• Reproductive and Developmental Biology
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• 제30권2호
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• pp.119-124
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• 2006

The present study compared the developmental potential of cloned porcine embryos with mesenchymal stem cells (MSCs), fetal fibroblasts (FFs) and cumulus cells (CCs) by assessing the cleavage and blastocyst rate, total cell number, inner cell mass (ICM) ratio and apoptosis. MSCs were isolated by ficoll gradients from femur of -6 month old female pig, and maintained for primary cultures. FFs from a female fetus at ${\sim}30$ day of gestation were established, and CCs were obtained from cumulus oocyte complexes (COCs) aspirated from $3{\sim}6$ mm follicles in diameter. Donor cells at $3{\sim}4$ passage were employed for nuclear transfer (NT). COCs were matured and fertilized in vitro(IVF) as control. Cleavage rate was significantly (P<0.05) higher in IVF than in NT embryos with MSCs, FFs and CCs ($82.7{\pm}8.9%\;vs\;70.6{\pm}5.4,\;68.7{\pm}5.1\;and\;63.4{\pm}5.6%$, respectively). However, blastocyst rates in IVF and NT embryos derived from MSCs ($24.5{\pm}2.8\;and\;20.4{\pm}8.3%$) did not differ, but were significantly (P<0.05) higher than NT derived from FFs and CCs ($10.6{\pm}2.7\;and\;9.8{\pm}2.1%$). Total cell number and the ratio of ICM to total cells among blastocysts cloned from MSCs ($35.4{\pm}5.2\;and\;0.40{\pm}0.09%$, respectively) were significantly (P<0.05) higher than those from FFs and CCs ($24.9{\pm}6.2%\;vs\;0.19{\pm}0.16,\;23.6{\pm}5.5\;and\;0.17{\pm}0.16%$, respectively). Proportions of TUNEL positive cells in NT embryos from FFs and CCs ($6.9{\pm}1.5\;and\;7.4{\pm}1.7%$, respectively) were significantly (P<0.05) higher than in MSCs ($4.8{\pm}1.4%$) and IVF ($2.3{\pm}0.9%$). The results demonstrate that MSCs have a greater potential as donor cells than FFs and CCs in achieving enhanced production of cloned porcine embryos.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.98-98
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• 2002

Recruitment of primordial follicles(PMF) is crucial for female fertility. however, factors and mechanisms that regulate this process is poorly understood. The present study was conducted to obtain an inclusive view of the gene expression and to identify novel factors and their pathways of regulating PMF arrest and/or growth initiation. Ovaries from one-day neonatal(consists of oocyte and PMF) and five-day old(consists of PMF and primary follicles, PRIF) mice were collected, either total RNA or mRNA was isolated, and suppression subtractive hybridization(SSH) was used to isolate and clone genes that differentially expressed in day 1 and day 5 ovaries. Confirmation that some of these genes are differentially expressed in PMF and/or in PRIF was accomplished by using laser captured microdissection(LCM), RT-PCR. in situ hybridization(ISH) and/or immunohistochemistry(IHC). In toto, 357 clones were sequenced and analyzed by BLAST and RIKEN program. Sequences of 330 clones significantly matched database entries while 27 clones were novel. Forty-two and 47 different genes were identified as differentially expressed in day 1 and day 5 ovaries, respectively, while 7 genes were expressed in both stages of ovaries. Day 5-subtracted library included several genes known as markers far growing follicles, such as ZP2, MATER, and fetuin. Among the genes with assigned functions, 23.8% was associated with cell cycle/apoptosis regulation, 7.1% with cellular structure, 11.9% with metabolism, 26.2% with signal transduction, and 31.0% with gene/protein expression in day 1; while 10.6%, 17.0%, 23.5%, 25.5%, and 23.4% in day 5, respectively. Genes such as GDF-8, Lats2, Septin2, and Weel were the highly expressed genes in PMF, while HSP84, Laminin2, MATER, MTi7, PTP, and Wrn were highly expressed genes in PRIF. We have successfully discovered list of genes expressed in day 1 and day 5 ovaries and confirmed that some of them are differentially expressed in PMF and/or PRIF. Gene expression profile from the present study would provide insight for the future study on the mechanism(s) involved in primordial-primary follicular transition. This work was Supported by Korean Health 21 RND Project, Ministry of Health and Welfare, Korea (01-PJ10-PG6-01GN13-0002).

• 한국수정란이식학회지
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• 제19권3호
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• pp.283-289
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• 2004

동결 과정 중 필수적인 단계중 하나인 냉각(cooling)과 냉각 후 배양시간이 생쥐 난자의 방추체의 형태와 염색체의 배열에 미치는 영향을 알아봄으로서 냉각 후 손상되었던 난자의 방추체와 염색체가 정상적으로 회복하는데 필요한 최적의 배양시간을 알아보기 위해 본 실험을 실시하였다. 생후 4-6주령의 암컷 B6C3Fl 생쥐를 과배란 처리하여 metaphase II상태의 난자를 회수하여 다음과 같이 처리하였다. 대조군은 난자를 냉각처리하지 않았으며 실험군은 난자를 $0^{\circ}C$에서 30분간 냉각한 후 37$^{\circ}C$에서 가온하여 즉시 일부 난자는 면역형광 염색을 실시하고 나머지 난자는 5% $CO_2$ 37$^{\circ}C$가 유지된 배양기내에서 Ml6 배지에 각각 5분, 15분, 30분, 60분, 120분간 배양한 후 면역 형광염색을 실시하였다. 난자의 방추체와 염색체를 평가하기 위한 면역형광염색은 Zenes 등의 방법(2001)에 준하여 실시하였다. 냉각처리하지 않은 생쥐 난자를 면역형광 염색하여 방추체와 염색체를 관찰한 결과 생쥐 metaphase II 상태의 난자는 대칭성의 원통모양의 방추체 형태를 보였으며 염색체는 metaphase plate위에 분리된 다발모양으로 밀집되어 보였다. 냉각 직후 미세관의 소실에 의한 방추체 형태의 이상과 형광성의 소실이 나타났으며 염색체는 다발모양의 밀집된 형상에서 벗어나 비정상적인 배열상을 보였다. 냉각 처리된 난자를 37$^{\circ}C$에서 가온하고 배양하였을 때 미세관의 재중합이 일어나 미세관의 형광성을 회복하기 시작하였고 방추체는 정상적인 배열상으로 회복되었다. 생쥐 난자를 냉각처리한 후 배양시간에 따른 방추체 미세관의 형광성(FIS), 염색체의 배열, 방추체의 형태를 비교하였다. 배양 5분에서 60분까지 FIS, 정상 염색체 배열을 보인 난자의 비율, 정상 방추체의 형태를 보인 난자의 비율이 점진적으로 증가하였으나 120분 배양에서는 감소하였다(P<0.05). 위의 세 가지 평가를 기준으로 하여 냉각 후 난자의 회복율을 관찰하였을 때 배양 60분에서 최상의 회복율을 나타냈다.

• 한국수정란이식학회지
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• 제19권3호
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• pp.229-237
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• 2004

본 연구에서는 소 체외수정란 생산에 있어서 체외성숙용 배지에 첨가하는 혈청과 호르몬의 효과를 검토하기 위하여 제1극체 출현율과 배발달율을 조사하였고, 생산된 배반포의 품질을 평가하기 위하여 세포수를 검토하였다. 1. 체외성숙용 배지에 혈청 및 성선자극호르몬의 첨가에 따른 한우 난포란의 제1극체 출현율은 비슷한 경향이었다. 배반포까지의 발달율은 혈청 및 성선자극호르몬 공동 첨가군(26.0％)이 대조군과 성선자극호르몬 단독 첨가군보다 유의하게 높았다(P<0.05). 혈청 및 성선자극호르몬 공동 처리군에서 생산된 배반포의 ICM, TE, 총 세포수가 가장 많았으며, ICM 세포는 혈청 첨가로 유의하게 증가하였다(p<0.05). 2. 체외성숙용 배지에 혈청의 첨가시기에 따른 한우 난포란의 제1극체 출현율은 체외성숙 18시간 동안의 처리군과 체외성숙 9시간째부터 첨가한 처리군이 미첨가군보다 높았으며, 체 외 성숙 후 9시간까지의 처리군보다는 유의하게 높았다(p<0.05). 체외성숙용 배지에 혈청의 첨가시기에 따른 한우 난포란의 배발달율은 전군에서 비슷한 수준이었다. 배반포의 TE와 총세포수는 비슷한 경 향이었으나, ICM 세포수는 체외성숙 18시간 동안 처리군이 혈청 미첨가군보다 유의하게 높았다(p<0.05).

• 한국수정란이식학회지
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• 제19권3호
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• pp.275-282
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• 2004

본 연구는 체외성숙/체외수정 유래의 돼지 난자를 이용하여 체외발달시 배양액의 종류나 교체에 따른 영향을 구명하고자 수행하였다. mNCSU-23에서 체외성숙시킨 다음 mTBM에서 체외수정시킨 난자를 목적에 따라 두 가지로 나누어 실험한 결과는 다음과 같다. 1. 체외성숙/체외수정란을 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 CZB에서 4일 배양한 다음 Pig-MEM으로 옮겨서 나머지 3일간 배양한 결과, 난분할율은 배양액간 차이를 보이지 않은 반면, 추정수정란대 배반포 발달을(P<0.05)과 분할란대 배반포 발달율은 NCSU-23에서 배양된 것이 유의적으로 높았다(P<0.01). 그러나 배 반포의 ICM 세포수, TE 세포수 및 총세포수에서는 모두 차이가 없었다. 2. 체외성숙/체외수정란를 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 체외배양 5일째에 신선한 동일 배양액이나 0.4％ BSA를 10％ FBS로 대체한 배양액(mNCSU-23F)으로 완전히 교체하여 배양한 결과, 난분할율, 배반포발달율, 배반포의 ICM 세포수, TE 세포수 및 총세포수 모두 처리간 유의적인 차이를 보이지 않았다. 결과적으로, NCSU-23이 CZB/Pig-MEM보다 체외성숙/체외수정 유래의 난자를 체외발달시키는데 적합한 것으로 사료되며, 체외발달배양 과정에 신선한 배양액이나 일부 변경된 배양액으로의 교체에 대해서는 더 많은 연구가 필요할 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제14권8호
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• pp.1188-1195
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• 2001

A tetraparental chimeric bull was successfully produced by aggregating bovine IVF embryos of F1 (female Holstein${\times}$male Japanese Black) and F1(female Japanese Brown${\times}$male Limousin) and culturing in vitro without the zona pellucida at Yamaguchi Research Station in Japan. In the microsatellite genotyping, 12% (28/228) microsatellite primer sets ware potentially useful for this parentage analysis in the chimeric bull, 78.6% (22/28) of microsatellite present in the chimeric bull were uniquely contributed from the Japanese Black and 21.4% (6/28) from Limousin. This chimeric bull semen was used in producing IVF embryos. The chromosome preparations were made from peripheral lymphocytes. Based on chromosome analysis the Chimera had apparently normal chromosomes (29 acrocentric pairs, one large sub metacentric X chromosome and one small sub metacentric Y chromosome). The proportion of acrosome reacted spermatozoa after 1 h of incubation was higher (p<0.01) with the Chimera than with the Holstein and in Japanese Brown bulls. But did not differ from Japanese Black and Limousin bull sperm. Fertilization rates observed after 5 h of sperm-oocyte incubation with Chimera sperm were higher (p<0.05) than with Japanese Brown and (p<0.01) than with Holstein sperm, but did not differ from Japanese Black and Limousin sperm. The cleavage rates of IVF oocytes inseminated with Chimera sperm were also higher (p<0.001) compared with Holstein, (p<0.01) Japanese Brown and (p<0.05) Limousin, but did not differ from Japanese Black sperm. The blastocyst rates of IVM oocytes inseminated with sperm were higher (p<0.05) than in Limousin, Japanese Brown and Holstein, but did not differ from Japanese Black. Chimeric cattles were produced by aggregation of parthenogenetic (Japanese Brown) and in vitro fertilized (Holstein) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus) and in vitro fertilized (Holstein) embryos at the St. Gabriel Research Station in Louisiana. The aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding. The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derieved embryos cultured without agar than when cultured with agar. The development rate to blastocysts, however, was not different among the treatment. To verify parthenogenetic and the cells derieved from the male IVF embryos in blastocyst formation, 51 embryos were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zonafree chimeric embryos at 24 h following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP. Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transfer of 40 chimeric embryos at the Louisiana station. Two pregnancies were Jost prior to 4 months and one phenotypically chimeric viable male born.

• Asian-Australasian Journal of Animal Sciences
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• 제22권7호
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• pp.969-976
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• 2009

Use of oocytes from prepubertal animals for in vitro embryo production holds potential application for reducing generation intervals and increasing genetic progress through embryo transfer. The objective of these studies was to compare the effect of three sperm pretreatments (prior to in vitro fertilization) and seven embryo culture protocols on fertilization rate and (or) subsequent development of in vitro fertilized embryos derived from oocytes harvested from ovaries of 1-6 month old prepubertal Boer goats in China. Cleavage rates were highest for embryos fertilized with heparin-treated versus calcium ionophore- or caffeine-treated sperm. Similar rates of blastocyst development were observed using heparin- and ionophore-treated sperm, which were higher than obtained with caffeine-treated sperm. No differences in cleavage or blastocyst rates were observed following embryo culture in basal medias (synthetic oviductal fluid (SOF), Charles Rosenkrans 1 (CR1) or tissue culture medium-199 (TCM-199)) containing 10% fetal bovine serum (FBS). Cumulus or oviductal cell co-culture did not enhance cleavage or blastocyst rates relative to culture in SOF+10% FBS. Replacement of FBS in SOF medium with 0.3% BSA increased cleavage rates, but did not increase rates of blastocyst development. Sequential culture in SOF+0.3% BSA followed by SOF+10% FBS increased blastocyst yield versus continuous culture in SOF+10% FBS and tended to increase blastocyst yield versus continuous culture in SOF+0.3% BSA. These results demonstrate a pronounced effects of sperm pretreatments and in vitro embryo culture systems on rates of blastocyst development and provide a potential protocol (sperm pretreatment with heparin and sequential embryo culture in SOF+0.3% BSA followed by SOF+10% FBS) for generation of the significant numbers of in vitro produced blastocysts from oocytes of prepubertal Boer goats necessary for application of embryo transfer in rural regions of China for distribution of Boer goat genetics.

• 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.

• 한국수정란이식학회지
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• 제18권2호
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• pp.157-161
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• 2003

본 연구는 소형 고양이의 불임 해결과 체외수정란을 생산하기 위한 방안으로서 난자의 형태, 번식주기, 배양시간 및 활성화 처리가 난포란의 체외수정 및 체외발생에 미치는 영향을 조사하였다. 1. 신선 및 salt에 보존한 난소로부터 회수한 난구세포부착 및 나화 난자를 각각 배양했을때 체외수정율 및 분할율은 65.7%와 17.1%, 28.6%와 8.6% 및 57.1%와 13.3%, 23.3%와 3.3%로서 난구세포 부착 신선난자가 나화 난자에 비해 높은 체외발생률을 나타냈다. 2. 휴지기, 발정기 및 황체기 단계로 구분하여 채취한 난포란을 성숙배양 후 수정시켰을 때 체외수정율은 각각 68.9%, 44.4%, 48.9%였으며, 분할율은 각각 17.8%, 8.9%, 12.8%로 나타났다 3. 24, 36 및 48시간 각각 배양한 난포란을 성숙 배양 후 수정시켰을 때 체외수정율은 각각 66.7%, 46.7%, 48.9%였으며, 분할율은 각각 17.8%, 11.1%, 8.5%로 나타났다. 4. 난자를 활성화처리 및 비활성화처리 후 각각 체외수정시켰을 때 체외수정율은 각각 57.4%와 31.4%였고, 체외분할율은 22.9%와 11.4%로서 활성화 처리를 한 난자가 높은 체외발생율을 나타냈다.

• 한국수정란이식학회지
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• 제32권4호
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• pp.311-317
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• 2017

Cryopreservation of oocytes by vitrification technique may contribute a lot in the field of reproductive biotechnology. The objectives of the present study were to evaluate the effectiveness of two cryo-devices for vitrification of immature oocytes of indigenous zebu cows. Slaughter house derived immature cumulus-oocyte-complexes (COCs) of cows were vitrified using 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA) with 0.5 mol sucrose in TCM 199 supplemented with 20% FBS. Vitrification of COCs was completed after immediate plunging of COCs loaded cryotop or French mini straw into the liquid nitrogen ($LN_2$). Then the COCs containing cryotop or French mini straws were warmed in 0.25 mol sucrose and 20% FBS supplemented TCM 199 followed by in vitro culture in $50{\mu}l$ droplets of bicarbonate buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 hrs at $39^{\circ}C$ with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. Denuded oocytes were also stained by whole mount technique using 1% orcein to examine the maturation by presence of MII chromosomes. The in vitro maturation rate was significantly (p<0.05) higher in oocytes vitrified and warmed using crytop ($47.1{\pm}6.9%$) than that of French mini straw ($15.9{\pm}12.5%$). Moreover, in vitro maturation rate was significantly (p<0.05) highe r in control oocytes (not vitrified) ($84.5{\pm}14.2%$) than that of vitrified oocytes. In conclusion, cryotop is better than French mini straw as cryo-device for vitrification of bovine immature oocytes.