• 대한생식의학회:학술대회논문집
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• 1996.11a
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• pp.60.2-61
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• 1996

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.213-217
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• 1999

In our previous study, we observed that hydrosalpingeal fluid (HSF) adversely effect mouswe embryo development and hatching. The aim of this study was to evaluate the effect of HSF as assessed by the blastocyst development rate (BDR) and by cell counting in vitro. HSF was collected from ninie patients undergoing salpingoneostomy to correct hydrosalpinx. Two-cell embryos were obtained from superovulated ICR mice. T6 medium and $T6{\pm}0.4%$ bovine serum albumin were used as control media. T6 medium containing 10% or 50% HSF and 100% HSF from each patient were used as test media. Nine to 15 embryos were cultured in micro drops prepared from each of these media. To assess the total cell number within each blastocyst, the blastocysts were fixed and stained with Hoechst 33342 to facilitate cell counting. The mean BDR in two control media were 88.89% and 85.40%. The mean BDR in media containing 10%, 50%, 100% HSF were 85.87%, 89.58% and $75.57%^*$, respectively ($^*$: p<0.05). The overall mean cell count $({\pm}SEM)$ in control media were $87.6{\pm}9.65\;and\;90.12{\pm}11.38$. The BDR was affected adversely only by 100% HSF and not in media containing 10% or 50% HSF. Mean cell counts were decreased significantly only in blastocysts cultured 100% HSF ($63.8{\pm}13.66$; p<0.01) but not in blastocysts cultured in 10% or 50% HSF ($91.3{\pm}12.44\;and\;82.9{\pm}18.27$, respectively). Thus, it is concluded that HSF has no embyotoxic effect but has a mildly negatively effect on embryonic growth and development.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.275-282
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• 2000

Objective: This study was to determine the effect of different concentration of calcium III medium on the preimplantational development of zygotes and early 2 cell embryos. Methods: Female mice of ICR strain ($5{\sim}8$ weeks old) were superovulated and mated with fertile males. Zygotes or early 2-cell embryos were collected by flushing the oviducts $31{\sim}32$ hours after hCG injection. The embryos were cultured in various concentrations of $Ca^{2+}$ in medium or with EDTA, EGTA and $Ni^{2+}$. Result and Conclusion: Treatment of high concentraion of $Ca^{2+}$ (3.42 mM $(2X){\sim}17.l$ mM (10X)) in medium didn't develop well compared to the control. Low concentrations of $Ca^{2+}$ (0.214mM $(1/8X){\sim}0.855$ mM (1/2X) were deterimental to development beyond 2-cell stage. EDTA, $Ca^{2+}$ chelating agent was treated with ranged concentrations of EDTA (0.014 $mM{\sim}0.107$ mM) to medium contaning 1.71 mM $Ca^{2+}$ showed beneficial effect to development to blastocyst compared to the control. EGTA, extracellular $Ca^{2+}$ chelator, was treated with ranged concentrations of EGTA ($0.014{\sim}0.107$ mM) to the medium contaning 1.71 mM $Ca^{2+}$. There is no significant difference with the control. $Ni^{2+}$ (50 ${\mu}M$), T-type $Ca^{2+}$-channel blocker was treated to medium contaning low concentration of $Ca^{2+}$. It overcame 2-cell block significantly. Rate of degenerated embryos decreased and developmental rate to morula and blastocyst increased more than low $Ca^{2+}$ concentration alone. Further studies are needed for the overcoming effect of 2-cell block by $Ni^{2+}$.

• Genomics & Informatics
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• v.10 no.1
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• pp.16-22
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• 2012

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of multiple fluid-filled cysts that expand over time and destroy renal architecture. The proteins encoded by the $PKD1$ and $PKD2$ genes, mutations in which account for nearly all cases of ADPKD, may help guard against cystogenesis. Previously developed mouse models of $PKD1$ and $PKD2$ demonstrated an embryonic lethal phenotype and massive cyst formation in the kidney, indicating that $PKD1$ and $PKD2$ probably play important roles during normal renal tubular development. However, their precise role in development and the cellular mechanisms of cyst formation induced by $PKD1$ and $PKD2$ mutations are not fully understood. To address this question, we presently created $Pkd2$ knockout and $PKD2$ transgenic mouse embryo fibroblasts. We used a mouse oligonucleotide microarray to identify messenger RNAs whose expression was altered by the overexpression of the $PKD2$ or knockout of the $Pkd2$. The majority of identified mutations was involved in critical biological processes, such as metabolism, transcription, cell adhesion, cell cycle, and signal transduction. Herein, we confirmed differential expressions of several genes including aquaporin-1, according to different $PKD2$ expression levels in ADPKD mouse models, through microarray analysis. These data may be helpful in $PKD2$-related mechanisms of ADPKD pathogenesis.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.109-113
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• 1998

Objective: To ananlyze the direct effect of nitric oxide (NO), generated from sodium prusside (SNP) on the embryo developments in reproductive process. Design: Ova from mouse were treated to allow fertilization in in vitro culture. And the samples of fertilized ova were alloted into five alliqutos. Each alliquot was cultured in media treated with either concentration at 0 (n=92), $25{\mu}M$ (n=84), $50{\mu}M$ (n=80), $100{\mu}M$ (n=77), $500{\mu}M$ (n=54) of SNP. Main Outcome Measure: Rates of embryonal cell cleavages, viability and cell morphology were assessed during in vitro fertilization and culture. Results: As analyse the cell cleavage at 24 hours after in vitro culture of fertilised egg in variuos NO concentration, all of egg cells of each alliquot were developed to $2\sim4$ cell stage. But the alliquot of egg cells treated with $50{\mu}M$, which were totally degenerated. And also all embryonal cells of each alliquot were developed to 8 cell stage and morula stage on culture continuosly. And the embryonal cells of each alliquot were analysed at 24 and 48 hours following the in vitro culture. The rates of cell fragmentation and fusion were $4.2{\pm}3.4%$ in control group which is not treated with NO, while experimental groups was high, as rated $23.4{\pm}6.2%$ in $25{\mu}M$, $28.2{\pm}5.7%$ in $50{\mu}M$ and $32.1{\pm}6.4%$ in $100{\mu}M$ concentration of NO. Accordingly the rate of abnormal morphology of embryonal cell in control was lower significantly than that in each alliquot of experimental groups (p<0.05). And the degenerated rates of embryonal cells were 0% in control, $17.8{\pm}6.7%$ in $25{\mu}M$, $23.6{\pm}4.7%$ in $50{\mu}M$ and $26.8{\pm}11.2%$ in $100{\mu}M$ at 8 cells and morula on culture of 48 and 72 hours. On the examination of embryonal cells developed to blastocyst through in vitro culture, the rates of degenerated cells were $16.8{\pm}7.2%$ in control, $37.5{\pm}6.2%$ in $25{\mu}M$, $73.4{\pm}4.6%$ in $50{\mu}M$, 100% in $100{\mu}M$. Conclusion: This results suggeted that the NO in any concentrations is harmful on embryos in view of morphology as well as viability of cell, and the toxicity of NO on embryo is stronger at condition in higher concentration of NO.

• The Korean Journal of Zoology
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• v.32 no.4
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• pp.404-411
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• 1989

In this study, we attempted to determine the precise patterns of protein synthesis during the second cleavage in mouse. F1 hybrid 2-cell embryos showing a highly synchronized cell cycle and outbreed ICR strain 2-cell embryos were used. The patterns of protein synthesis during the second cleavage showed the sequential changes in the F1 hybrid and ICR strain 2-cell embryos. Moreover, we examined the effects of mitotic and transcriptional inhibitors such as colcemid and a-amanitin on the protein synthesis in the late 2-cell embryos of ICR strain. Treatment of colcemid (0.1mg/ml) blocked the second cleavage, but did not affect on the change of protein synthesis. However, treatment of a-amanitin induced the synthesis of two set of polypeptides without affecting on synthesis of other proteins and cleavage. It thus seems that the appearance of a-amanitin-sensitive proteins may be not involved in the second cleavage. Therefore, these results indicate that the second cell cycle in mouse embryos appears to be regulated at post transcriptional level, presumably independent on the expression of embryonic genome. 본 연구는 생쥐 배아의 2세포기 분열과정중 단백질 합성양상과 단백질합성에 미치는 colcemid와 $\alpha$-amanitin의 영향을 조사하였다 이를 위하여 체내 수정된 ICR strain의 2세포기 배아와 매우 일치된 초기배아 분열양상을 보여주는 체외 수정된 F1 (C57BL x CBA) hybrid 2세포기 배아를 사용하였다. 두 종류의 2세포기 배아에서 단백질 합성은 분열단계에 따라서 매우 일치된 변화를 보여 주었다. 또한 유사분열 억제제인 colcemid (0.1mg/ml)의 처리는 2세포기 배아분열을 억제하였으나, 단백질 합성에는 아무런 변화를 주지 못하였다. 그리고 후기 2세포기 배아에 전사 억제제인 a-amanitin (100mg/ml)을 처리하였을 때 세포분열이나 다른 단백질의 합성에는 아무런 영향이 없이 단지 두개의 단백질의 합성만을 유도하였다. 이는 아마도 a-amanitin의 stress효과에 기인하는 것으로 추측된다. 따라서 생쥐 2세포기 배아의 분열과정은 배아게놈의 유전자 발현과는 무관하게 이미 합성되어 존재하는 mRNA에 의하여 조절되는 것으로 사료된다.

• The Korean Journal of Zoology
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• v.39 no.3
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• pp.239-247
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• 1996

An embryonic stem (ES) cell-based in vitro model system was examined to determine whether a simple differentiation of embryoid bodies (EB) in the suspension medium is useful to dissect early erythropoiesis. Characteristics of the differentiating EBs were monitored for their differentiation potential to generate hematopoietic cell types by general morphology, benzidine staining and two-step colony assays, and expressivity of several erythroid marker genes by the RT-PCR analysis for total cellular RNA prepared from the differentiating EBs. Every ematopoietic lineage cells were generated from the differentiating EBs with reproducible frequencies, similar to the other sophisticated differentiation protocols. Furthermore, the globin gene switching in differentiating ES cells paralleled the sequence of events found in the mouse embryo, and such that their expression was activated by at least 12 hrs later than those of erythroid-specific transcription factors, GATA-1 and Tal-1 The erythropoietic differentiation program initiated reproducibly and efficiently in this simple differentiation system in a suspension culture, such that this system may be useful for dissection of the molecular events of early erythropoiesis.

• Reproductive and Developmental Biology
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• v.28 no.4
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• pp.227-233
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• 2004

This study was conducted to investigate the effect of various addition and exclusion time of glucose (Control: no addition, A: 24～72 h, B: 24～48 h, C: 48～72 h, D: 0～72 h, E: 0～48 h, F: 0～24 h and 48～72 h, G: 0～24 h) on embryonic developmental capacity of 2-cell embryos in mice. Developed blastocysts were assessed for mean cell number by differential staining. The zona-intact blastocyst (ZiB) rates were higher (p<0.05) in group B than control. However, the zona-escape blastocyst (ZeB) rates were not significantly different in all groups. At 72 h, total blastocyst (ZiB + ZeB) formation rates were not significantly different in all groups. The mean cell number was not significantly different among all groups. The inner cell mass (ICM) cell number was higher (p<0.05) in group F than control, group A, B and G. The trophectoderm (TE) cell number was higher (p<0.05) in control than group A and D. The %ICM was higher (p<0.05) in group C, D and F than control. The ICM : TE ratio was not significantly different in all groups. Between control and glucose group, no significant difference was observed in the total blastocysts (ZiB + ZeB) formation rates. Also, no significant difference was observed in the mean cell number, ICM cell number and ICM : TE ratio. However the TE cell number was higher (p<0.05) in control than glucose group and %ICM was higher (p<0.05) in glucose group than control. In conclusion, glucose added in culture medium was not inhibitory on blastocyst formation but glucose added for 48 ～72 h in culture medium increases %ICM of blastocysts in mice.

• Molecules and Cells
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• v.20 no.3
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• pp.423-428
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• 2005

Immediately after fertilization, a chromatin remodeling process in the oocyte cytoplasm extracts protamine molecules from the sperm-derived DNA and loads histones onto it. We examined how the histone H3-lysine 9 methylation system is established on the remodeled sperm chromatin in mice. We found that the paternal pronucleus was not stained for dimethylated H3-K9 (H3-$m_2K9$) during pronucleus development, while the maternal genome stained intensively. Such H3-$m_2K9$ asymmetry between the parental pronuclei was independent of $HP1{\beta}$ localization and, much like DNA methylation, was preserved to the two-cell stage when the nucleus appeared to be compartmentalized for H3-$m_2K9$. A conspicuous increase in H3-$m_2K9$ level was observed at the four-cell stage, and then the level was maintained without a visible change up to the blastocyst stage. The behavior of H3-$m_2K9$ was very similar, but not identical, to that of 5-methylcytosine during preimplantation development, suggesting that there is some connection between methylation of histone and of DNA in early mouse development.

• Current Research on Agriculture and Life Sciences
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• v.8
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• pp.51-58
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• 1990

The study was carried out to investigate the viability of mouse embryos bisected at the stages of 8-cell, morula and blastocyst and, also, to find out the feasibility of offspring production by transfer of the bisected blastocysts with Quick-splitting. The results obtained are summarized as follows: 1. The mouse embryos were bisected with bio-cut blade at the stages of 8-cell and morula, and cultured in Medium 2. The bisected embryos were developed to blastocyst stage by 64% and 81%. respectively. 2. The blastocyts which were cultured in Medium 2 after being bisected at the stages of 8-cell and morula were observed normal outgrowth in Ham's F-10 medium by 86% and 90%, respectively. 3. The blastocyts which were Quick-splitted in Medium 2 were observed normal outgrowth by 97%. however, no offspring was obtained by transfer of Quick-splitted blastocysts.