• Clinical and Experimental Reproductive Medicine
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• 제27권3호
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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 $Ni^{2+}$.

• Clinical and Experimental Reproductive Medicine
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• 제40권4호
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• pp.148-154
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• 2013

Objective: To compare the mouse oocyte vitrification outcomes of the CryoLogic vitrification method (CVM) and the conventional open method using a Cryotop. Two CVM methods (original CVM and modified CVM) were tested. Methods: Mature oocytes obtained from female BDF-1 mice were vitrified by two-step exposure to equilibrium and vitrification solutions. Three vitrification protocols were tested on three groups: the CVM-kit, modified CVM, and Cryotop groups. After exposure to the two solutions, the oocytes were vitrified. After warming, the oocytes were fertilized in vitro, and the embryo development was assessed. Blastomeres positive for caspase were counted using an in situ assay kit. The spindle morphology and chromosome configurations of warmed vitrified oocytes were also assessed. Results: The modified CVM and Cryotop groups showed similar developmental capacities, and similar proportions of cells with intact spindles and chromosome configurations. The modified CVM protocol was superior to the original CVM protocol for developmental competence and intact spindle preservation. However, the CVM group showed a relatively higher number of apoptotic cells in blastocysts. Conclusion: Closed vitrification using the modified CVM protocol may be used as an alternative to the conventional open method, but strategies to decrease apoptosis in the blastomere need to be investigated.

• 한국동물생명공학회지
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• 제35권1호
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• pp.21-27
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• 2020

Somatic cell nuclear transfer derived embryonic stem cells (NT-ESCs) have significant advantages in various fields such as genetics, embryology, stem cell science, and regenerative medicine. However, the poor establishment of NT-ESCs hinders various research. Here, we applied fasudil, a Rho-associated kinase (ROCK) inhibitor, to develop somatic cell nuclear transfer (SCNT) embryos and establish NT-ESCs. In the study, MII oocytes were isolated from female B6D2F1 mice and performed SCNT with mouse embryonic fibroblasts (MEFs). The reconstructed NT-oocytes were activated artificially, and cultured to blastocysts in KSOM supplemented with 10 μM fasudil. Further, the blastocysts were seeded on inactivated MEFs in embryonic stem cell medium supplemented with 10 μM fasudil. A total of 26% of embryos formed into blastocysts in the fasudil treated group, while this ratio was 44% in the fasudil free control group. On the other hand, 30% of blastocysts were established NT-ESCs after exposure of fasudil, which was significantly higher than the control group (10%). The results suggest that fasudil reduced blastocyst development after SCNT due to inhibition of 2 cell cleavage while improved the establishment of NT-ESCs through the anti-apoptotic pathway.

• 한국가축번식학회지
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• 제20권3호
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• pp.233-238
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• 1996

This study was conducted to investigate the effects of cryoprotective agents and thawing temperature on the survival rate of the bisected embryos of mouse. The results of this study were summairzed as follows: 1. In in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed, the rates of normally developed bisected morula which was denuded were 31.7, 39.1, 28.0 and 23.1%, respectively. 2. The survival rates of bisected morula encased into the zona pellucida in in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed were 72.4, 68.7, 64.0 and 59.5%, respectively. 3. The survival rates of bisected denuded blastura in in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed were 48.3, 44.8, 32.1 and 28.6%, respectively. 4. The survival rates of bisected blasturae encased into the zona pellucida in in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed were 73.6, 67.4, 53.0 and 49.1%, respectively. 5. In in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed at room temperature, the rates of the normally developed bisected morula which was encased into the zona pellucida were 67.1, 62.3, 57.7 and 53.0%, respectively, and those of the bisected blasturae encased into the zona pellucida were 70.8, 65.4, 56.6 and 52.1%, respectively. 6. The survival rates of bisected morula which was encased into the zona pellucida in in vitro culture after forzen with DMSO, glycerol, ethylene glycerol or propanediol and thawed at 37$^{\circ}C$ were 74.3, 71.3, 63.9 and 57.4%, respectively, and those of bisected blastura encased into zona pellucida were 76.0, 69.1, 61.1 and 56.1%, respectively.

• Biomolecules & Therapeutics
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• 제20권3호
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• pp.280-285
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• 2012

The developing embryo naturally experiences relatively low oxygen conditions in vivo. Under in vitro hypoxia, mouse embryonic stem cells (mESCs) lose their self-renewal activity and display an early differentiated morphology mediated by the hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$). Previously, we demonstrated that histone deacetylase (HDAC) is activated by hypoxia and increases the protein stability and transcriptional activity of HIF-$1{\alpha}$ in many human cancer cells. Furthermore HDAC1 and 3 mediate the differentiation of mECSs and hematopoietic stem cells. However, the role of HDACs and their inhibitors in hypoxia-induced early differentiation of mESCs remains largely unknown. Here, we examined the effects of several histone deacetylase inhibitors (HDACIs) on the self-renewal properties of mESCs under hypoxia. Inhibition of HDAC under hypoxia effectively decreased the HIF-$1{\alpha}$ protein levels and substantially improved the expression of the LIF-specific receptor (LIFR) and phosphorylated-STAT3 in mESCs. In particular, valproic acid (VPA), a pan HDACI, showed dramatic changes in HIF-$1{\alpha}$ protein levels and LIFR protein expression levels compared to other HDACIs, including sodium butyrate (SB), trichostatin A (TSA), and apicidin (AP). Importantly, our RT-PCR data and alkaline phosphatase assays indicate that VPA helps to maintain the self-renewal activity of mESCs under hypoxia. Taken together, these results suggest that VPA may block the early differentiation of mESCs under hypoxia via the destabilization of HIF-$1{\alpha}$.

• Clinical and Experimental Reproductive Medicine
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• 제23권1호
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• pp.103-108
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• 1996

Through the previous studies(I,II), it was observed that human follicular fluid(HFF) was more effective than human fetal cord serum(HFCS) on promoting meiotic resumption of oocytes and improving embryonic development of mouse in vitro. On the basis of these results, we have gradually exchanged HFCS with HFF as protein supplement in human ART. This study was performed to investigate the efficiency of HFF on improving the pregnancy rate in ART. Oocytes were retrieved transvaginally from patients treated with pituitary suppression with GnRH-agonist and ovarian stimulation with human menopausal gonadotro-pin(HMG) and pure follicle stimulating hormone(FSH). Aspirated oocytes were rinsed and cultured in TCM-199 containing HFF, and the concentrations of HFF were adjusted to 10, 20, and 30% according to the use for insemination, embryo growth and embryo transfer, respectively. As possible as, we tried to do embryo transfer into fallopian tube to mimic the coincidence of the cell stage with the place of sojourn in vivo, so we performed various ART programs(IVF & ET; in vitro fertilization, ZIFT; zygote intra fallopian-tube transfer, ZIFT & ET) according to the tubal conditions of patients. On the while, intra cytoplasmic sperm injection(ICSI) was used to assist IVF of the patients who had shown poor standard IVF results by immunological or severe male factor. Of the 255 cycles of ART programs using HFF as protein supplement, 118 cycles were turn out to be succeeded in pregnancy(46.2%, per cycle, p<0.05), while 21 pregnancies were achieved in the 69 cycles using HFCS(30.4%). The 255 cycles using HFF were subdivided into cycles with the type of ART programs, and each pregnancy rate of the ART programs were 44.7% (IVF & ET, 76/170 cycles), 53.4%(ZIFT, 31/58 cycles) and 40.7% (ZIFT & ET, 11/27 cycles), respectively. In the 61 ICSI cycles using HFF, 28 cycles succeed in pregnancy(45.9%), while 7 pregnancies were obtained in the 17 ICSI cycles using HFCS. Also the ongoing pregnancy rate in the group using HFF(78.8%, 93/118 cycles) was higher than that in the group using HFCS(61.9%). Therefore, we found that the use of HFF as protein supplement was more suitable and effective than the use of HFCS to improve the pregnancy rate in ART.

• Current Research on Agriculture and Life Sciences
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• 제9권
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• pp.29-36
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• 1991

본(本) 연구는 생쥐의 4세포기배(細胞期胚)에서 하나의 할구(割球)를 뽑아내는 새로운 할구분유기술(割球分維技術)인 biopsy에 대한 효율성과 분리(分離)된 수정란(受精卵)과 분리(分離)한 할구(割球)의 생존성 및 새끼생산율을 검토한 결과(結果)이다. 그 결과(結果)를 요약(要約)하면 다음과 같다. 1. 4세포기배(細胞期胚)에서 분리(分離)한 단일해구(單一害球)와 4세포기배(細胞期胚)를 M2 배양액에 배양한 결과(結果), 단일해구(單一害球)는 82.6%가 영양배엽을 형성한 할구(割球)로 발생(發生)하였고 4세포기배(細胞期胚)로 89.5%가 배반포기배(胚盤胞期胚)로 발생(發生)하였다. 2. 4세포기배(細胞期胚)를 biopsy하여 하나의 할구(割球)가 분리(分離)된 수정란(受精卵)과 대조구인 4세포기배(細胞期胚)를 M2배양액에서 배양한 결과 각각 83.3%와 90.4%가 배반포기배(胚盤胞期胚)로 발생(發生)하였다. 3. Biopsy하여 4세포기배(細胞期胚)에서 분리(分離)한 단일해구(單一害球)와 대조구인 4세포기배(細胞期胚)에서 4개의 할구(割球)로 분리한 단일해구(單一害球)를 M2 배양액에서 배양한 결과 각각 80.8% 와 83.3%가 영양배엽을 형성한 할구(割球)로 발생(發生)하였다. 5. 4세포기배(細胞期胚)에서 하나의 할구(割球)를 뽑아낸 수정란(受精卵)과 대조구인 4세포기배(細胞期胚)를 수란생쥐에 이식한 결과 각각 36.0%와 48.6%의 새끼쥐 생산율(率)을 얻었다.

• Reproductive and Developmental Biology
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• 제29권1호
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• pp.1-7
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• 2005

텔로미어란 염색체 말단부에 (TTAGGG)n의 반복 염기서열이 단백질과 결합된 형태를 말하는데 이의 역할은 염색체의 안정성에 본질적으로 작용하여 세포의 노화, 사멸 및 암의 발생과 관련이 있다고 알려져 있다. 반면 텔로머레이스는 telomeric DNA 합성에 직접 관여하는 ribonucleoprotein이다. 본 연구에서는 마우스 염색체의 텔로미어 분포 양상을 제시하고, 초기 배 발생단계별 수정란의 텔로미어 함량과 이들 수정란의 텔로머레이스 활성도를 분석하고자 하였다. 본 분석에는 마우스의 섬유아세포, 생식세포, 정자, 난자 및 1세포기, 2세포기, 4세포기, 8세포기, 상실배와 배반포배의 각 단계별 수정란을 대상으로 하였다. 텔로미어의 양적 분석은 human telomeric DNA probe를 이용한 Q-FISH 방법을 이용하였고, 텔로머레이스 활성도는 TRAP 방법을 이용하였다. 분석 결과 마우스 염색체의 텔로미어는 성 염색체를 포함한 모든 염색체의 앙 말단부에 분포되어 있고, 염색체별 다소의 양적 차이를 보이나 대부분의 염색체에서 q-arm 말단이 p-arm 말단에 비해 높은 텔로미어의 함량을 나타내었다. Q-FISH를 이용한 마우스 초기 배 발생단계별 수정란의 텔로미어의 양적 분석에서 수정 직후 1세포기에서부터 상실배까지 거의 비슷한 텔로미어 함유율을 나타내고 있으나 배반포기에서 월등히 증가된 양상을 나타내었다. TRAP 분석을 이용한 초기배아의 텔로머레이스 활성도는 초기 배 발생 모든 단계에서 이의 활성도를 나타내었으며, 특히 상실배 및 배반포기에서 점진적으로 강한 활성을 보였다. 이상의 분석 결과로부터 마우스의 초기 배 분열단계의 각 세포들에 있어 텔로미어의 함유율과 텔로머레이스 활성도는 높은 상관관계가 있는 것으로 나타났다. 따라서 포유동물의 초기 배자에 있어 텔로미어의 함유율과 텔로머레이스 활성도는 배 발생 및 배자의 세포 분화와 매우 밀접한 관련이 있는 것으로 사료되어 텔로미어의 양적 분석 및 텔로머레이스 활성도 분석은 발생학적 연구를 위한 또 다른 좋은 자료로 생각된다.

• BMB Reports
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• 제40권5호
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• pp.656-661
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• 2007

In this report, we describe an optimized method for generation of ephA8 BAC transgenic mice expressing the lacZ reporter gene under ephA8 regulatory sequences. First, we constructed a targeting vector that carries a 1.2 kb ephA8 DNA upstream of its first exon, a lacZ expression cassette, a kanamycin cassette, and a 0.7 kb ephA8 DNA downstream of its first exon. Second, the targeting vector was electroporated into cells containing the ephA8 BAC and pKOBEGA, in which recombinases induce a homologous recombination between the ephA8 BAC DNA and the targeting vector. Third, the FLP plasmid expressing the Flipase was electroporated into these bacteria to eliminate a kanamycin cassette from the recombinant BAC DNA. The appropriate structures of the modified ephA8 BAC DNA were confirmed by Southern analysis. Finally, BAC transgenic mouse embryos were generated by pronuclear injection of the recombinant BAC DNA. Whole mount X-gal staining revealed that the lacZ reporter expression is restricted to the anterior region of the developing midbrain in each transgenic embryo. These results indicate that the ephA8 BAC DNA contains most, if not all, regulatory sequences to direct temporal and spatial expression of the lacZ gene in vivo.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2002년도 가을 학술발표논문집
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• pp.96-97
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• 2002

Primordial germ cells (PGCs) are the progenitors of the sperms or eggs of adult. Evidence suggests that the specification of primordial germ cells (PGCs) in the mammalian embryo does not depend on maternal determinants. Recent previous studies in the mouse has shown that several bone morphogenetic proteins (BMPs) are required for the formation of PGCs. However, there is no study about the effect of BMPs on avian PGCs. Here, we studied the effects of recombinant human BMP-2 (rhBMP-2) and recombinant human BMP-4 (rhBMP-4) on chicken blastodermal cells in culture. As a results, the addition of rhBMP-2 and rhBMP-4 increased the number of SSEA-1 positive cells in dose-dependent manner. However, there is no synergic effect by using both rhBMP-2 and rhBMP-4.