Distribution of 5-chloromethylfluorescein diacetate staining during meiotic maturation and fertilization in vitro of mouse oocytes

The aim of this confocal microscopy study was to determine whether the pattern of CellTracker Green 5-chloromethylfluorescein diacetate (CMFDA) staining changes during meiotic maturation and fertilization in vitro of mouse oocytes. At different times during meiotic maturation and fertilization, oocytes, zygotes and two-cell embryos were stained with CMFDA to demonstrate intracellular glutathione S-transferase activity. After washing in CMFDA-free medium, most oocytes, zygotes and embryos were stained with dihydroethidium (HE) to visualize DNA structures. Meiotic maturation and fertilization in vitro of mouse oocytes were associated with changes in the pattern of intracellular CMFDA staining. In particular, accumulations of CMFDA-positive membranes were observed around the nucleus of germinal vesicle (GV) oocytes, overlaying the sperm nucleus as well as overlaying the first mitotic spindle if this approached the plasma membrane. Staining of oocytes and zygotes with the probes 3,3'-dihexyloxacarbocyanine iodine [DiOC6(3)], which stains all the intracellular membranes, and rhodamine 123, which stains active mitochondria, demonstrated that the intracellular structures evidenced by CMFDA staining did not correspond to accumulations of mitochondria. Exposure of oocytes and zygotes to the microtubule-disrupting agent nocodazole or the actin-depolymerizing drug cytochalasin D revealed an autonomous microfilament-dependent transport and relocation of CMFDA-positive membranes during meiotic maturation and fertilization. Such a transport of CMFDA-positive membranes may be envisaged as a protective shield built to prevent damage to DNA from endogenous and exogenous mutagen metabolites.     

Metabolic toxicity of fluorescent stains on thawed cryopreserved bovine sperm cells

Several fluorescent probes, including derivatives of carboxyfluorescein, carbocyanine, ethidium, and rhodamine, have been used to assess sperm viability. However, the effects of these fluorescent dyes on the metabolic activity of sperm cells have not been systematically examined. This study was conducted to determine the effect of specific fluorescent stains on the metabolic processes of sperm. Cryopreserved bovine sperm cells were thawed, fluorescently stained, and examined using metabolic and flow cytometric techniques. Sperm were stained with either rhodamine 123 (Rhod-123), the aliphatic cell-tracking compound PKH2-GL, dihydro-ethidium (HED), the bisbenzimide stain Hoechst 33342 (Ho33342), or left unstained. The stained samples were compared for metabolic activity, cell staining pattern, and fluorescent intensity over a 180-min period. Samples stained with HED, Ho33342, and PKH2-GL had less oxygen uptake when compared with the unstained sperm samples (p greater than 0.05). Unstained samples and samples stained with Rhod-123 had similar oxygen consumption. The carbon dioxide produced during the 180 min was not different between controls and stained samples. Therefore, some fluorescent probes inhibit the oxygen metabolism of thawed, cryopreserved bovine sperm cells.     

Characterizing the silica deposition vesicle of diatoms

Summary The present study on a centric diatom,Ditylum brightwellii, includes two parts: detection of sugars in the silica deposition vesicle (SDV) with lectins and labeling the developing siliceous cell wall in the SDV with rhodamine 123. Cells with developing valves are treated with SDS to remove all the cytoplasmic contents, then either stained with fluorescein labeled lectins or thin-sectioned and stained with colloidal gold labeled lectins. The results show that mannose is part of the organic matrix in the SDV. Rhodamine 123, a non-toxic fluorescent laser dye, enters the cell immediately and is trapped in the SDV probably by the high reducing potential of the SDV. Silica is co-deposited with rhodamine 123 in the SDV, and the resulting valves and girdle bands become fluorescent. Implications of this study for the mechanism of silicification are discussed.Abbreviation SDV Silica deposition vesicle     

Staining of living bacteria with rhodamine 123

Abstract It is possible to stain live bacteria with rhodamine 123 (R123). The stained fluorescent cells still keep the ability to replicate ( Staphylococcus aureus, Bordetella pertussis ) and to swim (e.g., Salmonella minnesota ). Dead cells or cells with a dissipated transmembrane potential showed markedly diminished fluorescence. Gram-negative strains were stained with different efficiency, presumably reflecting the different constitutions of the outer membrane.     

Inhibitory effect of rhodamine 123 on the growth of the rodent malaria parasite, Plasmodium yoelii

The effect of the cationic permeant fluorescent dye, rhodamine 123 (R123), on the in vivo growth of Plasmodium yoelii was examined. Plasmodium yoelii-infected mouse erythrocytes were incubated in vitro with R123 and injected intravenously into mice. Examination of daily parasitemias showed that R123 delayed parasite growth whereas rhodamine 110, a neutral compound, and fluorescein, a negatively charged fluorescent dye, did not. Infected erythrocytes treated with R123 were not cleared from the circulation even 7 h after injection. Quantitation of cell-associated R123 by spectrophotometry revealed that infected cells with increased levels of R123 considerably prolonged the 2% prepatent period, the time required for the parasite to develop a 2% parasitemia. Degenerating parasites within and outside the host erythrocytes were observed on day 1 of infection in the mice. Thus it follows that R123, which accumulated in infected erythrocytes, inhibits the growth of P. yoelii; moreover, when R123-labeled infected erythrocytes were treated with 1-10 microM carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton ionophore, to release R123 from the cells, the inhibitory effect on the growth rate of P. yoelii was partially reversed.     

Culture of bovine embryos in intermediate host oviducts with emphasis on the isolated mouse oviduct

The oviduct provides the optimal environment for the transport of sperm and oocyte at the earliest stages of mammalian embryo development. During the early postfertilization period, several major developmental events occur in the embryo including (i) the first cleavage division, (ii) activation of the embryonic genome, (iii) compaction of the morula, and (iv) formation of the blastocyst. Most of these events are initiated in the oviduct. The absence of assistance from the oviduct may compromise the developmental ability of the cattle embryo under in vitro culture conditions. The oviducts of several mammalian species, including rabbits, cow, sheep (in situ), and mice (organ culture), can sustain early bovine embryos and yield blastocysts of better quality compared with those of culture conditions in vitro, leading to normal pregnancy rates in recipient animals. This review focuses on the use of oviducts in vitro or in vivo as intermediate hosts for postfertilization culture environment of bovine in vitro-produced zygotes with emphasis on the mouse model.     

Rhodamine 123 as a probe of in vitro toxicity in MDCK cells

The effect of mercuric chloride on Madin-Darby Canine Kidney (MDCK) cells grown in culture was assayed by the mitochondrial-specific fluorescent probe, rhodamine 123. Treatment of cells with mercuric chloride resulted in a dissipation of rhodamine fluorescence from the mitochondria into the cytoplasm, followed by a release into the medium bathing the cells. Toxicity was assayed either by determining the proportion of cells with delocalized rhodamine fluorescence, or by measuring the rhodamine released from or retained in the cells. Quantifying the release or retention of rhodamine 123 is semi-automated and represents a highly sensitive method of using a vital fluorescent dye for in vitro toxicity analysis.     

Estradiol decreases retention of rhodamine 123 fluorescence in GH4C1 pituitary tumor cells

Rhodamine 123 is a lipophilic cationic fluorescent dye that localizes in mitochondria. We found that 17 beta-estradiol changes the ability of GH4C1 cells, clonal rat pituitary tumor cells, to retain rhodamine 123. Cells incubated with 10 micrograms/ml rhodamine 123 for 30 min at 37 C took up about equal amounts of rhodamine 123, as determined by fluorescence microscopy, regardless of whether they had been treated with estradiol. After three 5-min washes at 37 C, cells treated with 1 nM estradiol for 7 days before incubation with rhodamine 123 had lost more fluorescence than untreated cells. We further characterized the effect by flow cytometry. The difference in fluorescence between control and treated cells ranged from 50- to 500-fold. The effect of estradiol was maximal at 10(-10) M and took a week to develop fully. The effect is specific for estradiol, because estradiol and diethylstilbestrol reduced retention of rhodamine 123 fluorescence at 10(-10) M, but the same concentrations of dihydrotestosterone, progesterone, dexamethasone, and cholesterol did not. To test if the effect on rhodamine 123 fluorescence was caused by activation of the multidrug resistance transport system, we examined the effect of estradiol on the retention of daunomycin, a known substrate of the transport system. Estradiol treatment caused a 3-fold decrease in daunomycin fluorescence. We isolated clones resistant to estradiol-induced loss of rhodamine 123 fluorescence by flow cytometry and found that two clones still showed an estradiol-induced decrease in daunomycin fluorescence equivalent to that of the parent line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Species-specific differences in the toxicity of rhodamine 123 towards cultured mammalian cells

The toxicity of cationic fluorescent dye, rhodamine 123, towards a number of independently established cell lines from three different species, namely human, mouse, and Chinese hamster, has been examined. All of the cell lines from any one species that were examined were found to exhibit similar sensitivities towards rhodamine 123 and no appreciable differences were observed between the normal and transformed cell types. However, in comparison to the cells of human origin, mouse and Chinese hamster cell lines exhibited about 10-fold and 70-fold higher resistance, respectively, and these differences appeared to be species related. In contrast to rhodamine 123, no differences in relative toxicities for these cell lines were observed for the structurally related neutral dye, rhodamine B. Fluorescence studies with rhodamine 123 show that in comparison to mouse and Chinese hamster cells, the more sensitive human cells show much higher uptake/binding of the drug, and a good correlation was seen in these studies between the extent of dye uptake/binding and the relative sensitivities of cell lines to rhodamine 123. These results provide evidence that the observed species-related differences in cellular toxicities are due to differences in the cellular uptake/binding of the dye.     

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.     

Inhibitory Effect of Rhodamine 123 on the Growth of the Rodent Malaria Parasite,Plasmodium yoelii1

The effect of the cationic permeant fluorescent dye, rhodamine 123 (R123), on the in vivo growth of Plasmodium yoelii was examined. Plasmodium yoelii-infected mouse erythrocytes were incubated in vitro with R123 and injected intravenously into mice. Examination of daily parasitemias showed that R123 delayed parasite growth whereas rhodamine 110, a neutral compound, and fluorescein, a negatively charged fluorescent dye, did not. Infected erythrocytes treated with R123 were not cleared from the circulation even 7 h after injection. Quantitation of cell-associated R123 by spectrophotometry revealed that infected cells with increased levels of R123 considerably prolonged the 2% prepatent period, the time required for the parasite to develop a 2% parasitemia. Degenerating parasites within and outside the host erythrocytes were observed on day 1 of infection in the mice. Thus it follows that R123, which accumulated in infected erythrocytes, inhibits the growth of P. yoelii; moreover, when R123-labeled infected erythrocytes were treated with 1–10 μM carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton ionophore, to release R123 from the cells, the inhibitory effect on the growth rate of P. yoelii was partially reversed.     

Fluorochroming Nuclei of Gold Chloride-Stained Motor Endings

Four millimeter cubes of muscle were soaked in 1% citric acid, 10 min; stained 60 min in 1% Au Cl₃,; then reduced 24 hr in 20% formic acid. Small pieces of muscle were teased on a slide and flooded with a single fluorescent dye or combination of fluorochromes. Single saturated solutions of berberine, acridine orange, coriphosphine, and entozon granulate gave adequate results. Berberine in a dilution of 1:5; acridine orange, berberine, coriphosphine and rhodamine B in a dilution of 1:10 were suitable. Combinations of acridine orange and berberine, berberine and coriphosphine, rhodamine B and berberine, entoion granulate and berberine, primulin and berberine and a combination of berberine, coriphosphine and rhodamine B in dilutions of 1:10 in equal parts were successful. Dilutions were volumetric measurements from saturated solutions. After the application of one or more fluorochromes, the preparations were washed with distilled water and observed with a fluorescence microscope. The plasmodesmata of the motor endings and the axon appeared dark purple or black when stained with gold chloride alone or in combination. The nuclei appeared as light bodies throughout the field and the color varied according to the dyes used. There was no color differentation between connective tissue, nerve or muscle nuclei. The method quickly and consistently permits identification of the cellular elements of the motor end plate, connective tissue and muscle fibers.     

Patterns of organelle distribution in mouse embryos during preimplantation development

We have evaluated the distribution of mitochondria and acidic organelles using, respectively, the specific vital fluorescent dyes rhodamine 123 and acridine orange during preimplantation embryonic development in the mouse. Under conditions used to visualize organelles in living embryos, staining with either dye was found to have no effect on either the rate or extent of in vitro development of five- to eight-cell embryos up to the blastocyst stage. Mitochondria were randomly distributed throughout the cytoplasm and located around nuclei in blastomeres of uncompacted embryos. During compaction, mitochondria initially reorganized to the blastomere cortex; however, these organelles were later confined to the perinuclear region in the trophectoderm (TE) of expanded blastocysts. Acidic organelles were randomly distributed in the cytoplasm of uncompacted embryos, but following compaction, they were concentrated in cortical and perinuclear locations. Moreover, in TE cells of expanded blastocysts, acidic organelles were found exclusively in a tight perinuclear pattern. Microtubules and microfilaments in TE cells were localized in fixed embryos stained with antitubulin antibodies and rhodamine phalloidin, respectively; these structures were found primarily in the cortical cytoplasm at areas of cell-cell contact and secondarily in a perinuclear location. Thus mitochondria and acidic organelles undergo stage-specific redistributions from a diffuse or cortical pattern at the eight-cell stage to a tight perinuclear localization in the TE. We conclude that the polarized distributions of some organelles and cytoskeletal proteins during compaction may not be reliable permanent markers of the mature TE.     

In vitro and in vivo development of bovine embryos from zygotes and 2-cell embryos microinjected with exogenous DNA

The objectives of these experiments were: 1) to determine an effective culture method for production of transferable bovine embryos following exogenous DNA microinjection; 2) to determine the effect of these methods on the ability of the injected zygotes and 2-cell embryos to develop in vivo; and, 3) to compare development of embryos microinjected as zygotes or 2-cell embryos. DNA fragments encoding bovine growth hormone (bGH), bGH-10Delta6, and a bGH antagonist, bGH-M8 (5) were used. A total of 639 zygotes and 153 2-cell embryos were injected. Zygotes and 2-cell embryos microinjected with bGH-M8 were incubated for 6 days in oviducts of intermediate recipients (rabbits or sheep) or co-cultured in vitro with bovine oviduct epithelial cells. Zygotes and 2-cell embryos microinjected with bGH-10Delta6 were co-cultured in vitro only. The most effective method for the production of transferable bovine embryos following exogenous DNA microinjection was via in vitro co-culturing with bovine epithelial cells. For example, 32.3% of the bGH-M8 and 33.5% of the bGH-10Delta6 microinjected zygotes reached the morula/blastocyst stage while 48.4% and 63.0% of the 2-cell embryos injected with bGH-M8 and bGH-10Delta6, respectively, developed to the morula/blastocyst stage. The percentage of blastocysts obtained for control, non-injected zygotes and 2-cell embryos was 34.5% and 69.6%, respectively. The developmental rate to the morula/blastocyst stage was approximately 20% greater for embryos obtained from microinjected 2-cell embryos relative to microinjected zygotes. However, there was no significant difference in pregnancy rates following transfer of these blastocysts to cow uteri.     

Purification of cybrids by fluorescence-activated cell sorting

A general method to isolate and purify substantial numbers of viable cybrids from cultured mammalian cells immediately following cytoplast-cell fusion is described. This method uses cytoplasts whose mitochondria are selectively stained in vivo by the cationic fluorescent rhodamine dye, rhodamine 123. Large numbers of highly purified, rhodamine-stained cytoplasts are fused to appropriate recipient cell lines and then the fusion mixture is sorted based on forward angle scatter and fluorescence parameters. Plating the positively sorted population in culture for as short as 12 h eliminates contaminating cytoplasts which, lacking a nucleus, are unable to adhere or survive. The resultant population, based on an analysis of genetic markers, is 75-100% cybrids, an enrichment of 1000- to 10,000-fold over the initial fusion mixture. Cybrids purified by cell sorting may be useful for detailed molecular studies of mitochondrial DNA gene expression and in the specific induction of new mitochondrial DNA mutants.     

Microplate screening of the differential effects of test agents on Hoechst 33342, rhodamine 123, and rhodamine 6G accumulation in breast cancer cells that overexpress P-glycoprotein

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 microM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.     

Complement effects on the infectivity of Plasmodium gallinaceum to Aedes aegypti mosquitoes. I. Resistance of zygotes to the alternative pathway of complement

Gametocytes are the intraerythrocytic stages of malaria parasites that infect mosquitoes. When gametocytes of the chicken malaria parasite Plasmodium gallinaceum are ingested by a mosquito they become extracellular in the mosquito midgut, form gametes, and fertilize within 10 to 15 min after the insect has taken a blood meal. Gametocytes of P. gallinaceum were infectious when fed to Aedes aegypti mosquitoes in blood meals containing native serum from chickens or from the non-host species, man or sheep. Gametocytes stimulated to undergo gametogenesis and to fertilize in vitro were also infectious when fed to mosquitoes in native chicken serum. However, native serum from most non-host species, including sheep and man, suppressed the infectivity of newly fertilized zygotes to mosquitoes and lysed the zygotes in vitro. These effects were shown to be due to the activity of the alternative pathway of complement (APC) in the serum of the non-host species. After mild trypsin treatment, the zygotes of P. gallinaceum no longer infected mosquitoes in the presence of native chicken serum, although in heat-inactivated chicken serum their infectivity was normal. We conclude that trypsin-sensitive components on the zygotes surface protect them from destruction by the APC of their native host. The ability of gametocytes of P. gallinaceum to infect mosquitoes in the presence of native human serum is probably due to proteases that inactivate the APC of human serum before the gametes and zygotes emerge as extracellular parasites in the blood meal.     

Spatial and developmental changes in the respiratory activity of mitochondria in early Drosophila embryos.

Mitochondria of early Drosophila embryos were observed with a transmission electron microscope and a fluorescent microscope after vital staining with rhodamine 123, which accumulates only in active mitochondria. Rhodamine 123 accumulated particularly in the posterior pole region in early cleavage embryos, whereas the spatial distribution of mitochondria in an embryo was uniform throughout cleavage stages. In late cleavage stages, the dye showed very weak and uniform accumulation in all regions of periplasm. Polar plasm, sequestered in pole cells, restored the ability to accumulate the dye. Therefore, it is concluded that the respiratory activity of mitochondria is higher in the polar plasm than in the other regions of periplasm in early embryos, and this changes during development. The temporal changes in rhodamine 123-staining of polar plasm were not affected by u.v. irradiation at the posterior of early cleavage embryos at a sufficient dosage to prevent pole cell formation. This suggests that the inhibition of pole cell formation by u.v. irradiation is not due to the inactivation of the respiratory activities of mitochondria. In addition, we found that the anterior of Bicaudal-D mutant embryos at cleavage stage was stained with rhodamine 123 with the same intensity as the posterior of wild-type embryos. No pole cells form in the anterior of Bic-D embryos, where no restoration of mitochondrial activity occurs in the blastoderm stage. The posterior group mutations that we tested (staufen, oskar, tudor, nanos) and the terminal mutation (torso) did not alter staining pattern of the posterior with rhodamine 123.     

重组谷糠源过氧化物酶的克隆、表达及逆转结肠癌化疗耐药活性

本课题组前期首次从谷糠中获得具抗肿瘤活性的过氧化物酶FMBP。为了该蛋白质后期在体外的规模化生产,本研究以谷子cDNA为模板,利用基因工程手段构建了pMal-s-FMBP融合质粒,并优化诱导条件进行原核表达与纯化,最终获得纯度大于90 %的具抗肿瘤活性的重组FMBP（Re-FMBP）。研究发现,Re-FMBP可以逆转HCT-8/5-Fu耐药细胞对5-Fu的耐药性,耐药逆转倍数达到9.6倍。通过Annexin V/碘化丙啶双染色流式细胞仪检测显示：Re-FMBP能够诱导HCT-8/5-Fu耐药细胞发生凋亡;通过罗丹明123染色,流式细胞仪检测结果显示,Re-FMBP处理后,HCT-8/5-Fu耐药细胞内的荧光强度增强。说明Re-FMBP能够增加5-Fu药物在HCT-8/5-Fu耐药细胞内的蓄积。结果揭示,Re-FMBP蛋白可通过抑制耐药细胞增殖,诱导耐药细胞凋亡,抑制耐药细胞对5-Fu药物外排功能来增加HCT-8/5-Fu耐药细胞对5-Fu药物的敏感性。因此,谷糠来源的Re-FMBP蛋白可以作为肿瘤化疗辅助药物来开发。     

Membrane potential of Plasmodium falciparum gametocytes monitored with rhodamine 123

The membrane potential of Plasmodium falciparum gametocytes was monitored with the cationic permeant fluorescent dye rhodamine 123 (R123) as a probe. Epifluorescence microscopy revealed that R123 at 1 microgram/ml rather selectively partitioned into structure resembling large mitochondria. Treatment of R123-loaded gametocytes with various inhibitors including those of respiration resulted in disappearance of fluorescence from what appeared to be the mitochondria, but not from the cytosol. These results indicate that P. falciparum gametocytes have the mitochondrion maintaining an inside negative membrane potential.