• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.13-24
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• 2001

Objective: In muscle and neuronal cells, calcium channels have been classified by electrophysiological and pharmacological properties into (1) voltage-dependent $Ca^{2+}$-channel (1) P/Q-type $Ca^{2+}$-channel (2) N-type $Ca^{2+}$-channel (3) L-type $Ca^{2+}$-channel (4) T-type $Ca^{2+}$-channel (5) R-type $Ca^{2+}$-channel. The present study was done in order to investigate whether there is any difference in $Ca^{2+}$-channel distribution between activated and normally fertilized embryos. Methods: The immunocytochemical method was used to identify the existence of voltage-dependent $Ca^{2+}$-channels in parthenogenetically activated 2-cell embryos by ethanol and $SrCl_2$ treatment. These 2-cell embryos were obtained by exposure to 6% ethanol for 6 min and to 10 mM $SrCl_2$ for 2h. Results: P/Q-type $Ca^{2+}$-channels and L-type $Ca^{2+}$-channels have been identified. Whereas, three type of $Ca^{2+}$-channel P/Q-type, N-type, L-type have been identified in 2-cell embryos fertilized in vivo. Conclusion: Activation by ethanol was faster than those by $SrCl_2$. However, there was difference in DAB staining of the embryos between ethanol and $SrCl_2$ treatment (87.7% and 54.1 %). Intensity of staining was also different between ethanol- and $SrCl_2$-treated group. However, it has not been known why there was some difference in DAB staining and staining intensity in the present study.

• Molecules and Cells
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• v.37 no.1
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• pp.59-65
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• 2014

Eph receptors and their ligands ephrins have been implicated in guiding the directed migration of neural crest cells (NCCs). In this study, we found that Wnt1-Cre-mediated expression of ephrinA5-Fc along the dorsal midline of the dien- and mesencephalon resulted in severe craniofacial malformation of mouse embryo. Interestingly, expression of cephalic NCC markers decreased significantly in the frontonasal process and branchial arches 1 and 2, which are target areas for the migratory cephalic NCCs originating in the dien- and mesencephalon. In addition, these craniofacial tissues were much smaller in mutant embryos expressing ephrinA5-Fc. Importantly, EphA7-positive cephalic NCCs were absent along the dorsal dien- and mesencephalon of mutant embryos expressing ephrinA5-Fc, suggesting that the generation of cephalic NCCs is disrupted due to ephrinA5-Fc expression. NCC explant experiments suggested that ephrinA5-Fc perturbed survival of cephalic NCC precursors in the dorsal midline tissue rather than affecting their migratory capacity, which was consistent with our previous report that expression of ephrinA5-Fc in the dorsal midline is responsible for severe neuroepithelial cell apoptotic death. Taken together, our findings strongly suggest that expression of ephrinA5-Fc decreases a population of cephalic NCC precursors in the dorsal midline of the dien- and mesencephalon, thereby disrupting craniofacial development in the mouse embryos.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.139-146
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• 1997

This experiment was carried out to evaluate the effect of mouse early embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells(BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined. For a comparative study of in vi패 and in vitro development, the fresh blastocyst which developed in vivo for 120 hours after hCG injection was collected from the uterus, and their numbers of nuclei were also counted. The higher developmental rates of blastocyst formation was a, pp.ared from 91% to 97% when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal eptithelial cells. The number of nuclei in the embryos cultured for 72 hours under each conditions was significantly reduced it than blastocyst in vitro conditions. The number of nuclei in embryos cultured in TCM 199, Ham's F-10 and Medicult IVF medium were counted 68.1$\pm$6.00, 67.3$\pm$4.49, 66.4$\pm$5.64, and 94.3$\pm$8.61, 92.5$\pm$7.60, 92.1$\pm$6.10 with BOEC and 93.3$\pm$5.80, 92.9$\pm$6.53, 92.3$\pm$7.35 with POEC coculture, respectively. These numbers were lowered than 107.2$\pm$7.43 in vivo conditions. In conclusions, the coculture between the mouse early embryos, and oviductal epithelial cells of BOEC and POEC give to improve the developmental and hatching rates of blastocyst but in vivo culture systems for the growth of nuclei were ineligible than in vitro conditions.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.151-156
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• 2003

The objective of this study was to investigate response of corpus luteum, recovery rate of embryos and production of transferable embryos according to superovulation by PEG 30% FSH in Hanwoo. Cows were selected as recipient, subsequent were superovulated with a total of 400 mg NIH-FSH-P1(Folltropin-V, Canada) given by one shot subcutaneously. At the time of five days after Folltropin injection, 25mg of a PGF$_2$a was injected and cows were inseminated 12 and 24h after the onset of estrus. Seven days after insemination, embryos were collected non-surgically and were cryopreserved by direct transfer methods. The results are summarized as follows : 1. Response of corpus luteum following the superovulation in Hanwoo, right ovary were 52.8%(271/513) and left ovary were 47.1%(242/513), respectively (P<0.05). 2. Recovery rate of embryos following the number of corpus luteum were 83.0%(426/513). 3. Mean number of embryos recovered and transferable embryos were 7.74 and 6.43, respectively (P<0.05). 4. In the total number of transferable embryos per flush were collected 6.4 and all saved transferable embryos were 355.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.315-317
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• 2015

In vitro culture of murine embryos is an important step for in vitro production systems including in vitro fertilization and generations of genetically engineered mice. M16 is widely used commercialized culture media for the murine embryos. Compared to other media such as potassium simplex optimization medium, commercial M16 (Sigma) media lacks of amino acid, glutamine and antibiotics. In the present study, we optimized M16 based embryo culture system using commercialized antibiotics-glutamine or amino acids supplements. In vivo derived murine zygote were M16 media were supplemented with commercial Penicillin-Streptomycin-Glutamine solution (PSG; Gibco) or MEM Non-Essential Amino Acids solution (NEAA; Gibco) as experimental design. Addition of PSG did not improved cleavage and blastocyst rates. On the other hand, cleavage rate is not different between control and NEAA treated group, however, blastocyst formation is significantly (P<0.05) improved in NEAA treated group. Developmental competence between PSG and NEAA treated groups were also compared. Between two groups, cleavage rate was similar. However, blastocyst formation rate is significantly improved in NEAA treated group. Taken together, beneficial effect of NEAA on murine embryos development was confirmed. Effect of antibiotics and glutamine addition to M16 media is still not clear in the study.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.301-307
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• 1996

This study was carried out to produce twin calves by embryo transfer in Hanwoo and investigate the pregnancy and twin rate by recipient's conditions. All recipients were bred at estrus by artificial insemination with Hanwoo semen and then transfered an additional embryo produced in vivo or in vitro to tbe uterine horn contralateral to the corpus luteum on Day 7. The results obtained were as follows ; 1. The pregnancy rate was higher in young recipients of 3 years (68.8%) than in old ones of 10 years and greater(36.4%). And for CL size pregnancy rate was 57.9, 45.4 and 60.1% in large, medium and small size of CL of recipients, respectively. 2. 447recipients were transferred an additional embryos at 7th day after Al and average pregnancy rate was 57.5% and twin production rate was 22.2%. 3. Average pregnancy and twin production rate by direct transfer methods of frozenthawed IVF embryos was 56.0 and 16.7%. 4. The ratio of male to female twin in a total of 55 twin pairs was 54.6%, and average gestation lengths of male to female and female to female twin were 280.6$\pm$5.4 and 279.715.4 days, respectively. Average birth weight of twins was beavior in male and male twin(23.2i5.8kg) than in male and female twin(20.5$\pm$2.6kg).

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.66-66
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• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.20-21
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• 2003

Insulin-like growth factors (IGFs) are mitogenic peptide hormones that regulate embryonic development, postnatal growth and cellular differentiation in vertebrates IGFs are initially translated as pre-pro-peptides and then proteolytically processed to yield the mature IGFs and E-peptides. Like the C-peptide of pro-insulin, the E-peptides of pro-IGFs are generally believed to possess little or no biological activity other than their potential roles in the biosynthesis of the mature IGFs. Like human IGF-1, previous studies in our laboratory showed that the recombinant trout Ea4-peptide of pro-IGF-1 exhibited a dose-dependent mitegenic activity in cultured BALB/3T3 fibroblasts and other non-oncogenic transformed cells (Tian et al., 1999) We have also shown by in vitro and in vivo studies that Ea4-peptide possessed novel anti-tumor activities (Chen et al., 2002, Kuo and Chen, 2002; Kuo and Chen 2003). Recent results of studies conducted in chorionicallantoic membrane of developing chicken embryos revealed that Ea4-peptide of trout pro-IGF-1 also possesses a dose-dependent antiangiogenic activity. Together these results raised the question whether Ea4-peptide of trout pro-IGF-1 may affect heart and blood vessel development and hematopoiesis in fish embryos. (중략)

• BMB Reports
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• v.45 no.10
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• pp.577-582
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• 2012

single-minded (sim) is a master regulatory gene that directs differentiation in the central nervous system during Drosophila embryogenesis. Recent identification of the mesectoderm enhancer (MSE) of sim has led to the hypothesis that two Snail (Sna)-binding sites in the MSE may repress sim expression in the presumptive mesoderm. We provide evidence here that three Sna-binding sites proximal to the sim promoter, but not those of the MSE, are responsible for the mesodermal repression of sim in vivo. Using transgenic embryos injected with lacZ transgenes, we showed that sim repression in the mesoderm requires the three promoter-proximal Sna-binding sites. These results suggest that Sna represses the mesectodermal expression of sim by directly repressing the nearby promoter, and not by quenching adjacent transcriptional activators in the MSE. These data also showed how the MSE, lacking the three proximal Sna-binding sites, reproduced the endogenous pattern of sim expression in transgenic embryos.

• Molecules and Cells
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• v.38 no.11
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• pp.975-981
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• 2015

Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity.