• Reproductive and Developmental Biology
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• v.32 no.2
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• pp.111-115
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• 2008

The aim of this study was to examine more effective cryoprotectant for the cryopreservation of mouse preantral follicles. Enzymetically isolated preantral follicles from 12-day-old mice were cryopreserved by a slow freezing protocol with 1.5 M propanediol (PROH), dimethyl sulphoxide (DMSO) or glycerol (GLY) and then grown and matured in vitro for 11 days after thawing. The survival of preantral follicles immediately after freezing and thawing was not different among the PROH (68.2%), DMSO (72.4%) and GLY (72.1%). After grown and matured in vitro, the rates of survival and metaphase II oocytes were 54.9% and 36.6% for PROH which was significantly higher rates (p<0.05) compared with the rates obtained from DMSO (16.9% and 9.0%) and GLY (16.3% and 7.5%). The diameter of metaphase II oocytes from pre antral follicles frozen in PROH ($67.4{\pm}1.8\;{\mu}m$) was significantly (p<0.05) smaller than that of the fresh preantral follicles ($69.1{\pm}2.3\;{\mu}m$). The results from the present study revealed that PROH is more suitable cryoprotectant for the cryopreservation of mouse preantral follicles.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.1
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• pp.111-119
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• 1994

"Spontaneous" hardening of the zona pellucida of mouse oocytes during in vitro culture is most likely due to cortical granules exocytosis. Thus the purpose of the present study was to determine whether the exocytosis factor is involved in spontaneous zona pellucida hardening during in vitro culture of the mouse. The results obtained form these experiments were summarized as follows; 1. When a protein synthesis inhibitor(100${\mu}g$/ml puromycin) was added to the culture medium, it did not prevent spontaneous ZPH of mouse oocyte during in vitro culture. 2. Calmodulin antagonists (trifluoperazine and chlorpromazine) and calcium channel blocker (verapamil) had no inhibitory effect in spontaneous ZPH. 3. A microtubule assembly inhibitor, colcemid had some inhibitory effect on spontaneous ZPH. 4. Treatment with a microfillament formation blocker(cytochalasin-B) at 1${\mu}g$/ml concentration, resulted in the excellent inhibitory effect on spontaneous ZPH. However cytochalasin-B did not inhibit ethanol-induced ZPH.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.115-115
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• 2002

To evaluate the correlations between the expression of cyclin B1 mRNA and protein after stimulation and oocyte activation and development of nuclear transferred mouse embryos, this study was performed. The oocyte activation was induced by 7% ethanol or 10$\mu\textrm{g}$/$m\ell$ Ca-ionophore without (single) or with (combined) 10$\mu\textrm{g}$/$m\ell$ cycloheximide (CH). Cyclin B1 mRNA and protein in mouse oocytes was evaluated by PCR and western blot. The activation and blastocyst development in both single (P<0.05) and combined (P<0.01) stimulation was higher than in non-activated group. The cyclin B1 mRNA and protein levels were significantly reduced in both single and combined stimulation groups (P<0.05), respectively. Cyclin B1 mRNA expression showed a negative correlation between activation and blastocyst development in both single and combined stimulation groups. And also the expression of cyclin B1 protein showed a negative correlation with between oocyte activation and blastocysts development in both single and combined stimulation groups. In conclusion, it may suggest that single and combined stimulation increases the oocyte activation and blastocyst development of nuclear transferred embryos, because it induces the degradation of cyclin B1 mRNA and protein after activation in enucleated mouse oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.1
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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.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.3
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• pp.319-327
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• 2009

Follicular fluid meiosis-activating sterol (FF-MAS) has been suggested as a positive factor which could improve the oocyte quality and subsequent embryo development after in vitro fertilization. However, FF-MAS is a highly lipophilic substance and is hard to detect in studying the relationship between MAS and quality of oocyte maturation. The present study focused on the expression of lanosterol 14${\alpha}$-demethylase (LDM), a key enzyme that converts lanosterol to FF-MAS, on mouse oocyte maturation and its potency on development. LDM expression was strong in gonadotropin-primed germinal vesicle stage oocytes, weak after germinal vesicle breakdown (GVBD), and then strong in MII stage oocytes. The LDM-specific inhibitor azalanstat significantly inhibited oocyte fertilization (from 79.4% to 68.3%, p<0.05). Also, azalanstat (5 to 50 ${\mu}M$) decreased the percentage of blastocyst development dosedependently (from 78.7% to 23.4%, p<0.05). The specific inhibition of sterol ${\Delta}14$-reductase and ${\Delta}7$-reductase by AY9944 accumulates FF-MAS and could increase blastocyst development rates. Additionally, in the AY9944 group, the rate of inner cell mass (ICM)/ total cells was similar to that of in vivo development, but the rate was significantly decreased in azalanstat treatment. In conclusion, LDM, the key enzyme of FF-MAS production, may play an important role in fertilization and early development of the mouse embryo, especially in vitro.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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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.

• Development and Reproduction
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• v.20 no.4
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• pp.359-365
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• 2016

Intact germinal vesicle (GV) arrest and release are essential for maintaining the fertility of mammals inducing human. Intact germinal vesicle release, maturation of oocytes is maintained by very complex procedures along with folliculogenesis and is a critical step for embryonic development. Cyclic guanosine monophosphate (cGMP) has been suggested a key factor for meiotic arrest but so far its mechanisms are controversy. In this study we examine the effects of cGMP on germinal vesicle breakdown in cumulus-enclosed oocytes and denuded oocytes. Spontaneous maturation was inhibited by a cGMP agonist, 8-Br-cGMP with concentration dependent manners both in cumulus-enclosed oocytes and denuded oocytes. The inhibitory effect was more severe in denuded oocytes than cumulus-enclosed oocytes. The Rp-8-Br-cGMP and Rp-pCPT-8-Br-cGMP did not severely block GVB compared to 8-Br-cGMP. The spontaneous GVB inhibitory effects were different by the existence of cumulus. Based on them it is suggested that the cumulus modulates the role of cGMP in GV arrest.

• Development and Reproduction
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• v.2 no.2
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• pp.197-203
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• 1998

DNA methylation seems to play an important regulatory role in gene expression and cell differentiation during postimplantation embryonic development. However, the significance of DNA methylation which is maintained by the DNA MTase during preimplantation embryonic development, is not fully understood. In order to study the role of DNA methylation in the preimplantation embryos, the expression of DNA MTase transcripts was monitored in the oocytes and preimplantation embryos. The mRNA of DNA MTase was detected in the oocytes and pleimplantation embryos. The relative mRNA levels of DNA MTase were high from the stages of GV-oocytes and pronuclear embryos, and thereafter decreased gradually. By the treatment of $\alpha$-amanitin, it was confirmed that the transcripts presented in pronuclear embryos was derived from the maternal genome. The presence of transcripts of DNA MTase in the oocytes and pronuclear embryos suggests that the maintenance of DNA methylation may be necessary and seems to play an important role in gene expression and cell differentiation during preimplantation embryonic develop-ment in mouse.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.2
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• pp.157-160
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• 1993

The human follicular f1uids(F.F.) may be considered to contribute the maturation of the oocytes on the in vitro culture. To investigate the effects of epidermal growth factor (E.G.F.), which is present in mature and immature follicular fluids, we had experiments of mouse oocytes maturation in vitro. The endpoints assayed were rated as percentage of oocytes undergoing germinal vesicle breakdown(G.V.B.D.) and polar body(P.B.) formation at 12 hours after in vitro culture. The rates of G.B.B.D. were 87% in mature F.F. 68% in immature F.F. and 78% in Ham's F-10 medium respectively. And overall the mature F.F. seem to stimulate on in vitro oocyte maturation compared with either immature F.F. or Ham's F-10 medium. As the concentration of addition of E.G.F. in immature F.F., the rates of G.V.B.D. and P.B. formation were 82 %, 23% in addition with 2 ng/ml while 84%, 32% in addition with 4 ng/ml respectivly. And at the concentration of addition of E.G.F. in Ham's F-10 media as well, the rates of G.V.B.D. and P.B. formation were 84%, 40% and 82%, 44% in addition with each 2ng, 4ng. AccordinglY there was no influence on the oocytes maturation at the addition of E.G.F. to each immature F.F. and Ham's F-10 medium. In conclusion, the E.G.F. is not able to induce oocyte maturation independent of it's effects in immature F.F. and Ham's F-10 media.

• Biomedical Science Letters
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• v.7 no.4
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• pp.173-179
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• 2001

To investigate the ability to decondense sperm head penetrated into cytoplasm of the oocytes and the relationship between this ability and the level of glutatione (GSH) in mouse oocyte at various maturing stages. The fertilizability of oocytes at various stages of maturation the decondensation of sperm nucleus and the formation of male pronucleus, were observed and the levels of GSH were measured in oocyte at same stages. Besides, the relation between fertilizability and level of GSH in oocyte cytoplasm treated with L-buthionine-S, R-sulfoxmine (L-BSO), the inbitor of biosynthesis of GSH, was determined. The decondensation of sperm head was not found in GV stage and L-BSO treated oocytes. In maturing oocytes (GVBD, MI), the decondensation was found, but the formation of male pronucleus was not. The levels of GSH in oocyte cytoplasm were measured; 2.2 pmol per oocyte in the ovulated and the matured in vitro each, 1.0 pmol in GV intact oocyte, 1.3 pmol in GVBD, and 1.5 pmol in MI phase oocyte. In L-BSO treated oocytes the levels of CSH were measured 0.08~o.09 pmol per oocyte, slightly lower than GV stage oocyte. In conclusion, GSH in oocyte is supposed to be synthesized and storaged in cytoplasm during maturation. The failure of decondensation in the cytoplasm of GV stage and L-BSO treated is suggested that GSH is an essential factor in decondensing the sperm head and that the a certain level of GSH, more than in GV oocyte cytoplasm, is required in decondensation.