• The Korean Journal of Zoology
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• v.29 no.1
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• pp.13-22
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• 1986

In order to investigate the 'In Vitro 2-Cell Block' phenomenon found in certain mouse strains such as ICR, the present studies have been done. Fertilized eggs (1-cell) and 2-cell embryos recovered from the oviducts of the ICR mouse at the various time intervals after hCG injection to induce ovulation were cultured for 3 or 4 days to examine the capability for further cleavage beyond 2-cell stage. Consequently, it was found that some proportions of the 1-cell or 2-cell embryos recovered at 30 hours post hCG showed their cleaving capability and if the embryos were obtained after 48 hours of hCG injection, they were all at 2-cell stage and most of them developed to the blastocysts in vitro. It was also found that the embryos obtained at 27 hours post hCG showed their stronger capacity of further development in the groups cultured for shorter period than 24 hours in vitro before transferring to the oviduct. Based on the results, it can be inferred that mouse fertilized eggs should be remained inside the oviduct for a certain length of period after fertilization, or they should be cultured for a short period than 12 hours before returning back to the oviduct in order to develop to blastocysts. It was also found that though the embryos under the 2-cell block in culture showed normal feature up to 24 hours under the microscopical observation, they had already lost their capacity for the normal development, and if the culture of the 2-cell embryos was extended to 48 hours, they showed nuclei with heteropyknosis, and the vacuoles were were detected in the cytoplasm of embryonic cell if they were cultured for 72 hours.

• Journal of Genetic Medicine
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• v.5 no.2
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• pp.100-104
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• 2008

DNA methylation is one of many epigenetic mechanisms that regulate gene expression in the human body. From the view of epigenetics, there are two phases of development, one for germ cell development and another for embryo development. This review will discuss the basic mechanism of methylation, its role in gene expression, and the role of methylation in embryonic reprogramming. Methylation of genes is very critical to embryo development and should be explored further in order to increase our understanding of development.

• Korean Journal of Environmental Biology
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• v.31 no.2
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• pp.69-77
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• 2013

Toxic effects of arsenic (As) and chromium (Cr) has been investigated using the sea urchin (Hemicentrotus pulcherrimus) germ cell and pluteus-larvae. The gametotoxic and embryotoxic effects of As and Cr on H. plucherrimus were each investigated at 6.25, 12.5, 25, 50, 100. Spawning was induced by 0.5 M KCl solution and the normal fertilization and embryogenesis rates were performed for 10 min and 64 hrs after fertilization, respectively. The normal fertilization and embryogenesis rates in the control condition (not including As and Cr) were greater than 94% and 93%, respectively. The fertilization rate was not significantly changed compared with control but embryogenesis rate was significantly decreased with concentration-dependent manner. As and Cr reduced normal embryogenesis rates and a significant reduction occurred at concentration greater than 6.25 ppb (P<0.01) and 25 ppb (P<0.05), respectively. The lowest-observedeffect- concentration (LOEC) of normal embryogenesis rate in As and Cr were each 6.25 and 25 ppb, respectively. From these results, normal embryogenesis rate of H. pulcherrimus have toxic effect at greater than the 6.25 ppb concentration of As and 25 ppb concentration of Cr in marine ecosystems. These results suggest that the normal embryogenesis rates of H. pulcherrimus are very useful test method for the toxicity assessment of heavy metal as As and Cr in marine ecosystems.

• Development and Reproduction
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• v.14 no.4
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• pp.253-259
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• 2010

DNA methylation is one of the major epigenetic regulations of gene expression. The DNA methylation patterns are dramatically changed during gametogenesis and embryogenesis, and especially, it has been known that embryonic stem cells show a distinct methylation pattern. In this study, we examined the methylation patterns of imprinting genes, H19, Igf2r, and Snrpn, in stem cells induced from fertilized embryo (fES) and somatic cell nuclear transferred embryo (ntES). The methylation pattern of H19 gene in both fES and ntES were similar. However, the methylation patterns of Igf2r and Snrpn in ntES (hypermethylated) were slightly different from fES cells.

• Development and Reproduction
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• v.3 no.1
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• pp.69-74
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• 1999

Insulin-like growth factors (IGF-1 and IGF-2) play an important regulatory role in premplantation embryonic development. To study the role of IGF-1 during premplantation embryonic development in mouse, the presence of mRNA transcripts for IGF-1 and IGF-lR in the oocytes and preimplantation embryos was examined. In this study, the transcripts of IGF-1 was detected in oocytes using primers for IGF-1. The PCR products were identified by Msp I restriction enzyme digest. We revealed that the transcripts of IGF-1 and IGF-1R were presented in the oocytes and preimplantation embryos. The highest mRNA levels in GV stage oocytes were decreased at 4- or 8-cell stage and then reincreased upto blastocyst. The presence of IGF-1 and IGF-lR in GV-oocytes suggests that the transcripts in the early stage embryos were derived from maternal genome. Additionally, the presence of IGF-1 and IGF-lR in the oocytes and preimplantation embryos suggests that IGF-1 plays an autocrine role during preimplantation embryonic development through IGF-lR as a signalling pathway.

• 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.

• Development and Reproduction
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• v.5 no.1
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• pp.17-21
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• 2001

This study was conducted to investigate the expression pattern of Trypsin-like enzyme and the effect of a trypsin inhibitor(benzimidine) on hatching process during in-vitro culture of mouse preimplantation embryos. The Trypsin-like enzyme was identified by rhodamine-conjugated Trypsin substrate probe. The expression of trypsin-like enzyme was firstly detected at the late morula stage, and the enzyme was uniformly localized in the trophectoderm of late blastocysts. Especially, intense fluorescence was observed in the blebbing area of hatching blastocysts. Bisbenzamidine, contained in culture media, did not alter embryonic development from 4-cell stage to the expanded blastocyst but decrease the hatching rate in ImM concentration (15.8% vs 89.7%, p<0.02). In the treatment of bisbenzimidine (5mM) for 12 hours according to the embryonic stage of mouse, the hatching rate of control (83.0%) and treatment in late blastocysts (8.7%) were significantly (p<0.01) different. From these results, we suggested that the hatching enzyme having trypsin-like activity was localized from the late morula stage, and the hatching process by this enzyme was activated in the late blastocyst stage of mouse embryos.

• Development and Reproduction
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• v.2 no.1
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• pp.29-37
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• 1998

To investigate the role of oviductal environment in early mammalian development, we examined the effects of bovine oviductal fluid (bOF) on the development of mouse 2-cell embryos in vitro. All of the embryos cultured in medium containing 5% or more of bOF underwent degeneration after 48 hr, whereas only 5% of embryos cultured in the absence of bOF degenerated. When bOF was heated at 65 \circ C for 30 min and then added to the culture medium, the embryotoxic effect of bOF was not removed at all such that none of the embryos remained alive after 48 hr. However, when bOF heated at 90 \circ C for 30 min was added to the culture, nearly most (95%) of embryos was alive. Similarly, pretreatment of bOF with 0.1% chymotrypsin for 1 hr or overnight following heating at 65 \circ C resulted in the development of 95.5% of mouse 2-cell embryos to early blastula after 48 hr culture in the presence of treated bOF. Interestingly addition of an anti-oxidant removed the evbryotoxic effect of bOF so that 91.0% of 2-cell embryos developed to morulae or blastulae in the presence of both 5% bOF and 10 mM of glutathione (GSH) after 48 hr culture. Neither oxidized form of GSH (GSSG) nor other antioxidants, however, could support the embryonic development in the presence of bOF. From these results, it is suggested that bOF contains a protein-like factor(s) which becomes embryotoxic by exposing in vitro, probably via oxidation reaction.

• Development and Reproduction
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• v.5 no.2
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• pp.101-106
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• 2001

Present study was aimed to verify the role of insulin and TNF-$\alpha$ in development of preimplantation embryos. Mouse morula were cultured for 40 hr in the presence or absence of insulin(400 ng/ml) and TNF-$\alpha$ (50 ng/ml). The morphological development, cell number of blastomeres per blastocyst, and mitogen activated protein kinase(MAPK) activity were examined. The developmental rate and cell number per embryo were the highest in insulin treatment group and the lowest in TNF-$\alpha$ treatment group. There was no significant difference in developmental rate between control and insulin plus TNF-$\alpha$ group. Taken together, it suggested that TNF-$\alpha$ impaired embryonic development and that insulin rescued developmental impairment imposed by TNF-$\alpha$. In blastocysts, insulin treatment significantly increased MAPK activity. TNF-$\alpha$ decreased the MAPK activity in a concentration-dependent manner. In the TNF-$\alpha$(50 ng/ml) -primed embryos, activation of MAPK by insulin was attenuated. In conclusion, these results suggest that there was a cross talk between insulin and TNF-$\alpha$ by means of activation of MAPK in preimplantation embryos and that insulin might rescue damage of embryos exposed to TNF-$\alpha$.

• Development and Reproduction
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• v.4 no.2
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• pp.161-173
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• 2000

The aim of this study was to assess the effect of the frequency of the L$N_2$ infusion on the ultrastructure, metabolism and development of the embryo after freezing and thawing by computerized cell freezer. Two-cell embryos of ICR mouse were randomly allocated into fresh (control), high-frequency freezing (group 1) and low-frequency freezing (group 2). For fresh and frozen-thawed intact 2-cell embryos, total ceil number in the blastocyst was counted by fluorescent microscope after Hoechst 33258 staining. Relative amount of $H_2O$$_2$ was measured by DCHFDA. Intracellular location and membrane potential of the mitochondria were evaluated by staining with rhodamine 123 and JC-1. The structure of actin filament was also evaluated by confocal microscope. DNA fragmentation was assessed by TUNEL method after development into blastocyst. The survival rate of intact embryo was higher in group 1 than group 2 (50.7％ vs. 34.6％ respectively, p<0.05). The blastocyst developmental rate was significantly low in group 2 (86.7％, 76.7％ vs. 44.0％ for control, group 1 and group 2 respectively, p<0.05). Total cell number in the blastocyst was also significantly lower in group 2 than control (79.5$\pm$12.9, 71.6$\pm$8.0, and 62.5$\pm$4.7 for control, group 1 and group 2 respectively, p<0.05). The relative amount of $H_2O$$_2$ was higher in group 2 than other groups (15.3$\pm$3.0, 16.6$\pm$1.6 vs. 23.4$\pm$1.8, p<0.05). After JC-1 staining, relative intensity of mitochondria with high membrane potential was significantly lower in group 2 than control and group 1 (17.2$\pm$3.8, 17.4$\pm$1.3 vs. 13.2$\pm$2.0, p<0.05). In group 2, partial deletion and aggregation of the actin filament was found. DNA fragmentation rate was also hieher for group 2 versus other groups (30.8％, 36.0％ vs. 65.6％, p<0.05). The frequency of the L$N_2$ infusion is an important factor for the development of frozen-thawed mouse embryo. High-frequency infusion may prevent damages of cytoskeleton and mitochondria in the embryo probably by preventing the temperature fluctuation during dehydration phase. We speculate that the application of high-frequency infusion method in human embryo may be promising.