Recovery of mouse embryos after short-term in vitro exposure to toxic nickel chloride

The development of preimplantation mouse embryos in vitro was adversely affected by the addition of nickel chloride (NiCl₂·6H₂O) to the culture medium. For day 3 (4–8 cell) embryos developmental cessation occurred after 48 h in culture, in NiCl₂·6H₂O-containing medium. However, transfer to NiCl₂·6H₂O-free medium after 5 min, 1 h, and 3 h exposure, resulted in regaining of the developmental capacity for a proportion of the exposed embryos.The in vivo development, in pseudopregnant recipients, of in vitro nickel-exposed embryos was not significantly different from that in control embryos.The results indicated that the effect of NiCl₂·6H₂O on the development of day 3 mouse embryos in vitro was reversible after a short exposure period.     

Effect of microcystin‐LR on human placental villous trophoblast differentiation in vitro

Microcystin‐LR is a cyanobacterial toxin found in surface and recreational waters that inhibits protein phosphatases and may disrupt the cytoskeleton. Microcystins induce apoptosis in hepatocytes at ≤2.0 µM. Nothing is known about the effects of microcystins on human placental trophoblast differentiation and function. The differentiation of villous trophoblasts to form syncytiotrophoblast occurs throughout pregnancy and is essential for normal placental and fetal development. To investigate the effects of microcystin, villous cytotrophoblasts were isolated from term placentas using an established method and exposed to microcystin‐LR. Microcystin‐LR below the cytotoxic dose of 25 µM did not cause cell rounding or detachment, had no effect on apoptosis, and no effect on the morphological differentiation of mononucleated cytotrophoblasts to multinucleated syncytiotrophoblast. However, secretion of human chorionic gonadotropin (hCG) increased in a microcystin‐LR dose‐dependent manner. When incubated with l ‐buthionine sulphoximine (BSO) to deplete glutathione levels, trophoblast morphological differentiation proceeded normally in the presence of microcystin‐LR. Microcystin‐LR did not disrupt the trophoblast microtubule cytoskeleton, which is known to play a role in trophoblast differentiation. Immunofluorescence studies showed that trophoblasts express organic anion transport protein 1B3 (OATP1B3), a known microcystin transport protein. In comparison to hepatocytes, trophoblasts appear to be more resistant to the toxic effects of microcystin‐LR. The physiological implications of increased hCG secretion in response to microcystin‐LR exposure remain to be determined. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 427–439, 2016.     

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.     

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.     

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.     

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 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.     

PPARalpha agonists clofibrate and gemfibrozil inhibit cell growth, down-regulate hCG and up-regulate progesterone secretions in immortalized human trophoblast cells

We studied effects of PPARα agonists clofibric acid and gemfibrozil on cell growth and functions of immortalized human extravillous trophoblast cells. Levels of DNA and protein gradually increased during incubation for 4 days. Gemfibrozil (>0.25 mM) and clofibric acid (2.5 mM) suppressed the rate of increase in DNA and protein. Specific activities of fatty acyl-CoA oxidase and catalase were increased to about 1.2-2.0 times the control value by 0.05 mM gemfibrozil and 1.0 and 2.5 mM clofibric acid after incubation for 3 days. Acid phosphatase activity showed a small increase in response to both agents, but esterase activity changed little. The secretion of progesterone from the cells into the medium was increased by 0.25 mM gemfibrozil and 1.0 and 2.5 mM clofibric acid after incubation for 3 days, but that of human chorionic gonadotropin (hCG) was decreased by 0.35 mM gemfibrozil and 2.5 mM clofibric acid. The specific activity of lactate dehydrogenase in the cells was hardly changed at all after incubation for 3 days.These results suggest that gemfibrozil and clofibric acid inhibit the proliferation of trophoblast cells. Cell metabolism is probably affected by both agents. The two agents may down-regulate hCG and up-regulate progesterone secretions.     

Effect of acute chloroquine treatment on prostaglandin- and gonadotropin-stimulated testosterone secretion of rat testis

The effect of chloroquine injection to rats on in vitro-testosterone secretion stimulated by human chorionic gonadotropin (hCG) and prostaglandin E1 (PGE1) was studied. Rats were injected for 5 days with chloroquine phosphate. Testosterone secretion was stimulated by hCG or PGE1 for 3 h in the removed testis and was measured by radioimmunoassay. Chloroquine treatment in vivo does not seem to have any demonstrable effect on testosterone secretion stimulated by hCG or PGE 1 in prepubertal as well as in the postpubertal rats. However, in pubertal rats chloroquine treatment inhibited testosterone secretion in hCG-stimulated testis.     

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+.     

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.     

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.     

300例异位妊娠的疗法分析

目的探讨异位妊娠的疗法分析。方法回顾性分析300例异位妊娠患者的诊治情况。结果 300例异位妊娠中采用非手术治疗65例,成功51例,成功率78.46%,失败14例;手术治疗249例,成功率100%。经病史、症状、体征、超声及血人绒毛膜促性腺激素检查来综合确定,手术病例均经病理证实,诊断符合率达100%。结论在目前的医疗条件,患者就诊及时,除少数需剖腹手术治疗外,绝大多数采用非手术治疗及熟练的腹腔镜手术治疗,效果良好。     

The combined treatment of preimplantation mouse embryos in vitro with cadmium (CdSO4, CdF2) and X-rays

The influence of cadmium (up to 10^?4 M), fluoride (up to 10^?4 M), and X-rays (up to 18.8 Gy) on different variables of preimplantation mouse embryos in vitro has been examined. The agents were applied either singly or in combination (0.94 Gy X-rays +3×10^?7 M CdSO₄ or CdF₂; 0.94 Gy X-rays +3×10^?6 M CdSO₄ or CdF₂). The following variables were determined:

1. the microscopic visible development until 144 h post conceptionem (=144 h p.c.),
2. the average cell numbers (48 h p.c., 56 h p.c., 72 h p.c., 96 h p.c., 120 h p.c., 144 h p.c.),
3. the number of micronuclei (72 h p.c.),
4. the distribution of cell nuclei within the cell cycle (72 h p.c.).

Nearly all results of the combination experiments correspond to the sum of the single effects. Only two values (out of about 40) significantly exceed the value obtained after addition of the single effects; both values lie within the envelope of additivity.     

The effect of herbicide BASTA 15 on the development of mouse preimplantation embryos in vivo and in vitro

The aim of this study was to evaluate the possible effect of maternal poisoning by BASTA-15 on developmental capacities and quality of preimplantation embryos in a mouse model. During in vivo tests, fertilized mice were fed with various doses of BASTA-15 for several days. During in vitro tests, isolated embryos were cultured in a medium with the addition of herbicide or its main compound glufosinate ammonium. Stereomicroscopic evaluation of embryonic pools obtained from treated dams showed that BASTA-15 at dose 58 μl/kg bw negatively affected their ability to reach the blastocyst stage. Moreover, as shown by morphological evaluation, based on cell counting and cell death assay, even the application of herbicide at the lowest dose (approx. 1/100 LD50) had a negative effect on obtained embryo quality. In vitro tests proved the direct ability of BASTA-15 to negatively affect embryo growth and quality. On the other hand, the addition of glufosinate ammonium at equivalent concentrations (from 0.015 to 15 μg/ml) had almost no damaging effect on embryos. It was harmful only at very high doses. Results show that maternal intoxication with BASTA-15 might affect the development of preimplantation embryos and suggest that the responsibility for this effect lies probably not solely with glufosinate ammonium, but in combination with the herbicide’s secondary compounds.     

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.     

氯米芬联合HCG对不孕症患者内分泌指标及子宫内膜容受性的影响

目的:探讨氯米芬联合人绒毛膜促性腺激素(HCG)对不孕症患者内分泌指标及子宫内膜容受性(ER)的影响。方法:回顾性分析某院妇产科2018年11月~2019年11月接收的96例不孕症患者临床资料,将采用氯米芬联合HCG治疗的患者归为观察组(49例),将单独采用氯米芬治疗的患者归为对照组(47例),对比两组患者治疗前、治疗3个月内分泌指标、ER及卵巢体积变化情况以及治疗期间不良反应发生情况。结果:治疗3个月后,两组促卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_2)及孕酮(P)水平均较治疗前高,且观察组FSH、LH、E_2及P水平高于对照组,差异有统计学意义(P<0.05);治疗3个月后,两组子宫内膜厚度(Em)指标较治疗前上升,卵巢体积较治疗前缩小,且观察组Em指标较大,卵巢体积较小,差异有统计学意义(P<0.05);两组不良反应发生率比较,差异无统计学意义(P>0.05)。结论:不孕症患者采用氯米芬联合HCG治疗效果良好,可有效改善内分泌指标,提升ER指标,且安全性较高,值得临床推广。     

Extraction of the hormonal substance from hop

Substances that suppressed a gain in the weight of pregnant mare's serum gonadotropin (PMSG)-primed immature rat ovaries, were obtained from dried powder of the hop cone. The substances were designated F_1a-I and F_1a-II. In immature rats at 4 days after PMSG priming, ovarian weights decreased by 58.0 ± 3.7 and 66.9 ± 6.6% the control by injections with 4 mg each of F_1a-I and F_1a-II, respectively. Apparent M^rs of the partially purified fractions were nearly 80 000 (F_1a-I) and 66 000–80 000 (F_1a-II). They were water-soluble. Acidic and neutral sugars were detected in the hydrolysate of the substance.     

Nicotine and cotinine effects on development of two-cell mouse embryos in vitro

The independent and interactive effects of nicotine and cotinine on the development of cultured two-cell embryos were investigated. Cultures were maintained for 120 h and developmental stages of embryos were scored after 72 h and at the termination of culture. Concentrations of nicotine at or below 0.5 mM, and concentrations of cotinine at or below 0.008 mM, did not adversely affect development. In addition, neither nicotine nor cotinine produced synergistic effects at higher concentrations at which both independently impaired development. These data show, therefore, that nicotine and its major metabolite, cotinine, significantly interfere with preimplantation development of mouse embryos only at concentrations far in excess of those anticipated to be present in the blood of an “average” smoker. Thus, we conclude that the well documented adverse effects of smoking during pregnancy are unlikely to be attributable to a direct effect of nicotine or cotinine on the preimplantation embryo.