To investigate the effect of placental isoferritin (PLF) on mouse embryo development in vitro, mice 2-cell embryos were co-cultured with human first trimester decidual cells at different concentrations of PLF in vitro. The following changes of the above system were observed under an invert microscope and the number of embryos were recorded and the embryos were classified. The results showed there was no significant difference in the percentage of embryos development to 4-cell, 8-cell and morula (P>0.05). PLF at the doses of 10 and 100 U/mL significantly enhanced more embryos development to the blastocyst and hatching blastocyst (P<0.05). PLF at the dose of 1000 U/mL depressed more embryos development from 2-cell to hatching blastocyst, meanwhile such phenomena as cell degeneration and irregular cleavage were observed in part of embryos, but there was no significant difference in statistics (P>0.05). It was concluded that PLF at the concentration of 10-100 U/mL had no significant effects on the early development of mice embryos, however, PLF could promote the growth, differentiation, and hatching of preimplantation blastocysts.
Coculture Techniques ; Decidua/*cytology ; Embryo, Mammalian/*cytology ; Embryo, Mammalian/drug effects ; Embryo, Mammalian/embryology ; Embryonic Development/*drug effects ; Ferritins/isolation & purification ; Ferritins/*pharmacology ; Placenta/*chemistry ; Tissue Culture Techniques

To investigate the method of RNA isolation from human embryonic tissues and the factors influencing the quality of RNA, the RNA from human embryonic tissues obtained with drug-induced labor or non-drug induced labor were isolated by using grind with liquid nitrogen or homogenizer without liquid nitrogen. The results showed that the positive rates of RNA integrity in grind with liquid nitrogen group and in homogenizer without liquid nitrogen group were 68.42% and 29.79% respectively, and there was significant difference between these two groups; however, there was no statistic difference in positive rate of RNA integrity, OD(260)/OD(280) ratio and beta-actin gene expression level between the drug-induced labor group and non-drug induced labor group. It is concluded that pulverize of tissue in liquid nitrogen remains the integrity of RNA isolated and may be applied for RNA isolation from human embryonic tissues. The quality of RNA is not affected by different methods of induction of maternal labor.
Embryo, Mammalian ; metabolism ; Freezing ; Humans ; Nitrogen ; pharmacology ; RNA ; isolation & purification ; RNA Stability ; drug effects

The purpose of this study is to evaluate the embryotoxicity of alkaloids in Senecionis Scandentis Hebra on in vitro cultured mouse embryos. Mouse whole embryo culture (WEC) was applied in this study. Post-implantation (8.5 d) mouse embryos were isolated from their mothers, and cultured in medium of immediately centrifuged serum (ICS) with different concentrations of seneciphylline (target concentrations were 100, 50, 25 and 12.5 μg x mL(-1)) or senkirkine (target concentrations were 50, 25 and 12.5 μg x mL(-1)) for 48 h. After culturing completed, the development and organic morphodifferentiation of the cultured embryos were evaluated microscopically. Treatment with seneciphylline and senkirkine had adverse effects on the development and organic morphodifferentiation of embryos. The effect also had clear dose-response. Alkaloidals in Senecionis Scandentis Hebra had embryotoxicity on cultured embryos, which indicated that pregnant people exposed to Senecionis Scandentis Hebra may get potential risk on fetus.
Animals ; Embryo Culture Techniques ; Embryo, Mammalian ; drug effects ; Female ; Mice ; Pregnancy ; Pyrrolizidine Alkaloids ; toxicity ; Senecio ; chemistry ; Teratogens ; toxicity

OBJECTIVETo investigate the fetotoxicity of monocrotaline.

METHODMouse whole embryo culture (WEC) was applied. Post-implantation (8.5 d) mouse embryos were isolated from their mothers and put into the medium of immediately centrifuged serum (ICS) prepared from rats. Different concentrations of monocrotaline (100, 50, 25, 12.5 mg x L(-1)) were added into the WEC. Development (yolk sac diameter, crown-rump length, head length, somite number) and organic morphodifferentiation (yolk sac circulation, allantois, embryonic flexion, heart, brain, optic-otic-olfactory organ, branchial arch, maxillary, mandible, bud) of embryos were observed at 48 h after treatment.

RESULTObvious fetotoxicity could be observed in various monocrotaline treatment groups in a dose-dependent manner. Development of embryos was delayed significantly at dose 12.5-100 mg x L(-1). Malformations were shown in all organic morphodifferentiation indice, especially in opti-otic organ, mandible and bud.

CONCLUSIONMonocrotaline had obvious fetotoxicity in vitro WEC, indicating that exposure of pregnant mice to monocrotaline may have potential risk on fetus.

Animals ; Cell Differentiation ; drug effects ; Culture Media ; Embryo, Mammalian ; drug effects ; physiology ; Female ; Male ; Mice ; Monocrotaline ; toxicity

OBJECTIVETo study the effect of different concentration of phytohemagglutinin (PHA) on mouse embryo development.

METHODIn experiment 1, crude and purified PHA extracted from Yunnan white kidney bean with different concentration were added into M16 culture medium, the final concentration of PHA were: 50, 100, 200, 500, 1 000, 2 000 and 5 000 mg x L(-1) respectively. 2-cell stage embryos were collected and cultured in PHA containing or control medium for 72-96 h and their development were recorded. In experiment 2, different stage of embryos from 1-cell to blastocyst were treated by different concentrations of PHA same as experiment 1 and 10 000 mg x L(-1) in culture medium for 24 h before washing and cultured in M16 + PVA without PHA to blastocyst or hatching blastocyst stage.

RESULTLow concentrations PHA at 50-100 mg x L(-1) promoted embryo development and increased the number of blastocyst stage embryos. In contrast, high concentrations of PHA (> 1 000 mg x L(-1)) blocked the embryos development from 1-cell to blastocyst stage and showed apoptosis morphology or death.

CONCLUSIONDepending on the concentrations, PHA from white kidney bean shown promotion or inhibition on mouse embryo development. 1-cell stage embryo shown more sensitive to PHA treatment than that of later stage embryos. Pretreatment 24 h in PHA containing medium can influence the further development of embryos. Low concentrations of PHA is benefit to embryo development, but high concentrations of PHA (> 1 000 mg x L(-1)) will block of the development of embryos.

Animals ; Embryo, Mammalian ; drug effects ; Embryonic Development ; drug effects ; Female ; Male ; Mice ; Phaseolus ; chemistry ; Phytohemagglutinins ; pharmacology ; Pregnancy

OBJECTIVETo investigate the fetotoxicity of retrorsine.

METHODMouse whole embryo culture (WEC) was applied. Post-implantation (8.5 d) mouse embryos were isolated from their mothers and put into the medium of immediately centrifuged serum (ICS) prepared from rats. Different concentrations of retrorsine (12.5, 25, 50, 100 mg x L(-1)) were added into the WEC culture. Development (yolk sac diameter, crown-rump length, head length, somite number) and organic morphodifferentiation (yolk sac circulation, allantois, embryonic flexion, heart, brain, optic-otic-olfactory organ, branchial arch, maxillary, mandible, bud) of embryos were observed at 48 h after treatment.

RESULTObvious fetotoxicity could be observed in various retrorsine treatment groups in a dose-dependent manner. Development of embryos was delayed significantly at dose 12.5-100 mg x L(-1). Malformations were shown in all organic morphodifferentiation indexes, especially in otic-olfactory organ, branchial arch, maxillary, mandible, bud.

CONCLUSIONRetrorsine had obvious fetotoxicity in vitro WEC culture, indicating that exposure of pregnant mice to retrorsine may have potential risk on fetals.

Animals ; Dose-Response Relationship, Drug ; Embryo, Mammalian ; drug effects ; Female ; Male ; Mice ; Pregnancy ; Pyrrolizidine Alkaloids ; toxicity ; Rats ; Toxicity Tests ; methods

In order to enhance the efficiency of sheep somatic cell nuclear transfer, we used a chemically assisted enucleation with colchicine to study the effects of the concentration of colchicine, the incubation time of oocytes in colchicine and the maturation time of oocytes on the enucleation rates and the development of reconstructed embryos. The results showed that 1) there were no significant differences in the rates of cytoplast protrusion and enucleation between oocytes that were incubated in colchicine (0.4 microg/mL) for 0.5 h and oocytes that were incubated in colchicine (0.4 microg/mL) for 1 h, and the rate of cytoplast protrusion can be 85.4% while the rate of cytoplast enucleation is 100%. 2) There was no significant difference in oocyte enucleation between oocytes treated with medium containing 0.2 microg/mL colchicine for 0.5 h and oocytes treated with medium containing 0.4 microg/mL colchicine for 0.5 h. 3) A maturation time of 18-23 h did not affect the rates of cytoplast protrusion and enucleation by chemically assisted enucleation, whereas the rate of enucleation of oocytes by blind enucleation was found to decrease with a prolonged incubation time. 4) The development rates of reconstructed embryos could not be influenced by these two enucleation methods, increased from oocytes matured for 21-23 h. These results demonstrate that sheep oocytes can be enucleated fast and effectively by optimized colcholine chemically assisted enucleation, which can enhance the enucleation rate of sheep oocytes and the early development of reconstructed embryos in vitro.
Animals ; Cloning, Organism ; methods ; Colchicine ; pharmacology ; Embryo, Mammalian ; embryology ; Female ; Nuclear Transfer Techniques ; veterinary ; Oocytes ; cytology ; drug effects ; Sheep

OBJECTIVETo investigate the protective effect of reactive oxygen species (ROS) scavenger, N-acetyl-L-cysteine (NAC), against H9c2 cardiomyocytes from injuries induced by chemical hypoxia.

METHODSH9c2 cells were treated with cobalt chloride (CoCl2), a chemical hypoxia-mimetic agent, to establish the chemical hypoxia-induced cardiomyocyte injury model. NAC was added into the cell medium 60 min prior to CoCl2 exposure. The cell viability was evaluated using cell counter kit (CCK-8), and the intercellular ROS level was measured by 2', 7'- dichlorfluorescein-diacetate (DCFH-DA) staining and photofluorography. Mitochondrial membrane potential (MMP) of the cells was observed by Rhodamine123 (Rh123) staining and photofluorography, and the ratio of GSSG/ (GSSG+GSH) was calculated according to detection results of the GSSG kit.

RESULTSExposure of H9c2 cardiomyocytes to 600 micromol/L CoCl2 for 36 h resulted in significantly reduced cell viability. Pretreatment with NAC at the concentrations ranging from 500 to 2000 micromol/L 60 min before CoCl2 exposure dose-dependently inhibited CoCl2-induced H9c2 cell injuries, and obviously increased the cell viability. NAC at 2000 micromol/L obviously inhibited the oxidative stress induced by CoCl2, decreased the ratio of GSSG/(GSSG+GSH), increased ROS level, and antagonized CoCl2-induced inhibition on MMP.

CONCLUSIONNAC offers obvious protective effect on H9c2 cardiomyocytes against injuries induced by chemical hypoxia by decreasing in the ratio of GSSG/(GSSG+GSH) and ROS level and ameliorating MMP.

Animals ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Embryo, Mammalian ; Free Radical Scavengers ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Oxidative Stress ; drug effects ; Rats ; Reactive Oxygen Species ; metabolism

OBJECTIVETo investigate the characteristics of embryonic toxicity of Senecio scandens, its total alkaloid and Qianbai Biyanpian so that to provide guidance for the safety of medication during pregnancy.

METHODTwo hundred and twenty pregnant SD rats were divided into 11 groups: control group, positive group (cyclophosphamide 10 mg x kg(-1)). Water extract of S. scandens (doses: 7.5, 15.0, 30.0 g herb of S. scandens per kilogram body weight respectively). Qianbai Biyanpian and total alkaloid at the same doses levels with the water extract of S. scandens (doses were expressed as herb of S. scandens per kilogram body weight). The test articles were given to the pregnant rats by gavage from day 6 to day 15 of pregnancy. Body weight and the food consumption of pregnancy rats, and fetal weight and length were measured. The number of absorbed and dead embryos was recorded. Fetuses were examined in viscus and bones.

RESULTWeight and the food consumption of pregnancy rats in high-dose of Qianbai Biyanpian and total alkaloids decreased. All treatment groups had no significant change in the number of absorbed embryos, but the stillbirths were significantly increased in high-dose groups of water extract and total alkaloids as compared with control group. Bone deformities such as fontanel expanding, hypoplasia of parietal bone, occipital bone and cervical arch were observed. Rib abnormality could also be seen in some rats. All water extract of S. scandens, Qianbai Biyanpian and total alkaloid could cause the bone abnormalities, but the percentage of bone deformities of total alkaloids was the highest (up to 80%).

CONCLUSIONS. scandens and its total alkaloids, its formula Qianbai Biyanpian can cause rat skeletal deformities in fetuses when they were given during pregnancy. It is suggested that S. scandens and the product containing S. scandens should not be used during pregnancy.

Alkaloids ; chemistry ; toxicity ; Animals ; Body Weight ; drug effects ; Drugs, Chinese Herbal ; toxicity ; Eating ; drug effects ; Embryo, Mammalian ; drug effects ; Female ; Male ; Pregnancy ; Rats ; Rats, Wistar ; Senecio ; chemistry

OBJECTIVETo establish a suitable protocol for activating mouse somatic cell nuclear transfer embryos with strontium chloride (SrCl2).

METHODSWe constructed and identified mouse nuclear transfer (NT) embryos. After nuclear injection, we activated the NT embryos using the following chemical activation methods: exposing the NT embryos to 5 and 10 mmol/L SrCl2 strontium for 1 -8 h, activating the NT embryos with 1-20 mmol/L SrCl2 strontium at 4 and 6 h, treating the NT embryos with 10 mmol/L SrCl2 strontium in different activating media, and exposing the NT embryos to 10 mmol/L SrCl2 strontium combined with 6-dimethylaminopurine (6-DMAP) and cytochalasin B (CB). After activation, the NT embryos were cultured in vitro in the cleavage medium.

RESULTSWhen the NT embryos were treated with SrCl2 at the concentration of 5 mmol/L, the cleavage rate was remarkably higher at 6 h (38.9%) than at 1 h (6.7%), 2 h (22.8%), 3 h (22.8%) and 4 h (25.6%) (P < 0.05), but with no significant differences from those at 5 h (28.9%), 7 h (34.4%) and 8 h (28.9%) (P > 0.05). When the NT embryos were treated with SrCl2 for 6 h, the rates of cleavage and blastulation were 68.9% and 7.2% at 10 mmol/L, markedly higher than at 1 mmol/L (28.3% and 0%), 2.5 mmol/L (35.6% and 0%), 5 mmol/L (37.8% and 1.1%), 7.5 mmol/L (60.6% and 2.2%), 15 mmol/L (51.7% and 1.1%), and 20 mmol/L (41.7% and 1.1%) (P < 0.05). The cleavage rate of the NT embryos cultured in the Ca2+ and Mg2+ KSOM medium was 27.8%, significantly lower than in the Ca(2+)-free KSOM (69.4%), Ca2+/Mg(2+)-free KSOM (66.1%), and Ca2+/Mg(2+)-free + EDTA KSOM (68.3%) (P < 0.05). The total cell blastocyst number was significantly larger in the NT embryos treated with SrCl2 + CB (45.40 +/- 2.23) than in those treated with SrCl2 (30.15 +/- 1.12), 6-DMAP (34.95 +/- 1.38), and 6-DMAP + CB (37.45 +/- 1.43) (P < 0.05).

CONCLUSIONSix-hour treatment with 10 mmol/L SrCl2 in Ca2+ alone or in combination with CB can well activate NT embryos in mice.

Animals ; Blastocyst ; cytology ; drug effects ; Embryo Culture Techniques ; Embryo, Mammalian ; cytology ; drug effects ; Embryonic Development ; drug effects ; Female ; Mice ; Mice, Inbred C57BL ; Nuclear Transfer Techniques ; Oocytes ; cytology ; drug effects ; Strontium ; pharmacology