• International Journal of Oral Biology
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• 제33권3호
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• pp.91-95
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• 2008

Embryonic stem cells have a pluripotency and a potential to differentiate to all type of cells. In our previous study, we have shown that embryonic stem cells (ESCs) lines can be generated from murine parthenogenetic embryos. This parthenogenetic ESCs line can be a useful stem cell source for tissue repair and regeneration. The defect in full-term development of parthenogenetic ESCs line enables researchers to avoid the ethical concerns related with ESCs research. In this study, we presented the results demonstrating that parthenogenetic ESCs can be induced into osteogenic cells by supplementing culture media with ascorbic acid and $\beta$-glycerophosphate. These cells showed morphologies of osteogenic cells and it was proven by Von Kossa staining and Alizarin Red staining. Expression of marker genes for osteogenic cells (osteopontin, osteonectin, alkaline phosphatase, osteocalcin, bone-sialoprotein, collagen type1, and Cbfa1) also confirmed osteogenic potential of these cells. These results demonstrate that osteogenic cells can be generated from parthenogenetic ESCs in vitro.

• International Journal of Oral Biology
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• 제30권4호
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• pp.131-134
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• 2005

In the present study, performances of several in vitro maturation (IVM) systems for porcine follicular oocytes were evaluated, and an efficient chemically defined IVM system for porcine oocytes was proposed. The proposed one-step culture system supplemented with polyvinylalcohol (PVA) gave competitive efficiencies in terms of oocyte maturation and blastocyst development after parthenogenetic activation and in vitro culture, compared with the conventional two-step culture system by a supplementation of porcine follicular fluid (pFF). Additionally, it is identified that the proposed chemically defined one-step culture system yielded the comparable level of blastocyst production to the conventional maturation system in porcine somatic cell nuclear transfer (SCNT). Therefore, one can eliminate un-expected effects accompanied by supplementation of pFF. No medium replacement during whole maturation period is an additional benefit by applying this new system. Thus, these data support that the developed PVA supplemented chemically defined one-step IVM system for porcine follicular oocyte might be used in porcine SCNT program.

• 한국수정란이식학회지
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• 제33권4호
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• pp.237-243
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• 2018

Hedgehog (Hh) pathway plays a key role in development from invertebrate to vertebrate. It is known to be involved in cell differentiation, polarity, proliferation, including the development of vertebrate limb and the establishment of flies' body plan. To investigate how the regulation of Hh pathway affects the development of parthenogenetic murine embryos, the parthenogenetically activated murine embryos were treated with either cyclopamine (Cyc), an antagonist of Hh pathway, or purmorphamine, an agonist of Hh pathway. While Cyc did not affect the blastocyst formation and its total cell number, the chemical reduced the hatching rate of embryos and the expression levels of Fn1 mRNA. The results of the present study show the possibility that Cyc may affect the development of embryos at blastocyst stage by blocking Hh pathway and this may cause detrimental effect to the embryos at peri-, and post-implantation stages.

• Asian-Australasian Journal of Animal Sciences
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• 제20권8호
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• pp.1190-1195
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• 2007

This study was conducted to improve efficiency of a follicle culture system without reducing developmental competence of intrafollicular oocytes. Preantral follicles (100 to $125{\mu}m$ in diameter) of F1 hybrid (B6CBAF1) mice were cultured singly for 216 h in modified ${\alpha}$-MEM-glutamax medium, to which 2.5 IU/ml hCG and epidermal growth factor was added 16 h prior to the end of culture. Medium change was either performed three times (54 h interval), twice (72 h interval), once (108 h interval), or not at all (216 h interval). Maturation (progression to the metaphase II stage) of intrafollicular oocytes was detected from 4 days after culture in the three-times change treatment, while all treatments yielded mature oocytes from day 5 of culture. Compared with the three-times change, decreasing the change frequency to once did not reduce the capacity to begin maturation (germinal vesicle breakdown of 82 to 86%), to mature (78 to 79%) and to develop into blastocysts after parthenogenetic activation (29 to 32%). Morphological parameters were similar among these treatments. Except for the no medium change treatment, similar colony-forming activity of inner cell mass cells after culturing of blastocysts in leukemia inhibitory factor-containing medium was detected, while the morphology of the colony-forming cells deteriorated in the change-once treatment compared with the change twice or three-times. In conclusion, the efficiency of the preantral follicle culture system could be improved by reducing frequency of medium change up to a 72 h interval (three times in total 216 h culture) without decreasing developmental competence of oocytes.

• 한국수정란이식학회지
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• 제27권2호
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• pp.121-126
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• 2012

The objective of this study was to examine the effect of in vitro culture media on embryonic development of in vitro-matured (IVM) oocytes after parthenogenetic activation (PA) in pigs. Immature pig oocytes were matured in TCM-199 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 22~26 h. IVM oocytes were activated by electric pulses and cultured in porcine zygote medium-3 (PZM-3) and North Carolina State University-23 supplemented with essential and non-essential amino acids (NCSU-23aa). These media were further modified by supplementing 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and $10{\mu}M$ ${\beta}$-mercaptoethanol (designated as mPZM-3 and mNCSU-23aa, respectively). Culture of PA embryos in mPZM-3 significantly increased development to the blastocyst stage than culture in NCSU-23aa (36.2% vs. 24.8%, p<0.05). Modified PZM-3 showed a significantly higher blastocyst formation than NCSU-23aa in both groups of embryos that were activated at 44 h and 48 h of IVM (51.0% vs. 35.5% and 49.0% vs. 34.2% in oocytes activated at 44 h and 48 h of IVM, respectively). Irrespective of the follicle diameter where oocytes were collected, embryonic development to the blastocyst stage was increased (p<0.05) by the culture in mPZM-3 compared to culture in NCSU-23aa (25.9% vs. 34.2% and 32.9% vs. 44.8% in embryos derived from small and medium size follicles, respectively). Our results demonstrated that culture media had significant effect on preimplantation development PA embryos and that mPZM-3 was superior to mNCSU-23 in supporting development to the blastocyst stage in pigs. This beneficial effect of mPZM-3 on embryonic development was not impaired by other factors such as time of oocyte activation and origin of immature oocytes (small and medium size follicles).

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.77-82
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• 2007

This study was conducted to investigate the effects of oocyte maturational age and activation condition on in vitro development of porcine parthenogenetic embryos (parthenotes). Porcine follicular oocytes were matured in vitro for 30 to 44 hr. Maturation rate was examined during in vitro maturation (IVM) every 2 hr interval. The cdc2 kinase activity was measured at 36 and 44 hr of IVM. Some oocytes were activated at 36 or 44 hr of IVM by three different conditions; 1) single electric stimulation (1.5 kV/cm for $30{\mu}sec$; ES), 2) double electric stimulations (1.5 kV/cm for $30{\mu}sec$, followed by 1.0 kV/cm for $50{\mu}sec$ after 1 hr; ES+ES) or 3) ES+ES followed by culture in 6-dimethlyaminopurine (6-DMAP) for 4 hr (ES+ES+D), and cultured for 6-7 days. Maturation rate was significantly increased as culture period was increased to 36 hr (66.9%, p<0.05), and then gradually increased to 87.1% at 44 hr of IVM. The cdc2 kinase activity was decreased (p<0.05) with culture period prolonged from 36 hr to 44 hr. Lower blastocyst formation rate (4.3%, p<0.05) were obtained by ES in 36 hr-matured oocytes compared to other treatments (16.5 and 20.5%) in the same age and the same treatment in 44 hr-matured oocytes (15.0%). High blastocyst formation rate (23.6%) was obtained by ES+ES+D in 44 hr-matured oocytes (p<0.05). These results demonstrate that porcine oocyte activation and in vitro development of parthenotes can be affected by interactions between oocyte maturational age and activation condition.

• 한국수정란이식학회지
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• 제25권1호
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• pp.1-7
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• 2010

The objective of this study was to examine the effect of vitamin B (pantothenic acid, folic acid, and myo-inositol) that was supplemented to embryo culture medium on in vitro development of parthenogenetically activated (PA) pig embryos. Cumulus-oocyte complexes derived from slaughtered ovaries were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (hCG and eCG) for the first 22 h and then further cultured in hormone-free medium for an additional 22 h. After maturation culture, metaphase II oocytes that extruded 1st polar body were electrically activated and treated with $5.0\;{\mu}g/ml$ cytochalasin B for 4 h. Then, PA embryos were cultured for 7 days in a modified NCSU-23 that was supplemented with pantothenic acid, myo-inositol, or folic acid at different concentrations ($3{\sim}300\;{\mu}M$) according to the experimental design. Myo-inositol added to culture medium did not show any beneficial or inhibitory effects on embryo cleavage and blastocyst formation. However, $300\;{\mu}M$ pantothenic acid significantly inhibited blastocyst formation compared to control (no addition) (24% vs. 36%, p<0.05). Folic acid ($300\;{\mu}M$) significantly (p<0.05) increased blastocyst formation (56%) compared to control (41%). Our results demonstrated that in vitro development of PA embryos was significantly influenced by vitamin B and addition of $300\;{\mu}M$ folic acid to culture medium improved in vitro development of pig PA embryos.

• 한국수정란이식학회지
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• 제23권4호
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• pp.245-250
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• 2008

This study was designed to investigate the effect of essential amino acids (EAA) and/or non-essential amino acids (NEAA) on the development of parthenogenetic and somatic cell nuclear transfer (SCNT) porcine embryos in vitro. To evaluate the timing of amino acids supplementation, activated oocytes were cultured in NCSU23-PVA with EAA, NEAA or NEAA+EAA (AAs) during specific periods as below: EAA, NEAA or AAs were supplemented during Day 0 to 6 (whole culture period: ALL), Day 2 to Day 6 (post-maternal embryonic transition period: POST-MET), Day 5 to Day 6 (post-compaction period: POST-CMP), Day 0 to Day 2 (pre-maternal embryonic transition period: PRE-MET), or Day 0 to Day 4 (post-compaction period: PRE-CMP). Supplementation of NEAA decreased cleavage rates in PRE-MET and PRE-CMP and also decreased blastocyst rates in POST-CMP. On the other hand, EAA significantly enhanced blastocyst formation rate in POST-MET and no detrimental effect on embryonic development in other groups. Interestingly, NEAA and EAA had synergistic effect when they were supplemented to the medium during whole culture period. Supplementation of AAs also enhanced SCNT porcine embryo development whereas BSA-free medium without AAs could not supported blastocyst formation of SCNT embryos. In conclusion, existence of EAA and NEAA in defined culture medium variously influences the development of parthenogenetic and SCNT porcine embryos, and their positive effect are only occurred when both EAA and NEAA are supplemented to the medium during whole culture period. Additionally, AAs supplementation enhances the blastocyst formation of SCNT porcine embryos when they are cultured in the defined condition.

• The Plant Pathology Journal
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• 제25권3호
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• pp.247-255
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• 2009

Root-knot nematode species, such as Meloidogyne hapla, M. incognita, M. arenaria, and M. javanica are the most economically notorious nematode pests, causing serious damage to a variety of crops throughout the world. In this study, DNA sequence analyses were performed on the D3 expansion segment of the 28S gene in the ribosomal DNA in an effort to characterize genetic variations in the three Meloidogyne species obtained from Korea and four species from the United States. Further, PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), SCAR (Sequence Characterized Amplified Region) PCR and RAPD (Randomly Amplified Polymorphic DNA) were also utilized to develop methods for the accurate and rapid species identification of the root-knot nematode species. In the sequence analysis of the D3 expansion segment, only a few nucleotide sequence variations were detected among M. incognita, M. arenaria, and M, javanica, but not M. hapla. As a result of our haplotype analysis, haplotype 5 was shown to be common in M. arenaria, M. incognita, M. javanica, but not in the facultatively parthenogenetic species, M. hapla. PCR-RFLP analysis involving the amplification of the mitochondrial COII and large ribosomal RNA (lrRNA) regions yielded one distinct amplicon for M. hapla at 500 bp, thereby enabling us to distinguish M. hapla from M. incognita, M. arenaria, and M. javanica reproduced via obligate mitotic parthenogenesis. SCAR markers were used to successfully identify the four tested root-knot nematode species. Furthermore, newly attempted RAPD primers for some available root-knot nematodes also provided some species-specific amplification patterns that could also be used to distinguish among root-knot nematode species for quarantine purposes.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.145-147
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• 2005

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/${\mu}L$ significantly improved development to the blastoryst stage ($72{\%}{\leq}$) compared with culture at the lower ($0.2{\~}1$e mbryos/${\mu}L,\;0\~37.5\%$) and the higher ($5{\~}6$ embryos/${\mu}L,\;30\~53\%$) concentration for 120 h when the oocytes were cultured in a 5 ${\mu}L$ drop under mineral oil In experiment 2, the embryos cultured at a concentration of $2{\~}4$ embryos/${\mu}L$ in a 10 ${\mu}L$ drop ($81.1{\%}$) showed significantly higher blastocyst rates than those in a 5 ${\mu}L$ 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.