• 대한한방부인과학회지
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• 제23권4호
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• pp.20-34
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• 2010

Purpose: These experiments were undertaken to evaluate the effect of administration of Palmulgunjatang gamibang on ovarian functions in female mice. Methods: We administered the Palmulgunjatang gamibang to 6-week-old female CF-1 mice for 4, 8, or 12 days. After administration of Palmulgunjatang gamibang with different concentration, the female mice were injected PMSG and hCG for ovarian hyperstimulation. The mice were divided into 3 different groups for each experiment. To compare the differences, we set a control group treated with plain water at the same volume by the same way. Results: In case of 4-day, 8-day, 12-day administration of Palmulgunjatang gamibang, the mean number of total ovulated oocytes and the number of morphologically normal oocytes were increased compared with control group. We were also examined the embryonic developmental competence in vitro. In case of 4-day administration of Palmulgunjatang gamibang, the rates of blastocyst formation from 2-cell stages were higher than control group. Conclusion: From our results suggested that the medication of Palmulgunjatang gamibang has beneficial effect on reproductive functions of female mice via promotion of cell proliferation.

• Reproductive and Developmental Biology
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• 제36권3호
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• pp.167-172
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• 2012

The key regulators of apoptosis are the interacting protein of the Bcl-2 family. Bcl-2, an important member of this family, blocks cytochrome C release by sequestering pro-apoptotic BH3-only proteins such as Bid, Bad, Bax and Bim. The pro-survival family members (Bcl-2, Bcl-XL, Bcl-W) are critical for cell survival, since loss of any of them causes cell death in certain cell type. However, its role during early porcine embryonic development is not sufficient. In this study, we traced the effects of Bcl-2 inhibitor, ABT-737, on early porcine embryonic development. We also investigated several indicators of developmental potential, including gene expression (apoptosis-related genes) and apoptosis, which are affected by ABT-737. Porcine embryos were cultured in the PZM-3 medium with or without ABT-737 for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without ABT-737 ($14.7{\pm}3.0$ vs $30.3{\pm}4.8%$, p<0.05). TUNEL assay showed that the number of containing fragmented DNA at the blastocyst stage increased in the ABT-737 treated group compared with control (4.7 vs 3.7, p<0.05). The mRNA expression of the pro-apoptotic gene Bax increased in ABT-737 treated group (p<0.05), whereas expressions of the anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-XL, Bcl-W) decreased (p<0.05). Also, expressions of the ER stress indicator genes (GRP78, XBP-1 and sXBP-1) increased in ABT-737 treated group (p<0.05). In conclusion, Bcl-2 is closely associated with of apoptosis- and ER stress-related genes expressions and developmental potential in pig embryos.

• Molecules and Cells
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• 제40권11호
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• pp.871-879
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• 2017

Levels of maturation-promoting factor (MPF) in oocytes decline after vitrification, and this decline has been suggested as one of the main causes of low developmental competence resulting from cryoinjury. Here, we evaluated MPF activity in vitrified mouse eggs following treatment with caffeine, a known stimulator of MPF activity, and/or the proteasome inhibitor MG132. Collected MII oocytes were vitrified and divided into four groups: untreated, 10 mM caffeine (CA), $10{\mu}M$ MG132 (MG), and 10 mM caffeine + $10{\mu}M$ MG132 (CA+MG). After warming, the MPF activity of oocytes and their blastocyst formation and implantation rates in the CA, MG, and CA+MG groups were much higher than those in the untreated group. However, the cell numbers in blastocysts did not differ among groups. Analysis of the effectiveness of caffeine and MG132 for improving somatic cell nuclear transfer (SCNT) technology using cryopreserved eggs showed that supplementation did not improve the blastocyst formation rate of cloned mouse eggs. These results suggest that maintaining MPF activity after cryopreservation may have a positive effect on further embryonic development, but is unable to fully overcome cryoinjury. Thus, intrinsic factors governing the developmental potential that diminish during oocyte cryopreservation should be explored.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.75-75
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• 2002

The present study was conducted for the production of transgenic cloned cows by somatic cell nuclear transfer (SCNT) that secrete human prourokinase into milk. To establish an efficient production system for bovine transgenic SCNT embryos, the offset was examined of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos. An expression plasmid far human prourokinase (pbeta-ProU) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected using a lipid-meidated method. In Experiment 1, developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In Experiment 2, the effect of cellular senescence in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed. In Experient 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 ${\mu}{\textrm}{m}$) or small cell (<30 ${\mu}{\textrm}{m}$)] were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.

• Clinical and Experimental Reproductive Medicine
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• 제37권4호
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• pp.307-319
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• 2010

목적: 유리화 동결액의 조성조절과 냉각속도 증진을 통한 동결보호제의 농도를 낮추는 전략을 통해 세포에 미치는 독성을 감소시켜 유리화 동결 및 융해 후 생쥐 배아의 생존율 및 발생률을 증진시키고, 궁극적으로 배아의 유리화 동결법을 개선하고자 하였다. 연구방법: 생쥐 배아와 포배기를 그리드를 이용한 유리화 동결법을 이용하여 동결/융해하였다. 동결액 내 ethylene glycol와 dimethylsulphoxide (DMSO)의 농도와 당의 농도를 조절하여 생쥐 배아의 융해 후 생존율과 발생률을 관찰하였고, 냉각속도의 증가와 동결억제제의 농도와의 상관관계를 포배기의 융해 후 생존율과 발생률에 따라 비교하였다. 또한 융해 후 배아를 대리모에 이식하여 산자를 생산함으로 냉각속도의 효율성을 알아보았다. 결과: EG를 단독으로 사용한 동결보존액 보다는 DMSO와 혼합된 동결보존액의 사용이 보다 유리하다는 결과를 얻을 수 있었다. 슬러시 질소에 의한 냉각속도의 증가가 동결보존의 대상의 상해를 줄여 유리화 동결의 효율성을 증진시키는 것으로 생각된다. 결론: 혼합된 동결보호제를 사용하였을 때 생쥐 배아의 유리화 동결 후 생존과 발생률이 증진되었다. 슬러시 질소를 이용한 유리화 동결의 도입은 냉각속도의 증가를 통해 기존 유리화 동결방법의 효율을 증진시켜 생존율과 융해후 발생률, 임신율 증진에 기여하였다. 또한 냉각속도의 증진은 유리화 동결의 필수요건인 고농도의 동결억제제에 대한 노출을 감소시킬 수 있었다. 이러한 노력은 생식력의 보전을 위한 유리화 동결법의 효율 향상에 기여할 것이다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.68-68
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• 2001

Blastocyst formation, consisting of the inner cell mass (ICM) and trophectoderm (TE), is the first differentiation process during embryonic development in mammals. It has been hypothesized that the proportion of ICM to TE in the blastocyst may be crucial for subsequent developmental competence of early embryos, which it may be expressed as a sensitive indicator for evaluating in vitro systems. In this study ICM/total cell ratio of nuclear transfer (NT) embryos was compared with IVF-derived and in vivo embryos. Somatic cell nuclei obtained from a fetus at Day 40 of gestation were transferred into the enucleated oocyte and then cultured in NCSU 23 medium for 6 days as previously described (Koo et al., Biol. Reprod. 2000; 63:986-992). ICM and TE cells of blastocysts were determined by using a differential staining method (Han et al., Biol. Reprod. 1999; 60:1110-1113). Development rate (9.8$\pm$2.5%, 23/225) to the blastocyst stage of NT embryos was lower than IVF embryos (23.8$\pm$2.7%, 53/223). Thus, a difference was detected in the in vitro developmental rate to blastocyst stage between NT and IVF-derived embryos (P<0.05). In the next experiment, we investigated ICM and TE nuclei to assess the quality of blastocysts that produced by NT, IVF and in vivo, respectively. NT blastocysts (27.6$\pm$8.3) showed a smaller total cell number than IVF-derived (42.6$\pm$17.4) and in vivo embryos (283.9$\pm$103.5) (P<0.05). Ratios of ICM/total cells in NT, IVF and in vivo blastocysts were 15.1$\pm$ 18.6% (n=56), 12.3$\pm$9.2% (n=57) and 30.4$\pm$6.8% (n=40), respectively. Individual blastocysts for the ratio of ICM/total cells were assigned to 3 groups (I; <20%, II; 20 to 40% and III;>40%). As the results, most in vivo blastocysts (97.5%, 39/40) were distributed into group II while most NT (78.6%, 44/56) and IVF-derived blastocysts (82.5%, 47/57) were allocated to group I. Thus, our data show that NT or IVF-derived embryos have aberrant morphology during early development in vitro systems, suggesting that these anomalies may result in developmental failures of the NT embryos to term.

• 한국수정란이식학회지
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• 제24권1호
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• pp.65-69
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• 2009

The aim of the present study was to investigate the cleavage pattern, its developmental ability and apoptosis of porcine embryo in vitro. Morphology data on a total of 919 embryos were analyzed retrospectively. Forty-eight hours after insemination, embryos were classified into five groups based on the cleavage state as follows; 1 cell, 2 cell, 4 cell, 5 to 8 cell and fragmentation. These groups were cultured another 120 hours and then evaluated for blastocyst formation. Blastocyst formation rates were significantly higher in 4 cell (42.5%) and 5 to 8 cell (48.6%) cleaving groups than in other groups (p<0.05). On the other hand, 2 cell and fragmentation groups produced 4.9% and 3,9% blastocysts, respectively. And we could verify that in the event of 2 cell block and fragmentation of embryo. To analyze the apoptotic frequency in preimplantation development of porcine IVF embryos, all cells of each blastocyst were performed by TUNEL assay. There were no significantly differences in the total cell numbers of embryos and apoptotic cell rate in blastocysts among the each classified groups. Data suggest that 4 cell and 5 to 8 cell cleaving embryos at 48 hour after insemination have high developmental competence, and may be an useful parameter to predict the development of preimplantation embryos and to study using preimplanation embryonic research.

• 한국수정란이식학회지
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• 제33권4호
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• pp.211-220
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• 2018

Nitric oxide (NO) has an important role in oocyte maturation and embryonic development in mammals. This study examined the effect of exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP) in a maturation medium on meiotic progression and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) in pigs. When oocytes were exposed to $0.1{\mu}M$ SNAP for first 22 h of in vitro maturation (IVM) in Experiment 1, SNAP significantly improved blastocyst development in both defined and standard follicular fluid-supplemented media compared to untreated control (48.4 vs. 31.7-42.5%). SNAP treatment significantly arrested meiotic progression of oocytes at the germinal vesicle stage at 11 h of IVM (61.2 vs. 38.7%). However, there was no effect on meiotic progression at 22 h of IVM (Experiment 2). In Experiment 3, when oocytes were treated with SNAP at 0.001, 0.1 and $10{\mu}M$ during the first 22 h of IVM to determine a suitable concentration, $0.1{\mu}M$ SNAP (54.2%) exhibited a higher blastocyst formation than 0 and $10{\mu}M$ SNAP (36.6 and 36.6%, respectively). Time-dependent effect of SNAP treatment was evaluated in Experiment 4. It was observed that SNAP treatment for the first 22 h of IVM significantly increased blastocyst formation compared to no treatment (57.1% vs. 46.2%). Antioxidant effect of SNAP was compared with that of cysteine. SNAP treatment significantly improved embryonic development to the blastocyst stage (49.1-51.5% vs. 34.4-37.5%) irrespective of the presence or absence of cysteine (Experiment 5). Moreover, SNAP significantly increased glutathione (GSH) content and inversely decreased the reactive oxygen species (ROS) level and mitochondrial oxidative activity in IVM oocytes. SNAP treatment during IVM showed a stimulating effect on in vitro development of SCNT embryos (Experiment 7). These results demonstrates that SNAP improves developmental competence of PA and SCNT embryos probably by maintaining the redox homeostasis through increasing GSH content and mitochondrial quality and decreasing ROS in IVM oocytes.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.287-294
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• 2011

Autophagy is a process of intracellular bulk protein degradation, in which the accumulated proteins and cytoplasmic organelles are degraded. It plays important roles in cellular homeostasis, apoptosis, and development, but its role during early embryo development remains contentious. Therefore, in the present study, we investigated the effects of 3-methyladenine (3-MA) on early embryonic development in pigs, we also investigated several indicators of developmental potential, including mitochondrial distribution, genes expressions (autophagy-, apoptosis- related genes), apoptosis and ER-stress, which are affected by 3-MA. After in vitro maturation and fertilization, presumptive pig embryos were cultured in PZM-3 medium supplemented with 3-MA for 2 days at $39^{\circ}C$ 5% $CO_2$ in air. Developmental competence to the blastocyst stage in the presence of 3-MA was gradually decreased according to increasing concentration. Thus, all further experiments were performed using 2 mM 3-MA. Blastocysts that developed in the 3-MA treated group decreased LC3-II intensity and expressions of autophagy related genes than those of the untreated control, resulting in down-regulates the autophagy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 3-MA treated group compared with control ($6.0{\pm}1.0$ vs $3.3{\pm}0.6$, p<0.05). Also, the expression of the pro-apoptotic gene Bax increased in 3-MA treated group, whereas expression of the anti-apoptotic gene Bcl-XL decreased. Mito Tracker Green FM staining showed that blastocysts derived from the 3-MA treated group had lower mitochondrial integrity than that of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. Then, the expression of the spliced form of pXBP-1 product (pXBP-1s) increased in 3-MA treated group, resulting increase of ER-stress. Taken together, these results indicate that inhibition of autophagy by 3-MA is closely associated with apoptosis and ER-stress during preimplantation periods of porcine embryos.

• 대한수의학회지
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• 제57권2호
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• pp.89-95
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• 2017

This study was conducted to determine the effects of biophoton treatment during in vitro maturation (IVM) and/or in vitro culture (IVC) on oocyte maturation and embryonic development in pigs. An apparatus capable of generating homogeneous biophoton energy emissions was placed in an incubator. Initially, immature pig oocytes were matured in the biophoton-equipped incubator in medium 199 supplemented with cysteine, epidermal growth factor, insulin, and gonadotrophic hormones for 22 h, after which they were matured in hormone-free medium for an additional 22 hr. Next, IVM oocytes were induced for parthenogenesis (PA) or provided as cytoplasts for somatic cell nuclear transfer (SCNT). Treatment of oocytes with biophoton energy during IVM did not improve cumulus cell expansion, nuclear maturation, intraoocyte glutathione content, or mitochondrial distribution of oocytes. However, biophoton-treated oocytes showed higher (p < 0.05) blastocyst formation after PA than that in untreated oocytes (50.7% vs. 42.7%). In an additional experiment, SCNT embryos produced from biophoton-treated oocytes showed a greater (p < 0.05) number of cells in blastocysts (52.6 vs. 43.9) than that in untreated oocytes. Taken together, our results demonstrate that biophoton treatment during IVM improves developmental competence of PA- and SCNT-derived embryos.