• Asian-Australasian Journal of Animal Sciences
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• 제27권2호
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• pp.266-277
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• 2014

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with $4{\mu}g/mL$ of digitonin for 2 min at $4^{\circ}C$ in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at $18^{\circ}C$ was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제22권4호
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• pp.500-506
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• 2009

A modified Tris-buffered medium (mTBM) has been widely used as an insemination medium for porcine in vitro fertilization (IVF). We examined whether mTBM could be used for bovine IVF. Bovine cumulus-oocyte complexes (COCs) were cultured in a serum-free medium containing 30 ng/ml EGF for 22 h. After culture, COCs were inseminated with spermatozoa for 12 h in mTBM containing 5 mM caffeine and 10 g/ml heparin. The penetration of oocytes increased significantly (p<0.05) as the sperm concentration increased from 0.1 (30%) to 1-10 $(87-100%){\times}10^6$ cells/ml. This was significantly different from values obtained at 1 (87%) and 10 $(100%){\times}10^6$ cells/ml. However, when COCs were inseminated with spermatozoa from different bulls, the proportions (62-100%) of oocytes penetrated varied according to the bull. The proportion (18%) of oocytes penetrated was significantly (p<0.05) lower in a fertilization medium without caffeine and heparin but increased with the addition of caffeine and/or heparin to the medium, and the proportion (93-96%) of oocytes penetrated increased significantly (p<0.05) when the medium was supplemented with heparin and caffeine. In this medium, sperm penetration was first observed at 3 h after insemination. Irrespective of the presence of glucose in the fertilization medium, the proportion (93-97%) of oocytes penetrated and the proportion (83-84%) of embryos at the ${\geq}2$-cell stage cultured in a chemically defined medium were not significantly different. However, the proportion of embryos developing to the blastocyst stage was significantly (p<0.05) higher in the presence (11%) of glucose in the fertilization medium than in its absence (2%). In conclusion, the present study demonstrated that bovine oocytes penetrated in vitro in mTBM can develop to the blastocyst stage and mTBM may be used for the in vitro production of bovine embryos.

• 한국가축번식학회지
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• 제26권3호
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• pp.215-221
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• 2002

본 연구는 일정량의 항산화제(NAC, ebselen 및 GSH)와 성장인자(EGF, PDGF)의 혼합첨가가 돼지 체외수정란의 체외배양에 미치는 영향을 검토하였다. 1. NCSU 23 배양액에 NAC 1nm과 NAC에 EGF 100ng/$m\ell$비 및 PDGF 5ng/$m\ell$를 각각 혼합 첨가하여 체외배양을 실시한 결과, 상실배기 이상 발육된 체외발육율은 각각 28.1%, 32.3% 및 35.3%로서 NAC와 PDGF 혼합처리구가 대조 구보다 다소 높은 체외발육율을 나타냈으나 통계적 유의성은 없었다(P>0.05). 2. NCSU 23 배양액에 ebselen 10$\mu\textrm{m}$과 ebselen에 EGF 100ng/$m\ell$ 및 PDGF 5ng/$m\ell$를 각각 혼합첨가하여 체외발육율을 조사한 결과, 상실 배기 이상 발육된 수정란의 체외발육율은 각각 17.8%, 36.9% 및 40.3%로 ebselen과 성장인자의 혼합처리구가 대조구보다 통계적 유의하게 높은 체외발육율을 나타냈다(P<0.05). 3. NCSU 23 배양액에 GSH 100$\mu\textrm{m}$과 GSH에 EGF 100ng/$m\ell$ 및 PDGF 5ng/$m\ell$를 각각 혼합 첨가배양하여 상실배기 이상 발육된 수정란의 체외발육율은 각각 24.4%, 30.5% 및 27.7%로 GSH과 EGF 혼합처리구가 여타구보다 다소 높은 체외발육율을 나타냈지만 통계적 유의성은 없었다(P>0.05). 4. 모든 처리구에서 배반포기 수정란의 세포수는 커다란 차이가 인정되지 않았으나(P>0.05), 체외배양액에 ebselen과 성장인자를 혼합첨가하여 체외배양한 처리구에서는 대조구에 비해 통계적으로 유의하게 높은 세포수를 나타냈다.(P<0.05).

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.80-80
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• 2002

This study was carried out that to investigate the effects of amino acids supplemented with culture medium on development of porcine embryos cultured in vitro. Cumulus oocyte complexes (COCs) were cultured in the maturation medium containing hormones (0.5$\mu\textrm{g}$/$m\ell$ LH, 0.5$\mu\textrm{g}$/$m\ell$ FSH and 1$\mu\textrm{g}$/$m\ell$ estradiol-17${\beta}$) for 20-22 h at 39$^{\circ}C$ in an atmosphere of 5% CO$_2$in air. Subsequently, COCs were cultured in hormone-free maturation medium for 20-22 h. After maturation for 40-44h, oocytes were removed cumulus cells by pipetting and cultured with epididymal sperm for 5 h in the mTBM. Embryos obtained were divided in 4 groups (1) cultured in NCSU 23 containing 0.4% BSA to blastocyst stage(Control), (2) essential amino acids (EA), (3) non-essential amino acids (NA), (4) mixture of essential and non essential amino acid (EA+NA). All treated groups(2-4) were used a glucose free NCSU 23 medium supplemented with pyruvate (0.33 mM), lactate (4.5 mM) to morula stage. From morula to blastocyst stage embryos of all treated groups were cultured in NCSU 23 containing 0.4% BSA. The rates of cleaved oocytes at 48 h after IVF were from 82% to 88% in the groups of control, EA, NA and EA+NA, respectively. The in vitro developmental rates into blastocysts in the groups of EA and EA+NA were significantly (P<0.05) higher than those of group of control (35.1, 35.4 vs. 19.4%, respectively), however, no significant (P<0.05) between control and NA. In conclusion, supplemented with essential amino acid or mixture of essential and non essential amino acid in the culture medium at morula stage increased the rate of development to blastocyst on in vitro produced porcine embryos.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.84-84
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• 2002

The advantages of the OPS techniques(Vajta G et al, Mol Reprod Dev 51: 53-58,1998) give 1) high survival rates of various types of eggs, 2) quick and simple process, 3) inexpensive equipment and reduced chilling injury. The efficiency of IVM/IVF technique in the porcine species is relatively lower than that obtained in other species such as ruminants. Two experiments were designed to investigate the effects of in-vitro fertilization of porcine oocytes matures using different OPS protocol for chilling and warming of vitrification. Porcine oocytes from ovaries collected at abattoir were matured for 44 hours in TCM199 Earle's salt supplemental with pyruvate, pff, L-cysteine, hormones and gentamycin. Oocytes were denuded and fertilized with frozen boar semen by common method. Porcine embryos produced routinely by in-vitro culture system of NCSU23 medium. The vitrification and the warming were conducted by OPS method with the glass micropipette instead of straw vessels and modified the protocol of G.Vajta(1999). In Exp 1, Chilling/Warming:Holding Medium(HM)+EG+DMSO/HM +sucrose Medium(SM) at 39$^{\circ}C$ warm stage. In Exp 2, : PBS+CS+EG+Ficoll+ Trehalose/PBS+Trehalose at 25$^{\circ}C$ stage. Filling, freezing, packing, thawing out and further culturing were performed to follow the basic protocol of G Vajta. During IVM-lVC and post-warming, fertilization parameter and developmental potential were compared to and statistically analysed. It was not significantly different from Exp 1 and Exp 2 but 25$^{\circ}C$ of stage was slightly higher on the morula/blastocyst forming rate and better atmosphere for worker than that at 39$^{\circ}C$ stage.

• 한국가축번식학회지
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• 제26권2호
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• pp.143-151
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• 2002

본 연구는 돼지의 미성숙 난포란을 체외에서 성숙, 수정시킨 후, 생산된 체외수정란을 NCSU23 체외배양액에 항산화제(NAC, ebselen 및 glutathione)와 성장인자(EGF와 PDGF)의 첨가가 돼지 체외수정란의 체외 발육에 미치는 영향을 검토하였다. NAC을 0, 0.5, 1.0 및 2.0mM 첨가하여 체외배양한 결과, 상실배기 이상 발육된 체외발육성적은 1.0mM과 2.0mM 첨가구가 여타구보다 높은 체외발육성적을 나타냈다. 한편, ebselen을 0, 5, 10 및 25$\mu$M을 첨가하여 배양한 경우 상실배 이상 발육된 체외발육 성적은 각각 17.9, 22.5, 36.9 및 10.8%로서 ebselen 10$\mu$M 첨가구가 여타구에 비해 다소 높은 체외발육성적을 얻었으나 통계적 유의차는 인정되지 않았다(P＞0.05). 또한 glutathione을 0, 50, 100 및 200$\mu$M 첨가시 상실배기 이상 발육된 체외발육성적은 각각 21.8, 17.4, 28.3 및 15.8%로서 100$\mu$M 처리구가 여타구보다 다소 높은 체외발육성적을 얻었으나 통계적 유의차는 인정되지 않았다(P＞0.05). 돼지 체외수정란의 체외배양시 체외배양액에 growth factor의 첨가배양이 돼지수정란의 체외발육에 미치는 영향을 검토한 결과, NCSU 23배양액에 EGF를 0, 10, 50 및 100ng/$m\ell$ 첨가시 상실배기이상 발육된 체외 발육 성적은 각각 19.7, 29.0, 26.6 및 40.4%로서 EGF 100ng1m1 처리구가 여타구보다 통계적으로 유의하게 높은 체외발육 성적을 나타냈으며(P＜0.05), PDGF를 0, 1, 5 및 10ng/$m\ell$ 첨가하여 배양한 경우 상실배기 이상 발육된 체외발육성적은 각각 22.0, 21.6, 33.9 및 15.9%로서 PDGF 5ng/$m\ell$ 처리구가 여타구에 비해 높은 체외발육성적을 얻었다. 본 실험에서 체외수정후 생산된 배반포기 수정란의 세포수는 처리구간 커다란 차이는 인정되지 않았다.

• 한국수정란이식학회지
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• 제19권3호
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• pp.265-273
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• 2004

본 연구는 mNCSU-23에서 체외성숙시킨 난자를 이용하여 돼지 체외수정시 적절한 배양액과 정자농도를 구명하고자 수행하였다. 정액은 액상정액을 이용하였고 체외수정배양액으로 mTBM과 mTLP-PVA, 정자농도는 운동정자수 5${\times}$$10^4$, 1${\times}$$10^{5}$ , 5${\times}$$10^{5}$ sperm/$m\ell$로 체외수정 한 다음 NCSU-23에서 체외발달을 유도한 결과는 다음과 같다. 1. 돼지 체외성숙란을 mTBM 또는 mTLP-PVA에서 운동정자수 1${\times}$$10^{5}$ sperm/$m\ell$로 체외수정시킨 다음 체외발달시킨 결과, 정자침투율, 웅성전핵형성율, 다정자수정발생율, 난분할율, 배반포 발달율 등 어느 것도 두 배양액간 차이를 나타내지 않았다. 2. 체외성숙란을 mTBM에서 운동정자수 5${\times}$$10^4$, 1${\times}$$10^{5}$ , 5${\times}$$10^{5}$ sperm/$m\ell$l로 체외수정시킨 다음 체외발달시킨 결과, 정자침투율, 웅성전 핵형성율, 다정자수정발생율 등은 모두 정자 농도간 유의적인 차이를 보였고(P<0.05), 난 분할율과 배 반포발달율은 5${\times}$$10^4$이 1${\times}$$10^{5}$ , 5${\times}$$10^{5}$ sperm/$m\ell$보다 유의 적으로 낮았으나(P<0.01), 1${\times}$$10^{5}$ 와 5${\times}$$10^{5}$ sperm/$m\ell$ 간에는 차이가 없었다. 따라서, 액상정액을 이용한 돼지 체외성숙란의 체외수정배양액으로 mTBM과 mTLP-PVA간 차이는 없으며, mTBM에서 체외수정시킬 때 정자농도는 운동정자수로 1${\times}$$10^{5}$ sperm/$m\ell$가 적절할 것으로 사료된다.

• Reproductive and Developmental Biology
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• 제32권4호
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• pp.237-243
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• 2008

The objective of this study was to examine the effect of caffeine and sodium bicarbonate in a fertilization medium on the fertilizability of boar spermatozoa that were frozen in straws. Boar spermatozoa were extended with Beltsville F5 extender and frozen in 0.25-ml straws. In vitro matured porcine oocytes were fertilized in vitro (IVF) with frozen-thawed boar spermatozoa for 6h in a modified tris-buffered medium (mTBM) or in its modified medium by substituting the tris with 25mM sodium bicarbonate (modified bicarbonate-buffered medium; mBBM). Some of inseminated oocytes were fixed and stained for examination of sperm penetration. IVF embryos were cultured in a North Carolina State University-23 medium for embryo development. The percentage of live sperm was $47{\pm}4%$ and morphological abnormality of acrosome was found in $14{\pm}3%$ of spermatozoa. Optimal sperm concentration for IVF was $0.75{\sim}1.0{\times}1.0{\times}10^6$ sperms/ml when mTBM containing 5mM caffeine was used as the fertilization medium. Sperm penetration was significantly (p<0.05) stimulated by increasing caffeine concentration in the IVF medium. In addition, mBBM significantly (p<0.05) increased sperm penetration (92%) compared to mTBM (65%). More (p<0.05) blastocysts (22% vs. 32%) developed from the oocytes that were fertilized in mBBM containing 1mM caffeine than from those fertilized in mTBM with 5mM caffeine. Our results indicate that boar spermatozoa can be frozen successfully in straws with holding their normal fertilizability and that caffeine and sodium bicarbonate stimulates sperm penetration in vitro.

• 한국수정란이식학회지
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• 제16권3호
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• pp.203-211
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• 2001

성간별에 이용할 돼지 수정란을 체외수정 방법으로 생산하기 위하여 돼지 난소에서 채취한 난자를 NCSU 23 배지에서 eCG, hCG를 첨가한 상태에서 22시간, 첨가하지 않은 상태에서 22시간 체외성숙을 시킨 후 mTBM을 이용하여 여러가지 정자농도(5$\times$$10^4$, 2.5$\times$$10^{5}$ , 6.0$\times$$10^{5}$ 그리고 10.0$\times$$10^{5}$ )에 따라 6시간 수정시켰고, NCSU 23 배지에서 배양시켰다. 배양 후 44시간에 수정란의 분할률을 관찰하였고, 144시간에 배반포 형성율을 확인하였다. 성감별 방법은 체외수정으로 생산된 배반포를 돼지 Y-specific DNA primer를 이용하여 PCR 처리를 한 후 전기영동으로 491 bp에서 증폭된 band의 유무에 따라 암컷과 수컷의 성을 판정하였다. 1. 돼지 체외수정에서 정자농도 5$\times$$10^4$, 2.5$\times$$10^{5}$ , 6.0$\times$$10^{5}$ 및 10.0$\times$$10^{5}$ 에 따른 수정란 분할률은 각각 55.95, 67.88, 60.18, 47,60%이었다. 2. 정자농도 5$\times$$10^4$, 2.5$\times$$10^{5}$ , 6.0$\times$$10^{5}$ 에 따른 배반포 형성율은 각각 16.03, 22.40, 21.41, 12.37%이었다. 3. 31개의 체외수정으로 생산된 배반포에 대한 PCR을 이용한 성감별 결과는 18개가 수컷, 13개가 암컷으로서 각각 58.1%과 41.9%를 나타내었다. 이상의 결과로 보아 돼지의 체외수정으로 생산된 배반포에 대한 성감별시 PCR 기법이 신속하고 정확하게 성감별을 실시할 수 있는 방법으로 판단되었다.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.281-285
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• 2011

In the present study, we identified differentially methylated region (DMR) upstream of Dnmt1o and Dnmt1s gene in early porcine embryos. Porcine Dnmt1o had at least one DMR which was located between -530 bp to -30 bp upstream from transcription start site of the Dnmt1o gene. DNA methylation analyses of Dnmt1o revealed the DMR to be hypomethylated in oocytes, whereas it was highly methylated in sperm. Moreover, the DMR upstream of Dnmt1o was gradually hypermethylated from oocytes to two cells and dramatically changed in the methylation pattern from four cells to BL stages in an in vivo. In an IVF, the methylation status in the DMR upstream of Dnmt1o was hypermethylated from one cell to eight cells, but demethylated at the Morula and BL stages, indicating that the DNA methylation pattern in the Dnmt1o upstream ultimately changed from stage to stage before the implantation. Next, to elucidate whether DNA methylation status of Dnmt1s upstream is stage-by-stage changed in during porcine early development, we analyzed the dynamics of the DNA methylation status of the Dnmt1s locus in germ cell, or one cell to BL cells. The Dnmt1s upstream was highly methylated in one and eight cells, while less methylated in two, four, morula, and BL cells. Taken together, our data demonstrated that DNA methylation and demethylation events in upstream of Dnmt1o/Dnmt1s during early porcine embryos dramatically occurred, and this change may contribute to the maintenance of genomewide DNA methylation in early embryonic development.