• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.96-96
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• 2003

Acyl-CoA synthetase 4는 생쥐에 있어서 거의 모든 조직에서 발현하며 아라키돈산에 특이적인 효소이다. 아라키돈산은 세포막의 인지질로부터 cPLA2에 의하여 유리되고 cyclooxygenase-1, -2에 의하여 eicosanoid로 변환된다. 이렇게 생산된 prostaglandin과 같은 eicosanoid는 배란, 수정, 임신에 있어서 중요한 기능을 수행하고 있다. 그러나 세포막으로부터 유리된 아라키돈산은 acyl-CoA synthetase 4에 의하여 다시 세포막으로 재에스테르화되어 eicosaniod의 생산을 조절하는 것으로 생각되어지고 있다. 또한 acyl-CoA synthetase 4 유전자 한쪽이 knock-out된 heterozygote mouse에서는 사산, 유산과 난소에 있어서 황체 수의 증가 등을 보고하고 있다. 그러므로 본 연구에서는 정상 생쥐 (C57BL/6) 임신 기간 중 acyl-CoA synthetase 4 유전자의 발현을 확인하기 위하여 자궁, 난소, 태아에서 RT-PCR을 수행하였다. 또한 cPLA2, cyclooxygenase-1, cyclooxygenase-2 유전자의 발현 양상을 분석하여 eicosanoid 생산에 관여하는 유전자 상호간의 발현 을 확인하였다. acyl-CoA synthetase 4는 임신 0 day에서부터 19.5 day까지 자궁과 난소에서 모두 발현하고 있었다. 또한 5.5 day에서부터 19.5 day까지의 태아에서도 그 발현이 확인되었다. 그리고 cPLA2와 cyclooxygenase-1은 acyl-CoA synthetase 4와 유사한 양상을 보였으나 cyclooxygenase-2는 임신기간 중의 자궁, 난소, 태아에서 전혀 발현하지 않았다. 그러므로 임신 중 생쥐 자궁, 난소, 태아에 있어서 eicosanoid 생산에는 cPLA2, cyclooxygenase-1, acyl-CoA synthetase 4 유전자가 관여하고 있는 것으로 생각된다.

• Development and Reproduction
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• v.7 no.1
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• pp.49-56
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• 2003

There have been reports that administrated high-dose gonadotropin-releasing hormone-agonist(GnRH-Ag) suppresses endogenous gonadotropin production and inhibits function of ovary. In human IVF-ET program, however, GnRH-Ag is employed in large amounts during superovulation induction resulting to luteal phase defects which must be supported with progesterone. To elucidate the reason of luteal phase defects by GnRH-Ag, the aim of this study was to investigate the apoptosis changes in the ovary and the hormonal changes in the serum after GnRH-Ag and PMSG administration in adult mice in a method similar to human superovualtion induction. GnRH-Ag(10 ${\mu}$g) or saline was injected every 12h beginning 48h prior to PMSG injection until 48h at)or PMSG injection when blood sampling and ovary collection was performed. In results, the ovary weight in the GnRH-Ag only injection group was significantly lower when compared with the other two groups, PMSG only or PMSC + GnRH-Ag injection. The ratio of preantral follicles in the ovary are increased in the GnRH-Ag only group, while the ratio of antral follicles are decreased and the corpus luteum ratio is increased in the PMSG + GnRH-Ag group. The proportion of all follicles showing apoptosis in the GnRH-Ag only in.iection group was seen to be more than twice the proportion seen in the PMSC only injection group, and such increased apoptosis is decreased after addition of PMSC. The serum levels of both estradiol and progesterone were significantly lower in the CnRH-hg only group compared to those in the other two groups. When the administration of GnRH-Ag were followed by PMSG in;ection, however, estradiol concentration was completely recovered compared to the serum level of PMSG group, but not progesterone level. In conclusion the use of GnRH-Ag in human IVF-ET program may induce the apoptosis and the suppression of hormone production by ovary leading to luteal phase defects, thus adequate progesterone support seems to be necessary against them.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.243-247
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• 1994

최근 의약적으로 유용한 단백질을 대량 생산키 위한 실현 가능한 방법이 유전자변환 가축의 이용과 관련되어 발전되어 왔다. 이러한 유전자 변환동물은 이종의 단백질을 유즙속으로 분비시키는 생체반응기로서 이용되고 있다. 이러한 전략적 목적을 위해 현재 유전자 변환동물의 생산을 위한 이용에 있어 여러 가지 방법들이 보고되고 있다. 그러나 ES 세포의 사용이 이러한 방법들 사이에서 가장 실질적인 것으로 추정되고 있다. 본 실험에서는 유전자 구축을 위해 사람 황체 호르몬(human luteinizing hormone; hLH)의 전사를 유도하기 위해 각각 2.2 및 0.5 kb의 토끼 $\beta$-casein pronoter 단편을 이용하여 생쥐의 유선에 hLH를 발현시키도록 조절하고 발현이 thynidine kinase(TK) pronoter에 의해 좌우되는 neo 유전자를 selectable marker로서 plasnid속에 삽입하였다. 그 결과 생긴 구축 유전자는 각각 pCas 2.2와 pCas 0.5로 명명하였다. 구축된 유전자로 2$\times$107의 TT-2 ES세포를 170V, 550$\mu$F로 100$\mu$g의 선상 plasmid에 의해 electroporation 시켰다. 감염된 colony들은 250$\mu$g/$m\ell$ G418을 함유하는 ESM 배양액에서 선별 7일 이후에 회수하여 성공적으로 감염된 ES세포는 PCR 및 Southern blot에 의해 확인되었고 그들 중 나머지는 trypsin 처리 후 각각 미세조작과 공배양 기술을 사용하여 ICR 생쥐의 8세포기 수정란 속에 도입하였다. 결국 24시간 동안 37$^{\circ}C$, 5% $CO_2$에서 배양된 배반포를 chimera의 생산을 위해 위임신 유기된 G418 선발처리 이후 400 및 275개의 ES 세포 colony가 생존하였으며, 3개의 ES 세포으 colony 의 genome 속에 임의적으로 plamid가 삽입된 것을 Southern blot에 의해 확인되었다. 총 13 chimera 생쥐가 3 colony로부터 생산되었으나 germ-line chimera는 현재 조사중이다. chimera 생산빈도는 공배양 기술보다 주입방법에서 현저히 높았다.

• Journal of Embryo Transfer
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• v.22 no.1
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• pp.53-61
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• 2007

This study was investigated factors affecting the pregnancy rates after transfer of pronuclear microinjected embryos for the production of transgenic Korean black goats. Embryo transfer was carried out in 343 recipient Korean black goats from September 1999 to June 2000. Estrus was induced by the insertion of intravaginal progesterone devices $CIDR^(R)$ for 2 weeks. A single injection of 400 IU equine chorionic gonadotropin was administered at 48h before $CIDR^(R)$ removal to increase the proportion of does cycling and ovulation rate. Good quality embryos were prepared by microinjection of DNA into the pronuclei of fertilized goat oocyte and cultured in vitro. Pronuclear microinjected $1{\sim}8$ cell stage embryos were surgically transferred into the oviducts of the recipient at day 4 or 5 following $CIDR^(R)$ removal, and morula to blastocyst stage embryos were surgically transferred into uterus at day 9. Pregnancy was diagnosed by transrectal ultrasound scanning at $20{\sim}30d$ and 8 weeks following embryo transfer. The pregnancy rate was affected by several factors, such as estrus induction, the number of previous transfer, transfer site, stage of CL (corpus luteum), the number of recipient CL, stage of embryos and the number of transferred embryo. The pregnancy rate was significantly higher in recipients that came into estrus naturally than recipients that induced to come into estrus with $CIDR^(R)$(59.1% vs. 36.8%; P<0.05). The pregnancy rate was higher when the embryos were transferred into the left oviduct than transferred into the right oviduct (42.9% vs. 35.3%; P<0.05). The pregnancy rate of recipients with $CH_1$ (early) stage corpus hemorrhagicum in ovary was hi틴or than recipient with $CH_3$ (late) stage hemorrhagicum (47.5% vs. 17.9%; P<0.01). Higher pregnancy rates were obtained by transfer of 1-cell stage embryos into oviduct while late blastocysts (51.6% vs. 66.7%; P<0.01) into uterus. The pregnancy rates when 3 embryos were transferred to recipients were significantly higher than when 2 embryos we.e transferred (47.6% vs. 27.0%; P<0.05). Although there were no significant difference among the group, adhesion of reproductive organs, uterine size, ovulation rate of recipients, presence of large follicle and difficulty of transfer affected pregnancy rate of recipient. Higher pregnancy rates were obtained in the recipients with $8{\sim}15m$ diameter uterine horn as compared to the recipients with <5m diameter or >20mm diameter uterine hem (38.9%, 20% vs. 18.2%), in the recipients with large follicle in the ovulated ovary ipsilaterally (53.6% vs. 37.1%) and in the transfer which was carried out easily (39.2% vs. 27.8%, 0%). In conclusion, the high rate of pregnancy was achieved following transfer of pronuclear microinjected embryos when three or four 1-cell stage embryos were transferred into oviduct with $CH_1$ stage corpus hemorrhagicum in the ovary of recipient which came into estrus naturally.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.2
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• pp.101-111
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• 2005

배 경: Pituitary adenylate cyclase-activating polypeptide (PACAP)은 양의 시상하부에서 추출된 신경펩타이드 호르몬으로 난소에도 존재하여 배양된 과립막 세포에서 스테로이드합성과 cyclic AMP 형성을 촉진함이 보고되었다. 목 적: 흰쥐 난소를 실험 모델로 사용하여 배란시 황체화호르몬 (luteinizing hormone; LH)에 의해 유도된 PACAP과 PACAP 수용체의 유전자 발현양상과 신호 전달경로를 규명하고자 하였다. 재료 및 방법: 미성숙 흰쥐의 배란전 난포를 체외 배양하면서 LH로 처리하고 PACAP 및 PACAP수용체의 유전자 발현을 보기 위해서는 Northern blot 분석과 in situ hybridization (ISH)을, 그리고 단백질 수준의 PACAP 검색을 위해서는 enzyme linked immunosorbent assay (ELISA) 분석을 이용하였다. 결 과: LH 처리 후 Northern blot상의 PACAP 유전자 발현은 6~9시간에 일시적으로 최고치에 도달하였으며 ISH로 보아 과립막 세포에서 발현됨을 알 수 있었다. ELISA 분석 상 PACAP 단백질도 LH처리 후 6~12시간에 최고치를 나타내었으며, PACAP 수용체 mRNA 역시 3~9시간에 최고치로 과립막 세포에서 발현되었다. Adenylate cyclase (AC) 억제제인 MDL12330A 처리시 LH로 발현된 PACAP mRNA가 감소되며, AC의 활성제인 forskolin 처리에는 LH시와 유사한 PACAP mRNA의 발현양상을 나타내었다. 그러나 protein kinase C (PKC)의 억제제인 chelerythrine과 2-0-tetradecanolphorbol-13-acetate (TPA) 처리로는 PACAP 의 유전자 발현에 영향을 주지 못하였다. 5-lipoxygenase의 억제제인 MK886이나 nordihydroguaiaretic acid (NDGA)로 처리한 결과 LH로 유도된 PACAP 유전자의 발현이 감소되었으나, cyclooxygenase의 억제제인 indomethacin은 별로 영향을 주지 못하였다. MEK와 p38의 억제제인 PD98059와 SB203580도 LH로 촉진 된 PACAP의 유전자 발현을 농도 의존적으로 억제하였다. 결 론 : 배란전 난포에서 PACAP과 PACAP 수용체의 유전자 발현은 모두 LH의 폭발적 분비에 의해 유도되어 일시적으로 과립막 세포에서 나타나 배란을 위한 국소적인 조절 작용을 할 것으로 추정되며, LH로 촉진된 PACAP 유전자 발현을 위한 신호전달은 cAMP-PKA, lipoxygenase 및 MAP kinase 경로를 통하는 것으로 사료된다.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.119-124
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• 2001

This study was performed to investigate the effects of the peptide to carrier ratio on the immune and biological functions to inhibin immunization in Hanwoo. A peptide sequence kom the alpha -subunit (19~32 peptide) of porcine inhibin was synthesized for antigen and conjugated to human serum albumin(HSA) for carrier protein. Anti-inhibin sera(AI) were produced 52 day later from rabbit after injection of inhibin-$\alpha$ -subunit peptide conjugator for antigen with the interval of 2 weeks. Immune-blotting analysis using antibody specific fur inhibin-$\alpha$ subunits revealed that the inhibin was detected at 1.0 cm bovine follicular fluid(bFF). However, each stage of corpus lutea and 0.1 cm of follicular fluid were not detected. The maximal contents of estradiol-17 $\beta$ in Hanwoo ovarian follicular fluid were detected at 2.0 cm of follicular size(diameter), but the mean total contents of these hormone decreased significantly with decreasing diameter of follicles. However, progesterone contents of follicular fluid were high at 1.0 cm of follicle. Progesterone secretion by Hanwoo granulosa cell cultured for 48 hr in vitro was significantly (p＜0.05) inhibited in 5% bFF and 5% bFF + 5% AI addition group compared with control group. Estradiol-17 $\beta$ secretion by Hanwoo granulosa cell cultured for 48 hr in vitro was significantly (p＜0.05) increased in 5% AI and 5% AI + 5% bFF addtion group compared with control group. However, the groups added 5% AI were not changed compared to control groups in progesterone and estradiol-17 $\beta$. Taken together, we suggested that inhibin in the mature FF plays a pivotal role on the biosynthesis of steroid hormone of follicular cells during follicular development.

• Journal of Embryo Transfer
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• v.24 no.1
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• pp.57-64
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• 2009

This study was carried out to investigate expression of apoptosis-related differentially expressed gene (DEG) in ovaries of Korean cattle with follicular and luteal cysts and to identify the relationship between cyst and apoptosis using microarray, real-time PCR, TUNEL staining, and Western blot analysis. Microarray data showed that PIK3R2 and AKT1 were significantly up-regulated in follicular cyst, and TNF-RAF2, PRLR, FOXL2, STK4, and COL4A3 were up-regulated whereas INHA, CIDEB, BCL10, and FASLG were down-regulated in luteal cyst. Real-time PCR was performed to validate DEGs altered in luteal cyst. Of nine DEGs, four DEGs down-regulated in luteal cyst showed a positive corelation between microarray data and real-time PCR data. In this study, we focused on INHA, among many DEGs, which was highly down-regulated in both follicular and luteal cysts. Real-time PCR and micro array data showed that INHA was down-regulated by 12.3-fold and by 1.4-fold, respectively, in the bovine follicular cyst. TUNEL assay and Western blot analysis for ERK, JNK, p38, PI3K, and Akt, which were used to detect whether apoptosis is occurred, showed no significant changes in cystic ovaries (p>0.05). In the expression and activity of caspase-3, Bax, Bel-2, and Bel-xL, there was no significant changes between follicular cystic ovary and normal ovary. Rather, the expression levels of PI3K and p-Akt were decreased in follicular cystic ovary. These results suggest that deficiency of apoptosis in cystic ovary is associated with decreased expression of apoptotic effectors.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.37-41
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• 2005

The aim of this study was to improve efficiency and quality of the production of Korean Native Cow embryos. We examined effects of ovarian morphology and maturation vessel on the development and cell number of blastocysts. The development rates to the 2-cell embryos from oocytes collected from the ovaries of different morphological statues were similar ranging between 70.3 and 84.1%. The development rate to the 8 cell- and blastocyst-stage embryos was the highest in the group without both corpus luteum (CL) and follicle. The inner cell mass (ICM), trophectoderm (TE) and total cell number (TCN) were significantly higher in the groups of follicular cyst and regressive CL than other treatment groups, and the same pattern was observed in the ICM/TCN ratio. The development rate to the 2-cell stage was significantly higher in 0.5-㎖ straw group than 0.25-㎖ straw group. However, the development rates to the blastocyst stage were similar between the dish and the straw group. There were no differences in the number of ICM and TE cells, TCN and ICM/TCN ratio of blastocysts from oocytes matured in the different vessels.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.109-113
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• 2003

The study was carried out to investigate the effects of morphology, preservation and reproductive cycle of oocytes in vitro maturation of cats oocytes and development of IVM embryos. The results were summarized as follows : 1. Nuclear status of GV and MI of in vitro cultured(24 h) oocytes with and whithout cumulus cells were 74.3% and 25.7%, 28.6% and 11.4%, 77.1% and 5.7%, respectively, The rate of oocytes with cumulus cells was higher than that of denuded oocytes. 2. Nuclear status of GV and MI of in vitro cultured(24 h) oocytes recovered from ovaries collected at different stages of the reproductive cycle(inactive, follicular and luteal) were 88.6% and 6.5%, 60.0% and 11.4%, 77.1% and 5.7%, respectively. 3. Nuclear status of fresh and salts-stored oocytes with and whithout cumulus cells were 74.3%, 25.7% and 37.1%, 11.4% and 57.1%, 13.3%, 17.1%, 3.3%, respectively. The rate of oocytes with cumulus cells(13.3%∼74.3%) was higher than that of denuded oocytes(3.3%∼57.1%).

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.29-34
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• 2006

본 연구는 개 체외성숙 난자를 안정적으로 생산하기 위하여 채취시기, 난구세포 부착 여부 및 배양액에 EGF와 호르몬을 첨가 후 배양했을 때 체외성숙율에 미치는 영향을 조사하였다. 1. 미성숙 난포란을 TCM-199 배양액에서 24, 45시간 배양했을 때 체외성숙율은 각각 7.93%, 8.94%로서 48시간 배양했을 때 가장 높은 체외성숙율을 나타냈다. 2. 휴지기, 난포기, 황체기에 채취한 난소로부터 회수한 난자를 20 ng/ml의 EGF가 첨가된 TCM-199 배양액에서 배양했을 때 체외성숙율은 14.3%로서 0, 10 ng/ml의 EGF 첨가군(3.1%, 7.5%)에 비해 높은 체외성숙율을 나타냈다. 3. 난구세포 부착 및 미부착 난자를 48시간 배양했을 때 체외성숙율은 각각 18.8% 및 7.5%로서 난구세포 부착 난자가 미부착 난자보다 높은 체외성숙율을 나타냈다. 4. 난자의 체외성숙 배양 시 0.5 mg/ml FSH, 5 mg/ml LH, 1 mg/ml $E_2$와 FSH+LH, $FSH+LH+E_2$를 첨가한 TCM-199 배양액에서 배양했을 때 체외성숙율은 각각 1.2%, 10.0%, 2.0%와 10.0%, 31.2%로서 호르몬의 병용처리군이 높은 체외성숙율을 나타냈다. 5. 난자의 체외성숙 배양 시 EGF와 FSH, LH, $E_2$ 및 EGF와 FSH+LH, $FSH+LH+E_2$를 첨가한 TCM-199 배양액에서 배양했을 때 체외성숙율은 32.3%, 27.0%, 3.0%와 36.2%, 69.4%로서 EGF와 호르몬 병용 처리군이 높은 체외성숙율을 나타냈다.