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.     

Embryotoxicity in vitro with extract of Indigofera suffruticosa leaves

Aqueous extract of leaves of Indigofera suffruticosa (AELIs) were studied for adverse effects in preimplantation mouse embryos. Two-cell mouse embryos were cultured for 94 h in human tubal fluid medium (HTF), and AELIs at a concentration of 5 or 10 mg/ml. On Day 4 of culture, morulae and blastocysts were collected for morphological analysis of blastomeres. We found that embryos exposed to the higher concentration of AELIs (10 mg/ml) did not develop and all embryos persisted at the two-cell stage. Those embryos exposed to the lower concentration (5 mg/ml) showed development until morula, blastocyst and hatched blastocyst stages that were similar to the controls. These results suggest that use of AELIs may be hazardous to humans who make use of it in folk medicine.     

不同的培养方法对鼠胚体外发育的影响

目的比较鼠胚在不同的培养体系中的体外发育情况,探讨培养液的体积对体外培养的影响。方法把收集的小鼠2-细胞胚胎分成两组,A组于20μl培养液的微滴中培养,B组与50μl培养液的微滴中培养,观察胚胎发育情况。结果 96h后A、B组的囊胚形成率分别为83.64%和81.63%,差异无统计学意义(P>0.05)。结论适当体积的培养滴可促进胚胎的体外发育。     

Cleavage and development in cultured preimplantation mouse embryos exposed to lidocaine

Two-cell preimplantation mouse embryos were exposed in vitro to lidocaine (0 to 1,000 μg/mL) for 72 h to determine the effects of this anesthetic on subsequent cleavage and development during prolonged exposures. Embryonic development was monitored each 24 h for 3 d. Lidocaine adversely affected the in vitro development of the mouse embryos, altering the distribution of the development stages at the evaluated culture tunes. The percentage of two-cell embryos that cleaved and developed to more advanced stages was decreased by the exposure to lidocaine. After 24 h of culture, two-cell embryos were arrested before completion of cellular division; this occured in 30% of the embryos at concentrations of 10 to 100 μg/mL and in 73.2% of the embryos with 1,000 μg/mL. After 48 h the blastomeres of the arrested embryos began to degenerate, showing lysis or fragmentation. At the lowest concentration, 14.9% of the arrested embryos exhibited the capacity to recover. These embryos continued their cleavage and normal development towards blastocyst formation. The cytotoxic effect and arrest at the two-cell stage were observed in a dose-dependent manner after 72 h of culture. We conclude that sensitivity to lidocaine embryotoxicity occurs during a window at the two-cell stage.     

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.     

Inhalation Toxicology and Histopathology of Ricin and Abrin Toxins

Abstract

The inhalation toxicology of ricin (supplied by Sigma) from the seed variety “Hale Queen” and abrin was examined following head-only exposure of rats to a range of concentrations of each toxin generated as an aerosol from solution using a constant-output nebulizer. The inhalation toxicity of an in-house preparation of ricin from a different seed type, Ricinus communis var. zanzibariensis (R. zanzibariensis), was also assessed for comparison. The approximate LCt50 values determined were very similar for the Sigma ricin and abrin (4.54–5.96 and 4.54 mg min m^-3, respectively). However, the LCt50 of ricin toxin prepared in-house from seeds of the R. zanzibariensis variety was assessed to be 12.7 mg min m^-3. Ricin prepared from this seed variety was therefore less toxic than Sigma ricin by a factor of almost threefold. Given that both ricin preparations were pure by silver-stained, sodium dodecyl sulfate polyacrylamide electrophoresis gels, the data must reflect differences in specific toxicity between seed varieties. The histopathology was studied in a separate group of experimental animals exposed to approximate LCt30 levels of each in-house toxin preparation and was found to be entirely restricted to the lung. The overall pattern and time course of damage observed were similar for ricin and abrin and were characterized by rapidly progressive and overwhelming pulmonary edema accompanied by acute destructive alveolitis and necrosis/apoptosis of the lower respiratory tract epithelium; severe intraalveolar edema and resulting hypoxia accounted for the majority of deaths in the decedent population. The resolution phase of the pulmonary damage in those animals destined to survive was heralded by a gradual disappearance of edema fluid accompanied by generalized, focally florid, hyperplasia of type II pneumocytes and striking transitory consolidation of the lung parenchyma by chronic inflammatory cells. Despite many similarities in histopathology between abrin and ricin there were some differences. Although by systemic administration abrin is several times more toxic than ricin, when delivered by inhalation there was no significant difference in potency between abrin and the commercial preparation of ricin (Sigma).     

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.     

生长抑制因子2在植入前小鼠胚胎发育中的作用

目的探讨生长抑制因子2(Ing2)在小鼠植入前胚胎中的表达及生物学功能。方法使用ICR小鼠进行促进排卵以获得植入前胚胎。采用免疫荧光方法对Ing2蛋白在小鼠植入前胚胎中的定位进行研究,采用显微注射Ing2-esiRNA实验对其在早期胚胎中的功能进行研究。抽提小鼠囊胚期合子的RNA反转录为cDNA,进行实时PCR分析。结果免疫荧光结果显示Ing2蛋白在小鼠植入前胚胎中定位于胞浆及胞核。从4-cell期胚胎开始,胞核中的表达量明显增高。Ing2-esiRNA显微注射实验显示在小鼠合子中干扰Ing2后,各期胚胎形成率均显著低于对照组(4-cell期胚胎比率:82.7%vs.94.7%;桑葚胚比率:67.3%vs.89.4%;囊胚形成率:48.7%vs.75.8%)(P<0.01)。结论 Ing2的下调,引起植入前小鼠胚胎发育阻滞。     

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.     

Differentiation,Quantification and Identification of Abrin and Abrus precatorius Agglutinin

Abrin, the toxic lectin from the rosary pea plant Abrus precatorius, has gained considerable interest in the recent past due to its potential malevolent use. However, reliable and easy-to-use assays for the detection and discrimination of abrin from related plant proteins such as Abrus precatorius agglutinin or the homologous toxin ricin from Ricinus communis are sparse. To address this gap, a panel of highly specific monoclonal antibodies was generated against abrin and the related Abrus precatorius agglutinin. These antibodies were used to establish two sandwich ELISAs to preferentially detect abrin or A. precatorius agglutinin (limit of detection 22 pg/mL for abrin; 35 pg/mL for A. precatorius agglutinin). Furthermore, an abrin-specific lateral flow assay was developed for rapid on-site detection (limit of detection ~1 ng/mL abrin). Assays were validated for complex food, environmental and clinical matrices illustrating broad applicability in different threat scenarios. Additionally, the antibodies turned out to be suitable for immuno-enrichment strategies in combination with mass spectrometry-based approaches for unambiguous identification. Finally, we were able to demonstrate for the first time how the developed assays can be applied to detect, identify and quantify abrin from a clinical sample derived from an attempted suicide case involving A. precatorius.     

囊胚培养的相关因素分析

目的探讨体外受精(IVF)和精子卵胞浆内注射技术(ICSI)治疗周期中体外培养囊胚形成的影响因素。方法将卵裂期第3d(D3)冷冻后剩余胚胎进行体外延长培养至囊胚期,观察囊胚形成情况。结果共收集7438个冷冻后剩余胚胎,经序贯培养,形成1382个优质囊胚(18.58%)。D3胚胎中6个细胞的优质囊胚形成率(10.34%)显著高于4-5个细胞的优质囊胚形成率(5.45%),差异有统计学意义(P=0.011);7-9个细胞的优质囊胚形成率(19.95%)显著高于6个细胞的优质囊胚形成率(10.34%),≥10个细胞的优质囊胚形成率(27.47%)显著高于7-9个细胞的优质囊胚形成率(19.95%),差异均有统计学意义(P<0.001)。D2胚胎中4个细胞的优质囊胚形成率(25.40%)显著高于3个细胞、5个细胞的优质囊胚形成率(10.26%,15.41%),差异有统计学意义(P<0.001)。碎片评级I-II级D2、D3胚胎的囊胚形成率均显著高于III-IV级的胚胎,差异有统计学意义(P<0.001)。结论体外延长培养能有效筛选具有发育潜力的胚胎,优质囊胚形成与D2、D3胚胎的卵裂球数及碎片评级密切相关。     

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。     

Knockdown of p66Shc by siRNA injection rescues arsenite-induced developmental retardation in mouse preimplantation embryos

Two-cell arrest plays a principal role in the elevated levels of embryo loss during the first week of development in mouse. Previously, we have shown that arsenic can apparently induce 2-cell arrest in mouse preimplantation embryo and the expression of oxidative stress adaptor protein p66^Shc is up-regulated in this process. In the present study, we demonstrated that microinjection of p66^Shc siRNA into the pronucleus of zygotes resulted in a markedly decrease in both mRNA and protein levels of p66^Shc. The arsenite-induced 2-cell arrests, along with a reduction in the levels of reactive oxygen species (ROS), were significantly inhibited and the number of embryos developing to morula stage concurrently increased upon p66^shc siRNA microinjection. These findings indicate that knockdown of p66^shc improves the developmental competence of arsenite-exposed embryos in vitro by increasing the resistance to oxidative stress. In addition, we highlight the utility of single-embryo analysis in preimplantation embryos.     

Protective effects of certain pharmaceutical compounds against abrin induced cell death in Jurkat cell line

Abrin is a plant glycoprotein toxin from the seeds of Abrus precatorius, and shares the structure and properties with ricin. Abrin is highly toxic, with an estimated human fatal dose of 0.1–1 μg/kg, causing death after accidental and intentional poisoning. It is a potent toxin warfare agent. There are no antidotes available for abrin intoxication. It is becoming increasingly important to develop countermeasures for abrin by developing pre- and post-exposure medical therapy. The present study involves the screening of certain pharmaceutical agents for their potential to counter abrin toxicity in Jurkat T lymphocytes and the probable mechanism of action of the compounds with protective effect.The compounds studied are: Prednisolone, Minocycline, Amifostine, DRDE-07 (amifostine analog), Melatonin, Ebselen, N-Acetyl-l-cysteine (NAC) and Trolox. Among them, only NAC and trolox were found to confer significant protection in Jurkat cells by restoring antioxidant enzymes depleted by abrin treatment. Abrin also shown to increase in stress factor associated proteins SAPK/JNK, c-fos and c-jun levels which were effectively suppressed by NAC and trolox. In addition to this, both compounds significantly inhibit abrin induced inflammation and caspase-3 activity.These data suggest that NAC and trolox may serve as potential candidates for management of abrin-induced poisoning.     

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.     

CYP2C76 deficiency is embryonic lethal in cynomolgus macaques: The potential role of CYP2C76 in early embryogenesis

Cynomolgus macaques are an important primate species for drug metabolism studies; however cynomolgus CYP2C76, an important drug-metabolizing enzyme, accounts for drug metabolism differences to humans, so that CYP2C76-null animals might show drug-metabolizing properties more similar to humans. In this study, attempts were made to produce CYP2C76-null animals by assisted reproduction technology. Oocytes and sperm collected from the heterozygotes for the null allele (c.449TG > A) were subjected to intracytoplasmic sperm injection, and the embryos produced were cultured in vitro through the blastocyst stage. Preimplantation genetic diagnosis using a biopsied portion of the blastocyst revealed that none of the 32 blastocysts analyzed were homozygotes. In contrast, 2 of the 20 embryos analyzed were homozygotes at the 8-cell stage, indicating that CYP2C76-null embryos most likely stop developing between the 8-cell and blastocyst stage. By polymerase chain reaction, expression of CYP2C76 mRNA was detected in oocytes and blastocysts, but not in 2-, 4-, 8-, or 16/32-cell stage embryos. Metabolic assays showed that CYP2C76 metabolized progesterone. These results indicated that CYP2C76 null was likely embryonic lethal, suggesting its potential role during early embryogenesis in cynomolgus macaques.     

Comparison of ribosome-inactivating proteins in the induction of apoptosis

The aim of this study was to evaluate the ability of verocytotoxin-1 (VT1), VT1 B chain alone, ricin and a hybrid toxin (RASTA2) consisting of ricin A chain linked to VT1 B chain to inhibit protein synthesis and to induce apoptosis. The lethal effects of the toxins were compared using vero cells (originating from green African monkey kidney tissue). As previously described cell death occurred through apoptosis which was quantified using the diphenylamine assay. DNA fragmentation was seen with VT1 at 10 pg/ml but there was no effect with B chain alone. Fragmentation with ricin was seen at 10 ng/ml and with RASTA2 at 1 ng/ml. Protein synthesis inhibition was measured by [³⁵S]methionine incorporation. VT1 had an IC50 of 0.0024 ng/ml, B chain alone was ineffective at inhibiting protein synthesis. Ricin had an IC₅₀ of 0.39 ng/ml and RASTA2 of 1.7 ng/ml. In vero cells the B chain of these toxins does not participate in cell killing.     

用实时荧光定量RT-PCR检测单个小鼠种植前胚胎中特异性基因表达

目的用单细胞实时荧光定量RT-PCR,检测单个小鼠种植前胚胎中特异性基因表达。方法采集小鼠卵母细胞和原核期胚胎,后者经体外培养至囊胚。采用实时荧光定量RT-PCR对多个及单个卵母细胞和2-细胞期胚胎中Tcl1基因的表达进行定量检测;用同样方法对小鼠种植前各发育阶段的胚胎进行Tcl1基因的表达检测。结果在多个及单个卵母细胞和2-细胞期胚胎中,均检测出Tcl1的表达;多个卵母细胞、2-细胞期胚胎中Tcl1的表达量显著高于单个卵母细胞、2-细胞期胚胎;此方法也适用于胚胎早期发育的各个时期,Tcl1在单个卵母细胞、原核期胚胎和2-细胞期胚胎中表达量最高,在4-细胞期胚胎、8-细胞期胚胎、桑葚胚和囊胚中表达量极低。结论应用单细胞实时荧光定量RT-PCR可对单个小鼠种植前胚胎中基因的表达进行定量检测。     

Silibinin ameliorates abrin induced hepatotoxicity by attenuating oxidative stress,inflammation and inhibiting Fas pathway

Abrin is a toxin from the seeds of Abrus precatorius. Abrin is considerably more toxic than ricin and a potent bio-warfare agent. The mechanism of abrin induced hepatotoxicity remains unclear. Silibinin has antioxidant, anti-inflammatory and hepatoprotective activities. But, its therapeutic potential in abrin toxicity is unknown. In view of these facts, the purpose of this study was to delineate the mechanisms and ameliorative role of silibinin against abrin induced hepatotoxicity. Parameters related to liver functions, oxidative stress, inflammation, Fas pathway and histopathology were evaluated in the liver of BALB/c mice after abrin exposure. Abrin intoxication resulted in hepatotoxicity, oxidative stress, inflammation, altered histopathology and increased Fas pathway signaling. Silibinin improves survival of abrin-exposed mice by decreasing serum liver enzymes and reinstating the antioxidant capacity. Silibinin also inhibits abrin-induced inflammation and Fas pathway. Present study for the first time demonstrates the hepatoprotective potential of silibinin against abrin toxicity.