• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.47-57
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• 2000

Objective: The effects of cryopreservation with or without coculture on the in vitro development of blastomere-biopsied 8-cell mouse embryos were investigated. This experimental study was originally designed for the setup of a preclinical mouse model for the preimplantation genetic diagnosis (PGD) in human. Methods: Eight-cell embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BL(표현불가)/CBA(표현불가)). Using micromanipulation, one to four blastomeres were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acid Tyrode's solution (ATS). A slow-freezing and rapid-thawing protocol with 1.5M dimethyl sulfoxide (DMSO) and 0.1M sucrose as cryoprotectant was used for the cryopreservation of blastomere- biopsied 8-cell mouse embryos. After thawing, embryos were cultured for 110 hours in Ham's F-10 supplemented with 0.4% bovine serum albumin (BSA). In the coculture group, embryos were cultured for 110 hours on the monolayer of Vero cells in the same medium. The blastocyst formation was recorded, and the embryos developed beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. Results: The survival rate of embryos after cryopreservation was significantly lower in the blastomere-biopsied (7/8, 6/8, 5/8, and 4/8 embryos) groups than in the non-biopsied, zona intact (ZI) group. Without the coculture, the blastocyst formation rate of embryos after cryopreservation was not significantly different among ZI, the zona drilling only (ZD), and the balstomere-biopsied groups, but it was significantly lower than in the non-cryopreserved control group. The mean number of cells in embryos beyond blastocyst stage was significantly higher in the control group ($50.2{\pm}14.0$) than in 6/8 ($26.5{\pm}6.2$), 5/8 ($25.0{\pm}5.5$), and 4/8 ($17.8{\pm}7.8$) groups. With the coculture using Vero cells, the blastocyst formation rate of embryos after cryopreservation was significantly lower in 5/8 and 4/8 groups, compared with the control, 7/8, and 6/8 groups. The mean number of cells in embryos beyond blastocyst stage was also significantly lower in 4/8 group ($25.9{\pm}10.2$), compared with the control ($50.2{\pm}14.0$), 7/8 ($56.0{\pm}22.2$), and 6/8 ($55.3{\pm}25.5$) groups. Conclusion: After cryopreservation, blastomere-biopsied mouse embryos have a significantly impaired developmental competence in vitro, but this detrimental effect might be prevented by the coculture with Vero cells in 8-cell mouse embryos biopsied one or two blastomeres. Biopsy of mouse embryos after ZD with ATS is a safe and highly efficient preclinical model for PGD of human embryos.

• 한국수정란이식학회지
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• 제6권1호
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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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• 제25권1호
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

• Clinical and Experimental Reproductive Medicine
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• 제22권2호
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• pp.163-170
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• 1995

Growth factors (GFs) produced by the embryo or by the maternal reproductive tract have been reported to regulate the embryonic development and differentiation. Among GFs, EGF as a mitogen plays a role in mitosis and functional differentiation of trophectoderm cells in mouse. The present study was carried out to investigate the effect of EGF on development of mouse embryos and to localize EGF in the mouse oocytes and embryos, which has been reported to be detected in the reproductive tract in mammals. To investigate the effect of EGF on the development of the embryo, mouse 2-cell embryos were cultured to blastocysts stage in Ham's F10 medium, treated with EGF(10-50 ng/ml) for 72 hrs. Immunocytochemistry was performed from oocyte to blastocyst stage with anti-EGF and anti-Mouse IgG, in order to determine the stage which EGF would be expressed in mouse. Exogenous EGF (more than 10 ng/ml) in the culture medium improved the developmental and hatching rates in the mouse embryos. As a result of immunocytochemistry, the embryonic EGF was expressed after the late 4-cell stage. EGF is thought to enhance preimplantation embryonic development and hatching. Exogenous EGF in the culture medium is thought to activate EGF receptor in the late 4-cell embryos and to enhance blastulation and hatching in mouse embryos. It is concluded that EGF enhances the developmental and hatching rates in the mouse embryos.

• 대한수의학회지
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• 제29권2호
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• pp.123-128
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• 1989

Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at $37^{\circ}C$, 5% $CO_2$ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6~12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, $100{\mu}M$ 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

• 한국가축번식학회지
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• 제24권3호
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• pp.263-268
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• 2000

본 연구는 2-, 4- 또는 8-세포기 생쥐 수정란으로부터 분리된 단일 할구의 발달 및 완만동결 후 생존능력을 조사하기 위하여 실시하였다. 2-, 4-또는 8-세포기 생쥐 수정란에서 각각 223개, 60개, 188개의 할구를 분리하여 96시간 배양하였는데 이중 2-세포기 수정란으로부터 분리된 할구는 111개(49.8%)가 배반포기까지 발육하였으며, 4-세포기와 8-세포기 수정란으로부터 분리된 할구는 각각 12개(20.0%)와 31개(16.5%)가 배반포기까지 발육하였다. 분리된 할구를 완만 동결한 후 융해하여 배양했을 때 배반포기까지의 발육율은 2-세포기 할구는 27.1%(16/50), 4-세포기 할구는 36.4%(4/11) 그리고 8-세포기 할구는 17.6%(3/17)였으며, 융해 후 할구 회수율은 각각 54.2%(65/120), 46.4%(13/28), 24.3%(17/70)을 나타냈다. 2-세포기 할구를 동결 융해한 후 동기화시킨 생쥐 자궁에 이식하여 정상적으로 발달한 태아를 생산하였다. 본 실험의 결과로 완만동결방법이 생쥐의 할구를 동결 보존하는데 이용될 수 있음을 확인하였다.

• Clinical and Experimental Reproductive Medicine
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• 제26권1호
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• pp.9-20
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• 1999

The genetic defects in human gametes and embryos can cause adverse effects on overall reproductive events. Biopsy of embryos for preimplantation genetic diagnosis (PGD) offers a new possibility of having children free of the genetic disease. In addition, advanced embryo culture method may enhance the effectiveness of embryo biopsy for the practical application of PGD. This experimental study was undertaken to evaluate the effects of coculture on the development in vitro of biopsied mouse embryos as a preclinical model for PGD of human embryos. Embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale/CBAmale). Using micromanipulation, 1, 2, 3 or 4 blastomeres of 8-cell stage embryos were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acidic Tyrode's solution (ATS). After biopsy of blastomeres, embryos were cultured in vitro for 110 hours in Ham's F-10 supplemented with 0.4% BSA or cocultured on the monolayer of Vero cells in the same medium. The frequence of blastocyst formation were recorded, and the embryos beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. There was no significant difference in the blastocyst formation between the zona intact control group and the zona drilling (ZD) only, or biopsied groups. The hatching rate of all the treatment groups except 4/8 group was significantly higher than that of control group. In all the treatment groups, there was a significant reduction in the mean cell number of embryos beyond blastocyst stage ($50.2{\pm}14.0$ in control group vs. $41.2{\pm}7.9$ in ZD, $39.3{\pm}8.8$ in 7/8, $29.7{\pm}6.4$ in 6/8, $25.1{\pm}5.7$ in 5/8, and $22.1{\pm}4.3$ in 4/8 groups, p<0.05). When the same treatments were followed by coculture with Vero cells, a similar pattern was seen in the blastocyst formation and the hatching rate. However, in all the treatment groups, there was a significant increase in the mean cell number of embryos beyond blastocyst stage with coculture, compared with the parallel groups without coculture. In the cleavage rate of biopsied blastomeres cultured for 110 hours after IVF, there was no significant difference between coculture and non-coculture groups (87.2% vs. 78.7%). However, the mean cell number of embryos developed from the biopsied blastomeres was significantly higher in coculture group ($11.5{\pm}4.7\;vs.\;5.9{\pm}1.9$, p<0.05). In conclusion, biopsy of mouse embryos after ZD with ATS is a safe and highly efficient method for PGD, and coculture with Vero cells showed a positive effect on the development in vitro of biopsied mouse embryos and blastomeres as a preclinical model for PGD of human embryos.

• 대한생식의학회:학술대회논문집
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• 대한불임학회 2001년도 제41차 추계학술대회
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• pp.115-115
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• 2001

• Clinical and Experimental Reproductive Medicine
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• 제19권1호
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• pp.1-8
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• 1992

A fluorescence microscopy technique using flurescein diacetateCFDA) as a substract has been tested for the evaluation of the viability of early mouse embryos. Embryos were incubated in T6 containing FDA concentrations of 2.5 to $50{\mu}g/ml$ for 1 to 5min. Embryos were then examined by reflected light fluorescence using a KP 490 and 520 barrier filter in a Nicon Diaphot microscopy. The results were as follow. 1. The rate of fluorescein accumulation increased on the concentration on FDA from $2.5{\times}10^{-6}M$ to $20{\times}10^{-6}M$ 2. The rate at which intracellular fluorescein was lost from embryos was depended on the temperature at which are stored. 3. Embryos with 3 min exposure to FDA have the most intensity of fluorescence. 4. Exposure of 2 cell embryos to FDA ($2.5-5{\mu}g/ml$) for 1 min did not alter their ability to delope normally in vitro.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.109-109
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• 2003

Even though success in birth of live offspring from nuclear transfer(NT) using somatic cells in many species, detailed information on processes or mechanisms of development are not well known. Cytoplasm of bovine oocyte has been known to support the development of nuclear transferred embryos using nuclear donor cells from different species. Therefore, interspecies NT might be used to find answers of some questions in basic aspect of nuclear transfer In this study, we examined the developmental potential of reconstructed embryos when bovine oocyte as a cytoplasm recipient and mouse embryonic fibroblast as a nuclear donor were used. The nuclear transfer units were aliocated in Group 1 (murine block media and normal media) and Group 2. (bovine block media and normal media). NT units were not blocked at 2-cell stage regardless of types of medium. On mouse media, poor development of interspecies NT units was observed compared to bovine media. However, as NT units cultured in bovine normal medium, embryos developed over 8-cell stage. Further studies performed to increase the developmental rate in condition of antioxidant treatment. Despite low development, bovine-murine interspecies nuclear transferred embryos could develop to blastocysts and they showed that blastocyts rate of antioxidant group was superior to those of non-antioxidant group. Next, we investigated gene expression pattern which is carried out for zygotic activation. The Xist gene is expressed in female mouse embryo after zygotic activation of 4-cell stage. But interspecies nuclear transferred embryos do not express Xist gene at 4-cell stage. As a result, it is suggested that the bovine cytoplasm controls the early preimplantation development in interspecies NT However, the development of later stages might require genomic control from transferred donor nucleus. Therefore, even though the involvement of several other factors such as mitochondrial incompatibility, effective development of embryos produced by interspecies NT requires proper genomic activation of donor nucleus after overcoming the cytoplasmic control of recipient oocytes.