• Journal of Embryo Transfer
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• v.23 no.1
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• pp.31-36
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• 2008

We attempted to control the maturation promoting factors (MPF) activity and nuclear remodeling of somatic cell nuclear transfer (NT) bovine embryos. Bovine ear skin fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h and activated. The nuclear remodeling type of the reconstituted embryos was evaluated 1.5 h after activation. MPF activity was assessed in enucleated and chemical treated oocytes before the injection of a donor cell. Effect of chemicals on the embryonic development was evaluated with parthenogenetic embryos. MPF activity increased significantly by caffeine treatment, but decreased by vanadate treatment (p<0.05). Caffeine or vanadate had no deleterious effect on the parthenogenetic embryo development. In caffeine treated group, premature chromosome condensation (PCC) was occurred in 72.2% of NT embryos (p<0.05). In contrast, vanadate induced the formation of a pronucleus-like structure (PN) in a high frequency (68.9%, p<0.05) without PCC (NPCC). Blastocyst development of NT embryos increased by treating with caffeine (30.3%), whereas decreased by treating with vanadate (11.4%) compared to control (22.1%, p<0.05). The results indicate that caffeine or vanadate can control of MPF activity and remodeling type of NT embryos, resulting in the increased or decreased in vitro development.

• Molecules and Cells
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• v.40 no.11
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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.

• Reproductive and Developmental Biology
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• v.33 no.4
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• pp.223-228
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• 2009

We attempted to control the maturation promoting factor (MPF) activity and investigated the subsequent reprogramming of bovine somatic cell nuclear transfer (SCNT) embryos. Serum-starved adult skin fibroblasts were fused to enucleated oocytes treated with 2.5 mM caffeine or $150\;{\mu}M$ roscovitine. The MPF activity, nuclear remodeling patterns, chromosome constitutions and development of SCNT embryos were evaluated. Methylated DNA of embryos was detected at various developmental stages. The MPF activity was increased by caffeine treatment or reduced by roscovitine treatment (p<0.05). Blastocyst development was higher in the caffeine-treated groups (27.6%) than that of the roscovitine-treated group (8.3%, p<0.05). There was no difference in the apoptotic cell index among the three groups. However, the mean cell number of blastocysts was increased in the caffeine-treated group (p<0.05). Higher methylation levels were observed in the Day 3 embryos of the roscovitine-treated group (50.8%), whereas lower methylation levels were noted at Day 5 in the caffeine-treated group (12.5%, p<0.05). These results reveal that the increase in MPF activity via a caffeine-treatment creates a more suitable condition for nuclear reprogramming after SCNT.

• Animal cells and systems
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• v.2 no.1
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• pp.87-91
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• 1998

The present study investigated the involvement of the phospholipase C (PLC) and protein kinase C (PKC) signaling pathways during progesteroneinduced meiotic maturation in amphibian (Rana dybowskii) oocytes. Prosesterone-induced germinal vesicle breakdown (GVBD) of oocytes was significantly inhibited by a PKC inhibitor, staurosporine and a PLC inhibitor, U73122, in a dose-dependent manner. In contrast, U73343, an inactive analogue of U73122, was ineffective in suppressing GVBD. PKC activity in oocytes reached a maximum level at 30 min after progesterone stimulation and this elevated PKC activity was effectively suppressed by U73122 or staurosporine, suggesting that the activation of PKC enzyme is closely linked to PLC signaling during oocyte maturation. In addition, these inhib itors blocked the maturation promoting factor (MPF) activity which appeared in oocytes in response to progesterone, suggesting that PKC activation is an important signal for MPF activity. Therefore, this study demonstrates that the activation of PKC via PLC signaling is directly linked to an intracellular protein kinase cascade related to the appearance of MPF activity during meiotic maturation in amphibian (Rana dybowskii) oocytes.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.56-56
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• 1993

새로운 cell cycle 특이적 억제제의 스크리닝 방법의 확립과 이를 이용하여 cell cycle 억제제의 검색 및 세포분열 및 성장을 억제하는 작용의 분석과 이들의 항암작용 및 세포성장 및 분열 억제 작용의 signal transduction mechanism을 규명한다. 이상의 연구를 수행하기 위해 흰쥐 재생간 조직 및 흰쥐 일차 배양 간세포를 연구 모델로 하여 스크리닝 방법을 확립하고, 세포 분열 및 성장 억제제의 연구 대상 약물로는 기존의 천연물 및 미생물의 2차 대사 산물을 분리 정제한 물질등을 사용하여 그 작용 효능을 연구한다. 1) 흰쥐 부분 간 절제 수술 26시간 후 핵 단백질을 분리 2) MPF activity 측정 3) MPF 활성 저해제 생산 균주의 1차 탐색

• Journal of Embryo Transfer
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• v.30 no.1
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• pp.33-43
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• 2015

Successful somatic cell nuclear transfer (SCNT) has been reported across a range of species using a range of recipient cells including enucleated metaphase II (MII) arrested oocytes, enucleated activated MII oocytes, and mitotic zygotes. However, the frequency of development to term varies significantly, not only between different cytoplast recipients but also within what is thought to be a homogenous population of cytoplasts. One of the major differences between cytoplasts is the activities of the cell cycle regulated protein kinases, maturation promoting factor (MPF) and mitogen activated protein kinase (MAPK). Dependent upon their activity, exposure of the donor nucleus to these kinases can have both positive and negative effects on subsequent development. Co-ordination of cell cycle stage of the donor nucleus with the activities of MPF and MAPK in the cytoplast is essential to avoid DNA damage and maintain correct ploidy. However, recent information suggests that these kinases may also effect reprogramming of the somatic nucleus and preimplantation embryo development by other mechanisms. This article will summarise the differences between cytoplast recipients, their effects on development and discuss the potential role/s of MPF and or MAPK in nuclear reprogramming.

• The Korean Journal of Zoology
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• v.36 no.2
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• pp.277-284
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• 1993

A possible involvement of maturation promoting factor (nfPF) in the two-cell block phenomenon was studied by fusion experiments. Germinal vesicle (GlF) ooeyte was fused with a blastomore from late or blocked 2-cell mouse embryos. and germinal vesicle breakdoum (GVBD) of fused GV oocvtes in the presence of dbcAMP (100$\mu$g/ml) was scored as an index of MPF aniviD. GnD was induced approximately 30% by fusion of a blastomere derived from late 2-cell embryos, but not from blocked 2-cell embryos. The rate of GVBD was changed when GV oocyte was fused with a blastomere from late 2-cell embryos which were treated with u-amanitin, puromvcin or colcemid before and after hsion: Treatment of late 2-cell embryos with puromycin (50 Is/mll but not with u-amanitin (100 Is/ml) clearly inhibited GVBD, indicating that do novo protein synthesis maw be required for the appearance of MPF activity in late 2-cell embryos. Treatment of late 2-cell embryos w기h colcemid (0.1 Is/mll doubled GVBD, presumably due to the maintenance of metaphase or mitotic phase. SDS-PAGE and twoiimensional electrophoresis revealed that there was no difference in protein synthetic pattern in late and blocked 2-cell embryos, but three phosphoproteins with 27, 35 and 46 M)a, presumsblv M-phase components were phosphorylated in late 2-cell embryos but not in blocked 2-cell embryos. It seems then that MPF activity is closely related to phosphorylstion of M-phase components in late 2-cell embryos.

• Journal of Life Science
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• v.25 no.10
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• pp.1184-1195
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• 2015

The zebrafish is an emerging vertebrate model organism in reproductive biology. The oocyte maturation of zebrafish is triggered by maturation inducing hormone (MIH, 17α,20β-Dihydroxy-4-pregnen-3-one). In almost all animals, the oocyte maturation is governed by activation of pre-MPF which consists of cyclinB and inactive Cdk1. In the oocyte of Xenopus and mice, the activity of Cdk1 is regulated in two ways, one is the interaction with cyclinB and the other is phosphorylation/dephosphorylation of T14/Y15 residues on the Cdk1 by Wee1 and Cdc25. Unlike Xenopus and mice that have a sufficient amount of pre-MPF, pre-MPF is absent in GV oocyte of most teleost including zebrafish. Therefore, the activation of MPF during zebrafish oocyte maturation might totally depend on de novo synthesis of cyclinB proteins. It is reported that the translation of maternal mRNA is regulated by combination of several RNA binding proteins such as CPEB, Dazl, Pum1/Pum2, and insulin-like growth factor2 mRNA-binding protein 3 in the zebrafish oocytes. However, the definitive mechanism of these proteins to regulate the translation of stored maternal mRNAs remains to be elucidated. Therefore, the investigation of the maturation process of the zebrafish oocyte will provide new information that can help identify the role of translational control in early vertebrate oocyte maturation.

• Korean Journal of Applied Biomechanics
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• v.15 no.1
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• pp.177-195
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• 2005

The purpose of this study was to analyze the kinematic variables of ankle joints and EMG signal of the lower limbs muscle activity for the different walking speed. The subjects were 6 males of twenties. It was classified into three different walking speed-0.75m/s, 1.25m/s, 1.75m/s. The walking performances were filmed by high speed video camera and EMG signal was gained by ME3000P8 Measurement Unit. Tibialis anterior(TA), Gastrocnemius medial head(GM), Gastrocnemius lateral head(GL), Ssoleus(SO) were selected for the dorsiflexion and plantarflexion of the ankle joint. The result of this study were as follows: 1. In the gait cycle, The time parameters for the phases were showed significant difference without the terminal stance phase and terminal swing phase for the different walking speed. 2. The angle of ankle joint was no significant difference for each time point and MDF, MPF but increasing walking speed the angle had the increasing pattern slightly. 3. The angular velocity of ankle joint was showed the significant difference for LHC, RTO, RKC, LHU, MPF and MDF point along the walking speed. 4. TA was showed about 2-3 times muscle activity at the 1.75m/s than 1.25m/s in some phases. And it was showed the similar muscle activity between the 0.75m/s and 1.25m/s but, showed a little much muscle activity in the 0.75m/s. GM was showed about 2-3 times muscle activity in the 1.75m/s than 1.25m/s, and even much muscle activity at the 0.75m/s than 1.25m/s in some phases. GL was showed increasing pattern of muscle activity specially in the initial swing phase as the walking speed increased. SO was showed about 3 times muscle activity in the 1.75m/s than 1.25m/s during the plantarflexion of ankle joint. It was showed the similar muscle activity between the 0.75m/s and 1.25m/s but, showed a little much muscle activity in the 1.25m/s.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.131-138
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• 2017

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.