• Clinical and Experimental Reproductive Medicine
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• 제21권2호
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• pp.177-182
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• 1994

ICR female mice aged 3 to 4 weeks, were stimulated with 7.5 IU PMS injection. At 48-52h post-PMS injection, ovaries were dissected out and oocytes-cumulus complexes(OCCs) were divided into three groups, cumulus-free oocytes(O), cumulus-free oocyte cocultured with cumulus cells(O+C) and OCC. The oocyte were cultured in TCM199 containing various protein sources, FCS, BSA or PVP with gonadotropins(Gns) for 24h. Spermatozoa were collected from cauda epididymis and capacitated in T6 + BSA for 2h. After oocyte maturation in vitro(IVM) in different experimental groups, matured oocytes were inseminated with the capacitated spermatozoa in T6 + BSA for 6h. In the groups of IVM in TCM + BSA or PVP, fertilization(IVF) did not occur efficiently. However, increased fertilization was found in TCM+ FCS group. The oocytes groups, with cumulus cells showed decreased polyspermy in FCS group (O; 31.8 %, O + C; 12.2 %, OCC; 16%), the addition of Gns did not prevent polyspermy in all three groups. The rates of fertilization increased in zona-free oocytes in PVP group. This results showed that culture system for IVM and IVF could be improved. Furthermore, PVP can be used for the substitution of protein source during maturation, and its low rate of fertilization has been found due to zona hardening which occurred in FCS-free medium.

• 대한약침학회지
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• 제16권1호
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• pp.7-11
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• 2013

Objective: Pharmacopuncture, a new therapy in traditional medicine, has attracted significant attention since its introduction to the Western world. This short review article employs a database analysis to examine the profile of publication activity related to pharmacopuncture. Methods: Three databases were searched: PubMed, Embase, and Cochrane. About 300 papers related to the topic "pharmacopuncture" were found in these three most-commonly-used databases. Results: Fourteen papers are described in detail and are discussed in the context of the research performed at the Medical University of Graz, especially by the Frank Bahr Research Group "Auriculomedicine and Pharmacopuncture." Conclusion: High-tech research methods concerning future pharmacopuncture studies are briefly discussed.

• 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.

• 한국가축번식학회지
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• 제26권2호
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• pp.105-110
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• 2002

본 연구는 소형견의 불임 해결과 번식효율 증진을 위해 소형견 난소 난포로부터 채취한 난자를 활성화 처리후 부고환 정자로 ICSI시켰을 때 체외발생율을 조사하기 위하여 수행하였다. 1. 난포란을 회수 후 24, 48시간 배양하였을 때 배양시간에 따른 GV, MI, MII로의 체외발생율은 각각 14/30(46.7%), 2/30(6.7%), 8/30(26.7%)였고 48시간 배양 시간에 따른 GV, MI, MII로의 체외발생율은 각각 l1/30(36.7%), 3/30(10.0%), 9/30(30.0%)였다. 2. 난포란을 회수 후 48시간 배양하였을 때 배양액에 따른 MII로의 체외발생율은 SOF액(10/30, 30.3%)에서의 배양이 TCM-199액(7/30, 23.3 %)보다 높은 체외발생율을 나타냈다. 3. 활성화 처리 난자에 부고환 정자로 ICSI를 하였을 때 상실배와 배반포로의 체외발생율은 각각 3/16(18.8%), 4/16(25.0%)로서 비활성화 처리 난자군의 3/13(23.1%), 1/13(7.7%)에 비해 높은 체외발생율을 나타냈다. 4. 활성화 처리 난자에 신선정자, 부고환 정자 및 동결 융해한 부고환 정자로 ICSI를 하였을 때 체외발생율은 각각 8/18(44.4%), 5/16(31.3%), 2/14(14.3%)로서 동결 부고환 정자 처리군은 신선정자 처리군에 비해 낮은 체외발생율을 나타냈다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2002년도 춘계학술발표대회 발표논문초록집
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• pp.52-52
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, one group of oocytes was activated with 7% ethanol for 5 min, and second group was not activated. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24-30 hrs in a incubator with 5% CO₂ in air at 38.5℃. (omitted)

• Reproductive and Developmental Biology
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• 제28권3호
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• pp.181-185
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• 2004

본 연구는 동물복제 및 형질전환 동물생산 등의 연구에 기초자료를 제공하고자 재래산양의 oocyte를 단위발생을 유도하여 회수난자의 조건, 활성화방법, 배양조건 등이 단위발생란의 체외발달율에 미치는 영향을 조사하여 재래산 양의 최적의 배양조건을 확립하고자 실시하였다. 난자의 회수는 체중 15～25 Kg 전 ㆍ 후의 성숙한 미경산 재래산양에 FSH와 PMSG 를 사용하여 과배란을 유기하여 hCG 투여 후 제 35시간째에 외과적인 방법으로 in vivo (체내성숙) 난자는 난관을 관류하는 방법으로 회수하였고, in vitro (체외성숙) 난자는 난포로부터 흡입하여 난포란을 채취하여 약 22시간 체외성숙을 실시하였다. 활성화 처리는 전기자극법과 Ionomycin + 6-DMAP를 처리하여 단위발생을 유도하였다. 복제수정란의 배양은 M16, TCM-199 및 mSOF 배양액으로 6～7일 동안 체외배양을 실시하였다. 활성화를 유도하기 위하여 전기자극 및 ionomycin + 6-DMAP 처리를 하였을 때 분할율은 각각 64.1 및 76.5%로서 이들간에 차이는 없었다. 배반포기로의 발달율은 전기자극방법으로는 전혀 발달이 이루어지지 않았으나, ionomycin +6-DMAP 처리방법에서는 15.6%가 배반포로 발달하였다. In vivo 난자와 in vitro 난자를 활성화를 유도하였을 때 분할율은 86.8 및 69.0%로서 이들간에 유의적인 차이는 없었다. 4-세포기(93.9% vs 66.1%), 8-세포기(90.9% vs 37.0%) 및 상실배기(89.4% vs 23.6%)는 이들간에 유의적(P<0.05)인 차이가 있었으며, 배반포기로의 발달율은 체내성숙난자가 50.0%로서 체외성숙난자의 0.8%보다는 유의적으로 높았다. 활성화를 유도한 난자를 mSOF 배양액으로 체외배양을 실시하였을 때 분할율은 81.0%로서 TCM-199 +oviduct cell 의 64.3% 및 Ml6 배양액의 51.6%보다는 높게 나타났다. 배반포기로의 발달율은 mSOF 배양액에서는 3.4%가 발달하였으나, TCM-199+ oviduct cell 배양액과 M16 배양액에서는 전혀 발달이 이루어지지 않았다. 이상의 결과는 포유동물 난자의 단위발생의 유도 및 체외배양시 난자의 공급원, 난자의 활성화 방법 및 배양조건 등이 차후 단위발생란의 체외발생율에 크게 영향을 미칠 수 있는 근거를 제시해 준다.

• 한국가축번식학회지
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• 제26권4호
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• pp.353-360
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• 2002

Although successful pronuclear formation and apposition were seen in porcine oocytes following mouse sperm injection, little is known on the morphology of male and female pronuclei following sperm injection. The objective of this study is to describe the ultrastructure of porcine zygote following murine sperm injection in relation to the chronology of pronuclear S phase. At 40h ~ 44h following in vitro maturation, Cumulus cells were removed in TCM-HEPES with 0.1% hyaluronidase. Then, spermatozoa was injected into the cytoplasm of oocytes. After. injection, all oocytes were transferred to NCSU23 medium and cultured at 39$^{\circ}C$ under 5% $CO_2$ in air. Oocytes were fixed in 2% glutaraldehyde in Dulbeccos phosphate-buffered saline and observed by Transmission Electron Microscopy. Nuclear precursor bodies were observed in each pronucleus. A cluster of large and small granules was attached in the nucleolus precursor body. After the apposition of male and female chromatin, chromatin condensation was observed throughout the nucleoplasm and nucleolus precursor bodies and condensed chromatin in contact with clusters of small and large granules and the nuclear envelope were found in apposed pronuclear regions. These results suggest that non-species specific nuclear cytoplasmic interactions take place during pronuclear formation and apposition following sperm injection.

• 한국수정란이식학회지
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• 제17권1호
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• pp.55-59
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• 2002

ICSI시 동결 융해한 부고환 정자의 이용 가능성을 알아보고자 난자의 배양시 체외성숙율과 활성화 처리를 한 난자와 동결 융해한 부고환 정자로 ICSI시 체외발생율을 조사하였으며, 결과를 요약하면 다음과 같다. 1. 난포란을 회수 후 24시간 배양하였을 때 배양 시간에 따른 GV, MI, M II로의 체외성숙율은 각각 7/60(11.7%), 5/60(8.3%), 48/60(80.0%)였고 30시간 배양 시간에 따른 GV, MI, M II로의 체외성숙율은 각각 3/60(5.0%), 4/60(6.7%), 53/60(88.3%)였고 퇴화란은 각각 2/60(3.3%)와 1/60(1.7%)였다. 2. 동결 융해한 부고환 정자를 이용하여 활성화 처리를 한 난자에 ICSI를 하였을 때 상실배와 배반포로의 체외발생율은 각각 12/46(26.1%), 22/46 (47.8%)로서 비활성화처리 난자군 5/39 (12.8%), 10/39(25.6%)에 비해 높은 체외발생율을 나타냈다. 3. 활성화 처리를 한 난자에 신선정자, 부고환 정자 및 동결 융해한 부고환 정자로 ICSI시 체외 발생율은 각각 24/45(53.3%), 15/40(37.50%), l1/43 (25.6%)로서 신선정자에 비해 동결 융해한 부고환 정자처리군은 체외발생율은 약간 낮았지만 이용 가능성이 있음을 확인하였다.

• 한국발생생물학회지:발생과생식
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• 제3권1호
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• pp.39-44
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• 1999

성선자극호르몬으로 자극된 난소로부터 회수된 사람의 성숙난자의 전핵형성과 초기발생에 정자의 운동성과 성숙도가 미치는 영향을 조사하였다. 세포질내정자주입 (ICSI)은 HEPES-buffer mTCM-199 배양액에서 실시하였다. 체내에서 성숙된 난구세포부착난자를 ICS에 의하여 사정된 운동성 정자 또는 비운동성 정자로 수정하였을 때 운동성 정자로 수정된 난자가 비운동성 정자로 수정된 난자보다 전핵형성율이 높았다 (79.8% vs 51.7% ; p<0.002)). 그러나 체내에서 성숙된 난구세포부착난자를 ICS에 의해 정소 내 운동성 정자 또는 비운동성 정자로 수정하였을 때 운동성 정자와 비운동성 정자 사이의 전핵형성율은 차이가 없었다. 10.0 mM lactate, 0.5 mM pyruvate, 0.2 mM taurine, 1.0 mM glutamine, 2.22 mM MEM amino acids, vitamin 그리고 10% 사람 난포액이 포함된 수정 Tyrode 배지에서 전핵이 형성된 수정란의 초기 발생은 정자의 채취원과 운동성에 관계없이 9∼16세포기로 발생하였다.

• 한국수정란이식학회지
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• 제13권1호
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• pp.11-19
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• 1998

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 $\mu$M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 $\mu$sec) in 0.3 M mannitol solution supplemented with 100 $\mu$M CaCl$_2$ and MgCl$_2$. The nuclear donor embryos of 8-cell stage were synchronized to G$_1$ phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.