• 대한방사선기술학회지:방사선기술과학
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• 제18권2호
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• pp.75-86
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• 1995

Embryos and fetuses are more sensitive to various environmental agents than adults of children biological effects following the exposure, such as intrauterin, malformation, have intimate conception with the prenatal exposure. There have been many studies on radiation and other agent. However, imformation about the ultrasound effects is limited. It is very important to study the effect of ultrasound with these kinds of fatera in consideration of ultrasound protection and safty. In this study, embryonic and fefal effects of ICR mouse embryos irradiated on 24, 48, 12 and 192 hpc of preimplantation and organogenesis period at the intensity of $0.5{\sim}3\;W/cm^2$ were investigated. Many type of external malformation observed in mouse irradiated on 72 hpc and 192 hpc. However, the embryos irradiated on 24 hpc and 48 hpc, at witch embryos had less then 6 cells and were pre-compaction stage, had no sensitivity for external malformation. The threshold doses of external malformation in mouse irradiated on 72 hpc and 192 hpc, at which embryos were consisted of $16{\sim}32$ cells and neural formation stage, were $1\;W/cm^2$ and $0.5\;W/cm^2$.

• 한국환경보건학회지
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• 제24권2호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제30권2호
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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.

• Clinical and Experimental Reproductive Medicine
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• 제50권4호
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• pp.244-252
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• 2023

Objective: We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Methods: Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. Results: The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Conclusion: Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

• 한국발생생물학회지:발생과생식
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• 제3권1호
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• pp.59-67
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• 1999

초기배아의 발생과정동안 배아와 모체에서 발현되는 여러 cytokine은 착상을 위한 신호물질로 중요한 역할을 한다. 그 중 interleukin-1$\beta$ (IL-1$\beta$)는 배아와 모체간의 상호 신호전달체로서 성공적인 착상을 위한 필수적인 요소로 작용한다고 알려져 있다. 따라서 본 연구에서는 초기배아의 발생과정에 있어서 IL-1$\beta$ 유전자의 역할을 규명하기 위해 생쥐 초기배아에서의 IL-l$\beta$ 유전자의 발현양상을 역전사중합효소연쇄반응을 통해 조사하였고, IL-l$\beta$ 유전자의 발현에 미치는 수란관과 자궁내막세포의 영향을 밝혀보기 위해 공배양방법을 이용하였다. 그 결과 IL-l$\beta$ in vivo 에서는 4-세포기부터 포배기까지, in vitro에서는 상실배부터 부화중 포배기까지 발현하는 양상을 보였다. 또한 수란관과 자궁내막세포와의 공배양시 대조군과 비교하였을 때 실험군에서 IL-l$\beta$ 유전자의 발현이 촉진되었다. 이러한 결과는 IL-l$\beta$ 의 존재가 착상전 초기배아의 발생에 중요한 역할을 한다는 것을 의미한다. 또한 수란관과 자궁내막세포와의 공배양을 통해 IL-1$\beta$ 유전자의 발현이 수란관과 자궁요소에 의해 조절됨을 확인하였다.

• Clinical and Experimental Reproductive Medicine
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• 제28권3호
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• pp.199-207
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• 2001

Objective: The present study was undertaken to examine the effects of magnesium ion in the culture medium on the development of mouse fertilized oocytes either before or after pronuclear formation, and to investigate whether the effect of magnesium ion is related with the redistributional change of mitochondria. Methods : Fertilized oocytes obtained from the oviducts of mice at 15 hr after hCG injection before pronuclear formation (pre-PN) or 21 hr after hCG injection after pronuclear formation (post-PN) were used. The embryos were cultured for 3 days with basic T6 medium-magnesium free and various concentrations of magnesium ion, 0.0, 0.5, 1.0, 2.0, 4.0 or 8.0 mM, respectively. After culture, the developmental stages of embryos and the number of nuclei were evaluated. To observe the effects of magnesium ion on the mitochondrial distribution, fertilized oocytes were collected at 21 hr after hCG injection and cultured for 6 hr with various concentration of magnesium ion. As a control, fertilized oocytes with pronuclei at 27 hr after hCG injection were used. Results: The concentration of magnesium ion to accelerate the in vitro development of mouse fertilized oocytes appeared to be at 2.0 mM for the pre-PN and the post-PN stage embryos. In the mitochondrial redistribution patterns, the embryos cultured in 2.0 mM concentration of magnesium ion showed the highest percentage (22.6%) of distinct perinuclear clustering pattern comparing to other experimental group. Conclusion: The effect of magnesium ion may be related to the cytoplasmic redistribution of mitochondria. This relationship seems to connect the developmental competence of preimplantation mouse embryos in vitro. These results can suggest that higher concentration of magnesium ion (2.0 mM) than those of conventional culture medium ($0.2{\sim}1.2\;mM$) is more suitable for in vitro culture of preimplantation mouse embryos.

• 한국발생생물학회지:발생과생식
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• 제6권1호
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• pp.25-30
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• 2002

내분비계 장애물질은 다양한 동물의 생식과 배아의 발생에 위해한 영향을 미친다고 보고되었다. 이러한 초기 배아의 비정상 발생 중 대표적인 것이 할구파편이다. 본 연구에서는 착상 전 생쥐 초기 배아를 PCB, BPA, DDT에 체내, 체외에서 각각 노출시켰다. 내분비계 장애 물질에 노출시켰던 배아는 할구파편, 불규칙한 할구와 깨짐, 일부 파괴된 투명대 배아 등의 다양한 형태학적 비정상 양상을 보였다. 착상 전 생쥐 초기배아의 세포괴사에 관여하는 유전자를 조사하기 위하여 RT-PCR을 사용하여 mRNA수준에서 평가하였다. Bcl-2, bad 그리고 bax유전자 발현 정도를 대조군과 내분비계 장애물질 처리군으로 나누어 비교하였다. Bcl-2유전자는 내분비계 장애물질 처리군에서 낮아지는 경향을 보였고, bad와bix는 내분비계 장애물질 처리군에서 보다 높은 경향을 보였다. 이러한 연구 결과는 착상 전 생쥐 초기배아를 체내와 체외에서 내분비계 장애물질에 노출시킴으로써 발생적 독성물질을 신속히 판단할수 있는 유용한 수단이 될 수 있을 것으로 생각한다.

• 한국동물학회지
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• 제38권2호
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• pp.196-203
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• 1995

The c-myc proto-oncogene, one of the immediately earlY genes, is expressed in various mammalian cell types and heavily involved in the regulation of cell proliferation and differentiation. To determine endogeneous expression pattern of c-myc gene in preimpBantation mouse embwos, we employed a reverse transcription coupled to polvrnerase chain reaction (RT-PCR). Transcript of c-myc was detected at fertilized embryos as a maternal transcript. At the early two-cell stave, transcript of c-myc gene was hardly detected, bu, appeared at late two-cell embryos as a zygotic transcript. The level of c-myc expresion was increased at later stases and peaked at blastocvst stage. To examine the functional role of promoter region for c-myc gene transcription, we fused the 5'upstream region (1.8 kb) including econ 1 of c-myc genomic DNA with E. coli lacE gene fnamed as pcMYC-laczl. pcMYC-lacZ was microiniected into the pronscleus of mouse one-cell embryovs, and p·salactosidase activity was determined tv histochemical staining with X-gal at different stases. f-galactosidase activity was detected only at blastocyst, but not at the earlier stage embryos. This result indicates that c-myc gene is transcriptionallv active during mouse preimplantation development.

• Journal of Radiation Protection and Research
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• 제21권4호
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• pp.273-284
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• 1996

착상전기(着床前期}의 태아(胎兒)는 방사선(放射線)을 비롯한 많은 환경요인(環境要因)에 대하여 감수성(感受性)이 높은 개체(個體)임에도 불구하고 특히 이 시기는 임신부(姙娠婦)가 자각(自覺)적으로 임신을 감지할 수 없는 시기이기에 이러한 여러 환경유해요인으로부터 의도적으로 피할 수가 없다. 그러므로 착상전기의 영향을 충분히 검토한 후에 의료행위를 취할 것이며 이에 대한 방어(防禦)대책도 검토할 필요가 있다. 종래 까지 방사선에 대한 태아영향에 관한 많은 연구결과에 의하면 방사선 및 그 외의 유해요인에 대한 착상전기의 영향은 배사망(胚死亡)(유산(流産))만이 일어나며 기형(奇形)은 유발(誘發)하지 않는다고 하여 발생학(發生學)등 여러 교과서에서 기형은 기관형성기(器官形戚期)만이 국한(局限)해서 일어나는 영향이라고 단정되어 왔었다. 그러나 이 연구결과 착상전기에 있어서도 기형이 유발하여 오히려 기관형성기((器官形成期)보다도 감수성이 높다는 것이다. 또한 착상전기에서도 기형유발의 시기특이성을 가지며 여러 종류의 기형이 발생한다는 것이 본 연구로부터 밝혀졌다. 실험동물은 ICR Mouse를 사용했다. ICR Mouse는 일반적으로 태아사망 및 기형실험에 널리 사용되는 것이다. 사육조건은 Conventional 한 조건하에서 사육했으며 Mating 방법(方法)은 Female 마우스의 발정기(Sexual Excitement period)에 있는 mouse 질(膣)을 육안 적으로 관찰하여 $AM 6:00{\sim}AM 9:00$시까지 3시간만 mate시켰다. AM9:00시에 Vaginal Plug를 관찰하여 임신을 확인했다. Plug가 확인 된 마우스는 AM8:00시에 수정(Conception)된 것으로 가정하고 이 시점을 임신 0일 0시로 수정 난의 태아연령을 산정했다. 방사선조사는 $^{135}Cs\;{\gamma}-$선을 사용하였으며 임신 마우스의 전신조사를 실시하고 조사한 시기는 각 2, 48, 72, 96hpc이며 조사한 방사선 선량 군은 $0.1{\sim}2.5Gy$이다. 태아영향 관찰지표는 태아 연령은 mate일 오전 8:00시를 임신 0일 0시로 환산하여 태아연령 18일에 임신마우스를 Cervical vertebral dislocation에 의해 도살했다. 도살 후 해부하여 각 임신 마우스별로 관찰했다. 착상 율을 관찰하기 위하여 황체수를 세었고, 태아사망과 생존태아를 구별했다. 자궁 내 사망의 분류는 태아사망을 1) preimplantation death 2) Embryonic death 3) Fetal death로 분류했다. 착상전사망은 수정후 $0{\sim}4.5$일(1세포기${\sim}$배반포후기 부화까지)까지의 사망으로써 난소의 황체수(배란 수)와 착상태아(생존태아, 착상흔, 태반유잔, 흡수태아, 침연태아의 합계)로부터 구할 수 있다. Embonic death는 수정 후 $4.5{\sim}13$일까지의 사망으로써 Implantation sites, Placental remnants, Resorption of fetus로 관찰된 것이다. Fetal death는 수정후 $14{\sim}18$일까지의 사망으로써 Maceration of fetus로 관찰되는 것이다. 통계학적 분석은 각 Group의 착상 을과 자궁 내 사망 율을 산출할 때에는 각 임신마우스에 따라 발생빈도가 크게 다르기 때문에 통계처리에는 Non parametric 검정인 Kluskal Wallis 검정을 사용하여 분석하였다. 또한 개체 Level 영향인 착상을, 태아사망, 기형의 threshold dose의 산정에 대해서는 SAS-Logistic 검정에 따라 통계 분석을 하여 $5%(Ld_5,\;ED_5)$ 및 $10%{\times}2/3$점을 threshold dose로 판단했다. 태아체 중에 대해서는 parametric검정인 t-test검정에 의하여 분석했다. 그 결과 착상전기에서도 기형이 유발하며 특히 시기에 따라 일어나는 때와 일어나지 않는 때가 있음을 본 연구로부터 밝혀졌다. 또한 착상전기의 영향으로써 유발되는 기형은 여러 종류의 기형이 발생함이 밝혀졌다. 특히 이시기는 착상전 사망 및 배(胚)사망은 방사선 선량에 따라 크게 일어나나 태아사망(Fetal death) 및 태아체중은 유의차(有意差)가 없었다.

• 한국수정란이식학회지
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• 제11권1호
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• pp.35-40
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• 1996

This study was carried out to select the best cryoprotectant and to establish optimal concentration of the cryoprotectant in ultrarapid freezing of mouse 4-cell embryos and morulae, respectively. We investigated survival of ultrarapid frozen embryos according to various cryoprotectants such as glycerol, ethylene glycol, propylene glycol and dimethyl sulfoxide (DMSO). Suvival of the embryos frozen at different concentrations (3.0, 4.0 and 5.0 M) of indivisual cryoprotectant was also tested. Preimplantation developmental rate (96.3%, 83/86) of 4-cell mouse embryos treated with 4.0 M ethylene glycol after ultrarapid freezing and thawing was higher than those of other cryoprotectants (glycerol, propylene glycol and DMSO). In the ultrarapid freezing of mouse morulae, the highest developmental rate (98.8%, 89 /90) of the embryos to blastocysts was shown in the group of 5.0 M glycerol. Thus, these results demonstrate that 4.0 M ethylene glycol and 5.0 M glycerol are optimal for ultrarapid freezing of 4-cell mouse embryos and morulae, respectively.