Effect of coenzyme Q10 supplementation on post-vitrification mouse embryo development

Objective:To investigate the effects of coenzyme Q10(CoQ10)supplementation on post-vitrification embryo development and gross morphology.Methods:Balb/c mouse embryos were cultured in potassium simplex optimised medium(KSOM)with varying CoQ10 concentrations[0(control),20,40,and 60μM].The most effective CoQ10 concentration(40μM)was selected for subsequent post-vitrification morphology study.Embryos were randomly divided into four groups:Group A(non-vitrified without CoQ10),Group B(non-vitrified with CoQ10),Group C(vitrified without CoQ10),and Group D(vitrified with CoQ10),followed by vitrification at the 8-cell stage.Survival rates and development until the blastocyst stage were evaluated through morphological examinations using ASEBIR's system,distinguishing normal and abnormal embryos.Results:Supplementation of 40μM CoQ10 significantly increased blastocyst formation(95%)compared to the control group(92%),20μM(62%),and 60μM(56%)(P<0.001).Following vitrification,Group D exhibited a significant increase in blastocyst formation(92%)compared to Group C(82%)(P<0.05).Morphological assessments indicated superior embryo quality in Group B over Group D during the cleavage stage,morula,and blastocyst(P<0.05).Conclusions:CoQ10 supplementation exhibits promising potential to enhance preimplantation embryo development,increase blastocyst formation rates,and improve embryo quality post-vitrification.This offers a promising approach to mitigate oxidative stress on embryos,potentially improving overall assisted reproductive technology outcomes.

Effects of Alcohol and Liquid Paraffin on Development of Early Mouse Embryos in vitro

In culturing early mouse embryos in vitro,liquid paraffin and alcohol exert deleterious influence on the development of embryos. Some of light liquid paraffin produced by Chinese factories have proved harmful for early mouse embryos. As shown by our experiments, the nitronaphthalene contained and the specific gravity of liquid paraffin were not involved in the injurious effects.However,alcohol mingled in medium had harmful effects on the development of embryos. At the 0.1% concentration of alcohol in medium the proportion of embryos developing to blastocysts decreased to 73.9%. When the concentration of alcohol was increased to 0.8%, all embryos ceased developing. In our experiments, CO_2 which contained 0.13% alcohol had no visible effects on the development of embrvos in vitro.

Construction of pINC-lacZ Retroviral Vector and its Expression in Mouse Embryonic Stem Cells

A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.

Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson's disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.

Effect of PMSG/hCG Superovulation on Mouse Embryonic Development

Kunming mouse strain is widely used in China, and the superovulation was administrated with 10 IU PMSG combined with 10 IU hCG. In this study, the effects of the exogenous gonadotropins on superovulation of Kunming mice and embryo quality derived from the superovulated mice were assessed. Female mice at 6-8-wk old were superovulated with 0, 5, 7.5 and 10 IU PMSG/hCG and mated with male mice. The embryos were retrieved at 2.5 d post coitum. No statistic difference was observed for the number of 2-cell embryos collected per mouse between control and 5 IU PMSG/hCG treatment group, but the number significantly increased for 7.5 and 10 IU PMSG/hCG treatment group （P〈0.05）. The average number of 4- cell and 8-cell embryos collected from each mouse significantly differed between control and 5, 7.5, 10 IU PMSG/hCG treatment groups （P〈0.05）. When 8-cell embryos derived from mice administrated with 0, 5, 7.5 and 10 IU PMSG/hCG were cultured in KSOM, the blastocyst development rates were 88.1, 94.7, 96.1 and 94.3%, respectively, which were similar to control （P〉0.05）. This indicated that exogenous gonadotropins have no effects on development of Kunming mouse embryos. The quality of blastocyst was assessed by labelling with Hoechst and propidium iodide for inner cell mass and trophectoderm cells, the result showed that ICM/TE ratio significantly decreased for 10 IU PMSG/hCG treatment group compared with control, 5 and 7.5 IU PMSG/hCG treatment group （P〈0.05）. This suggested that the embryo quality of Kunming mouse has been affected by high dose of gonadotropins.

Effect of Clenbuterol Hydrochloride on the in vitro Development of Mouse Embryo

To investigate the effect of clenbuterol hydrochloride on the in vitro development of both 1 cell and 2 cell mouse embryos. Methods The cultural systems of both 1 cell and 2 cell mouse embryo were used to determine the effect of clenbuterol hydrochloride at doses of 1 ng/mL, 3 ng/mL, and 10 ng/mL on developmental rates of mouse embryos. Results When 1 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, developmental rates from the 4 cell stage to blastocyst stage were significantly lower than those in the control group (P<0.05), but on dosages of 3 ng/mL and 10 ng/mL, the inhibiting effects on embryo development were significantly increased (P<0.01). When 2 cell embryos cultured with 1 ng/mL of clenbuterol hydrochloride, obvious differences in developmental rates were not found between the 2 cell embryo group and the control (P>0.05). However, at levels of 3 ng/mL and 10 ng/mL, significant decrease of developmental rates in 2 cell embryos was observed from the 4 cell and from the 8 cell stage, respectively (P<0.05). Embryos cultured with clenbuterol hydrochloride appeared to have more granules, fragments and degeneration than those in the control. Conclusion Clenbuterol hydrochloride has a toxic effect on the mouse embryos, and the effect is in a dose dependent. 1 cell mouse embryos cultured with clenbuterol hydrochloride could be easily inhibited at 2 cell stage, but the effect of clenbuterol hydrochloride on development of the late 2 cell embryos would be reduced.

Transglutaminase 3 expression in C57BL/6J mouse embryo epidermis and the correlation with its differentiation

Epidermal-type transglutaminase 3 (TGM3) is involved in the cross-linking of structural proteins to form the cornifiedenvelope in the epidermis. In the present study, we detected the expression of TGM3 in the mouse embryo using RT-PCR.TGM3 mRNA is weakly presented from E11.5 to E14.5 and increases significantly from E15.5 to birth. Then wedetermined the spatial and temporal expression pattern of TGM3 in the skin and other organs by in situ hybridization. Wefound a deprivation of TGM3 in skin at E11.5, while a rich supply in periderm cells and a weak expression in basal cellsfrom E12.5 to E14.5. From the period of E15.5 to E16.5, after keratinization in the epidermis, TGM3 was expressed inthe granular and cornified layers. The electron microscopic observation of the C57BL/6J mouse limb bud skin develop-ment provided several morphological evidences for the epidermal differentiation. The above findings suggest that theexpression of TGM3 plays a important role in the epidermis differentiation in embryogenesis.

DDX3X regulates cell survival and cell cycle during mouse early embryonic development

DDX3X is a highly conserved DEAD-box RNA helicase that participates in RNA transcription, RNA splicing, and mRNA transport, translation, and nucleo-cytoplasmic transport. It is highly expressed in metaphase II （MII） oocytes and is the predominant DDX3 variant in the ovary and embryo. However, whether it is important in mouse early embryo development remains unknown. In this study, we investigated the function of DDX3X in early embryogenesis by cytoplasmic microinjection with its siRNA in zygotes or single blastomeres of 2-cell embryos. Our results showed that knockdown of Ddx3x in zygote cytoplasm led to dramatically diminished blastocyst formarion, reduced cell numbers, and an increase in the number of apoptotic cells in blastocysts. Meanwhile, there was an accumulation of p53 in RNAi blastocysts. In addition, the ratio of cell cycle arrest during 2-cell to 4-cell transition increased following microinjection of Ddx3x siRNA into single blastomeres of 2-cell embryos compared with control. These results suggest that Ddx3x is an essential gene associated with cell survival and cell cycle control in mouse early embryos, and thus plays key roles in normal embryo development.

A Novel Three-Dimensional Mouse Embryonic Implantation Model In Vitro

To regenerate three-dimensional endometrium in vitro as a novel model for studying the mechanism of implantation of embryos, the luminal epithelial cells and stromal cells of the rabbit uterus were separated and cultured in vitro. The type Ⅰ mouse tail collagen was used as scaffolding material. The stromal cells were inoculated in the type I mouse tail collagen, and the luminal epithelial cells were inoculated on the type i mouse tall collagen to regenerate the endometrium in vitro. The regenerated endometrium was cultured in DMEM-F/12 media containing 100 nmol L^-1 progesterone, 10 nM β-estradiol, and 10% fetal bovine serum （FBS） for 3 d. The media were then replaced with CZB containing 100 nM progesterone, 10 nmol L-1 β-estradiol, and 10% FBS, and the mouse blastulas were co-cultured with it. The results of scanning electronic micrography showed that the epithelial cells on the surface of the reconstructed endometrium were covered with numerous slender microvilli and some epithelial cells protruded pinopodes. After culturing for 12 h with the mouse blastula, the shedding, attachment, and implantation of the blastula were observed. The blastula can escape from zona pellucida and attach to the three-dimensional endometrium and is then implanted into it. This study showed that the reconstructed three-dimensional endometrium can serve as a robust embryo implantation model in vitro.

Establishment of Embryonic Stem Cell Lines Derived from Outbred Mouse Embryos and Production of Chimeras

The aim of the present investigation was to determine if embryonic stem (ES) cells could be isolated from outbred mouse embryos (KM) and if chimeras could be producedly using outbred ES cells. Three ES cell lines,designated KE1, KE2, and KE5,were isolated from 5 Kunming albino blastocysts. Normal diploid composition of these cell lines was above 70%. By using C57BL/6J and 615 blastocysts as host embryos, one chimera was obtained in living pups. It was shown for the first time that chimeras can be produced by using outhred ES cells. This work implies that to establish ES cell lines from other animal embryos is possible. More interestingly, white color dots from ES cells on the coat of this chimeric mouse enlarged a lot after half a year, indicated that ES cells were inhibited by cells from outbred mouse or the cells of outbred mouse grew vigiously.

Pattern of expression of the CREG gene and CREG protein in the mouse embryo

Background The cellular repressor of ElA-stimulated genes(CREG) is a secreted glycoprotein that inhibits cell proliferation and/or enhances differentiation.CREG is widely expressed in adult tissues such as the brain,heart, lungs,liver,intestines and kidneys in mice.We investigated the level of CREG expression during mouse embryogenesis and its distribution at 18.5 days post coitus(dpc).Methods Immunohistochemical staining with diaminobenzidine,western blotting and reverse transcription-polymerase chain reaction were used.Results CREG expression was rst detected in mouse embryos at 4.5 dpc.It was expressed at almost all stages up to 18.5 dpc.The level of CREG was found to increase gradually and was highest at 18.5 dpc.Western blotting showed that the CREG protein was expressed at higher levels in the brain,heart,intestines and kidneys than in the lungs and liver at 18.5 dpc.In 9.5 dpc embryos,CREG was expressed only in the endothelial cells of blood vessels,after the vascular lumen had formed.With advanced differentiation, vascular smooth muscle cells developed in the embryonic vascular structures;the expression of smooth muscle a-actin protein and CREG were positive and increased gradually in 10.5 dpc embryonic vessels.CREG expression in the embryonic blood vessels peaked at 15.5 dpc and was reduced slightly at 18.5 dpc.Conclusions These results indicate that CREG is expressed during mouse embryogenesis and might participate in the differentiation of these organs during embryogenesis.

Glucose Metabolism During Kunming Mouse Preimplantation Development:Analysis of Gene Transcription in Embryos in Vivo

In order to investigate glucose metabolism pathways and their changes in Kunming mouse preimplantation 1-,2-,4-,8-cell,and morula embryos,the mRNA level for the genes involved in glucose metabolism was tested by nested RT-PCR on embryos at different development stages in vivo.These genes were glucose 6-phosphate dehydrogenase(G6PDH),phospho-fructokinase(PFK),and phosphoglucomutase(PGM),representing pentose phosphate pathway(PPP),glycolysis,and glycogensis and glycogenolysis respectively.Three sets of inner and outer primers were designed and synthesized based on cDNA sequences of G6PDH,PFK and PGM.RT-PCR results revealed that G6PDH gene transcription was found in Kunming mouse 1-8 cell embryos,and not in morula embryos;it indicated that 1-8 cell embryos may metabolize glucose by pentose phosphate pathway,but morula embryos can not do so.PFK gene transcription was found in 1-8 cell and morula embryos;it is probable that there exists glycolysis in those embryos.PGM gene transcription was not found in 1-8 cell and morula embryos,so glycogenesis and glycogenolysis in these embryos were not present.

Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage

A full-length rabbit oviductin cDNA(1909bp) was cloned. It consists of a 5’-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3’-UTR of 483bp and has more than 80% homology with that of other mammal oviductins. N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively. Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified. NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one. In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared. Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct. The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mous e cultured in vitro. Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn’t. There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium. These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal.

Delay of ZGA initiation occurred in 2-cell blocked mouse embryos

One-cell mouse embryos from KM strain and B6C3F1 strain were cultured in M16 medium, in which 2-cell block generally occurs. Embryos of KM strain exhibited 2-cell block, whereas B6C3F1 embryos, which are regarded as a nonblocking strain, proceeded to the 4-cell stage in our culture condition. It is often assumed that the block of early development is due to the failure of/ygotic gene activation (ZGA) in cultured embryos. In this study we examined protein synthesis patterns by two-dimensional gel eleetrophoresis of [35 S] methionine radiolabeled 2-cell embryos. Embryos from the blocking strain and the nonblocking strain were compared in their development both in vitro and in vivo. The detection of TRC expression, a marker of ZGA. at 42 h post hCG in KM embryos developed in vitro suggested that ZGA was also initiated even in the 2-cell arrested embryos. Nevertheless, a significant delay of ZGA was observed in KM strain as compared with normally developed B6C3F1 embryos. At the very beginning of major ZGA as early as 36 h post hCG, TRC has already been expressed in B6C3F1 embryos developed in vitro and KM embryos developed in vivo. But for 2-cell blocked KM embryos, TRC was still not detectable even at 38 h post hCG. These evidences suggest that 2-cell-blocked embryos do initiate ZGA, and that 2-cell block phenomenon is due not to the disability in initiating ZGA. but to a delay of ZGA.

Effect of RNA Interference Hsp72 Gene Expression on Development of Mouse Preimplantation Embryos

The method of RNAi was used to inhibit the expression of induced heat shock protein 70 （Hsp72） in the 4-cell stage mouse embryos and the embryo development competence was analyzed to identify the functions of Hsp72 on embryonic heat resistance. The results indicated that the inhibition rates of siRNA1 for Hsp72 mRNA and Hsp72 protein were 87.1 and 78.5%, respectively. The blastocysts development rates were 41, 86, and 84% for the siRNA1 group, the LipofectamineTM 2 000 exposed group, and the 37℃ group, respectively, and the hatched blastocysts development rates for the above three groups were 35, 72, and 68%, respectively. The data suggest that the siRNAI has a significant inhibiting effect on Hsp72 gene, and Hsp72 gene silence reduces the blastocysts development rate and hatched blastocysts rate after heat shock during the development of mouse preimplantation embryos.

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neuroeyte in vitro. Methods Isolate the blastula o f 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass （inner cell mass, ICM） which were isolated by mechanical method on the mouse embryonic fibroblaste cell （MEF） feeder layer or 0.1% gelatin coated dishes. The stem ceils were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunoehemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 （ stage specific embryonic antigen 1 ）. Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated ceils presented the characters of ESCs. Then the isolated cells were able to differentiate into neuroeytes in vitro. Conclusion Mouse embryonic stem ceils isolation, culture and differentiation system has been established.

Expression of Angiopoietin-1/-2 in the Process of Mouse Embryo Implantation

This study examined the expression and distribution of angiopoietin-1/-2 （Ang-1/-2） in the endometrium of early pregnant mice. The expression of Ang-1/-2 was detected by immunohistochemical staining and in situ hybridization respectively. Computerized image analysis system was used to measure the average optical intensity of Ang-1/-2 in endometria at different time points after gestation. Mice were randomly divided into 5 groups： control group, D2 group （2 days after pregnancy）, D4 group （4 days after pregnancy）, D6 group （6 days after pregnancy） and D8 group （8 days after pregnancy）, each containing 15 mice. The results showed that the expression of Ang-1 and Ang-2 was very different among 4 groups （P〈0.01）. Immunohistochemical staining revealed that Ang-1 was localized in the cytoplasma of stromal cells 2 days after pregnancy （day 2）, and in luminal epithelial cells on day 4. The protein of Ang-2 was mainly expressed in the cytoplasma of glandular epithelia and stromal cells. With gestation time, the positive reactions of Ang-1/-2 were stronger in the endometria of the pregnant mice （P〈0.01）. In situ hybridization showed Ang-I mRNA in stromal cells on day 2. Hybridization signal was localized in both stromal cells and vessel epithelial cells on day 4; Ang-2 mRNA was expressed in stromal cells and glandular epithelia on day 2; high mRNA levels appeared in stromal cells, glandular epithelia and vascular endothelia on day 4; an increasing in mRNA expression of Ang-1/-2 was observed on day 6 and day 8 （P〈0.01）. It is suggested that Ang-1/-2 may play an important role in the cross-talk between blastocyst and maternal endometrium during the process of embryo implantation.

Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis- mutase 1 mutant （SOD1G93A） and wild-type （SOD1wv） mouse models were exposed to H202. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenos- ine monophosphate-activated protein kinase （AMPK） ct-subunit, paired box 3 （Pax3） protein, and p53 in western blot analyses. Compared with SOD1wr cells, SOD1~93A embryonic neural stem cells were more likely to undergo H202-induced apoptosis. Phosphorylation of AMPKct in SOD1G93A cells was higher than that in SOD1wr cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKct. p53 protein levels were also correlated with AMPKct phosphorylation levels. Compound C, an inhibitor of AMPKa, attenuated the effects of H20~. These results suggest that embryonic neural stem cells from SOD1C93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKa pathway.

Effect of 935-MHz Phone-simulating Electromagnetic Radiation on Endometrial Glandular Cells during Mouse Embryo Implantation

This study examined the impact of 935MHz phone-simulating electromagnetic radiation on embryo implantation of pregnant mice.Each 7-week-old Kunming (KM) female white mouse was set up with a KM male mouse in a single cage for mating overnight after induction of ovulation.In the first three days of pregnancy,the pregnant mice was exposed to electromagnetic radiation at low-intensity (150 μW/cm2,ranging from 130 to 200 μW/cm2,for 2-or 4-h exposure every day),mid-intensity (570 μW/cm2,ranging from 400 to 700 μW/cm2,for 2-or 4-h exposure every day) or high-intensity (1400 μW/cm2,ranging from 1200 to 1500 μW/cm2,for 2-or 4-h exposure every day),respectively.On the day 4 after gestation (known as the window of murine embryo implantation),the endometrium was collected and the suspension of endometrial glandular cells was made.Laser scanning microscopy was employed to detect the mitochondrial membrane potential and intracellular calcium ion concentration.In high-intensity,2-and 4-h groups,mitochondrial membrane potential of endometrial glandular cells was significantly lower than that in the normal control group (P<0.05).The calcium ion concentration was increased in low-intensity 2-h group but decreased in high-intensity 4-h group as compared with the normal control group (P<0.05).However,no significant difference was found in mitochondrial membrane potential of endometrial glandular cells between low-or mid-intensity groups and the normal control group,indicating stronger intensity of the electromagnetic radiation and longer length of the radiation are required to inflict a remarkable functional and structural damage to mitochondrial membrane.Our data demonstrated that electromagnetic radiation with a 935-MHz phone for 4 h conspicuously decreased mitochondrial membrane potential and lowered the calcium ion concentration of endometrial glandular cells.It is suggested that high-intensity electromagnetic radiation is very likely to induce the death of embryonic cells and decrease the chance of their implantation,thereby posing a high risk to pregnancy.

Effects of Colchicine on Distribution of Mitochondria in 2-cell and Compacted 8-cell Mouse Embryos

The distribution of mitochondria during early development of mouse embryos was visualized bymitochondria-specific vital fluorescent dye, rhodamine 123(Rh 123). Mitochondrial clusters wasmarkedly conceotrated to perinuclear area in blastomere of normal 2-ccll embryos. In blastomere ofuncompacted 8-cell embryos, mitochondria were randomly distributed throughout the cytoplasm, butthey were reorganizcd to the cytocortices beneath the apposed surfaces of blastomere duringcompaction. As demonstrated in our study, colchicine (10 μg/ml) produced marked effect onmitochondrial distribution in blastomcre of 2-cell and compacted 8-cell embryos: mitochondriabecame scattered throughout the cytoplasm ofblastomere. It is suggested that the spatial distributionof mitochondria in early mouse embryo are maintained by microtubule.