• Journal of Radiation Protection and Research
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• v.21 no.4
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• pp.273-284
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• 1996

Embryos and fetuses are more sensitive to various environmental agents than are adults or children. The biological effects such as intrauterine death and malformation are closely connected with prenatal exposure very various agents. The sensitivity of these embryonic/fetal effects depends on the stage of pregnancy. From the viewpoint of fetal development, embryonic and fetal stages can be divided into three stages : Preimplantation, organogenetic and fetal. Each stage corresponds to 0 to 4.5days, 4.5 to 13.5days, and 13.5days of gestation in mice, respectively. Many studies on the biologcal effects of mice irradiated by ${\gamma}-rays$ at various stages during organogenesis and fetal period have been performed. Based on these results, the dose-effect and dose-response relationships in malformations, intrauterine death, or retardation of the physical growth have been practically modeled by the ICRP(International Commission on Radiological Protection) and other international bodies for radiation protection. Many experimental studies on mice have made it clear that mice embryos in the preimplantation period have a higher sensitivity to radiation for lethal effects than the embryos/fetuses on other prenatal periods. However, no eratogenic effects of radiation at preimplantation stages of mice have been described in many textbooks. It has been believed that 'all or none action results' for radiation of mice during the preimplantation period were applied. The teratogenic and lethal effects during the preimplantation stage are one of the most important problems from the viewpoint of radiological protection, since the preimplantation stage is the period when the pregnancy itself is not noticed by a pregnant woman. There are many physical or chemical agents which affect embryos/fetuses in the environment. It is assumed that each agents indirectly effects a human. Then, a safety criterion on each agent is determined independently. The pregnant ICR mice on 2, 48, 72 or 96 hours post-conception (hpc), at which are preimplantation stage of embryos, were irradiated whole body Cesium-gamma radiation at doses of 0.1, 0.25, 0.5, 1.5, and 2.5 Gy with dose rate of 0.2 Gy/min. In the embryos from the fetuses from the mice irradiated at various period in preimplantation, embryonic/fetal mortalities, incidence of external gross malformation, fetal body weight and sex ratio were observed at day 18 of gestation. The sensitivity of embryonic mortalities in the mice irradiated at the stage of preimplantation were higher than those in the mice irradiated at the stage of organogenesis. And the more sensitive periods of preimplantation stage for embryonic death were 2 and 48 hpc, at which embryos were one cell and 4 to 7 cell stage, respectively. Many types of the external gross malformations such as exencephaly, cleft palate and anophthalmia were observed in the fetuses from the mice irradiated at 2, 72 and 96 hpc. However, no malformations were observed in the mice irradiated at 48 hpc, at which stage the embryos were about 6 cell stage precompacted embryos. So far, it is believed that the embryos on preimplantation stage are not susceptible to teratogens such as radiation and chemical agents. In this study, the sensitivity for external malformations in the fetuses from the mice irradiated at preimplantation were higher than those in the fetuses on stage of organogenesis.

• Animal cells and systems
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• v.2 no.1
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• pp.99-106
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• 1998

In this study, we have examined the role of cAMP in gap junctional communication (GJC) in preimplantation mouse embryos. GJC was monitored by Lucifer Yellow (LY) injected into one blastomere of compacted embryos. The speed of GJC was defined as the time taken for the last blastomere of the embryo to become visibly fluorescent. The median time for 8-cell embrvos (140 sec) was similar to that for 16-cell (135 sec). To determine whether cAMP and cAMP-dependent protein kinase (PKA) are involved in the regulation of GJC, the effects of PKA inhibitor (H8) and cAMP analogues (Rp-cAMP and 8-Br-cAMP) on dye transfer between blastomeres of compacted embryos were examined. Some of the embryos treated with either H8 or Rp-cAMP failed to transfer LY to all blastomeres within 10 min. In contrast, 8-Br-cAMP speeded up fluorescent dye transfer. The median time to fill all blastomeres with LY was 140 sec in untreated controls and 90 sec in siblings treated with 8-Br-cAMP. Inhibition of PKA by H8 or Rp-cAMP induced delay or arrest in embryo development after compaction, but the increase of intracellular cAMP showed no effect. These findings suggest that GJC in preimplantation mouse embryos is regulated by cAMP-PKA pathway and transient interference by PKA inhibitors induces the developmental delay beyond compaction.

• Journal of Environmental Health Sciences
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• v.24 no.2
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• pp.126-133
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• 1998

Effects of B(a)P on preimplantation mouse embryos in vitro were studied. Preimplantation mouse embryos were exposed to a concentration of 0.3, 1, 3 and 10 $\mu$M B(a)P for 72 hrs. The toxicological effects of B(a)P were evaluated by morphological observation of embryos up to the blastocyst stage, and by measuring DNA, RNA and protein synthesis by radioactive precursor incorporation. At 1 $\mu$M B(a)P did not affect preimplantation development but interfered with hatching and ICM formation. Suppressing effect of ICM formation was dose dependent. At the eight cell stage, the developmental rate was decreased at above 3 $\mu$M of B(a)P. At the blastocyst stage, attachment and trophoblast outgrowth were diminished at the 10 $\mu$M of B(a)P and ICM formation was decreased at 1 $\mu$M of B(a)P. Inner cell number of blastocyst was decreased dose dependently. So, number of ICM was one of the most sensitive and toxicological end point. The RNA incorporation rate of 0.1 $\mu ^3$H-uridine was dosedependent and the protein incroporation of 0.5 $\mu Ci ^{35}$S-methionine showed a significant decrease after 48 hrs. But the DNA incorporation rate of methyl-$^3$H thymidine was not affected. Our results suggested that B(a)P did not affect the DNA replication but transcription was inhibited by dose dependent manner. There delay of development during the blastocyst stage was mainly due to the inhibition of RNA synthesis followed by protein synthesis.

• Development and Reproduction
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• v.18 no.3
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• pp.153-160
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• 2014

Adaptive development of early stage embryo is well established and recently it is explored that the mammalian embryos also have adaptive ability to the stressful environment. However, the mechanisms are largely unknown. In this study, to evaluate the possible role of aquaporin in early embryo developmental adaptation, the expression of aquaporin (AQP) 5 gene which is detected during early development were examined by the environmental condition. To compare expression patterns between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP5 by whole mount immunofluorescence. At in vivo condition, Aqp5 expressed in oocyte and in all the stages of preimplantation embryo. It showed peak at 2-cell stage and decreased continuously until morula stage. At in vitro condition, Aqp5 expression pattern was similar with in vivo embryos. It expressed both at embryonic genome activation phase and second mid-preimplantation gene activation phase, but the fold changes were modified between in vivo embryos and in vitro embryos. During in vivo development, AQP5 was mainly localized in apical membrane of blastomeres of 4-cell and 8-cell stage embryos, and then it was localized in cytoplasm. However, the main localization area of AQP5 was dramatically shifted after 8-cell stage from cytoplasm to nucleus by in vitro development. Those results explore the modification of Aqp5 expression levels and location of its final products by in vitro culture. It suggests that expression of Aqp5 and the roles of AQP5 in homeostasis can be modulated by in vitro culture, and that early stage embryos can develop successfully by themselves adapting to their condition through modulation of the specific gene expression and localization.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.253-259
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• 2000

Objective: To verify the effect of Matrigel, a ECM complex from Engelbreth-Holm-Swarm (EHS) mouse sarcoma on the preimplantation development and apoptosis of mouse fertilized eggs. Method: Late pronucleus stage eggs were cultured through the blastocyst stage in the presence of Matrigel (0.5%, v/v). Characteristics of apoptosis and cell number assesed by Hoecst staining and TUNEL labeling at the blastocyst stage, respectively. Results: Morphological development, number of cells per embryo was significantly increased but rate and number of TUNEL positive nuclei of the embryo were decreased in the presence of Matrigel. Conclusion: This result suggested that at low concentration of Matrigel improves both viability and morphological development in the preimplantation mouse embryos.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.4
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• pp.381-385
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• 2000

Objective: To verify the effect of two forms (growth factor and growthfactor-reduced) of solubilized Matrigel on the development in mouse preimplantation embryos. Methods: Late 2-cell stage eggs were cultured through the blastocyst stage in the presence of GF- or GFR-Matrigel (0.5%, v/v). Morphological development, cell number and % apoptotic nuclei of blastocyst were measured by Roecst staining and TUNEL of nuclei. Results: Morphological development, number of cells per embryo was significantly increased in the presence of GF- or GFR-Matrigel. Culture of the embryos in the GF-Matrigel gave the best result. Conclusion: Low concentration of solubilized Matrigel improved development of mouse embryos regardless of growth factor content of the Matrigel. Growth factors and extracellular matrix protein included in the Matrigel synergistically potentiated the development of mouse embryos.

• Journal of Embryo Transfer
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• v.6 no.1
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• pp.33-40
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• 1991

In vitro developmental ability of early preimplantation monse embryos was shown to be depend on the embryonic stages, media and snpplements and their interaction(Experiment 1). The development of I-cell embryos were more promoted in the complex medinm(Ham's Fl0) than in the simple one(m-KRB), but that of 2-cell embryos showed the reverse effect. The bovine serum albumin(BSA) as a medium snpplement more promoted the development of I- and 2-cell embryos, compared with human fetal cord serum(HCS). On the other hand, the harmful effect of HCS was especially shown on the early cleavage in the embryonic development of the two stages. The effect of serum, in the respect of interaction between media and snpplements. was also more significantly appeared in m-KRB than Ham's Fl0. In the experiment 2, when the harmful effect of HCS was compared with that of fetal bovine serum(FBS), the former more promoted the development of l - and 2-cell embryos than the latter. The effect of HCS was more significantly shown in the development of I-cell than that of 2-cell embryos. Conclusively, as I- and 2-cell embryos were different in the requirements for the in vitro development. the optimal medium and supplement have to be selected for each embryonic stage. It is also respected to the better result if it take into consideration into the kinds of sera when serum is used for culture of early preimplantation embryos.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.2
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• pp.111-118
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• 2003

Objectives: The development of an useful method for obtaining metaphase chromosomes from a biopsied blastomere would allow differentiation between embryos with balanced and normal chromosome complements in the preimplantation genetic diagnosis for chromosomal translocations. This study was performed to evaluate the effects of microtubule depolymerizing agents (MTDAs) on the blastomeres of mouse and human preimplantation embryos, and to establish an effective method for obtaining metaphase chromosomes of biopsied blastomeres in human early embryos. Materials and Methods: Early embryos (2-4 cell stage) from superovulated mice (ICR strain) were collected and treated with single or mixture MTDAs, such as vinblastine, nocodazole and colcemid. After the treatment of MTDAs for 16 hours, the metaphase aquisition (MA) rates were evaluated by the observation of chromosome status with bis-benzimide or DAPI staining. The optimal condition from the above experiment was applied to human embryos, which were developed from abnormal fertilization (3-pronuclei). Fluorescence in-situ hybridization (FISH) with whole chromosome probes was conducted on the human metaphase chromosomes by the MTDAs. Results: In mouse embryos, the effective concentrations of each MTDAs for obtaining metaphase chromosomes were $1.0{\mu}M$ of vinblastine (20.3%), $5.0{\mu}M$ of nocodazole (28.1%) and $1.0{\mu}M$ colcemid (55.6%), respectively. The highest MA rate (91.2%) in the mouse embryos was obtained by a mixture of vinblastine ($1.0{\mu}M$) and nocodazole ($1.0{\mu}M$). In the human embryos, the metaphase chromosomes of blastomeres were obtained in 44 of 113 blastomeres (38.9%) by treatment of the mixture of vinblastine and nocodazole. FISH signals of the metaphase chromosomes were successfully observed in human individual blastomeres. Conclusions: The treatment of a mixture MTDAs for obtaining metaphase chromosomes was an efficient method, and the MA rate was above 90% in the mouse embryos. However, only a relatively small proportions of the blastomeres yielded metaphase chromosomes by the MTDAs in the human embryos. The inconsistent effects of MTDAs may be related to the variation of different species and the poor developmental potency of abnormally fertilized human embryos. We should develop more reliable and efficient methods for obtaining the metaphase chromosomes in the biopsied blastomeres of human preimplantation embryos.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.289-298
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• 2015

Edaravone (Eda) is a potent scavenger of inhibiting free radicals including hydroxyl radicals ($H_2O_2$). Reactive oxygen species (ROS) such as $H_2O_2$ can alter most kinds of cellular molecules such as lipids, proteins and nucleic acids, cellular apoptosis. In addition, oxidative stress from over-production of ROS is involved in the defective embryo development of porcine. Previous study reported that Eda has protective effects against oxidative stress-like cellular damage. However, the effect of Eda on the preimplantation porcine embryos development under oxidative stress is unclear. Therefore, in this study, the effects of Eda on blastocyst development, expression levels of ROS, and apoptotic index were first investigated in preimplantation porcine embryos. After in vitro fertilization, porcine embryos were cultured for 6 days in PZM medium with Eda ($10{\mu}M$), $H_2O_2$ ($200{\mu}M$), and Eda+$H_2O_2$ treated group, respectively. Rate of blastocyst development was significantly increased (P<0.05) in the Eda treated group compared with only $H_2O_2$ treated group. And, we measured intracellular levels of ROS by DCF-DA staining methods and investigated numbers of apoptotic nuclei by TUNEL assay analysis is in porcine blastocyst, respectively. Both intracellular ROS levels and the numbers of apoptotic nucleic were significantly decreased (P<0.05) in porcine blastocysts cultured with Eda ($10{\mu}M$). More over, the total cell number of blastocysts were significantly increased (P<0.05) in the Eda-treated group compared with untreated group and the only $H_2O_2$ treated group. Based on the results, Eda was related to regulate as antioxidant-like function according to the reducing ROS levels during preimplantation periods. Also, Eda is beneficial for developmental competence and preimplantation quality of porcine embryos. Therefore, we concluded that Eda has protective effect to ROS derived apoptotic stress in preimplantation porcine embryos.

• Development and Reproduction
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• v.6 no.1
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• pp.25-30
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• 2002

Endocrine disruptors have been reported to adversely affect reproduction and embryonic development in wild animals. One of the major abnormalities observed during early embryonic development is cellular fragmentation. In this study, we exposed mouse preimplantation embryos to PCB, BPA and DDT in vivo or in vitro. Embryos exposed to endocrine disrupter showed a variety of morphological abnormalities such as fragmentation, irregular blastomeres and cracked empty zonae pellucidae. To investigate the levels of gene expression related which genes contribute to apoptosis in preimplantation mouse embryos, we carried out the reverse transcription polymerase chain reaction to assess mRNA levels far apoptotic gene. Bcl-2, bad and bax expression levels were compared between control group and endocrine disrupter treated group. Expression level of bcl-2 gene tended to be lower in the treated group than control while expression levels of bad and bax genes were higher in the treated group. Results of this study may provide a useful tool for rapidly screening developmental toxicants in preimplantation embryos exposed to endocrine disruptors in vivo or in vitro.