Effect of nickel chloride and cadmium acetate on the development of preimplantation mouse embryos in vitro

Preimplantation mouse embryos were used to investigate the toxic effect of nickel chloride and cadmium acetate on early embryo development in vitro.Embryos at the 2- and 4–8 cell stage were cultured in approximately 0.05 ml of mouse embryo culture medium (No. 16), overlaid with paraffin oil and incubated in a humidified atmosphere of 5% CO₂ in air for 48 h. NiCl₂ · 6H₂O was added to the culture medium at concentrations of 10–1000 μM, Cd(CH₃COO)₂ · 2H₂O at concentrations of 10–50 μM. Morphological criteria were used to check embryonic development.Ten micromolars of nickel chloride affected adversely the development of Day 2 embryos (2-cell stage), whereas 300 μM was needed to affect Day 3 embryos (8-cell stage). Toxic effect of cadmium acetate on Day 2 embryos was observed at a concentration of 10 μM.     

Effects of cadmium on preimplantation and early postimplantation mouse embryos in vitro with special reference to their trophoblastic invasiveness

Cadmium chloride, at concentrations of 0.5 or 1 microgram/ml medium, did not affect the trophoblastic invasiveness of mouse embryos treated for 24 hours at 4-cell and morula stages. At higher concentrations of 5 or 10 micrograms/ml medium, most treated embryos in vitro underwent degeneration while a few survivors formed trophoblastic outgrowths with variable areas. Cadmium chloride, at a low concentration of 0.5 microgram/ml medium presented continuously to blastocysts after attachment in vitro, has significantly retarded the trophoblastic outgrowth areas and reduced the number of trophoblastic giant-cell nuclei, though the spreading blastocysts appeared morphologically normal. At higher concentrations of 1 or 5 micrograms/ml medium, cytoplasmic disintegration and detachment of trophoblasts were observed. It is suggested that cadmium may interfere with the cell division and/or the transformation of trophectoderm cells into giant cells, resulting in the retardation of the trophoblastic outgrowths.     

Induction of micronuclei formation in preimplantation mouse embryos after maternal treatment with 2-bromopropane

We examined effects of 2-bromopropane (2-BP), a chlorofluorocarbon replacement, on mouse embryonic mutagenicity. 2-BP was administered to pregnant mice intraperitoneally (i.p.) (300, 600, 900, and 1800 mg/kg) during the early preimplantation period. On day 3 of gestation, micronuclei (MN) frequency and embryo cell number were determined. 2-BP induced a dose-related significant increase in MN frequency and a treatment-related decrease in embryo cell number. Furthermore, the cell numbers were significantly smaller in the MN-positive embryos by two-way ANOVA taking it into account an interactive effect between 2-BP dose and the presence or absence of MN. A simultaneous decrease in cell number and increase in MN frequency may reflect an embryonic developmental disadvantage resulting from maternal treatment with 2-BP. Further study is needed to establish how 2-BP contributes to postimplantation embryonic development.     

Effects of 3H-thymidine on preimplantation mouse embryos in vitro

The effect of 3H-thymidine on in vitro development of preimplantation mouse embryos was studied. Two-cell and 4-8-cell embryos from B6CBA/F1 mice were continuously exposed to 3H-thymidine in medium containing 3H-thymidine in concentrations ranging from 10-500 nCi/ml. The effect of the radioactive precursor on embryo development to the blastocyst stage was studied by morphological observation, counting the blastocyst cell number and measuring 3H-thymidine incorporation. The continuous presence of 3H-thymidine significantly inhibited development of 2-cell and 4-8-cell embryos to the blastocyst stage. Embryos cultured from the 2-cell stage were more sensitive to 3H-thymidine than those exposed from the 4-8-cell stage. Even in morphologically normal blastocysts the cell number was significantly reduced. A 2 hr pulse of 100 nCi/ml 3H-thymidine at the blastocyst stage, did not affect the blastocyst formation or the blastocyst cell number and the amount of incorporated 3H-thymidine was sufficient to provide a reliable quantitation of DNA synthesis during the culture of preimplantation embryos in vitro. Continuous incubation with 3H-thymidine in order to measure DNA synthesis of preimplantation mouse embryos should be avoided when DNA synthesis is used as a means of evaluating toxic effect of an agent. Adverse radiation effects by 3H-thymidine on preimplantation mouse embryos during toxicity testing can be avoided by pulse labelling.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on preimplantation mouse embryos

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental contaminant that can exert developmental toxicity. To investigate the stage-specific effects of TCDD on preimplantation embryos, we exposed mouse embryos to TCDD at different stages (1-, 2-, and 8-cell) and collected them at different stages of development (the 1- or 2-, 8-cell, and blastocyst stage, respectively). Semiquantitative RT-PCR revealed increased constitutive gene expression of the arylhydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) at the 1-cell stage, decreased expression at the 2- to 8-cell stage, and increased expression again at the blastocyst stage, and addition of TCDD to media did not affect their mRNA levels. Interestingly, no cytochrome P4501A1 (CYP1A1) mRNA was detected in embryos at the 1-, 2-, and 8-cell stages after exposure to 10 nM TCDD for 12 or 24 h, whereas CYP1A1 mRNA was significantly increased at the blastocyst stage in response to TCDD, and its induction was found to be concentration-dependent on TCDD exposure from 0.01 to 10 nM for 24 h. In addition, no significant differences in development rate of preimplantation embryos, cell number of blastocyst embryos, or apoptotic indices, such as TUNEL-positive cell number or Bax/Bcl-2 expression ratios were observed at the blastocyst stage between TCDD-exposed groups and non-exposed group. These results suggest that the sensitivity to TCDD differs with the embryonic stage, which may reflect an ability of embryos to adapt to environmental stressors, such as dioxins.     

胰岛素与葡萄糖对ICR小鼠早期胚胎体外培养的影响

目的探讨胰岛素与葡萄糖对ICR小鼠早期胚胎体外发育的影响。方法同时添加胰岛素＋葡萄糖的CZB培养液对ICR小鼠胚胎进行体外培养,观察囊胚发育率、孵化胚率和囊胚细胞数的变化,并研究两者最佳的浓度搭配。结果在CZB培养液中同时加入葡萄糖与胰岛素可促进小鼠1-细胞胚胎体外培养,且0．05μg·ml^-1胰岛素与5mmol·L^-1葡萄糖添加组囊胚率、孵化胚率和囊胚细胞数均显著高于其他各浓度添加组。结论联合添加胰岛素和葡萄糖的培养液对小鼠胚胎体外发育具有促进作用,且胰岛素浓度为0．05μg／ml,葡萄糖浓度为5mmol／L时促进作用最大。     

Enhancement of radiation effects by mercury in preimplantation mouse embryos in vitro

Mercuric chloride (3 M or 10 M) increased several effects of ionizing radiation (1 Gy) on preimplantation mouse embryos in vitro. Blastocyst formation, hatching of blastocysts, and the number of cells per embryo were affected by this increase in radiation risk. The formation of micronuclei, however, was not influenced either in experiments using mercury alone or in combination experiments with X-rays.Not only was the sum of the single effects exceeded in some of the combination experiments, but the dose-pairs, which were necessary for obtaining a certain effect, clearly fell to the left of the envelope of additivity. That is, the enhancement of effects cannot be explained merely by the shape of the dose-response curves, but there is an interaction between mercury and ionizing radiation.     

The effects of lanthanum chloride on pregnancy in mice and on preimplantation mouse embryos in vitro

A single intraperitoneal injection of 10(-6.5) mol lanthanum chloride/g body wt (44 mg metal/kg body wt) into pregnant mice reduced the number of successful pregnancies and the average litter size. The most susceptible periods of pregnancy were peri-implantation (days 4 and 6) and near-term period (days 14 and 16). Injection of lanthanum during the peri-implantation period resulted in a cessation of pregnancy in 24-43% of females, and injection during near-term period produced the cessation of pregnancy in 36-46% of the females. The average litter size after injection of lanthanum during peri-implantation or near-term periods was reduced to about 75% of the average litter size in the control animals. No external malformations were observed among fetuses. Paradoxically, the exposure of 1-cell stage embryos to 10(-3.0) M and 10(-3.5) M lanthanum chloride in vitro resulted in a significant improvement of the proportion of embryos developing into blastocysts.     

Embryologic and cytogenetic effects of ethanol on preimplantation mouse embryos in vitro.

Ethanol and its primary metabolite acetaldehyde were studied in cultured preimplantation mouse embryos with respect to embryotoxicity, embryolethality, chromosome breaking activities, and ability to induce sister chromatid exchange (SCE). Analysis of differentiation and cell number of mouse morulae and blastocysts show that acetaldehyde is three orders of magnitude more toxic than ethanol, indicating that the metabolite is responsible for the embryotoxicity of ethanol in preimplantation embryos. Concentrations of ethanol that do not inhibit growth induce SCEs and chromosome aberrations. The SCE-inducing effect of ethanol disappears in the presence of 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase (ADH). These data suggest that preimplantation embryos are able to convert ethanol to acetaldehyde and that ADH is the enzyme involved. It is, furthermore, shown histochemically that mouse oocytes as well as morulae and blastocysts are able to oxidize ethanol in the presence of NAD+.     

Effect of tumour necrosis factor-alpha (TNF-alpha) on protein synthesis by mouse preimplantation stage embryos in vitro

Successful blastocyst implantation depends upon the synchronous dialogue between age- and stage-matched embryo and adequately primed maternal endometrium. Endometrial signals present in the uterine lumen influence the growth and the viability of preimplantation stage embryo. Thus, uterine secretion of embryotoxic cytokines may affect the preimplantation stage embryo. Our previous study in the rhesus monkey has indicated that tumor necrosis factor-alpha (TNF-alpha) is one such candidate present in the uterine lumen, which may act as an embryotoxic agent. In the present study, the effect of TNF-alpha on de novo protein synthesis by mouse morulae (n = 100) and blastocysts (n = 100) in vitro was investigated by 2D-polyacrylamide gel electrophoresis. A total of 35 and 40 protein spots were detected in lysates of control morulae and blastocysts, respectively. Exposure of embryos to TNF-alpha (50 ng/ml) reduced the number of protein spots to 15 and 17 compared to that of control morulae and blastocysts. Seven spots in morula and 13 protein spots in blastocyst flourograms showed quantitative changes in their expressions with exposure to TNF-alpha. Morulae and blastocysts exposed to TNF-alpha expressed 8 and 17 protein spots, respectively, that were not seen in control embryos. It appears from the present study that exposure of preimplantation stage embryos to TNF-alpha affects their protein synthesis both quantitatively and qualitatively.     

Pregnancy outcomes for mouse preimplantation embryos exposed in vitro to the estrogenic pesticide o,p'-DDT

Pregnancy outcomes were evaluated following uterine transfer of murine preimplantation embryos exposed in vitro to the estrogenic pesticide o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT). Single-cell embryos were incubated 72 h in medium droplets containing 0.1% ethanol (control) or 0.1 microg/ml o,p'-DDT (pesticide). Morula and preblastocyst embryos were transferred in groups of eight to right uterine horns of pseudopregnant mice (n=111) and pups (n=132) were evaluated at Caesarean-section (C-section). In vitro exposure to o,p'-DDT reduced development to morula (P<0.001) and modestly increased blastomere apoptosis (P=0.05). However, treatment differences were not detected for implantation rates (35% versus 39%; P=0.64), pup numbers per dam (2.3 versus 1.9; P=0.36), transfer efficiencies (16% versus 14%; P=0.53), fetal weights (1.56 g versus 1.57 g; P=0.91), skeletal abnormalities (55% versus 66%; P=0.47), or male ratios (54.8% versus 53.8%; P=1.0). In vitro exposure of preimplantation embryos to 0.1 microg/ml o,p'-DDT for 72 h resulted in no measurable effects on subsequent implantation or pup characteristics at C-section.     

Previous maternal chemotherapy by cyclophosphamide (Cp) causes numerical chromosome abnormalities in preimplantation mouse embryos

This study investigated the consequences of maternal cyclophosphamide treatment on fertilization rate, development and chromosomal integrity of embryos. It also evaluated efficiency of two methods of classic and metaphase induction for chromosomes assessment. Two different groups of NMRI mice 2–3 weeks and 6–7 weeks were injected intraperitonealy with 75 mg cyclophosphamide/kg. Six weeks later, oocytes were recovered, fertilized and incubated for 3 days in 5% CO₂ in air. Eight to 10 cell stage embryos were subjected to chromosomal study. Cyclophosphamide in both experimental groups reduced the oocyte fertilization rate and in the 6–7 weeks group it was significantly lower compared with control group (P < 0.01). Also there was a reduction in 8-cell stage embryos formation from 48 to 72 h post-fertilization in both treated groups (P < 0.05). Aneuploidy increased in the treatment groups compared with controls, which in the older group was significant (P < 0.001). The success rate of the classic method to analyze metaphase plates was 30.1% and the success rate of heterokaryons formation with analyzable chromosomes was 67.8% in the metaphase induction method. In regards to the adverse effects of cyclophosphamide on fertilization rate, embryo development and chromosomal integrity of the mouse embryos, using Preimplantation Genetic Diagnosis in addition to Assisted Reproductive Technique is suggested.     

Development of preimplantation mouse embryos after exposure to a 50 Hz magnetic field in vitro.

Effect of sinusoidal 50 Hz magnetic field (MF) on development of preimplantation CBA/S mouse embryos in vitro was studied. Superovulated and in vivo fertilized preimplantation embryos were collected at one cell stage and divided to control and MF-exposed groups. Sinusoidal 50 Hz MF with field strength of 10 A/m r.m.s., corresponding a flux density of 13 microT r.m.s., was used to expose the embryos in culture at 37 degrees C in a CO2-incubator. The developmental stage and abnormalities were recorded twice daily except once daily during weekends. The vitality and developmental stages of the embryos were similar in both groups although slightly more dead embryos were found during the 1st day in MF-exposed group (P<0.05) and the development of MF-exposed embryos was slightly impaired. In conclusion, the exposure to sinusoidal 50 Hz MF at field strength of 10 A/m did not significantly disturb the development of the mouse embryos in vitro up to the blastocyst stage.     

In vitro culture of preimplantation mouse embryos and day 12 limb-buds: Effects of serum and albumin

The effects of three different protein sources at different concentrations on the growth and development of preimplantation mouse embryos and day 12 mouse limb-buds in culture were studied. Mouse embryos and forelimb-buds were cultured with a range of concentrations (5.5 to 42%) of either donor bovine serum (DBS) or fetal bovine serum (FBS), or (0.2 to 0.8%) bovine serum albumin (BSA). After 48 h in culture, the rate of embryo development was significantly higher in 5.5% DBS than in all other groups (P < 0.05). The embryo hatching rate was higher in 21% FBS, 42% FBS, and all DBS groups than in serum-free medium, and all BSA groups (P < 0.05). Morphologic analysis of cultured limb-buds at 72 h revealed that total, paw, and cartilage area were greater (P < 0.05) in the serum-free medium than in all other groups. Shape factor analysis suggested that 5.5% DBS was most beneficial to mouse limb-bud development. No differences were seen in DNA or protein content of limb-buds among groups. Results suggest that mouse forelimb-buds can be succesfully cultured in serum-free medium and that high concentrations of FBS and DBS may be detrimental for preimplantation embryo and/or limb-bud growth and development.     

Effects of paraquat on development of preimplantation embryos in vivo and in vitro

Paraquat can cause oxidative stress through redox cycling, and preimplantation embryos are sensitive to oxidative stress in vitro. In this study, the effects of paraquat on preimplantation embryo development were examined. Exposure of preimplantation embryos (collected on the day after ovulation) to paraquat in vitro for 24 h at concentrations as low as 8 microM caused a significant decrease in the percentage of 8-cell embryos and an increase in the percentage of compacted morulae, but the content of reduced glutathione (GSH) in embryos was not changed. Altered embryo development was most likely due to premature compaction because a 42% decrease in cell number per compacted morulae was observed in embryos exposed to paraquat at 1 mM. Exposure of preimplantation embryos to paraquat in vitro for 4 days at 200 microM or higher eliminated development beyond the blastocyst stage. Exposure of bred female mice to paraquat at 30 mg/kg on day 2 after ovulation led to a small but significant decrease in the percentage of 8-cell embryos on day 3 without a detectable increase in the percentage of compacted morulae. No detectable change in preimplantation embryo development was found following paraquat exposure on the day of ovulation (day 0), although a significant decrease in embryo GSH was found on day 1. These data indicate that paraquat can adversely impact the development of preimplantation embryos in vitro and in vivo without consistent modulation of GSH level.     

Taxifolin mitigates oxidative DNA damage in vitro and protects zebrafish (Danio rerio) embryos against cadmium toxicity

Taxifolin (TAX) is a natural source of bioflavonoid found in various conifers. In this study, initially we investigated the antioxidant potential of TAX under in vitro assays such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), ferric-ion reducing power (FRAP) and hydroxyl radical (OH). The activities of DPPH, ABTS, FRAP and OH radical levels were significantly inhibited by TAX with an IC₅₀ values of 16.48, 66.34, 18.17 and 11.42 μg/ml, respectively. Secondly, TAX exhibited a strong protection against OH mediated DNA damage on pUC19 plasmid DNA at 1.0 μg/ml. Finally, we evaluated the protective mechanism of TAX against cadmium intoxicated zebrafish embryos (Danio rerio). We found that embryos exposed to 100 μM Cd exhibited significantly reduced survival, delayed hatching and phenotypic abnormalities at 24, 48, 72 and 96 hours post fertilization (hpf). Similarly, Cd intoxicated embryos showed significantly increased cardiac function (131 beats/min) at 60 hpf. Conversely, treatment with TAX (0.1, 1.0 and 10 μM) significantly enhanced the antioxidant enzyme levels (SOD, CAT, GPx and GR) by reducing the lipid peroxidation (MDA) in zebrafish embryos. Collectively, our results concluded that TAX could act as a potent redox scavenger against oxidative DNA damage and also functions as a crucial suppressor of Cd toxicity in zebrafish embryos.     

Cell survival after the combined action of manganese (MnCl2) and X-rays in synchronized Chinese hamster cells

The interactions between the effects of manganese chloride and X-rays were studied in synchronized populations of V79 Chinese hamster fibroblasts. The cells were selected by shaking off asynchronous cultures for detachment of mitotic cells which were plated in petri dishes and exposed to various treatments. Irradiation was carried out with a Philips RT-100 X-ray unit. A final concentration of 0.25 mM MnCl₂ was used. The main parameter was the colony forming ability of the surviving cell fraction. When MnCl₂ was administered over 1 h, its toxicity was low regardless of the phase of the cell cycle. Administered separately, 2 Gy irradiation produced only a slight decrease in survival, less marked in the S phase. However, the two agents together induced a synergistic inhibition of the surviving fraction in the S phase when the metal was given immediately after irradiation. If manganese was administered 3 h after irradiation the two inhibitory effects apparently remained only additive. It seems that MnCl₂ can impair some repair processes starting immediately after irradiation.     

In vitro culture of mouse embryos for toxicological evaluation. (3): Effect of carcinogens on development of the embryos

Mouse embryos were collected at the 2 cell or 8 cell stage. The embryos of each stage were exposed to 1 pM-10 nM 4-nitroquinoline-1-oxide (4-NQO) or 1 nM-10 microM N-methyl-nitro-nitrosoguanidine (MNNG) for 24 hr, and cultured to develop to blastocysts within clean medium. Since after exposure to 4-NQO, the appearance of early blastocysts and blastocysts were increased in exposure groups compared with controls, the growth to the 8 cell stage embryo (8-E) appeared to be late in development. Frequency of sister chromatid exchange (SCE) and mitotic index were increased with doses in the blastocysts (2-B) which derived from 2-E. There was little change in the SCE and mitotic index for blastocysts (8-B) which derived from the 8 cell stage embryo (8-E). Cessation of development in the 2 cell stage embryo (2-E) appeared in the 10 microM group during the period of exposure to MNNG. Development to the 8-E stage appeared to be slightly late in the other exposed groups. Thereafter at 48 hr after the initiation of culture, the exposed groups appeared to be more advanced in development than the controls. After this period, developmental rates to blastocysts were increased in the 10-100 nM groups. It appeared that the development of these groups was more advanced. In 2-B after exposure to MNNG, cell counts were dose-responsively increased in the 1 and 10 nM groups. The mitotic index in the 1 to 100 nM groups was higher than the controls.(ABSTRACT TRUNCATED AT 250 WORDS)