• 대한생식의학회:학술대회논문집
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• 2006.06a
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• pp.174.2-174.2
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• 2006

• Clinical and Experimental Reproductive Medicine
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• v.34 no.2
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• pp.67-74
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• 2007

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.6
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• pp.781-787
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• 2017

Objective: The early growth response (Egr) family consists of four members (Egr1, Egr2, Egr3, and Egr4) that are zinc finger transcription factors. Among them, Egr3 is involved in transcriptional regulation of target genes during muscle spindle formation and neurite outgrowth. We previously showed that the immunoreactive Egr3 is localized on oocyte spindle and accumulate near the microtubule organizing center during meiosis I in mice. Egr3 was also shown to be localized on spermatocytes. We herein investigated if Egr3 is expressed in mouse gonads and if Egr3 blockade results in any defect in oocyte maturation. Methods: Expression of Egr3 in mouse gonads was examined by reverse transcription-polymerase chain reaction. Full-length Egr3 and truncated Egr3 (${\Delta}Egr3$) complementary RNAs (cRNAs) with Xpress tag at N-terminus and DsRed2 at C-terminus, and small interfering RNA (siRNA) targeting Egr3 were microinjected into mouse oocytes at germinal vesicle stage. Localization of microinjected Egr3 was examined by confocal live imaging and immunofluorescence staining. Results: Egr3 mRNA was detected in mouse ovaries and testes from 1 to 4 week-old mice. An uncharacterized longer transcript containing 5'untranslated region was also detected in 3 and 4 week-old gonads. Microinjected Xpress-Egr3-DsRed2 or Xpress-${\Delta}Egr3$-DsRed2 localized to nuclei and chromosomes during meiotic progression. Microinjection of these cRNAs or Egr3 siRNA in oocytes did not affect meiotic maturation. Immunofluorescence staining of Egr3 in Xpress-${\Delta}Egr3$-DsRed2-injected oocytes showed a positive signal only on meiotic spindle, suggesting that this antibody does not detect endogenous or exogenous Egr3 in mouse oocytes. Conclusion: The results show that Egr3 localizes to chromosomes during meiotic progression and that certain antibodies may not faithfully represent localization of target proteins in oocytes. Egr3 seems to be dispensable during oocyte maturation in mice.

• Development and Reproduction
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• v.18 no.2
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• pp.117-125
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• 2014

Vitrification method is widely used in oocyte cryopreservation for IVF but the birth rates are lower than that of the fresh oocyte. One of the known main reasons is structural instability of meiotic spindle and chromosome systems of mature oocyte. To get the best way for keeping competence of matured oocytes, we studied the best conditions for vitrification focused on equilibration times. The mature oocytes were underwent vitrification with current popular method and analyzed the survival rates, microtubule stability and DNA integrity. The survival rates of recovered oocyte are almost same between groups and are more than 93%. The structural configuration of meiotic spindle was well kept in 10 min equilibration group and the stability rate was almost same with that of control. The chromosomal breakdown was observed in all experimental groups, but the chromosomal stability was higher in 10 min equilibration group than the other groups. The 10 min equilibration group showed best condition compared with the other groups. Based on these results, the equilibration time is one of the key factors in successful keeping for competence of mature oocyte. Although, more fine analysis about the effects of physical stress on oocyte during vitrification is needed to define the optimal condition, it is suggested that the optimal equilibration time to get competent oocyte in mouse is 10 min. Information acquired this study may provide insight into intracellular structural events occurring in human oocytes after vitrification and application for cryopreservation of human oocyte.

• Clinical and Experimental Reproductive Medicine
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• v.31 no.1
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• pp.75-81
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• 2004

Objective: The purpose of this study was to evaluate the efficacy and effect of various cryopreservation method on the survival and the cytoskeletal stability of metaphase II mouse oocyte. Methods: Mouse ovulated oocytes were collected and cryopreserved by a modified slow-freezing method with 1.5 M 1, 2-propanediol (PrOH)+0.1 M sucrose or by vitrification using cryo loop and EM grid with 40% ethylene glycol+0.6 M sucrose. Four hours after thawing, intact oocytes were fixed and stained with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-$\beta$-tubulin antibody to visualize spindle and propidium iodide (PI) to visualize chromosome. Spindle morphology was classified as follows: normal (barrel-shaped), slightly and absolute abnormal (multipolar or absent). Results: Survival rate of the frozen-thawed oocytes in vitrification group was significantly higher than that of slow-freezing group (62.7% vs. 24.4%, p<0.01). Vitrification with cryo loop showed significantly higher survival rate than that with EM grid (67.7% vs. 53.5%, p<0.05). On the other hand, proportion of normal spindle and chromosome configurations of the frozen-thawed oocytes between two vitrification group was not significantly different. Conclusion: For mouse ovulated oocytes, vitrification with cryo loop may be a preferable procedure compared to slow-freezing method. Further study should be needed to investigate developmental competency of frozen-thawed mouse oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.3
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• pp.293-301
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• 1996

In the present study, mouse follicular oocytes and 2-cell embryos(late -zygote stage embryos included) were cultured on the electric pad for electric stimulation in the culture incubator. In addition, follicular oocytes and embryos were tested for maturation and development under higher temperature condition($39^{\circ}C$).Mouse follicular oocyte maturation were not affected by anion electric stimulation and there is no significant difference in GBVD and MI between the control and experiment group after 4hr culture. In the embryo culture, it was found that more morula and blastocyst were found in the electric stimulation group rather than the control(96hr). This may seem to be caused with cytoplasmic $Ca^{2+}$ transient rise by electric stimulation(anion pad). On the other hand higher temperature incubation ($39^{\circ}C$) on the anion pad caused all the embryos degenerated within $12h{\sim}24hr$ culture. This was quite different from large animal embryos(bovine, pig, sheep), in which beneficial effect of high temperature incubation for oocyte maturation and embryo development were found.

• Korean Journal of Animal Reproduction
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• v.18 no.3
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• pp.217-228
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• 1994

These experiments were carried out to establish the optimal condition of electrostimulatin inducing cell fusion and oocyte activation for nuclear transplantation in mouse embryos. Eggs selected for cell fusion or activation by electrostimulation were equilibrated for 5~10 min. in 0.3M sucrose solution and electrostimulated for 60$\mu$sec using 1 pulse of 60, 70, 80, 90 or 100 volts DC with electrodes 0.2 mm apart. Then they were cultured in 20${mu}ell$ dropsof Tyrode's solution. The results of these experiments are as follows : 1. When one pulse of 60, 70, 80, 90 or 100 volts DC for 60$\mu$sec were applied to 2-cell embryos for fusion of blastomeres, fusion rates were 50.0, 81.7, 91.7, 100 and 100%, respectively ; and developmental rates of fused embryos to blastocyst were 76.7 to 81.5%. Higher fusion rates were observed in 90V and 100V. 2. The average cell number in fused embryos developed to blastocyst was about half of the cell number in diploid controls; and the cell number decreased with increasing of voltages. 3. When pulse numbers were increased, fusion rates improved, but developmental rates were not signficiantly different from the group for which the number of pulse was not increased. And the cell number of blastocyst decreased even more. 4. Oocytes aged for 6hrs after ovulation were electrostimulated for oocyte activation by the same method used for cell fusion. Rates of oocyte activated by electrostimulation were 45.3 to 60.4%, and fragmentation rates were 7.5~15.1%. The lysis rates were 17.0~34.0%. The results of these experiments indicate that the optimal condition for achieving cell fusion and activation is 1 pulse, duration 60$\mu$sec in 90 Volt. The results also show that this condition is suitable for nuclear transplantation using mouse eggs.

• The Korean Journal of Zoology
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• v.31 no.1
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• pp.21-28
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• 1988

The present study was carried out to examine the fertilizability of the mouse oocytes pre-ex-posed to dbcAMP which is a well-known inhibitor of the oocyte maturation. The oocytes once cultured in the dbcMP-containing medium for a certain length of, time were cultivated in the dbcMp-free medium to induced the maturation, then mixed with sperms, and observed following culture for 24 hours. The fertilization rate of cocytes was judged by the index of the number of 2-cell embryo developed 24hr following insemination. The fertilization rate of the oocyte previously incubated with dbcAMP (100 g/ml) for 2, 4, 8 16 hours was 32.3, 14.5, 4.7 and 8.8%, respectively, while that of the control was 53.3% indicating that the fertilizability was decreased as a function of time exposed to dbcAMP. The pretreatment of dbcMP, however, didn't affect the process of sperm penetration to egg. In addition, there is no prominent changes in the morphological architecture of fertielized eggs which has been exposed to dbcAMP as revealed by electron microscopic observation. Consequendy, it can be concluded that the mouse cocytes once inhibited their maturation by dbcMP may retain, in some extent, the fertilizability, although most of the fertilized egg may not proceed to further development because of the failure of pronucleus formation.

• The Korean Journal of Zoology
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• v.33 no.1
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• pp.45-52
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• 1990

In order to determine whether maturation inhibiting activity(MIA) in the cytoplasm of growing follicular oocytes would suppress the cleavage of the embryonal cells, the growing oocytes were fused with the 2 or 4 cell blastomeres and then examined for the nuclear phase of the fused giant cells 24 hr after culture. A significant number of the giant cells(60%) composed of growing mouse oocyte and 2 cell mouse blastomere(1/2) in interphase has contained 2 nuclei 24 hr after culture and most of the giant cells (90%) composed of the growing oocyte and 4 cell blastomere(1/4) also contained 2 nuclei after culture. The unfused blastomeres or the isolated blastomeres cultured without fusion treatment cleaved one cell cycle under the same culture condition. In contrast, the nucleus of the growing oocytes was disintegrated and the chromosome condensed when fused with 2 cell blastomere in mitosis. The growing rat oocytes also suppressed the nuclear disintegration of the mouse embryonal cells during culture. The data presented here showed that MIA in the growing mammalian oocyte inhibited the cleavage of the embryonal cells in interphase stage, but not in milosis stage.

• Clinical and Experimental Reproductive Medicine
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• v.34 no.2
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• pp.95-106
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• 2007

Objective: We previously identified differentially expressed genes (DEGs) between germinal vesicle (GV) and metaphase II (MII) mouse oocyte. The present study was accomplished as a preliminary study to elucidate the role of ribose 5-phosphate isomerase A (Rpia), the essential enzyme of the pentose phosphate pathway (PPP), in oocyte maturation. We observed expression of Rpia in the mouse and porcine oocytes. Methods: Expression pattern of the 11 MII-selective DEGs in various tissues was evaluated using RT-PCR and selected 4 genes highly expressed in the ovary. According to the oocyte-selective expression profile, we selected Rpia as a target for this study. We identified the porcine Rpia sequence using EST clustering technique, since it is not yet registered in public databases. Results: The extended porcine Rpia nucleotide sequence was submitted and registered to GenBank (accession number EF213106). We prepared primers for porcine Rpia according to this sequence. In contrast to the oocyte-specific expression in the mouse, Rpia was expressed in porcine cumulus and granulosa cells as well as in oocytes. Conclusion: This is the first report on the characterization of the Rpia gene in the mouse and porcine ovarian cells. Results of the present study suggest that the mouse and porcine COCs employ different mechanism of glucose metabolism. Therefore, the different metabolic pathways during in vitro oocyte maturation (IVM) in different species may lead different maturation rates. It is required to study further regarding the role of Rpia in glucose metabolism of oocytes and follicular cell fore exploring the regulatory mechanism of oocyte maturation as well as for finding the finest culture conditions for in vitro maturation.