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Mi-Rung Park,In-Sun Hwang,Joo-Hyun Shim,Hyo-Jin Moon,Dong-Hoon Kim,Yeoung-Kyu Ko,Hwan-Hoo Seong,Gi-Sun Im 한국동물생명공학회(구 한국동물번식학회) 2008 Reproductive & developmental biology Vol.32 No.3
This study was conducted to investigate the development and gene expression in miniature pig nuclear transfer (mNT) embryos produced under different osmolarity culture conditions. Control group of mNT embryos was cultured in PZM-3 for 6 days. Treatment group of mNT embryos was cultured in modified PZM-3 with NaCl (mPZM-3, 320 mOsmol) for 2 days, and then cultured in PZM-3 (270 mOsmol) for 4 days. Blastocyst formation rate of the treatment group was significantly higher than the control and the apoptosis rate was significantly lower in treatment group. Bax- and caspase-3 mRNA expression were significantly higher in the control than the treatment group. Also, the majority of imprinting genes were expressed aberrantly in in vitro produced mNT blastocysts compared to in vivo derived blastocyst H19 and Xist mRNA expression were significantly lower in the control than the treatment group or in vivo. IGF2 mRNA expression was significantly higher in the control than the treatment group or in vivo. IGF2r mRNA expression was significantly lower in the control. Methylation profiles of individual DNA strands in H19 upstream T-DMR sequences showed a similar methylation status between treatment group and in vivo. These results indicate that the modification of osmolarity in culture medium at early culture stage could provide more beneficial culture environments for mNT embryos.
Park, Mi-Rung,Hwang, In-Sun,Shim, Joo-Hyun,Moon, Hyo-Jin,Kim, Dong-Hoon,Ko, Yeoung-Gyu,Seong, Hwan-Hoo,Im, Gi-Sun 韓國受精卵移植學會 2008 한국동물생명공학회지 Vol.23 No.2
This study investigated the developmental ability and gene expression of somatic cell nuclear transfer embryos using ear skin fibroblast cells derived from miniature pig. When miniature pig (m) and landrace pig (p) were used as donor cells, there were no differences in cleavage (79.2 vs. 78.2%) and blastocyst rates (27.4 vs. 29.7%). However, mNT blastocysts showed significantly higher apoptosis rate than that of pNT blastocysts (6.1 vs. 1.7%) (p<0.05). The number of nuclei in pNT blastosysts was significantly higher than that of mNT (35.8 vs. 29.3) (p<0.05). Blastocysts were analyzed using Realtime RT-PCR to determine the expression of Bax-, Bcl-xl, H19, IGF2, IGF2r and Xist. Bax- was higher in mNT blastocyst than pNT blastocyst (p<0.05). There was no difference in Bcl-xl between two NT groups. Bax-/Bcl-xl was, however, significantly higher in mNT blastocyst compared to pNT. The expression of imprinting genes were aberrant in blastocysts derived from NT compared to in vivo blastocysts. H19 and IGF2r were significantly lower in mNT blastocysts (p<0.05). The expression of IGF2 and Xist was similar in two NT groups. However, imprinting genes were expressed aberrantly in mNT compared to pNT blastocysts. The present results suggest that the NT between donor cells derived from miniature pig and recipient oocytes derived from crossbred pig might affect reprogramming of donor cell, resulting in high apoptosis and aberrant expression patterns of imprinting genes.
The Imprinted Messenger RNA Expression in Cloned Porcine Pre-implantation Embryos
Park, Mi-Rung,Kim, Bong-Ki,Lee, Hwi-Cheul,Lee, Poong-Yeon,Hwang, Seong-Soo,Im, Gi-Sun,Woo, Jae-Seok,Cho, Chang-Yeon,Choi, Sun-Ho,Kim, Sang-Woo 韓國受精卵移植學會 2010 한국동물생명공학회지 Vol.25 No.2
The objective of this study was to determine the mRNA expression patterns of several putative imprinted genes in in vivo and in vitro fertilized, parthenogenetic, and cloned porcine preimplantation embryos. Both maternally (Dlk1, IGF2, Peg1/Mest and Ndn) and paternally (IGF2r, H19 and Xist) imprinted genes were selected. We have used reverse transcription polymerase chain reaction (RT-PCR) to investigate gene expression patterns in the porcine embryos. IGF2 transcripts were detected in the most of embryos. In nuclear transfer (NT), Peg1/MEST transcripts showed fluctuating pattern. Dlk1 was only expressed partially from the morula and blastocyst stage of NT embryos. Ndn gene expression was started somewhat early for in vivo embryos. However, the expressions of maternally imprinted genes were similar in all types of blastocysts (NT, in vivo and in vitro fertilized, and parthenogenetic embryos). The IGF2R gene expression level was somewhat irregular and varied among samples. However, for the majority samples of all types of embryos, IGF2R expression was diminished after one- to two-cell stages and reappeared at the morulae or blastocyst stage embryos. H19 gene was only expressed early in parthenogenetic and in vivo embryos. For NT embryos, H19 was only expressed in blastocysts. Xist expression was detected in all blastocysts with the earliest being in vivo 8-cell stage embryos and the last one being NT blastocysts. These putative imprinted genes appeared to have stage specific expression patterns with a fluctuating pattern for some genes (Peg/Mest, IGF2r, H19). These results suggest that stage specific presence of imprinted genes can affect the embryo implantation and fetal development.
PARK, In-Seok,KIM, Eun-Mi,WOO, Seon Rang,OH, Sung-Yong,KIM, Dong Soo,HUR, Jun Wook Blackwell Publishing Asia 2006 Fisheries Science Vol.72 No.4
<P>ABSTRACT: </P><P>The aim of this study was to establish effective procedures for chromosome manipulation in greenling <I>Hexagrammos otakii</I> Jordan et Starks, which has enormous aquacultural potential. To accomplish this, temperature-dependent measurements of the mitotic intervals (&tgr;<SUB>0</SUB>) were carried out. The &tgr;<SUB>0</SUB> in this fish was determined by averaging the duration of the first and third embryonic divisions at temperatures ranging 5–25°C. At higher temperatures, eggs developed faster and underwent more identical development. For greenling, &tgr;<SUB>0</SUB> were 341.1 ± 3.60 min at 5°C, 275.5 ± 4.53 min at 10°C, 189.7 ± 6.93 min at 15°C, 99.2 ± 8.27 min at 20°C and 34.2 ± 8.74 min at 25°C. There were strong, negative correlations between the &tgr;<SUB>0</SUB> and water temperatures at all temperatures studied (<I>Y</I> = −79.3<I>X</I> + 425.3, <I>R</I><SUP>2</SUP> = 0.9968, where <I>Y</I> is the mitotic interval and <I>X</I> is the temperature).</P>
Park, Mi-Rung,Hwang, In-Sun,Shim, Joo-Hyun,Moon, Hyo-Jin,Kim, Dong-Hoon,Ko, Yeoung-Kyu,Seong, Hwan-Hoo,Im, Gi-Sun The Korean Society of Animal Reproduction 2008 Reproductive & developmental biology Vol.32 No.3
This study was conducted to investigate the development and gene expression in miniature pig nuclear transfer (mNT) embryos produced under different osmolarity culture conditions. Control group of mNT embryos was cultured in PZM-3 for 6 days. Treatment group of mNT embryos was cultured in modified PZM-3 with NaCl (mPZM-3, 320 mOsmol) for 2 days, and then cultured in PZM-3 (270 mOsmol) for 4 days. Blastocyst formation rate of the treatment group was significantly higher than the control and the apoptosis rate was significantly lower in treatment group. Bax-$\alpha$ and caspase-3 mRNA expression were significantly higher in the control than the treatment group. Also, the majority of imprinting genes were expressed aberrantly in in vitro produced mNT blastocysts compared to in vivo derived blastocyst H19 and Xist mRNA expression were significantly lower in the control than the treatment group or in vivo. IGF2 mRNA expression was significantly higher in the control than the treatment group or in vivo. IGF2r mRNA expression was significantly lower in the control. Methylation profiles of individual DNA strands in H19 upstream T-DMR sequences showed a similar methylation status between treatment group and in vivo. These results indicate that the modification of osmolarity in culture medium at early culture stage could provide more beneficial culture environments for mNT embryos.