present in the CCs possibly because these contained some extraneous LHC polypeptides. The LHC IIb trimer and the monomeric LHC II a, c, and d pigment-proteins from dark-adapted plants each contained, in addition to lutein and neoxanthin, one violaxanthin molecule but little antheraxanthin and no zeaxanthin. Following illumination, each complex had a reduced violaxanthin content, but now more antheraxanthin and zeaxanthin were present. PSI had little or no neoxanthin. The pigment content of LHC I was deduced by subtracting the pigment content of CC I from that of PSI. Our best estimate for the carotenoid content of a LHC IIb trimer from dark-adapted plants is one violaxanthin, two neoxanthins, six luteins, and 0.03 mol of antheraxanthin per mol trimer. The xanthophyll cycle occurs mainly or exclusively within the light-harvesting antennae of both photosystems.     

Submarine pollination in the marine angiosperm Zostera marina (Zosteraceae). I. The influence of floral morphology on fluid flow

An understanding of the process of submarine pollination should provide insight into the evolutionary and reproductive ecology of the marine angiosperms (seagrasses). The flow around the reproductive organs of the seagrass Zostera marina L. (Potamogetonales) was, therefore, examined in a flow chamber. The phenological emergence of flowers during (1) pollen capture and (2) pollen release, and by fruit during (3) seed release, led to a reduction in flow rate toward the inflorescence. This change in flow due to floral emergence was associated with a 50% increase in the fluid shear stress [tau = (2.2 _ 0.3) x 10-3 Pa for an immature flower vs. tau = (3.1 _ 0.5) x 10-3 Pa for a receptive flower]. The Reynolds number (Re) and fluid shear stress around inflorescences and infructescences were comparable, indicating a dynamic similarity in the processes of pollen capture and fruit dehiscence [Re = 47 _ 5, tau = (1.6 _ 0.3) x 10-3 Pa for inflorescences; Re = 38 _ 5, tau = (1.3 _ 0.1) x 10-3 Pa for infructescences]. These results indicate that the emergence of reproductive organs leads to changes in fluid shear stress, which will affect the release, transport, and capture of particles including pollen. Theoretical considerations of these observations using aerosol-filtration theory suggest that pollen capture in Z. marina occurs through direct interception of pollen by stigmas.     

Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. I. Inhibitors of de novo purine synthesis and a comparison with the effects of caffeine

The effect of pre- and posttreatment incubation of UV-irradiated and ethyl methanesulphonate (EMS) treated cells with non-toxic concentrations of inhibitors of de novo purine synthesis (dnPS) on expression of potentially lethal and premutational damage at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 cells has been examined. The concentrations of inhibitors used were shown to profoundly perturb de novo DNA synthesis, by measurements of [¹⁴C]formate uptake, and cell cycle progression by flow cytofluorimetry. Postincubation in 6-methyl mercaptopurine ribonucleoside (MMPR) usually but not invariably potentiated the cytotoxic effects of UV and EMS but azaserine (AZS) and methotrexate (MTX) were without effect. No effects on mutant frequencies were observed on posttreatment with any of these agents. Caffeine produced the least effect on dnPS, but invariably potentiated lethal damage. This potentiation of lethal damage is not mediated by dnPS inhibition as has been suggested for Chinese hamster ovary (CHO) cells.     

Inhibition by caffeine of post-replication repair in Chinese hamster cells treated with cis platinum (II) Diamminedichloride: the extent of platinum binding to template DNA in relation to the size of low molecular weight nascent DNA.

Treatment of Chinese hamster lung V79-379A cells with the anti-tumour agent cis platinum (II) diamminedichloride, (cis Pt(II)), resulted in an immediate recuction in the rate of DNA synthesis. Sedimentation of newly synthesised DNA through alkaline sucrose gradients revealed it to be approximately the same size as that obtained from untreated cells. In contrast, in the presence of 0.75 mM caffeine, the rate of DNA synthesis rapidly returned to control levels, although sedimentation analysis showed the DNA synthesised in cis Pt(II)-treated cells to be of lower molecular weight than in untreated cells. The reduction in molecular weight was directly proportional to the initial dose of the platinum compound. Furthermore, the results of separate binding studies suggested that at several levels of reaction the new DNA was synthesised up to a size approximately equal to the interplatinum distance in the template strand. This has been interpreted as being the result of the formation of a gap in the daughter DNA strand opposite every DNA-platinum product in the template strand. If caffeine was removed from the culture medium, there was a rapid increase in the molecular weight of the nascent DNA strands. However, if caffeine remained in the medium, the DNA remained of lower molecular weight than in untreated cells. It is proposed that this effect of caffeine is the result of the inhibition of a post-replicative DNA repair process which allows the eventual synthesis of a continuous DNA strand on a template containing unexcised lesions. It is further proposed that inhibition of this post-replicative DNA repair process provides a molecular basis for the previously observed potentiation by caffeine of cis Pt(II)-induced chromosomal aberrations and lethality.     

Role of topoisomerase II in the response of mammalian cells to the action of ionizing radiation. I. Change in the mitotic cell cycle of Chinese hamster CHO K1, irradiated with x-rays, after the action of novobiocin at a high concentration]

Flow cytometry was used to study cell cycle recovery in X-irradiated Chinese hamster cells after action of novobiocin, an inhibitor of topoisomerase II. A prolonged treatment with 1 mM novobiocin (20-30 h) of intact cells results in the G2 + M delay. Novobiocin treatment of 5 Gy-irradiated cells results in a slight delay in cell exit from G1 into S phase and in a much longer G2-delay if compared with X-irradiated cells. These data allow to suggest an involvement of topoisomerase II in cell response to ionizing radiation.     

Calcium and the Ca-ATPase SPCA1 modulate plasma membrane abundance of ZIP8 and ZIP14 to regulate Mn(II) uptake in brain microvascular endothelial cells

Manganese (II) accumulation in human brain microvascular endothelial cells is mediated by the metal-ion transporters ZRT IRT-like protein 8 (ZIP8) and ZRT IRT-like protein 14 (ZIP14). The plasma membrane occupancy of ZIP14, in particular, is increased in cells treated with Mn²⁺, lipopolysaccharide, or IL-6, but the mechanism of this regulation has not been elucidated. The calcium-transporting type 2C member 1 ATPase, SPCA1, is a Golgi-localized Ca²⁺-uptake transporter thought to support Golgi uptake of Mn²⁺ also. Here, we show using surface protein biotinylation, indirect immunofluorescence, and GFP-tagged proteins that cytoplasmic Ca²⁺ regulates ZIP8- and ZIP14-mediated manganese accumulation in human brain microvascular endothelial cells by increasing the plasma membrane localization of these transporters. We demonstrate that RNAi knockdown of SPCA1 expression results in an increase in cytoplasmic Ca²⁺ levels. In turn, we found increased cytoplasmic Ca²⁺ enhances membrane-localized ZIP8 and ZIP14 and a subsequent increase in ⁵⁴Mn²⁺ uptake. Furthermore, overexpression of WT SPCA1 or a gain-of-function mutant resulted in a decrease in cytoplasmic Ca²⁺ and ⁵⁴Mn²⁺ accumulation. While addition of Ca²⁺ positively regulated ZIP-mediated ⁵⁴Mn²⁺ uptake, we show chelation of Ca²⁺ diminished manganese transport. In conclusion, the modulation of ZIP8 and ZIP14 membrane cycling by cytoplasmic calcium is a novel finding and provides new insight into the regulation of the uptake of Mn²⁺ and other divalent metal ions–mediated ZIP metal transporters.