• 한국수정란이식학회지
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• 제20권3호
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• pp.191-200
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• 2005

체외 배양 과정 중에 나타나는 생쥐 초기 2-세포 배의 "in vitro 2-cell block" 현상은 세포내 $Ca^{2+}$ 농도 변화와 밀접한 관련이 있다. 다양한 종류의 세포에서 acetylcholine은 세포막에 존재하는 muscarnic acetylcholine receptor를 통해 세포내 $Ca^{2+}$ 농도 증가를 유도한다. 본 실험에서는 생쥐 "in vitro 2-cell block" 현상에 있어서 ACh의 영향을 알아보기 위해 세포 내 $Ca^{2+}$ 농도 조절 물질을 처리한 후, 공초점 현미경을 이용하여 세포 내 $Ca^{2+}$ 농도 변화를 기록하였다. ACh은 세포 내에서 농도 의존적으로 $Ca^{2+}$ 농도 증가를 유도하며, "in Vitro 2-cell block" 현상을 극복하여 포배기로 발생을 유도하였다. ACh에 의한 $Ca^{2+}$ 농도 증가가 세포막에 존재하는 ACh receptor를 경유하여 나타나는 반응인지를 알아보기 위해 ACh receptor의 저해제인 atropine을 전처리한 결과, ACh에 의한 $Ca^{2+}$ 농도 증가가 완전히 저해되었다. 초기 2-세포 배에서 ACh이 결합하는 receptor의 종류를 확인하기 위하여 carbachol과 nicotin tartrate를 처리 하였다. Nicotinic AChR의 agonist인 nicotine tartrate 1 mM은 세포내 $Ca^{2+}$ 농도 증가를 보이지 않았다. 따라서 초기 2-세포 배의 세포막에는 muscarnic AChR가 기능적으로 작용함을 알 수 있다. ACh에 의한 세포내 $Ca^{2+}$ 농도 증가가 $Ca^{2+}$이 제거된 배양액에서도 나타나는 것으로 보아 ACh에 의한 세포내 $Ca^{2+}$ 변화는 주로 소포체와 같은 세포내 $Ca^{2+}$ 저장고로부터 분비됨을 알 수 있었다. 이러한 세포내 $Ca^{2+}$ 저장고로부터의 $Ca^{2+}$ 분비가 어떤 신호전달체계를 통해 나타나는 지를 조사하였다. 세포막의 PLC 저해제인 U73122를 전처리한 배는 ACh에 의한 $Ca^{2+}$ 농도 증가가 나타나지 않았으며, 세포 내 $Ca^{2+}$ 통로인 IP3R와 RyR의 저해제인 xestospongin과 heparin 혹은 dantrolene을 전처리한 결과 dantrolene에 의해 세포내 $Ca^{2+}$ 농도 증가가 억제되었다. 그리고 세포내 반복적인 $Ca^{2+}$ 농도 증가에 의해 활성도가 변화는 CaMKII의 작용을 확인하기 위하여 Ca MKII의 저해제인 KN-93을 전처리한 결과 $Ca^{2+}$ 농도 증가가 억제되는 것을 확인하였다. 이상의 결과로부터 ACh은 생쥐 초기 2-세포 배에서 ryano-dine receptor를 통하여 세포내 $Ca^{2+}$ 저장고로부터 $Ca^{2+}$ 분비를 유도하며, CaM KII에 의해서도 영향을 받는 것으로 보여진다. 생쥐 초기 2-세포 배에서 "in vitro 2-cell block"의 극복은 ACh에 의해 유도된 신호전달체계를 통해 세포내에 증가하는 $Ca^{2+}$ 농도 및 이에 따른 세포내 대사 작용의 활성화에 의하여 나타나는 것으로 생각된다.

• Asian-Australasian Journal of Animal Sciences
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• 제24권2호
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• pp.190-197
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• 2011

The objective of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co-culture on developmental capacity of bovine IVM/IVF embryos and to determine whether or not melatonin acts as an antioxidant in BOEC culture and subsequent embryo development. These studies examined the effects of melatonin against NO-induced oxidative stress on cell viability, lipid peroxidation (LPO) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl-2, Caspase-3 and Bax) during BOECs culture. We also evaluated the developmental rates of bovine IVM/IVF embryos with BOEC co-culture, which were pre-treated with melatonin ($1,000\;{\mu}M$) in the presence or absence of sodium nitroprusside (SNP, $1,000\;{\mu}M$) for 24 h. Cell viability in BOECs treated with SNP (50-$2,000\;{\mu}M$) decreased while melatonin addition (1-$1,000\;{\mu}M$) increased viability in a dose-dependent manner. Cell viability in melatonin plus SNP ($1,000\;{\mu}M$) gradually recovered according to increasing melatonin addition (1-$1,000\;{\mu}M$). The LPO products were measured by thiobarbituric acid (TBA) reaction for malondialdehyde (MDA). Addition of melatonin in BOEC culture indicated a dose-dependent decrease of MDA, and in the SNP group among BOECs treated with SNP or melatonin plus SNP groups MDA was significantly increased compared with SNP plus melatonin groups (p<0.05). In expression of apoptosis or antioxidant genes detected by RT-PCR, Bcl-2 and antioxidant genes were detected in melatonin or melatonin plus SNP groups, while Caspase-3 and Bax genes were only found in the SNP group. When bovine IVM/IVF embryos were cultured for 6-7 days under the BOEC co-culture system pre-treated with melatonin in the presence or absence of SNP, the highest developmental ability to blastocysts was obtained in the $1,000\;{\mu}M$ melatonin group. These results suggest that melatonin has an anti-oxidative effect against NO-induced oxidative stress on cell viability of BOECs and on the developmental competence of bovine IVM/IVF embryo co-culture with BOEC.

• 한국발생생물학회지:발생과생식
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• 제26권2호
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• pp.79-90
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• 2022

Krüppel-like factor 10 (KLF10) regulates various cellular functions, such as proliferation, differentiation and apoptosis, as well as the homeostasis of several types of tissue. In the present study, we attempted a loss-of-function analysis of zebrafish Klf11a and Klf11b, which constitute human KLF10 homologs. Embryos injected with klf11b-morpholino (MO) showed developmental retardation and cell death, whereas klf11a-MO-injected embryos showed normal development. In klf11b-MO-injected embryos, a dramatic increase in the amount of zebrafish p53 mRNA might be the cause of the increase in that of bax. The degree of apoptosis decreased in the klf11b-MO and p53-MO co-injected embryos. These findings imply that KLF10 is a negative regulator of p53-dependent transcription, suggesting that the KLF10/p53 complex may play an important role in apoptosis for maintenance of tissue homeostasis during embryonic development.

• Asian-Australasian Journal of Animal Sciences
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• 제14권9호
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• pp.1260-1266
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• 2001

The effect of protein supplementation, $O_2$ concentration and co-culture on the development of embryos produced by nuclear transfer using cultured cumulus cell was investigated. Recipient oocytes and cumulus cells were obtained from the ovaries of the slaughtered Hanwoo cows. Donor cumulus cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 5% $CO_2$ in air at $38.5^{\circ}C$. The 1 to 6 passages of cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One $15{\mu}s$ pulse of 180 volts was applied to induce the fusion between karyoplast and cytoplast. The fused embryos were activated with $10{\mu}M$ calcium ionophore for 5 min and 2 mM 6-dimethylaminopurine for 3 h. To examine the effect of protein supplementation, nuclear transfer (NT) embryos were cultured in one of the following 4 treatments : 1) CR1aa + 3 mg/ml BSA for 7 days ; 2) CR1aa + 10% FBS for 7 days ; 3) CR1aa + 1.5 mg/ml BSA + 5% FBS for 7 days ; and 4) CR1aa + 3 mg/ml BSA for first 3 days and then CR1aa + 1.5 mg/ml BSA + 5% FBS for 4 days. Culture took place at 5% $CO_2$, 5% $O_2$ and 90% $N_2$ at $38.5^{\circ}C$. Although there were no significant differences in cleavage rate among different protein supplements, the rates of blastocyst formation were significantly different. When NT embryos were cultured in the medium supplemented with only BSA, they could develop to only morula not to blastocyst. However, when FBS was supplemented, NT embryos developed to blastocyst stage. In order to investigate the effect of $O_2$ concentration and co-culture, NT embryos were cultured in CR1aa + 1.5 mg/ml BSA + 5% FBS with or without cumulus cell co-culture at an atmosphere of 5% $CO_2$ in air (20% $O_2$) or 5% $CO_2$, 5% $O_2$, 90% $N_2$ (5% $O_2$) at $38.5^{\circ}C$ for 7 days. The percentage of blastocyst development was significantly higher when the NT embryos were cultured at an atmosphere of 5% $O_2$ than that of 20% $O_2$ (p<0.05). However, there was no significant difference between with and without cumulus cell co-culture at an atmosphere of 5% $O_2$ or 20% $O_2$. Fifty embryos were transferred to 25 recipients and 5 recipients were pregnant at 100 days. From 5 pregnant cows, only one cow was delivered of female twin. In conclusion, the embryos reconstructed by enucleation of metaphase II oocytes and introduction of the cycling and quiescent cumulus donor cells in Hanwoo had developmental potential to term after embryo transfer to recipient cows.

• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.35-41
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• 1998

This study was carried out to investigate the development rates of human embryos co-cultured with cumulus cells to each blastocyst stage. Human zygotes were co-cultured on cumulus cell monolayer in YS medium supplemented with 20% hFF. On day 2, if patient had four or more "good" embryos (regular blastomeres without fragmentation), embryos were further cultured for 72hrs. Blastocysts on day 5 were classified into early blastocyst (ErB), early expanding blastocyst (EEB), middle expanding Blastocyst (MEB), and expanded blastocyst (EdB) on the basis of their morphological aspects of trophectoderm cells and blastocoele. Subsequently, maximum 3 of best blastocysts were transferred in 486 cycles. The results in this study were as follows: Patients who had four or more "good" embryos on day 2 were 498 persons, but patients whose embryos could not be transferred due to failure in development to the blastocyst stage on day 5 were 12 persons (2.4%). The development rate of embryos to the blastocyst stage was 58.2% (2,885/4,957) on day 5, and the rates that developed to the ErB, EEB, MEB, and EdB stage were 15.0% (743/4,957), 14.9% (739/4,957), 14.4% (714/ 4,957), and 13.9% (689/4,957), respectively. Total 1366 blastocysts were transferred in 486 cycles (mean number=2.81). The implantation rate and the ongoing implantation rate obtained by observing the number of G-sac and FHB were 29.9% (409/1,366) and 22.5% (308/1,366), respectively. The clinical pregnancy rate was 51.2% (249/486), and the ongoing pregnancy rate' was 39.1% (190/486). Among women showing ongoing pregnancy, women with singleton were 50% (95/190), women with twin were 37.9% (72/190), and women with triplet were 12.1% (23/190). Although triplet pregnancy rate in this study was high such as 12.1%, because many blastocysts with high viability were produced in our co-culture system using cumulus cells on day 5, we really believe that a multiple pregnancy except twin should not occur by selecting good embryos for maximum two blastocyst transfer. These results demonstrate that autologous cumulus cells may be used for the production of blastocysts with high developmental competence, and the use of autologous cumulus cells to be collected easily, and to be treated conveniently at OPU must be an effective means for obtaining high implantation and pregnancy rate.

• 한국수정란이식학회지
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• 제29권4호
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• pp.345-350
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• 2014

We investigate the effect of L-glutathione (GSH), an antioxidant, treatment during the somatic cell nuclear transfer (SCNT) procedures on the in vitro development and DNA methylation status of bovine SCNT embryos. Bovine in vitro matured (IVM) oocytes were enucleated and electrofused with a donor cell, then activated by a combination of Ca-ionophore and 6-dimethylaminopurine. The recipient oocytes or reconstituted oocytes were treated with $50{\mu}M$ GSH during these SCNT procedures from enucleation to activation treatment. The SCNT embryos were cultured for 7 days to evaluate the in vitro development, apoptosis and DNA methylation in blastocysts. The apoptosis was measured by TUNEL assay and caspase-3 activity assay. Methylated DNA of SCNT embryos at the blastocyst stages was detected using a 5-methylcytidine (5-MeC) antibody. The developmental rate to the blastocyst stage was significantly higher (P<0.05) in GSH treatment group ($32.5{\pm}1.2%$, 78/235) than that of non-treated control SCNT embryos ($22.3{\pm}1.8%$, 50/224). TUNEL assay revealed that the numbers of apoptotic cells in GSH treatment group ($2.3{\pm}0.4%$) were significantly lower (P<0.05) than that of control ($3.8{\pm}0.6%$). Relative caspase-3 activity of GSH treated group was $0.8{\pm}0.06$ fold compared to that of control. DNA methylation status of blastocysts in GSH treatment group ($13.1{\pm}0.5$, pixels/embryo) was significantly lower (P<0.05) than that of control ($17.4{\pm}0.9$, pixels/embryo). These results suggest that antioxidant GSH treatment during SCNT procedures can improve the embryonic development and reduce the apoptosis and DNA methylation level of bovine SCNT embryos, which may enhance the nuclear reprogramming of bovine SCNT embryos.

• 한국수정란이식학회지
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• 제9권2호
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• pp.181-188
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• 1994

• Asian-Australasian Journal of Animal Sciences
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• 제18권1호
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• pp.17-21
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• 2005

The effect of melatonin on in vitro embryo development and the expression of antioxidant enzyme gene in preimplantation porcine embryos was determined by modified semi-quantitative single cell RT-PCR. Porcine embryos derived from in vitro maturation /in vitro fertilization were cultured in 5% $CO_2$ and 20% $O_2$ at $37^{\circ}C$ in NCSU23 medium. Melatonin was added to medium at concentration of 1nM, 5 nM, and 10 nM. When treated with 1nM (39.0%) of melatonin, the developmental rate of embryos beyond the morula stage were higher than that of control group (31.0%) (p<0.05). Number of inner cell mass and tropectoderm cell in control (23.0${\pm}$0.5 and 17.3${\pm}$0.8), 1 nM (23.6${\pm}$0.6 and 19.0${\pm}$0.5), and 5 nM (23.3${\pm}$1.1 and 16.3${\pm}$0.8) treated with melatonin were higher than in 10 nM (20.0${\pm}$0.5 and 13.3${\pm}$0.8) treated with melatonin (p<0.05). To develop an mRNA phenotypic map for the expression of catalase, bax and caspase-3, single cell RT-PCR analysis were carried out in porcine IVM/IVF embryo. Catalase was detected in 0, 1 and 5 nM supplemented with melatonin, but bax and caspase-3 were detected in 10 nM treated with melatonin.

• 한국발생생물학회지:발생과생식
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• 제19권1호
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• pp.53-60
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• 2015

Environmental conditions during early mammalian embryo development are critical and some adaptational phenomena are observed. However, the mechanisms underlying them remain largely masked. Previously, we reported that AQP5 expression is modified by the environmental condition without losing the developmental potency. In this study, AQP11 was examined instead. To compare expression pattern between in vivo and in vitro, we conducted quantitative RT-PCR and analyzed localization of the AQP11 by whole mount immunofluorescence. When the fertilized embryos were developed in the maternal tracts, the level of Aqp11 transcripts was decreased dramatically until 2-cell stage. Its level increased after 2-cell stage and peaked at 4-cell stage, but decreased again dramatically until morula stage. Its transcript level increased again at blastocyst stage. In contrast, the levels of Aqp11 transcript in embryos cultured in vitro were as follows. The patterns of expression were similar but the overall levels were low compared with those of embryos grown in the maternal tracts. AQP11 proteins were localized in submembrane cytoplasm of embryos collected from maternal reproductive tracts. The immune-reactive signals were detected in both trophectoderm and inner cell mass. However, its localization was altered in in vitro culture condition. It was localized mainly in the plasma membrane of the blastocysts contacting with external environment. The present study suggests that early stage embryo can develop successfully by themselves adapting to their environmental condition through modulation of the expression level and localization of specific genes like AQP11.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.43-43
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• 2003

This study was conducted to produce transgenic pig harboring human tissue plasminogene activator (tPA) gene. Two different tPA genes containing bovine $\beta$-casein promoter and mouse uroplakin promoter were prepared for microinjection and confirmed the expression level of tPA protein from the CHO (Chinese hamster ovary) cell lines by gene transfection. Concentration of tPA expression from the six cell lines (all of CHO cells) were average 212.4 ng/ml. Reconstructed DNA to used the CHO cell were microinjected into the pronuclei of in vivo embryos The total of 2,307 zygotes were collected from 95 donors and 1,851 embryos were in 1-cell stage which were visualized the pronuclei for DNA microinjection. The concentration of linear DNA was 2.0 ng per microliter and injected into zygotes with two pronuclei on an inverted Nikon microscope equipped with narishige micromanipulator and modulation contrast optics. The 541 embryos injected with bovine $\beta$-casein promoter-tPA were transferred to 22 recipients. The 1,154 embryos injected with mouse uroplakin promoter-tPA were transferred to 51 recipients. Sixty nine offspring from 9 delivered sows were produced. We analysed the transgenes with PCR methods from 69 offsprings, but could not detect the PCR product from piglet tails DNA.