• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.95-95
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• 2003

Inositol은 세포의증식 및 정보전달과정에 관여하는 phosphatidylinositol (PI)의 구성성분으로서 중요한 세포내 기능을 수행한다. P1는 세포내에서 특이적 인산화효소에 의하여 Pl4-phosphate(PIP), Pl4,5-phosphate($PIP_2$)로 변환되며 PIP$_2$는 phospholipase C(PLC)에 의하여 세포내 second messengers인 1,2-diacylglycerol (DAG)와 inositol 1,4,5-triphosphate ($IP_3$)로 변환된다. 이렇게 생산된 DAG와 IP3는 각각 protein kinase C의 활성과 $Ca^{2++}$의 동원에 관여하여 다양한 세포내 신호전달에 관여하는 것으로 보고되고 있다. 또한 mouse에서 $IP_3$의 작용에 의한 $Ca^{2++}$의 상승은 난모세포의 성숙분열이 촉진되고 돼지 난포세포에 있어서도 PI대사가 일어나고 있는 것으로 보고되고 있다. 본 연구에서는 돼지 미성숙 난모세포의 성숙과 단위발생에 미치는 inositol의 영향을 확인하기 위하여 실시하였다. inositol의 농도가 난포란의 성숙에 미치는 영향을 검토하기 위하여 난포란을 각각 $150 \mu mole$, $250 \mu mole$, $350 \mu mole$, inositol을 포함하는 Whitten's 배양액에서 44시간 성숙시킨 결과 $93.57 \pm 4.21, 93.91 \pm 2.71, 92.96 \pm 3.58%$가 성숙되어 대조구의 $87.10 \pm 4.21$보다 유의적(P<0.05)으로 차이를 나타내었다. 난포란의 등급에 따른 inositol의 영향을 확인하기 위하여 형태적으로 난구세포가 치밀한 난포란과 난구세포가 치밀하지 않은 난포란을 $250 \mu mole$ inositol을 포함하는 Whitten's 배양액에서 성숙을 유도한 결과 난구세포가 치밀한 난포란과 난구세포가 치밀하지 않은 난포란에서 inositol을 첨가하였을 때 성숙율은 각각 $95.35 \pm 2.22와 63.55 \pm 8.12$로 inositol을 첨가하지 않은 대조구보다($89.21 \pm 3.69와 48.56 \pm 8.99$) 유의적인 차이를 보였다. 난구세포가 inositol에 의한 성숙에 미치는 영향을 확인하기 위하여 난구세포를 제거한 난포란을 inositol을 포함하는 배지에서 성숙을 유도한 결과 inositol을 첨가하지 않은 처리구보다 양호한 성숙율을 보였다.

• Applied Biological Chemistry
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• v.46 no.1
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• pp.50-54
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• 2003

Polygonum cuspidatum has been used as a fork medicine for a long time. Emodin was purified from the root of P. cuspidatum by thin layer chromatography (TLC) and preparative high perfomance liquid chromatography (HPLC). The effects of emodin on the migration of endothelial cells and in vitro angiogenesis stimulated with vascular endothelial cell growth factor (VEGF) were examined, using human umbilical vein endothelial cells (HUVECs) and porcine pulmonary arterial endothelial cells (PPAECs). Emodin potently inhibited the VEGF-induced migration of (HUVECs) at relatively low cocentrations $(0.1-10\;{\mu}g/ml)$; the inhibition of endothelial cells by emodin was 75.4% at $0.1\;{\mu}g/ml$ and about 90% at $1\;{\mu}g/ml$. Emodin also inhibited VEGF-induced sprout formation in vitro at concentrations of $0.1-10\;{\mu}g/ml$. Emodin was also evaluated for the inhibitory potential on in vivo angiogenesis in a growing chick embryo chorioallantoic membranes (CAM). At a concentration of $1.0\;{\mu}g/ml$ Per disc, emodin was able to induce avacular zone in the CAMs. These findings suggest that emodin is a potent angiogenesis inhibitor and P. cuspidatum is a useful herb in the development of therapeutics for angiogenesis dependent diseases.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.26-26
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• 2003

Hyaluronic acid (HA)-binding sites have been shown the diagnostic potential fur assessment of sperm maturity, which is related to male fertility. This study was designed to evaluate chromosomal patterns in porcine embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with non- or HA-binding sperm (HABS). For binding of sperm with HA, sperm incubated in 10 ${mu}ell$ drop containing HA (0.8 mg/ml)-agarose (0.8%) mixture for 15 min. IVF and ICSI with non- or HA-bound sperm examined with matured oocytes at 44 hr after in vitro maturation. Embryos were cultured in 50 ${mu}ell$ of NCSU 23 containing 0.5% BSA for 5 days and then in 50 ${mu}ell$ of NCSU 23 containing 10% FBS for 2 days. For the evaluation of chromosomal aneuploidies, chromosome 1 sub-metacentric specific probe was used in sperm and embryos by fluorescence in situ hybridization (FISH). The frequency of aneuploidy sperm for chromosome 1 was 6.25%. The significant differences following IVF and ICSI with non- or HA-bound sperm were not observed in blastocyst formation rates (18.6, 23.5, and 23.8%) and cell number (61.8 $\pm$ 12.5, 55.5 $\pm$ 7.3, and 59.3 $\pm$ 9.6). Moreover, the percentage of diploidy in 4-cell stage embryos was 57.1% (IVF), 68.8% (ICSI), and 76.3% (ICSI-HABS). These results suggest that HA-binding sites may be a material for selection of normal sperm for ICSI. Therefore HA selection of normal sperm may be reduce the loss to embryonic mortality prior to embryo transfer in pig.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.340-345
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• 2008

Oxidative stress is one of the major causes of failure of in vitro storage of boar semen. Reactive oxygen species (ROS) are one of the important mediators of oxidative stress during in vitro storage of boar semen. Our study examined the effects of taurine on sperm characteristic and on in vitro developmental embryos during in vitro storage of boar semen for 7 days. Semen was randomly aliquoted into 3 centrifuge tubes and treated with different concentrations of taurine (25-100 mM). The characteristics of boar sperm were analyzed for motility by light microscopy, viability by using a Makler counting chamber and membrane integrity by a hypoosmotic swelling test (HOST). The percentages of motile spermatozoa in taurine groups after 5 days were significantly higher compared to the control. Sperm viability in the control was lower than in taurine groups after 7 days irrespective of different taurine concentration. In the hyoosmotic swelling test (HOST), significantly higher results were obtained in taurine groups after 3 days. Also, the developmental rates of IVM/IVF porcine embryos from semen treated with pyruvate and taurine were significantly increased when compared with the control (p<0.05). These results indicate that supplementation of taurine as an antioxidant in boar semen extender can improve the semen quality.

• Development and Reproduction
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• v.15 no.1
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• pp.31-39
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• 2011

Change in intracellular $Ca^{2+}$-concentration ($[Ca^{2+}]_i$) is an essential event for egg activation and further development. $Ca^{2+}$ ion is originated from intracellular $Ca^{2+}$-store via inositol 1,4,5-triphosphate receptor and/or $Ca^{2+}$ influx via $Ca^{2+}$ channel. This study was performed to investigate whether changes in $Ca^{2+}$/calmodulin dependent protein kinase II (CaM KII) activity affect $Ca^{2+}$ influx during artificial egg activation with ethanol using $Ca^{2+}$ monitoring system and whole-cell patch clamp technique. Under $Ca^{2+}$ ion-omitted condition, $Ca^{2+}$-oscillation was stopped within 30 min post microinjection of porcine sperm factor, and ethanol-induced $Ca^{2+}$ increase was reduced. To investigate the role of CaM KII known as an integrator of $Ca^{2+}$- oscillation during mammalian egg fertilization, CaM KII activity was tested with a specific inhibitor KN-93. In the eggs treated with KN-93, ethanol failed to induce egg activation. In addition, KN-93 inhibited inward $Ca^{2+}$ current ($I_{Ca}$) in a time-dependent manner in whole-cell configuration. Immunostaining data showed that the voltage-dependent $Ca^{2+}$ channels were distributed along the plasma membrane of mouse egg and 2-cell embryo. From these results, we suggest that $Ca^{2+}$ influx during fertilization might be controlled by CaM KII activity.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.108-108
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• 2003

The purpose of this is to investigate the effects of vitrification in open pulled straws (OPS) on in vitro survival of porcine embryos. Blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed boar semen, and subsequent culture on granulosa cell monolayer. After frozen-thawing, embryos were culture in NCSU-23 medium with 5 mM hypotaurine, 4 mg/$m\ell$ BSA and 10 ng/$m\ell$ for 48 hrs to survival tests. When blastocysts were frozen-thawed by OPS methods, the embryos with normal morphology were 32.1, 34.5 and 38.9 % in early blastocyst, blastocyst and expanded blastocyat stages. The rates of partial damaged embryos were significantly (P<0.05) higher in early biastocysts than expanded blastocysts. In another experiment, the embryos frozen by OPS methods were cultured for 48 hrs for survival and developmental rates in vitro. The proportions of embryos hatched were 11.8, 20.2 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. On the other hand, The proportions of embryo with normal morphology after culture were 23.5, 25.0 and 33.3% in embryos frozen-thawed at stages of early blastocyst, blastocyst and expanded embryos. These finding indicate the possible broader application for OPS methods that this procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages.

• Korean Journal of Veterinary Research
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• v.63 no.1
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• pp.3.1-3.9
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• 2023

The efficiency of somatic cell nuclear transfer (NT) in pigs is low and requires enhancement. We identified the most efficient method for zona pellucida (ZP) removal and blastomere aggregation in pigs and investigated whether the aggregation of NT and parthenogenetic activation (PA) of blastomeres could reduce embryonic apoptosis and improve the quality of NT-derived embryos by investigating. Embryonic developmental competence after ZP removal using acid Tyrode's solution or protease (pronase E). The embryonic developmental potential of NT-derived blastomeres was also investigated using well-of-the-well or phytohemagglutinin-L. We analyzed apoptosis in aggregate-derived blastocysts. The aggregation rate of protease-treated embryos was lower than that of Tyrode's solution-treated embryos (69.2% vs. 88.3%). No significant difference was observed between phytohemagglutinin-L and well-of-the-well (35.7%-38.5%). However, 2P1N showed a higher number of blastocysts compared to 3N (73.8% vs. 24.3%) and an increased blastocyst diameter compared to the control and 1P2N (274 ㎛ vs. 230-234 ㎛). In blastomeres aggregated using phytohemagglutinin-L, the apoptotic cell ratio was significantly higher in 1P2N and 3N than in 3P (5.91%-6.46% vs. 2.94%, respectively). Our results indicate that aggregation of one NT embryo with two PA embryos improved the rate of blastocysts with increased blastocyst diameter.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.123-128
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• 2005

This study examines the effects of kinds and concentrations of cryoprotectants on the survival rate of vitrification-thawed porcine oocytes, together with the effects on survival, in vitro fertilization and development of immature oocytes. 1. The developmental rate of oocytes to MII and diploid stage when the vitrification-thawed of recovered immature oocytes cultured for 0, 15, 30 and 40h were cultured for 0, 15, 30 and 40h were $56.7\%,\;53.3\%,\;63.3\%,\;65.0\%\;and\;23.3\%,\;18.3\%,\;10.0\%,\; 3.3\%$, respectively. The in vitro development to MII stage were lower than the control group $(78.2\%)$, but higher fo. diploid stage $(5.5\%)$. 2. When the vitrification of immature oocytes after being culture for 0, 15, 30 and 40 hours, the survival rate were $34.0\%,\;26.0\%,\;18.0\%\;and\;10.0\%$ respectively. This result was lower than that of the control group $(60.0\%)$. 3. When the fertilization of the vitrified immature oocytes after being culture for 0, 15, 30 and 40 hours, the in vitro fertilization rate were $60.0\%,\;54.0\%,\;48.0\%,\;38.0\%$, and developmental rates were $26.0\%,\;18.0\%,\;8.0\%,\;4.0\%$, respectively. This results were lower than the control group $(78.0\%\;and\;38.0\%)$. 4. When the fertilization of the immature oocytes after being culture for $0\~15$ hours vitrified with EDS and ETS, the fertilization and developmental rates were $50.0\%,\; 22.0\%$ and $46.0\%,\;18.0\%$, respectively. This results were lower than the control group $(74.0\%\;and\;38.0\%)$.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.61-61
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• 2001

The main goal of this study was to produce transgenic piglets by the method of injection of sperm-mediated exogenous DNA. Spermatozoa (1$\times$106 sperm of final concentration) obtained from caudal epididymis were mixed with pBC1-hEPO (20 ng/${mu}ell$) or pcDNA3 LAC Z (20 ng/${mu}ell$), and followed by electroporation (500 V, 25 ㎌). Matured oocytes having the first polar body and dense cytoplasm were selected and centrifuged at 12,000g for 6 min. After sperm injection, the oocytes were activated electrically (1.7 ㎸/cm, 30 $\mu$ sec, single pulse) in 0.3 M mannitol solution. Eggs injected sperm were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air for 192 h. This study were comprised 3 experiments. Experiment 1 compared the developmental efficiencies between the sperm-injected oocytes (Group 1) and further activated electrically (Group 2). Experiment 2 compared the expression of pcDNA3 LAC Z in the embryos produced by Group 1 and Group 2. Finally, experiment 3 carried out transfer of embryos (1-8 cell stage) transfected with pBC1 -hEPO into surrogate recipients synchronized by injection of combination of PG600 with hCG. The rates of cleavage and development into blastocyst stage in Group 2 were significantly higher than those of Group 1 (71.3% and 28.1% vs. 43.3% and 10.3%, respectively, p<0.05). Thirty (24.2%) out of 124 embryos analyzed in Group 2 were positive by X-gal. Similarly, in Group 1, 16.3% (8/49) were positive. After transfer of 789 embryos to 7 recipient gilts, three out of them examined by ultrasound became pregnant. One recipient is in day 50 pregnancy. On day 54 of gestation, two were carried out uterotomy in order to confirm the pregnancy One had 7 and another had 2 fetuses. We conclude that injection of sperm-mediated gene transfer will be used as a valuable tool for the production of transgenic piglets.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.219-227
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• 2004

This study was conducted to develop a serum-free, defined medium of IVM of pig oocytes. The TCM-199 with supplemented with polyvinylalcohol(PVA), polyvinylpyrrollidone(PVP) and porcine follicular fluid(pFF) were used as basal medium. The effects of the these additives on the rates of maturity and development under in-vitro fertilization and in vitro culture were examined and subsequently considered on the possibilities be sustituted for the bovine serum albumin(BSA). Maturation rate of pig oocytes in IVM media containing PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly higher(P<0.05) than that of PVP(78.6％). Cleavage rate after IVF of PVP(64％) was significantly lower(P<0.05) than these of PVA(73％), pFF(77％) and BSA(73％) supplements. in vitro development rates to morulae and blastocyst on PVP(54％) were also significantly lower(P<0.05) than these of the supplements of PVA(63％), pFF(69％) and BSA(65％). In comparison of maturation and fertilization rates of pig oocytes in each supplements, the maturity rates of PVA(82.4％), pFF(89.4％) and BSA(90.0％) were significantly lower(P<0.05) than that of PVP(72.4％) and while, the fertilization rates of pFF(87.1％) and BSA(89.1％) were significantly higher(P<0.05) than these of PVA(78.0％) and PVP(70.6％). It may be concluded that PVA and pFF can be substituted far BSA in medium for culturing pig oocytes; however, it may be considered that PVP were limited to for BSA in the in vitro culture of the embryos.