Optimization of Embryo Density and the Volume of Culture Medium for an Improvement of Mouse Parthenogenetic Embryo Development

Roh Sangho,Choi Young-Joo,Min Byung-Moo 한국동물생명공학회(구 한국동물번식학회) 2005 Reproductive & developmental biology Vol.29 No.3

Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of 2~4 embryos/ uL significantly improved development to the blastoryst stage (72%≤) compared with culture at the lower (0.2~1 embryos㎕, 0~37.5%) and the higher (5~6 embryos㎕, 30~53%) concentration for 120 h when the oocytes were cultured in a 5㎕ drop under mineral oil In experiment 2, the embryos cultured at a concentration of 2~4 embryos㎕ in a 10㎕ drop (81.1%) showed significantly higher blastocyst rates than those in a 5㎕ drop (68.5%). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

In Vitro Development of Mouse Parthenogenetic Embryos: Effect of Temperature before Oocyte Activation

Roh Sangho,Won Cheolhee,Min Byung-Moo 한국동물생명공학회(구 한국동물번식학회) 2005 Reproductive & Developmental Biology(Supplement) Vol.29 No.2s

This study was conducted to establish the optimal temperature condition before oocyte activation in B6D2 F1 mouse. In experiment 1, two embryo culture media (CZB vs KSOM) were evaluated for the development of activated mouse oocytes. Parthenogenetic embryos cultured in KSOM showed better blastocyst development than ones cultured in CZB(56.2% vs 81.0%, p<0.01). Two-hour of pre-incubation before activation significantly reduced the number of hatched blastocysts in KSOM (22.0% versus 8.8%, p<0.05). In experiment 2, recovered oocytes were pre-incubated at different temperature conditions before activation. The experimental groups were divided by 5 as follows. Group A: pre-incubation for 120 min at 37℃, Group B: pre-incubation at 37℃ for 90 min then at 25℃ for 30 min, Group C: pre-incubation at 37℃ for 60 min then at 25℃ for 60 min, Group D: pre-incubation at 37℃ for 30 min then at 25℃ for 90 min, and Group E: pre-incubation at 25℃ for 120 min before activation. Group A (67.6%) and B (66.7%) showed better development to the blastocyst stage than other groups (Group C: 50.0%, Group D: 49.2%, Group E: 33.3%, p<0.05). The present study indicates that the temperature before activation affects the development of B6D2 F1 mouse parthenogenetic oocytes and exposure to room temperature should be limited to 30-min when the oocytes are left in HEPES-buffered medium for micromanipulation.

Somatic Cell Nuclear Transfer in Rodents, the Little Big Animals

Roh, Sangho 韓國受精卵移植學會 2012 한국동물생명공학회지 Vol.27 No.4

Transgenic rats and mice are useful experimental animal models for medical research including human disease model studies. Somatic cell nuclear transfer (SCNT) technology is successfully applied in most mammalian species including cattle, sheep, pig and mouse. SCNT is also considered to increase the efficacy of transgenic/knockout mouse and rat production. However, in the area of reproductive biotechnology, the rodent model is inadequate because of technical obstacles in manipulating the oocytes including intracytoplasmic sperm injection and SCNT. In particular, success of rat SCNT is very limited so far. In this review, the history of rodent cloning is described.

Effect of the Timing of Oocyte Activation on Development of Rat Somatic Cell Nuclear Transfer Embryos

Sangho Roh 한국동물생명공학회(구 한국동물번식학회) 2004 발생공학 국제심포지엄 및 학술대회 Vol.2004 No.1

Mental stress of animal researchers and ways to relieve it

Sangho Roh(노상호) 한국실험동물학회 2022 한국실험동물학회 학술발표대회 논문집 Vol.2022 No.1

Novel Culture Systems for In Vitro Production and Transfer of Mammalian Embryos

Sangho Roh 한국동물생명공학회(구 한국동물번식학회) 2011 발생공학 국제심포지엄 및 학술대회 Vol.2011 No.1

In the first part of this study, a novel culture device the named oil-free micro tube culture (MTC) system for in vitro culture (IVC) of murine and porcine embryos was introduced. Parthenogenetic mouse and porcine embryos were placed into 0.2-mL thinwall flat cap PCR tubes and cultured to the blastocyst stage. Conventional drop culture was used as the control. Murine embryos in MTC had a higher blastocyst formation rate and larger population of cells in the blastocysts. This was due to higher numbers of trophectoderm (TE) cells rather than inner cell mass cells. On the other hand, the 'MTC' system in the pig showed similar (in 20 μl medium volume) or lower (in 10 μl medium volume) blastocyst formation rate when compared with drop culture system. In the second part of this study, dexamethasone (DEX) and leukemia inhibitory factor (LIF), which suppress PGF2α, were directly supplemented into ET media, and transfer of the embryos to surrogate was followed. In the cattle industry, embryo transfer technology has been used to produce the most valuable cows or bulls. Numerous factors such as heat stress, mastitis, manipulating female reproductive tract may contribute to early embryonic loss through premature increases of uterine luminal concentrations of PGF2α in cows. Furthermore, addition of PGF2α to culture medium has been shown to inhibit the development and hatching of mammalian embryos. When DEX and LIF were supplemented, the pregnancy rate (6 month post-ET) was increased from 56.0% to 68.3%. In IVC experiment, DEX and LIF supplementation supported hatching of bovine embryos in the presence of PGF2α in the medium (from 16.9% to 40.6%). Additional ET experiments using alternative drugs are currently under investigation. The present work was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (MIFAFF; 109020-3).

In Vitro Development of Interspecies Nuclear Transfer Embryos: Effects of Culture Systems

Sangho. Roh 사단법인 한국동물생명공학회 2004 Reproductive & developmental biology Vol.28 No.4

Porcine fibroblasts were transferred into enucleated bovine oocytes for the interspecies nuclear transfer (NT). After NT, the embryos were cultured in three different culture systems. The media used for the experiment were CR1aa and NCSU23. The culture systems used for the experiment were: 1. Culture in CR1aa for 7 days (CR). 2. Culture in CR1aa for 2 days and subsequently in NCSU23 for 5 days (CR-NC). 3. Culture in NCSU23 for 7 days (NC). Bovine (intraspecies) NT group was used as a control. The oocytes in bovine NT group were treated the same as interspecies NT embryos except using bovine fibroblasts as nuclear donors. Regardless of their nuclear origin (interspecies vs bovine), the embryos in CR (68.4% vs 77.2%) and CR-NC (67.8% vs 70.5%) showed better developmental competence to the 2-cell stage (p 0.05) than those in NC (41.0% vs 10.0%). Bovine NT embryos in CR-NC did not develop over the 4-cell stage after the medium replacement, while interspecies NT embryos in CR-NC continued to develop and could reach over the 8-ell stage (12.2%). Blastocysts were only found in bovine NT group (17.4%), but no blastocyst was found in interspecies NT group. This study suggests that the development of interspecies NT embryos mostly depends on their recipient cytoplasm during the culture in vitro.

Effect of the Timing of Oocyte Activation on Development of Rat Somatic Cell Nuclear Transfer Embryos

Sangho Roh 한국동물생명공학회(구 한국동물번식학회) 2005 Reproductive & developmental biology Vol.29 No.4

Methods for activation of reconstructed oocytes were examined for the production of nuclear transfer (NT) rat embryos using fetal neural stem cells as donor. Neural stem cells were isolated from Day 14.5 rat fetuses, and the oocytes for recipient cytoplasm were recovered from 4-week old Sprague Dawley rats. After enucleation and nuclear injection, the reconstructed oocytes were immediately exposed to activation medium consisting of 10 mM SrCl₂ for 4 h (immediate activation after injection; IAI), or cultured in vitro for 2~3 h before activation treatment (injection before activation; IBA). Pre-activated oocytes were also used for NT to test reprogramming potential of artificially activated oocytes. The oocytes were grouped as IIA (immediate injection after activation) and ABI (activation 2~3 h before injection). Following NT, the oocytes were cultured in vitro. Development of the NT embryos was monitored at 44 and 119 h after activation. The embryos in groups IAI, mA, and IIA were cleaved to the 2-cell stage at the rates of 36.6%(15/41), 39.5% (17/43) and 46.3% (25/54), respectively. However, in the ABI group, only one embryo (1.8%, 1/55) was cleaved after activation. After in vitro culture, two NT embryos from IAI group had developed to the morula stage (4.9%, 2/41). However, no morula or blastocyst was obtained in the other groups. These results suggest that immediate activation after injection (IAI) method may be used for the production of rat somatic cell NT embryos.

Improvement of pregnancy rate by intrauterine administration of dexamethasone and recombinant human leukemia inhibitory factor at the time of embryo transfer in cattle

Sangho Roh,김세웅,Yeon-Gil Jung,박종임 대한수의학회 2016 Journal of Veterinary Science Vol.17 No.4

Bovine embryos (day 5) were cultured to day 10 with or without 100 ng/mL PGF2 in medium supplemented with control; 100 nM Dex;1,000 U/mL recombinant human leukemia inhibitory factor (rhLIF); or Dex+rhLIF. Although the rates to development to the blastocyst werenot significantly different among groups, the hatching rate after additional culture with Dex +/or rhLIF was significantly higher in allsupplemented groups than the control (p < 0.05). In the presence of PGF2, the hatching rate was significantly restored in all supplementedgroups relative to the group treated with only PGF2 and the control (p < 0.05). Embryo transfer (ET) was performed with blastocysts (day7). PGF2 levels of control recipient cows were significantly higher in the circulatory blood samples collected 60 min after ET than in samplescollected 60 min before ET (p < 0.005), and were decreased in cows injected with loading medium supplemented with Dex+rhLIF (p <0.005). Pregnancy rate was significantly higher in the ET group that received supplemented embryo-loading medium than in thenon-supplemented control (p < 0.05). The intrauterine administration of Dex and rhLIF at ET prevented increased PGF2 in circulatory bloodand resulted in enhanced pregnancy rate.

Reports on progress in Nuclear Transfer, Parthenogenesis and Embryonic Stem Cell Research

Sangho Roh 대한구강악안면병리학회 2007 대한구강악안면병리학회지 Vol.31 No.5

For many years, experience has been accumulated on embryo and gamete manipulation in mammals, The present work is an introduction of these techniques and their possible application in human embryology in s pecific cases, Mammalian c1on ing has been studied by many groups, but the success rate is sti ll low‘ Removal of maternal chromosomes from unfertil ized oocytes and injection of donor cells into enucleated oocytes are the most important factors for the improvement of cloning effi cien cy, Here, we introduce a novel one-step rnicromanipulation (OSM) system and laser-assisted zona pellucida piel'cing technique (LAZP) , 1n genera l, somatic cell nuclear transfer (SCNT) is completed by many processes including enucleation and donor cell fusion , Howevel', OSM is a simple method because donor cell is directly injected into ooplasm without fusion pl'ocess, 1n addition, chromosomal enucleation and donor cell inj ec tion are perfOl‘med simultaneously in OSM, While OSM was a pplied to porcine SCNT, LAZP was a pplied to murine SCNT, This rninirni zed the use of piezo-dri ven micromanipul ator (P1EZO) , I'educing chances 0 1' problems caused by P1EZO pulses, LAZP reduced time that took to pierce zona pellucida in removal of nucleus fl'om oocyte and somatic cell injection, which might have taken longer time with P1EZO, The simple , new OSM and LAZP system may help to enable large scale cloning by reduction of procedural steps, Pa l'thenogenesis de scribes the growth and development of an embryo without fertilization by a male Parthenogenetic ES cell s (PESCs) can be a useful cell source for tissue I'epail‘ and I'egeneration , Moreover , the defects in full-term developrnent of this PESCs enable researc hers to avoid the ethical concern , Here, the author showed that PESCs can differentiate into osteogenic lineage, The PESCs were induced osteogenic dlfferentlatlon The osteoblas t-specific gene expression such as osteocalcine, osteopontine, osteonectin, bone-sialo protein‘ coll agen type-l and alka line phos phatase showed osteogenic potential of differentiated PESCs, The author also focused on the neuronal induction of murine PESCs by simplified neurona l induction system to generate doparninergic (DA) neurons , As a result , PESCs were differentiated into nestin and Tuj-l positive cell s successfully, a lthough t he generation of DA neuron was Illruted For murine embryo cul ture, novel oil-free microtube cul tu re system was applied , This new culture system provides oil-free cu ltu re condi t ions and is easy to handle It was also associated with faster development and mOl'e t l'ophectodel'mal cells , which will enhance the development of murine embl'Yos to fur t hel' stages ,