• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.113-113
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• 2002

The stimulatory effect of EGF and FSH on oocyte maturation have been reported in various mammalian species. And some reports presented FSH enhanced the effect of EGF on oocyte maturation. But, the interaction between EGF and FSH on nuclear maturation of mammalian oocytes is not fully understood. We observed the effect of EGF and FSH on nuclear maturation during in vitro maturation of mouse oocytes. Also, we examined the interaction between EGF and FSH on nuclear maturation of mouse oocytes using the EGFR inhibitor or FSH inhibitor. Germinal vesicle (GV) stage oocytes were obtained from 3-4weeks PMSG primed BCFI hybrid mice and cultured in TCM-199 medium with 0.4%PVP supplemented with/without EGF (1ng/ml), FSH (1ug/ml), EGFR specific tyrosine kinase inhibitors: Tyrphostin AG 1478 (500nM), MAP kinase kinase inhibitor : U0126 (20uM) or PD 98059 (100uM) for 14-l5hr. Rapid staining method were used for the assessment of nuclear maturation. Nuclear maturation rates of EGF indjor FSH-treated group were significantly higher than those of control group. Treatment of EGFR inhibitor significantly block the nuclear maturation of GV oocyte in EGF-treated group, but it did not block those of GV oocyte in FSH-treated or FSH and EGF-treated group. Treatment of FSH inhibitor(U0126, PD98059) significantly block the nuclear maturation of EGF-treated group, FSH-treated and FSH and EGF-treated group. These results show that EGF has a stimulatory effect as well as different action pathway with FSH on in-vitro maturation of mouse oocyte in vitro. Therefore, further studies will be needed to find the signaling pathway of EGF associated with nuclear maturation.

• Reproductive and Developmental Biology
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• 제28권4호
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• pp.267-273
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• 2004

Maturation of oocytes is maintained by complex procedures along with follicular genesis and is a critical step for embryonic development. Purine known as an oocyte maturation regulator is present in follicular fluid. In this study, the roles of guanosine as a strong inhibitor of GVBD and a modulator of cyclic GMP concentration in ooyctes were revealed. Denuded immature oocytes were treated with guanosine, and the maturation rates and cGMP concentration of oocytes were measured. GVBD was blocked in a concentration dependent manner by guanosine, but this effect was reversible. However, GVBD was lagged yet not significant by adenosine. Both guanosine and adenosine modified cGMP concentration in oocytes. The characteristic of the guanosine-treated oocyte was significantly higher cGMP compared with the adenosine-treated oocyes at initial time of the maturation. Based these results, guanosine may be a strong and reversible GVBD inhibitor. Although the precise mechanism of guanosine presently is unclear, the results suggest that guanosine may lead the accumulation of cGMP in oocyte cytoplasm, which in turn suppresses GVBD.

• 한국동물학회지
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• 제33권1호
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• pp.53-62
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• 1990

양서류의 여포난자를 생체외에서 배양하면서 호르몬을 처리하면 난자의 성숙(핵붕괴)을 일으킨다. 본 연구는 북방산개구리의 여포난자를 배양하면서 난자내 단백질 분해효소들의 활성변화를 유도하여 이것이 난자의 성숙에 어떠한 영향을 미치는가를 조사하였다. chymotrypsin의 저해제로 알려진 N$\alpha$ -tosyl-L-phenylalanine-chloromethyl-ketone(TPCK)을 배양액에 처리하면 비교적 낮은 농도(0.001-1 $\mu$M)에서는 호르몬의 도움없이도 난자의 성숙을 유도하나 높은 농도 (100 $\mu$M)에서는 호르몬에 의한 난자의 성숙까지도 억제하는 이중적인 효과를 나타내었다. Trypsin의 저해제인 N$\alpha$ -tosyl-L-phenylalanine-chloromethyl-ketone(TPCK)은 성숙유도능력이 없을 뿐 아니라 progesterone에 의한 난자의 성숙을 억제하였다. Trypsin을 직접 처리했을 때에는 농도에 의존하여(0.001-1$\mu$g/2ml) 호르몬의 도움없이도 난자의 성숙을 유도함을 발견하였다. TLCK나 TPCK의 억제효과는 성숙 초기에만 나타났다. 본 결과는 양서류 난자의 성숙조절 과정에 몇종의 단백질 분해효소들이 참여한다는 것을 시사해주고 있다.

• 한국발생생물학회지:발생과생식
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• 제17권1호
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• pp.63-72
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• 2013

A specific inhibitor of RNA polymerase II, ${\alpha}$-amanitin is broadly used to block transcriptional activities in cells. Previous studies showed that ${\alpha}$-amanitin affects in vitro maturation of cumulus-oocyte-complex (COC). In this study, we evaluated the target of ${\alpha}$-amanitin, and whether it affects oocytes or cumulus cells (CCs), or both. We treated ${\alpha}$-amanitin with different time period during in vitro culture of denuded oocytes (DOs) or COCs in comparison, and observed the changes in morphology and maturation status. Although DOs did not show any change in morphology and maturation rates with ${\alpha}$-amanitin treatment, oocytes from COCs were arrested at metaphase I (MI) stage and CCs were more scattered than control groups. To discover causes of meiotic arrest and scattering of CCs, we focused on changes of cumulus expansion, gap junctions, and cellular metabolism which to be the important factors for the successful in vitro maturation of COCs. Expression of genes for cumulus expansion markers (Ptx3, Has2, and Tnfaip6) and gap junctional proteins (Gja1, Gja4, and Gjc1) decreased in ${\alpha}$-amanitin-treated CCs. However, these changes were not observed in oocytes. In addition, expression of genes related to metabolism (Prps1, Rpe, Rpia, Taldo1, and Tkt) decreased in ${\alpha}$-amanitin-treated CCs but not in oocytes. Therefore, we concluded that the transcriptional activities of CCs for supporting suitable transcripts, especially for its metabolic activities and formation of gap junctions among CCs as well as with oocytes, are important for oocytes maturation in COCs.

• Molecules and Cells
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• 제37권2호
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• pp.126-132
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• 2014

As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein ${\gamma}$-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.

• Clinical and Experimental Reproductive Medicine
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• 제13권2호
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• pp.201-206
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• 1986

These experiments were conducted to know whether RNA syntheis is involved in the cumulus expansion and oocyte maturation of mouse cumulus-oocyte complexes in vitro. Mouse cumulus-oocyte complexes(COC's) were cultured in the presence of cordycepin, an inhibitior of RNA synthesis and its effect on the cumulus expansion and oocyte maturation were examined. The results were as follows. 1. Continuous presence of cordycepin in the medium(200${\mu}g/ml$) inhibited the HCG-induced cumulus cell expansion of mouse complexes. This inhibition was reversible. 2. When the COC'S were preincubated with different concentration of cordycepin plus HCG for 3 hours and then transferred to the plain medium, the HCG induced cumulus expansion was suppressed at $100{\mu}g/ml$ of cordycepin. 3. When the COC'S were cultured with cordycepin after HCG stimulation(3hrs), the cumulus expansion were not suppressed by cordycepin. 4. Oocyte meiotic maturation did not appear to be affected by cordycepin. The data presented here imply that RNA synthesis is involved in the cumulus expansion process and that mouse oocytes are more resistant to RNA synthesis inhibitor than cumulus cells.

• Clinical and Experimental Reproductive Medicine
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• 제19권1호
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• pp.15-29
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• 1992

In the present study, it was aimed to find the role of calcium on the maturation of mouse follicular oocytes as well as for the role of calcium inhibitors, $Ni^{2+}$ and $La^{3+}$. Mouse follicular oocytes were cultivated in different media at $37^{\circ}C$, in 100% humidified $CO_2$ incubator for 3 and 17 hrs. The results were as follows; 1. There was no differences in GVBD between the control and experimental groups during the 3 hr culture. 2. Mouse oocytes were matured to higher rate in MHBS rather than HTF for 17 hr culture. 3. Maturation rate was significantly lower in $Ca^{2+}$-free and $Ca^{2+}$ 0.4 mM which were tested, compared to other calcium concentration used in the present study. 4. Calcium inhibitor, $Ni^{2+}$, it showed highest degeneration rate at all calcium concentrations and additionally in $Ni^{2+}$ $100{\mu}M$ treated group next. Maturation rate was significantly decrease as the $Ca^{2+}$ inhibitor concentration increased. 5. In all Lanthanum treated groups of calcium-free, degeneration were significantly high treated groups at 0.4 mM $Ca^{2+}$ concentrations degeneration rates of all group were significantly lower than that of the control but maturation rates were not significantly different in any group. In lanthanum $100{\mu}M$ treated group at 0.4 mM and 0.8 mM calcium concentration, its maturation rate was significantly higher than that of the control. Maturation rates of all groups of lanthanum treated at 1.71 mM calcium concentration were not significantly different among groups. 6. In the calcium treated group (0.4mM-1.7 mM), the presence of phosphate does not seem to be needed for oocyte maturation. However, the presence of phosphate at $Ca^{2+}$ 0.8 mM only seems to stimulated maturation.

• Animal cells and systems
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• 제2권1호
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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.

• 한국동물생명공학회지
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• 제34권3호
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• pp.212-221
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• 2019

Sestrin-2 (SESN2) as a stress-metabolic protein is known for its anti-oxidative effects as a downstream factor of PERK pathways in mammalian cells. However, the expression patterns of SESN2 in conjunction with the UPR signaling against to ER stress on porcine oocyte maturation in vitro, have not been reported. Therefore, we confirmed the expression pattern of SESN2 protein, for which to examine the relationship between PERK signaling and SESN2 in porcine oocyte during IVM. We investigated the SESN2 expression patterns using Western blot analysis in denuded oocytes (DOs), cumulus cells (CCs), and cumulus-oocyte complexes (COCs) at 22 and 44 h of IVM. As expected, the SESN2 protein level significantly increased (p < 0.01) in porcine COCs during 44 h of IVM. We investigated the meiotic maturation after applying ER stress inhibitor in various concentration (50, 100 and 200 μM) of tauroursodeoxycholic acid (TUDCA). We confirmed significant increase (p < 0.05) of meiotic maturation rate in TUDCA 200 μM treated COCs for 44 h of IVM. Finally, we confirmed the protein level of SESN2 and meiotic maturation via regulating ER-stress by only tunicamycin (Tm), only TUDCA, and Tm + TUDCA treatment in porcine COCs. As a result, treatment of the TUDCA following Tm pre-treatment reduced SESN2 protein level in porcine COCs. In addition, SESN2 protein level significantly reduced in only TUDCA treated porcine COCs. Our results suggest that the SESN2 expression is related to the stress mediator response to ER stress through the PERK signaling pathways in porcine oocyte maturation.

• 한국가축번식학회지
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• 제21권2호
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• pp.123-130
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• 1997

Normal maturation of the mammalian oocytes is prerequisite for the fertilization and the early embryonic development. We have been tested the effects of purine and its de novo synthetic inhibitor, azaserine(Aza) on the maturation of germinal vesicle(GV) and germinal vesicle breakdown(GVBD) mouse oocytes. Denude-immature oocytes were cultivated in the media containing adenosine, guanosine, and/or azaserine, and checked the matruation stage by monitoring the prominent morphological changes. In GV stage oocytes, GV was arrested temporarily by the adenosine(1.0%) and protractedly by the guanosine(65.9%, P<0.001). The regression was increased significantly at the adenosine(90%, P<0.001) but decreased at the guanosine(1.6%, P<0.05). Inhibiting the de novo synthesis of purine, nuclear maturation rate was increase(90.4% : 96.7%), but GV arrest was significantly increased by cotreatment with guanosine(P<0.001). Polar body extraction significantly was increased at the Aza(P<0.05), but not in others. In GVBD oocytes, adenosine itself did not affect GVBD arrest. Guanosine, on the other hand, elevated GVBD arrest rate(P<0.001), but co-treated with Aza, decreased GVBD arrest(P<0.001). Aza increased GVBD arrest rate(20.2%, P<0.05) compared with control. From those results, we know that guanosine shows more prominent effect on the inhibition of nuclear maturation at the GV stage, and of the 1st polar body extrusion at the GVBD stage. Adenosine showed the cytoplasmic toxicity at GV stage oocyte. Our data speculate that cytoplasmic cAMP level is auto-regulated by endogenous adenylate cyclase while GVBD is inhibited by guanosine, since purine toxicity is not observed in the GVBD stage. And it is showed that purine metabolism is concerned with nuclear maturation, that the amounts of purine metabolism is not even during the oocyte maturation.