• 한국수정란이식학회지
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• 제20권1호
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• pp.1-8
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• 2005

본 실험은 LEY에 다양한 당류를 첨가한 용액으로 동결보존한 돼지정자의 응해후 생존율과 정상 첨체율, 체외성숙한 난자와의 수정능력과 배 발달 능력 및 인공수정을 통한 수태율과 산자수에 미치는 효과를 조사하기 위해 실시하였다. 융해후의 정상 첨체율은 glucose 농도 증가와 함께 증가되었으나, 생존율에 있어서는 효과가 인정되지 않았다. LEY에 fructose를 단독 또는 glucose와 함께 첨가하면 융해후의 생존율이 유의하게 증가되었으며 (p<0.05), 정상 첨체율에 있어서는 LEY 보존액에 fructose와 glucose를 첨가한 구가 $81.4{\pm}2.3\%$로 control의 $41.6{\pm}0.6\%$에 비하여 유의하게 높았다(p<0.001). 체외성숙 난자와의 수정율, 분할율 및 배반포 발생율은 $70.8\~80.7\%$, $44.6\~45.7\%$ 및 $13.6\~16.0\%$로 glycerol과 fructose 농도 및 정자농도 간에 차이가 인정되지 않았다. 1회 발정당 2회의 인공수정을 하였을 때, 수태율은 $83.3{\pm}0.1\%$, 산자수는 $9.4{\pm}1.7\~10.4{\pm}0.7$두로서 SGI사의 동결정액의 $50.0{\pm}0.1\%(p<0.05)$과 $8.0{\pm}1.1$두에 비하여 높았다.

• 한국수정란이식학회지
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• 제30권4호
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• pp.319-325
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• 2015

Gangliosides exist in glycosphingolipid-enriched domains on the cell membrane and regulate various functions such as adhesion, differentiation, and receptor signaling. Ganglioside GM3 by ST3GAL5 enzyme provides an essential function in the biosynthesis of more complex ganglio-series gangliosides. However, the role of gangliosides GM3 in porcine oocytes during in vitro maturation and early embryo development stage has not yet understood clear. Therefore, we examined ganglioside GM3 expression patterns under apoptosis stress during maturation and preimplantation development of porcine oocytes and embryos. First, porcine oocytes cultured in the NCSU-23 medium for 44 h after $H_2O_2$ treated groups (0.01, 0.1, 1 mM). After completion of meiotic maturation, the proportion MII (44 h) was significantly different among control and the H2O2 treated groups ($76.8{\pm}0.3$ vs $69.1{\pm}0.4$; 0.01 mM, $55.7{\pm}1.0$; 0.1 mM, $38.2{\pm}1.6%$; 1 mM, P<0.05). The expressions of ST3GAL5 in $H_2O_2$ treated groups were gradually decreased compared with control group. Next, changes of ST3GAL5 expression patterns were detected by using immunofluorescene (IF) staining during preimplantation development until blastocyst. As a result, we confirmed that the expressions of ST3GAL5 in cleaving embryos were gradually decreased (P<0.05) according to the early embryo development progress. Based on these results, we suggest that the ganglioside GM3 was used to the marker as pro-apoptotic factor in porcine oocyte of maturation and early embryo production in vitro, respectively. Furthermore, our findings will be helpful for better understanding the basic mechanism of gangliosides GM3 regulating in oocyte maturation and early embryonic development of porcine in vitro.

• 한국수정란이식학회지
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• 제13권2호
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• pp.89-96
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• 1998

Anti-phospholipid antihodies (aPL) have important roles in various pregnancy complications such as recurrent miscarrige, growth retardation, placental abruption and stillbirth. However, their biological actions on preimplantation development of oocytes are still unclear. In this study, we investigated whether either aPL containing sera or phospholipids could affect in vitro fertilization and development of mouse oocytes. Sera used in this study were collected from three patients and the presence of aPL in the sera was confirmed by enzymatic-linked immunosorbent assay. When mouse oocytes were cultured in a serum-free, Chatot, Ziomek and Bavister (CZB) medium (Experiment 1), addition of aPL-containing sera (10%) to CZB medium did not. significantly (P>0.05) influence sperm penetration of oocytes. However, development to the blastocyst stage was significantly (P<0.05) inhibited by serum addition, and formation of morulae (16-23% vs. 58%) and blastocysts (0-4% vs. 21%) was markedly reduced compared with no addition (Experiment 2). In Experiment 3, pronuclear stage embryos were cultured for 96 h in GZB medium supplemented with 1 $\mu$g /ml phosphatidyl ethanolamine, 1 $\mu$g/ml phosphatidyl inositol or 1 $\mu$g /ml phosphatidyl choline. No increase in embryo development was found after addition of the phospholipids to CZB medium. These results suggest that 1) aPL have an inhibitory role in preimplantation development of mouse embryos, and that 2) the action of aPL may be related to a specific phospholid (s) rather than the tested phospholipids in the present study.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.313-317
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• 2011

This study was conducted to examine the effect of oocyte donor age and micromanipulation medium on the development of mouse cloned embryos receiving cumulus cells. Mouse oocytes were obtained from 6 to 11 week-old mice BDF1 female mice(experiment 1) and cumulus cells were used as donor cells. Micromanipulation procedures for nuclear transfer(NT) were performed in FHM, M2 or Hepes-buffered TCM199(TCM199) medium(experiment 2). After nuclear transfer, the reconstructed oocytes were activated by 10 mM $SrCl_2$ in Ca-free CZB medium in the presence of 5 II ${\mu}$g/ml cytochalasin B for 5 h and cultured in KSOM medium for 4 days. In experiment 1, the survival rate of oocytes after injection of cumulus cells were significantly(p<0.05) lower in oocytes from 6~7 week-old mice(53.3%) than in oocytes from 8~9(80.9%) and 10~11 week-old mice(77.1%). In experiment 2, the survival rate of oocytes after cell injection were significantly(p<0.05) higher in FHM and M2 medium(71.7% and 76.9%) than in TCM199 medium(51.2%). The activation rates of cloned embryos were not different among the micromanipulation media. However, the embryos developed to blastocyst stage were significantly(p<0.05) higher in FHM medium(13.9%) than in M2 and TCM199 medium(0.0% and 0.0%). In conclusion, the present study suggest that oocytes from above 8 week-old mice are superior to oocytes from 6~7 week-old mice as a source of recipient cytoplasm and FHM is superior to M2 and TCM199 as a micromanipulation medium for mouse somatic cell cloning.

• 한국수정란이식학회지
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• 제11권3호
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• pp.249-258
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• 1996

To improve the efficiency of in vitro production of embryos with follicular oocytes in Korean Native cows, the recovery rates, in vitro maturation, fertilization and development, and the time required for collecting and processing oocytes by aspiration with or without slicing were evaluated comparatively. The ovaries were obtained from a local abattoir and placed in physiological saline at 25~28$^{\circ}C$ and brought to the laboratory within 3 hrs. The oocytes were collected by aspiration of follicles(2~6mm) with or without slicing ovaries after aspiration, and classified into Grade I, Grade II, Denuded, Expanded oocytes by the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules. Also the time required for each step of collecting and processing oocytes were measured. The cumulus cells were removed in some Grade I oocytes to measure their size and nuclear configuration before and after in vitro maturation. The Grade I oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35$\mu$g /ml FSH, 10$\mu$g /ml LH, 1 $\mu$g /ml at 39$^{\circ}C$ under 5% C02 in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24hrs. and then the zygotes were cocultured in vitro (IVC) with bovine oviductal epithelial cells for 10 days. The results obtained were as follows: The number of oocytes recovered per ovary was averaged 6.6 by aspiration and 11.2 by slicing post aspiration, which summed to 17.8. The number of Grade I oocytes recovered per ovary was averaged 3.1 by aspiration and 3.6 by slicing, which summed to 6.7. The percentage of Grade I to total oocytes recovered was significantly(P<0.05) higher as 48.0 % in aspiration than 31.6% in slicing post aspiration. The time requlred for recovering a Grade I oocyte by aspiration and slicing was 1.1 and 2.5 min, respectively. The mean diameter of Grade I oocytes by aspiration and slicing was similar as 148.7 and 151.5$\mu$m, respectively. The percentage of Metaphase II stage oocytes after IVM for 24 hours was significantly (P

• 한국수정란이식학회지
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• 제29권2호
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• pp.141-148
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• 2014

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein $50{\mu}M$ for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and $50{\mu}M$ and genestein 1 and $50{\mu}M$ for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and $50{\mu}M$, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin $1{\mu}M$ for 3 hr incubation. In mitochondrial activities, addition of quercetin $50{\mu}M$ for 6 hr incubation increased mitochondrial activity but decreased at $100{\mu}M$ concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin ($1{\sim}10{\mu}M$), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin ($25{\sim}50{\mu}M$). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin $10{\mu}M$ (p<0.05). The developmental rates to morula or blastocysts at low ($0.01{\sim}1{\mu}M$) and high ($5{\sim}10{\mu}M$) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and $10{\mu}M$) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations ($0.01{\sim}10{\mu}M$).

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.102-102
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• 2002

Heparin-bindin epidermal growth factor (HB-EGF) is one of the EGF family to be expressed at the time of implantation in the mouse uterus. Although HB-EGF has been shown to stimulate the development of embryo and uterus in the mouse, its correlation between cell adhesion molecules remains undefined. Integrin $\alpha$$_{ν}$$\beta$$_3$, one of the cell adhesion molecules, is an important mediator of cell-substratum and cell-cell adhesion in implantation. In the present studies, we investigated the effects of HB-EGF on the embryonic development, initiation of implantation and expression of integrin $\alpha$$_{ν}$$\beta$$_3$ in in vitro culture, blocking of HB-EGF, RT-PCR and immunofluores cence analysis. The results showed that HB-EGF significantly improved the developmental rate of hatched embryos (24.1%, p<0.01) and outgrowth embryos (42.5%, p<0.01). On the other hand, this growth factor showed no offset before the hatching embryonic stage. Analysis of RT-PCR showed that HB-EGF upregulated the expression level of integrina $\alpha$$_{ν}$$\beta$$_3$ subunit genes on the preimplantation embryo and outgrowth of blastocyst (120hr and 144hr after hCG injection). Immunofluorescence analysis showed that the integrin $\alpha$$_{ν}$$\beta$$_3$ subunits localized at the pericellular borders and cell-cell contact areas. Increase in fluorescence intensity was observed in the HB-EGF treated embryos. Intrauterine injection of an anti-HB-EGF antiserum at day 3 significantly decreased the number of implantation sites (14.4, p<0.01) and significantly increased the number of recovered embryos(6.4, p<0.05) at day 5. From these results, it imply that HB-EGF improve the embryo development and accelerated the expression of integrin $\alpha$$_{ν}$$\beta$$_3$ in the preimplantation mouse embryos.

• 한국수정란이식학회지
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• 제28권3호
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• pp.251-256
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• 2013

The present study was performed to investigate the effect of Hh-Ag1.5, a small-molecule chemical agonist of SMOothened receptor, on the in vitro maturation and development of in vitro fertilized (IVF) embryos in pigs. Oocytes or fertilized embryos were cultured in a maturation or embryo culture medium supplemented with 0 (control), 25, 50 or 100 nM of Hh-Ag1.5, respectively. Although the maturation rate were not different among treatment groups, the blastocyst formation rate in the group treated with 25 nM Hh-Ag1.5 was significantly increased compared to other groups (P<0.05). While the highest dose of Hh-Ag1.5 (100 nM) did negatively affect to the embryo development and cell number in blastocysts compared to other groups (P<0.05), the apoptotic cell index in blastocysts was significantly lower in 25 and 50 nM groups than in control and 100 nM groups (P<0.05). The mRNA expression of the proapoptotic gene Bax and the ratio of Bax/Bcl-XL decreased in among treatment groups compared to control (P<0.05). The embryo quality related genes, Tert and Zfp42, were significantly decreased in 50 and 100 nM groups compared with control and 25 nM groups (P<0.05). In conclusion, the addition of 25 nM Hh-Ag1.5 to in vitro maturation and culture medium can enhance the developmental potential as well as quality of IVF embryos in pig.

• 한국수정란이식학회지
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• 제16권2호
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• pp.99-106
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• 2001

본 연구에서 도축된 한우 난소를 aspiration법과 면도날 장착으로 제작된 기구를 이용해서 slicing 법으로 난포란을 회수하여 그에 따른 난포란의 회수율과, 난포란을 체외에서 22시간 성숙시킨 후 난포란의 핵 성숙을 및 체외수정 후 수정율과 발달율을 요약하면 다음과 같다. 1. 난포란의 회수율에 있어서 각 난소당 회수는 aspiration법이 6.7개. slicing법이 15.1개의 결과를 나타내어 난소에 대한 난포란의 회수율은 slicing법이 유의적 (P<0.05)으로 높은 결과를 나타내었다. 2 Aspiration법과 slicing법으로 채란된 난자를 체외에서 22시간 성숙시킨 후, 제 2감수분열 중기까지의 핵 성숙율은 각각 83%와 62%로 유의적인(P<0.05) 차이를 보였으나, 난소 한 개당 제 2감수분열 중기까지 성숙된 난포란수는 aspiration과 slicing법이 각자 5.6개와 9.4개로 slicing법에 의해서 유의적 (P<0.05)으로 많았다. 3. Aspiration법과 slicing법으로 채란된 난포란의 발달을 조사결과를 살펴보면 분할율과 배반포기까지의 발달율에서 aspiration법이 유의적 (P<0.07)으로 높게 나타났으나, 이와 상반되게 난소 한 개당 생산된 배반포기 수정란의 수는 slicing법에 있어서 2.8개로 aspiration법의 2.1개보다 유의적인 (P<0.05) 증가를 보였다. 이상의 결과에 따라 많은 난자를 이용하기 위한 목적과 혹은 이식 가능한 다수의 수정란 생산을 위해서는 본 연구에서 고안 제작된 기구를 이용하여 slicing법으로 난포란을 채란하는 것이 효과적인 방법이 될 것으로 사료된다.

• 한국가축번식학회지
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• 제21권1호
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• pp.71-78
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• 1997

본 연구는 체외성숙 직후의 소 미수정란의 활성화 조건을 검토하기 위하여 실시하였다. 체외에서 22~24시간 성숙배양된 소 미수정란을 다양한 활성화 조건으로 처리하였다. 실험 1에서는 성숙직후 난자를 전기자극(1.25kV/cm, 70$\mu$sec$\times$2회), 에탄올(7%, 5분), Ca2+-ionophore(A23187; 10$\mu$M, 5분) 및 cycloheximide(10$\mu\textrm{g}$/ml, 6시간)로 각각 처리하였다. 활성화율은 전기자극, 에탄올 및 A23187 처리시 48.8~54.3%로 비슷하였으나, cycloheximide처리시는 15.9%로 유의적으로(P<0.05) 낮았고, 무처리구인 대조군은 전혀 활성화 되지 않았다. 실험 2에서 체외성숙 미수정란을 전기자극, 에탄올 및 A23187중 2개 또는 3개를 병용처리한 결과, 활성화율은 46.9~63.5%로 나타났다. 실험 3에서는 전기자극, 에탄올 또는 A23187과 cycloheximide를 병용처리한 결과, 대부분의 난자(98.0~100%)가 활성화되었다. 실험 4에서는 실험 3과 같은 조건으로 처리하여 할성화된 난자를 cytochalasin B로 처리하여 2배체배 형성을 유도한 후 체외배양하여 발육율을 검사하였다. 분할율은 79.8~90.4%였으며, 배반포 발육율은 32.1~42.6%로 나타났다. 이러한 결과는 전기자극, 에탄올 또는 A23187과 chcloheximide의 병용처리가 성숙직후 소 미수정란의 활성화에 효과적임을 확증한다.