• Clinical and Experimental Reproductive Medicine
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• v.44 no.1
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• pp.15-21
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• 2017

Objective: The aims of this study were to investigate whether fertilization could induce the resumption of meiosis in mouse oocytes arrested at metaphase I (MI) after in vitro maturation (IVM), and to investigate the effect of $Ca^{2+}$ chelator treatment at the time of fertilization on the transition from MI to metaphase II (MII). Methods: MII-stage and arrested MI-stage mouse oocytes after IVM were fertilized, and then embryonic development was monitored. Blastocysts from each group were transferred into 2.5 days post-coitum pseudo-pregnant ICR mice. MI oocytes after IVM were treated with a $Ca^{2+}$ chelator to investigate the effect of $Ca^{2+}$ oscillations on their maturation. Results: As insemination time increased, the number of oocytes in the MI group that reached the MII stage also increased. The blastocyst rates and total cell numbers in the MII group were significantly higher than in the MI group. No pregnancy occurred in the MI group, but 10 pregnancies were achieved (10 of 12) in the MII group. The proportion of MI oocytes that matured to MII oocytes after fertilization was significantly higher in the non-treated group than in the $Ca^{2+}$ chelator-treated group. Conclusion: The findings that a higher proportion of MI-arrested oocytes progressed to MII after fertilization and that the MI-to-MII transition was blocked by $Ca^{2+}$ chelator treatments before fertilization indicate that the maturation of MI oocytes to MII oocytes is associated with intracellular $Ca^{2+}$ oscillations driven by fertilization.

• The Korean Journal of Zoology
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• v.31 no.2
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• pp.81-86
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• 1988

The relationship between cumulus cell expansion, cocyte maturation and metabolic cooperativitiy was investigated by using mouse and pig cocyte-cumulus complexes in vitro. Cocyte germinal vesicle breakdown (GVBD) and cumulus expansion were manipulated with hormones or reagents which increase intracellular cAMP leveL Metabolic cooperativity between oocyte and cumulus cells was assessed by determination of the fraction of radiolabelled uridine marker that was transferred from the cumulus mass to the oocyte. Uptake of uddine marker by mouse and pig cumulus mass was increased by about fourfold of basal level with the stimulation of hormones (human choriononic gonadotrophin, HCG; follicle stimulating hormone, FSH) or cyclic AMP sttmulators (3-isobutyl-1-methylxanthine, IBMX; forskolin) during culture. However, the fraction of uridine that was transferred from the cumulus mass to the cocyte (transfer ratio) was gradually decreased during culture, irrespective with the presence of hormones or stimulators. The decrease of the transfer ratio was not correlated with the state of occyte whether they have GV or not, or with the degree of cumulus expansion. In mouse complexes, HCG induced more significant reducton of transfer ratio than other treatments. These results do not support the idea that modulations of metabolic cooperativity between cumulus cells and oocytes are important for the regulation of meiotic resumption in mammals.

• Development and Reproduction
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• v.2 no.1
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• pp.9-20
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• 1998

It has been wel known that phospholipase C(PLC) plays an important role in the intracellular signaling in a variety of cell types. However, involvement of PLC in mouse oocyte maturation and preimplantation embryo development remains unknown. The present study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturatio and preimplantation embryo development study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturation and preimplantation embryo development by the competitive reverse transcription-polymerase chain reaction (RT-PCR method). PLC \gamma 1 mRNA (0.1 fg) was readily detected in germinal vesicle (GV)-stage oocyte and its level was reduced as meiotic resumption proceeded. PLC-\beta 1 mRNA (<0.1 fg) as detected at low level at GV-stage oocytes and scarcely detected at germinal vescle breakdown (GVBD)-stage oocytes. After fertilization, both PLC \beta 1 and \gamma 1 mRNA levels began to increase at morula-stage embryos (0.2 fg) and were more prominent in blastocyst-stage embryos(1 fg). to elucidate the possible involvement of PLC via protein kinase C(PKC) pathway during oocyte maturation and preimplantation embryo development , the effects of sphingosine (PKC inhibitor), sn-$diC_{8}$(PKC activator) anc U73122 (PLC ingibitor) were examined. Treatment of GV-stage oocytes with sphingosine (20 \mu M) facilitated the meiotic resuption by 10-20 over the control within 1 h as judged by GVBD, whereas U73122 failed to show any significant effect. U73122 (10 \mu M) effectively blocked the compaction of morula, while sn-$diC_{8}$(50 \mu M). In summary, the present study shows that the mouse PLC \beta 1 and \gamma 1 are expressed in a developmental stage-specific manner and PLC-PKC pathway may be involved in early preimplantation embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.103-108
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• 1996

Through the previous studies(I,II), it was observed that human follicular fluid(HFF) was more effective than human fetal cord serum(HFCS) on promoting meiotic resumption of oocytes and improving embryonic development of mouse in vitro. On the basis of these results, we have gradually exchanged HFCS with HFF as protein supplement in human ART. This study was performed to investigate the efficiency of HFF on improving the pregnancy rate in ART. Oocytes were retrieved transvaginally from patients treated with pituitary suppression with GnRH-agonist and ovarian stimulation with human menopausal gonadotro-pin(HMG) and pure follicle stimulating hormone(FSH). Aspirated oocytes were rinsed and cultured in TCM-199 containing HFF, and the concentrations of HFF were adjusted to 10, 20, and 30% according to the use for insemination, embryo growth and embryo transfer, respectively. As possible as, we tried to do embryo transfer into fallopian tube to mimic the coincidence of the cell stage with the place of sojourn in vivo, so we performed various ART programs(IVF & ET; in vitro fertilization, ZIFT; zygote intra fallopian-tube transfer, ZIFT & ET) according to the tubal conditions of patients. On the while, intra cytoplasmic sperm injection(ICSI) was used to assist IVF of the patients who had shown poor standard IVF results by immunological or severe male factor. Of the 255 cycles of ART programs using HFF as protein supplement, 118 cycles were turn out to be succeeded in pregnancy(46.2%, per cycle, p<0.05), while 21 pregnancies were achieved in the 69 cycles using HFCS(30.4%). The 255 cycles using HFF were subdivided into cycles with the type of ART programs, and each pregnancy rate of the ART programs were 44.7% (IVF & ET, 76/170 cycles), 53.4%(ZIFT, 31/58 cycles) and 40.7% (ZIFT & ET, 11/27 cycles), respectively. In the 61 ICSI cycles using HFF, 28 cycles succeed in pregnancy(45.9%), while 7 pregnancies were obtained in the 17 ICSI cycles using HFCS. Also the ongoing pregnancy rate in the group using HFF(78.8%, 93/118 cycles) was higher than that in the group using HFCS(61.9%). Therefore, we found that the use of HFF as protein supplement was more suitable and effective than the use of HFCS to improve the pregnancy rate in ART.

• Development and Reproduction
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• v.4 no.1
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• pp.45-52
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• 2000

Membrane type matrix metalloproteinases(MT-MMPs) have been suggested to play an important role during structural remodeling of various tissue. Expression patterns of MT1-MMP and MT2-MMP mRNAs were investigated in oocytes, embryos, ovary and oviduct of mouse during their differentiation or periovulatory period using RT-PCR technique. Both cDNA products of MT1- and MT2-MMP of immature oocytes were barely discernable with a minimum amount but the expressions were distinct in mature oocytes regardless that they were matured in vivo or in vitro. MT2-MMP was not expressed by 2-cell embryos but was expressed by 4-cell stage embryos. From the morula stage untill hatched blastocyst stage, embyos showed intesnse expression of MT2-MMP with a sudden increase at blastocyst stage. While mouse ovarian tissues showed both expression of MT1- and MT2-MMP, there was no stage-specific difference throughout the estrous cycle. Mouse oviducts also exhibit constant amount of both MT1- and MT2-MMP expressions throughout periovulatory period, i.e., before or after ovulation. These observations lead to suggest that the differential expressions of maternal MT1- and MT2-MMP during meiotic resumption of mouse oocytes and embryonic expression of MT2-MMP particularly at blastocyst stage might play a role in the differentiation of mouse oocytes and／or embryos. The precise function of MT1- and MT2-MMP with regards to their participation in the remodeling of ovarian and oviductal tissues remains in a question.