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- 검색키워드
- 저자 : ( Joohyeong Lee ) (검색결과 51 건)
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Joohyeong Lee,Yongjin Lee,Hae Hong Jung,Seung Tae Lee,Geun-Shik Lee,Eunsong Lee 한국수정란이식학회 2018 한국동물생명공학회지 Vol.33 No.4
The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.
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Lee, Joohyeong,Lee, Yongjin,Jung, Hae Hong,Lee, Seung Tae,Lee, Geun-Shik,Lee, Eunsong The Korean Society of Embryo Transfer 2018 한국동물생명공학회지 Vol.33 No.4
The osmolarity of a medium that is commonly used for in vitro culture (IVC) of oocytes and embryos is lower than that of oviductal fluid in pigs. In vivo oocytes and embryos can resist high osmolarities to some extent due to the presence of organic osmolytes such as glycine and alanine. These amino acids act as a protective shield to maintain the shape and viability in high osmotic environments. The aim of this study was to determine the effects of glycine or/and alanine in medium with two different osmolarities (280 and 320 mOsm) during IVC on embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. To this end, IVC was divided into two stages; the 0-2 and 3-7 days of IVC. In each stage, embryos were cultured in medium with 280, 320, or 360 mOsm and their combinations with or without glycine or/and alanine according to the experimental design. Treatment groups were termed as, for example, "T(osmolarity of a medium used in 0-2 days of IVC)-(osmolarity of a medium used in 3-7 days of IVC)" T280-280 was served as control. When PA embryos were cultured in medium with various osmolarities, T320-280 showed a significantly higher blastocyst formation (29.0%) than control (22.2%) and T360-360 groups (6.9%). Glycine treatment in T320-280 significantly increased blastocyst formation (50.4%) compared to T320-280 only (36.5%) while no synergistic was observed after treatment with glycine and alanine together in T320-280 (45.7%). In contrast to PA embryonic development, the stimulating effect by the culture in T320-280 was not observed in SCNT blastocyst development (27.6% and 23.7% in T280-280 and T320-280, respectively) whereas the number of inner cell mass cells was significantly increased in T320-280 (6.1 cells vs. 9.6 cells). Glycine treatment significantly improved blastocyst formation of SCNT embryos in both T280-280 (27.6% vs. 38.0%) and T320-280 (23.7% vs. 35.3%). Our results demonstrate that IVC in T320-280 and treatment with glycine improves blastocyst formation of PA and SCNT embryos in pigs.
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Hanna Lee,Yongjin Lee,Bola Park,Fazle Elahi,Joohyeong Lee,Jung Hoon Choi,Seung Tae Lee,Choon-Keun Park,Sang-Hwan Hyun,Eunsong Lee 한국수정란이식학회 2014 한국동물생명공학회지 Vol.29 No.4
This study was designed to evaluate the effect of bovine serum albumin (BSA) in a maturation medium on oocyte maturation and embryonic development in pigs. Immature pig oocytes were matured for 44 h in a medium supplemented with 0.4% (w/v) BSA, 0.1% (w/v) polyvinyl alcohol (PVA), or 10% (v/v) pig follicular fluid (PFF). After IVM, oocytes reached metaphase II stage were activated for parthenogenesis (PA) or used as cytoplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation (89.5%, 90.7% and 91.3% for BSA, PVA and PFF, respectively) and intraoocyte glutathione contents (1.20, 1.16 and 1.00 pixels/oocyte for BSA, PVA and PFF, respectively) were not altered by the macromolecules added to maturation medium. IVM of oocytes in a medium containing BSA (21.4%) and PVA (20.7%) showed significantly lower blastocyst formation after PA than culture in medium with PFF (39.2%). After SCNT, oocytes matured in medium with BSA showed decreased embryonic development to the blastocyst stage (9.2%) compared to those matured in medium with PFF (28.9%), while 23.6% of SCNT oocytes matured in medium with PVA developed to the blastocyst stage. When the effect of BSA in a maturation medium during the first 22 h and the second 22 h of IVM in combination with PFF or PVA was examined, PVA-BSA showed a higher nuclear maturation (94.1%) than BSA-PFF (84.5%). However, there was no significant difference in the blastocyst formation among tested combinations (47.3, 52.2, 50.0, 44.4 and 49.0% for PFF-PFF, PFF-BSA, PVA-BSA, BSA-PVA and BSA-PFF, respectively). Our results demonstrate that BSA and PVA added to maturation medium can support oocyte maturation comparable to PFF-supplemented medium. However, maturation of oocytes in a BSA-containing medium decreases embryonic development after PA and SCNT when compared with the medium supplemented with PFF.
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Joohyeong Lee,Jung Hoon Choi,Seung Tae Lee,Sang-Hwan Hyun,Eunsong Lee 韓國受精卵移植學會 2013 한국동물생명공학회지 Vol.28 No.2
The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21~22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05<P<0.1) nuclear maturation when compared with oocytes matured in M199 (76.9% vs. 83.8%) but no significant differences were found in embryo cleavage, blastocyst formation, and mean number of cells in blastocyst (73.8% vs. 74.6%, 11.1% vs. 12.1%, and 28.4 cells vs. 30.1 cells, respectively). The oocyte degeneration was not reduced by CB treatment during ICSI (11.9%) when compared with no treatment control (11.3%) while the treatment showed detrimental effects (P<0.05) on embryonic cleavage (40.0%) and blastocyst formation (1.8%) rates when compared with control (60.0% and 11.5%, respectively). For activation of ICSI oocytes, additional electric stimulus has no positive or negative effect on in vitro development of preimplantation stage ICSI porcine embryos. Our results demonstrate that CB treatment during ICSI inhibits embryonic development of ICSI oocytes and additional electric activation after ICSI has no effect in improving ICSI embryonic development in pigs. Further studies are needed to improve ICSI efficiency by investigating factors influencing embryonic development after ICSI in pigs.
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Lee, Hanna,Lee, Yongjin,Park, Bola,Elahi, Fazle,Lee, Joohyeong,Choi, Jung Hoon,Lee, Seung Tae,Park, Choon-Keun,Hyun, Sang-Hwan,Lee, Eunsong The Korean Society of Embryo Transfer 2014 한국동물생명공학회지 Vol.29 No.4
This study was designed to evaluate the effect of bovine serum albumin (BSA) in a maturation medium on oocyte maturation and embryonic development in pigs. Immature pig oocytes were matured for 44 h in a medium supplemented with 0.4% (w/v) BSA, 0.1% (w/v) polyvinyl alcohol (PVA), or 10% (v/v) pig follicular fluid (PFF). After IVM, oocytes reached metaphase II stage were activated for parthenogenesis (PA) or used as cytoplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation (89.5%, 90.7% and 91.3% for BSA, PVA and PFF, respectively) and intraoocyte glutathione contents (1.20, 1.16 and 1.00 pixels/oocyte for BSA, PVA and PFF, respectively) were not altered by the macromolecules added to maturation medium. IVM of oocytes in a medium containing BSA (21.4%) and PVA (20.7%) showed significantly lower blastocyst formation after PA than culture in medium with PFF (39.2%). After SCNT, oocytes matured in medium with BSA showed decreased embryonic development to the blastocyst stage (9.2%) compared to those matured in medium with PFF (28.9%), while 23.6% of SCNT oocytes matured in medium with PVA developed to the blastocyst stage. When the effect of BSA in a maturation medium during the first 22 h and the second 22 h of IVM in combination with PFF or PVA was examined, PVA-BSA showed a higher nuclear maturation (94.1%) than BSA-PFF (84.5%). However, there was no significant difference in the blastocyst formation among tested combinations (47.3, 52.2, 50.0, 44.4 and 49.0% for PFF-PFF, PFF-BSA, PVA-BSA, BSA-PVA and BSA-PFF, respectively). Our results demonstrate that BSA and PVA added to maturation medium can support oocyte maturation comparable to PFF-supplemented medium. However, maturation of oocytes in a BSA-containing medium decreases embryonic development after PA and SCNT when compared with the medium supplemented with PFF.
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Joohyeong Lee,Donghyuk Hur,Seung Tae Lee,Eunsong Lee 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10
Spermatogonial stem cells (SSC) are promising resources for genetic preservation and restoration of male germ cells in human and animals. However, in our preliminary experiment, it was observed that SSC was fragile when used as nuclei donor for SCNT. This study investigated the potential of porcine SSC as donor nuclei for somatic cell nuclear transfer (SCNT) and developmental competence of SSC-derived cloned embryos. In addition, it was examined whether demecolcine could prevent rupture of SSC during SCNT. SCNT embryos were produced using a standard protocol of our laboratory. After electric activation, SCNT embryos were treated with demecolcine combined with 6-DMAP for 4 h, washed, and then cultured in a porcine zygote medium-3 at 39℃ in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 for 7 days. When SSC was compared with porcine fetal fibroblast (PFF) in the potential to support embryonic development after SCNT, SSC-derived SCNT embryos showed higher (p< 0.05) developmental competence to the blastocyst stage (53.7%) than PFF-derived embryos (28.6%). Treatment of SSC with demecolcine significantly (p<0.01) inhibited the rupture of SSC during SCNT (7.8% vs. 16.4%) and increased fusion of cell-ooplasm couplets compared to no treatment (71.9% vs. 62.8%). In addition, even after demecolcine treatment, SSC-derived SCNT embryos showed a higher blastocyst formation (46.4%) than PFF-derived embryos (27.8%). Our results demonstrate that porcine SSC are a desirable donor cell type for production of SCNT pig embryos and demecolcine increases production efficiency of cloned embryos by inhibiting rupture of nuclei donor SSC during SCNT.
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Joohyeong Lee,Ji Eun Park,Minji Kim,Hyeji Shin,Geun-Shik Lee,Seung Tae Lee,Eunsong Lee 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11
Poor embryo quality and low blastocyst formation have been major limitations in establishment of cloned embryonic stem cells and production of cloned animals through somatic cell nuclear transfer (SCNT). Aggregation of embryos is a promising method for improving developmental competence of blastocysts. The aim of this study was to improve the blastocyst formation and the quality of parthenogenetic (PA) pig embryos by the aggregation of blastomeres at the 4-cell stage that were cultured in various type of culture dishes with or without phytohemagglutinin (PHA). The PA embryos were produced by the general method of our laboratory. On Day 2 after PA, the zona pellucida of 4 cell-stage embryos were removed by treatment with 0.5% (wt/vol) pronase solution. The 3x zona-free blastomere (ZFB) were randomly distributed in each of the following treatments for aggregation. ZFB were cultured for 5 days at 39℃ in an atmosphere 5% CO2, 5% O2, and 90% N2. In Experiment 1, effect of culture dishes on the aggregation efficiency and developmental competence of PA embryos were investigated. ZFB were cultured on non-coated (control) culture dish or dishes coated with 1% (wt/vol) agarose substrate (AS) or Well of the Well in dishes coated with 1% (wt/vol) agarose substrate (WAS). The ZFB cultured in WAS showed significantly higher (P<0.05) aggregation (81.2%) than AS and control (21.6-45.5%). The mean cell number in blastocysts derived from AS and WAS (81.4-89.3 cells/blastocyst) was significantly higher (P<0.05) than that of control (63.8 cells/blastocyst). In Experiment 2, effects of 150 ug/ml PHA treatment on the aggregation efficiency and developmental competence of embryos were investigated. The ZFB cultured in AS with PHA showed a higher (P<0.05) aggregation rate (90.0%) than that in AS without PHA, control with PHA, and control (39.2%, 57.9% and 17.5%, respectively). In conclusion, aggregation of porcine ZFB treated with PHA and agarose substrate could be a useful technique for producing improving blastocyst development with increased mean cell number of blastocysts in pigs.
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