• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.117-120
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• 2005

This study was conducted to establish the optimal temperature condition before oocyte activation in B6m F1 mouse. In experiment 1, two embryo culture media (CZB vs KSOM) were evaluated for the development of activated mouse oocytes. Parthenogenetic embryos cultured in KSOM showed better blastocyst development than ones cultured in CZB $(56.2\%\;vs\;81.0\%\;p<0.01)$. Two-hour of pre-incubation before activation significantly reduced the number of hatched blastocysts in KSOM $(22.0\%\;versus\;8.8\%\;p<0.05)$. In experiment 2, recovered oocytes were pre-incubated at different temperature conditions before activation. The experimental groups were divided by 5 as follows. Group A: pre-incubation for 120 min at $37^{\circ}C$, Group B: pre-incubation at $37^{\circ}C$ for 90 min then at $25^{\circ}C$ for 30 min, Group C: pre-incubation at $37^{\circ}C$ for 60 min then at $25^{\circ}C$for 60 min, Group D: pre-incubation at $37^{\circ}C$ for 30 min then at $25^{\circ}C$ for 90 min, and Group E: pre-incubation at $25^{\circ}C$ for 120 min before activation. Group A $(67.6\%)$ and B $(66.7\%)$ showed better development to the blastocyst stage than other groups $(Group\;C:\;50.0\%\;Group \;D:\;49.2\%\;Group\;E:\;33.3\%,\;p<0.05)$. The present study indicates that the temperature before activation affects the development of B6D2 F1 mouse parthenogenetic oocytes and exposure to room temperature should be limited to 30-min when the oocytes are left in HEPES-buffered medium for micromanipulation.

• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.173-181
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• 1993

In order to demonstrate whether ovoperoxidase hardens the zona of oocytes activated by incubating in M-S buffer supplemented with 20$\mu$M of Ca-ionophore A 23187, the effect of peroxidase inhibitors(250mM pheylhydrazine, 28mM sodilum sulfite, 350mM glycine ethyl ester and 50mM sodium azide), tyrosine analogue(12.5mM tyramine) and exogeneous peroxidase(50$\mu\textrm{g}$/ml horseradishperoxidase ; HRP) on zona hardening in ionorphore-treated oocytes were investigated. The results obtained from thses experiments were summarized as follows : 1. The zona solubility (t50) of ionophore-activated and DMSO-treated oocytes at 1, 2 and 3 hr of culture were 25.0, 31.6 and 40.6min., and 9.7, 10.8 and 15.5 min., respectively. The longest time required for zona lysis of ionophore activated oocytes at 1 hr after onset of ionophore treatment. The diferences int50 for zona was significantly greater as compared to DMSO-treated controls(P<0.01). 2. The inhibition rates of hardening in the oocytes treated with the phenylhydrazine, sodium sulfite, glycine ethyl ester and sodium azide, were 23.8, 61.9, 95.2 and 23.8%l, respectively, and the tyramine, was 14.3%. Several known peroxidase inhibitors and tyrosine analogue were blocked zona hardening in ionophore activated oocytes. 3. The treatment of exogeneous peroxidase promoted the zona hardening of activated oocytes but not unactivated oocytes. These resuls indicate that the ovoperoxidase apparently catalyzes the hardening of the zona following ionophore activation of mouse oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.3
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• pp.331-340
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• 1998

We determined the incidence of activation, male pronuclear formation and apposition of pronuclei in porcine oocytes following intracy-toplasmic injection of various porcine sperm components and foreign species spermatozoa, such as mouse, human or cattle. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. Neither isolated sperm tail nor perinuclear material removed sperm head activated oocytes. Because injection of mouse, bovine or human spermatozoon activated porcine oocytes, the sperm born activation factors is not strict species specific. Male pronuclear formation and pronuclear apposition were observed in the porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. The electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation nor pronuclear apposition comparent with sperm cell injection alone (p>0.1). Mitosis and two cell division in some oocytes were observed at 20 to 24 h after injection of porcine spermatozoon. However, none of oocytes following injection of mouse, bovine or human spermatozoa developed to the mitotic metaphase or normally divided to the two cell stage. These results suggested that the oocyte activating factor(s) presented in the perinuclear material and it is not species specific for the porcine oocyte.

• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.19-27
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• 1999

This study was carried out to investigate the effect of intracytoplasmic injection of $Ca^{2＋}$ and human sperm extract on the parthenogenetic activation of mouse oocytes. The results obtained were as follows: 1. The mouse oocytes were injected with 10 pl of PBS medium containing 0, 1.7 and 5 mM calcium concentrations, respectively. Activation rate of the oocytes with formation of pronuc1ei and extrusion of the second polar bodies was 14.5, 9.8 and 14.9% at the above calcium concentrations, respectively. There were no significant differences in the activation rates among the calcium concentrations. 2. The mouse oocytes were injected with 10 pl of non-heated human sperm extract, and cultured for 12~15 h in the PBS media with the 0, 1.7 and 5 mM calcium concentrations, respectively. Activation rate(51.8%) of the oocytes at the 1.7 mM calcium concentration was significantly higher than those at the 0 and 5 mM calcium concentrations. 3. The mouse oocytes were injected with 10 pl of heated human sperm extract, and cultured for 12~15 h in the PBS media with the 0, 1. 7 and 5 mM calcium concentrations, respectively. No significant differences were found in the activation rates (11.8~17.0%) among the calcium concentrations. 4. The mouse oocytes were injected with 10 pl of PBS medium, non-heated sperm extract and heated sperm extract, and cultured for 12~15 h in the PBS media with 1.7 mM calcium concentrations, respectively. Activation rate (54.5%) of the oocytes injected with the non-heated sperm extract was highest. There were significant differences in the activation rates among the above injection materials (P＜0.05). 5. The mouse oocytes were injected with 10 pl of 1 and 6 days old non-heated sperm extracts, and cultured for 12＜15 h in the PBS media with 1.7 mM calcium concentrations, respectively. Activation rate(60.0%) of the oocytes injected with 1 days old sperm extract was significantly higher than that (11.1%) injected with 6 days old sperm extract. The results obtained in this study suggest that non-heated human sperm extract may contain sperm-associated oocyte-activating factor such as oscillin.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.427-432
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• 1997

These studies were performed to approach the precise pathway inducing the meiotic inhibitory action of hypoxanthine on mouse follicular oocytes and to identify the cause of detrimental effect of hypoxanthine on viability of the oocyte in vitro. In addition, a correlation between the meiotic inhibitory effect and the detrimental effect of hypoxanthine was investigated. Mouse follicular oocytes at germinal vesicle(GV) stage were collected from the ovaries of ICR mice by puncturing the antral follicles with a fine needle, at 48 hours after PMSG injection. Oocytes were cultured in Modified Whittingham's T6 media containing hypoxanthine and several materials that involved in metabolism of hypoxanthine, and the effects of the materials on the actions of hypoxanthine were investigated by observing germinal vesicle breake down (GVBD), 1st polar body (PB) extrusion and viability of the oocytes. Phophodiesterase significantly reduced the meiotic inhibitory effect of dbcAMP but did not influence on the inhibitory effect of hypoxanthine. Allopurinol and 6-MP significantly enhanced the meiotic inhibitory effect of hypoxanthine, but the materials themselves also showed the meiotic inhibitory action like hypoxanthine. Hypoxanthine-guanine phosphoribosyltransferase significantly enhanced the meiotic inhibitory effect of hypoxanthine, on the contrary HGPRT itself promoted meiotic resumption of the oocytes. Catalase did not induce any change in the meiotic inhibitory effect of hypoxanthine, but SOD increased the GVBD rate suppressed by hypoxanthine. The detrimental effect of hypoxanthine on viability of the oocytes was significantly reduced by allopurinol and catalase, but SOD increased the GVBD rate suppressed by hypoxanthine. The detrimental effect of hypoxanthine on viability of the oocytes was significantly reduced by allopurinol and catalase, but SOD did not reduce the deterimental effect of hypoxanthine. In conclusion, the meiotic inhibtory effect of hypoxanthine may be caused by guanyl dervartives converted from hypoxanthine via salvage pathway, and superoxide anion may partially participate in the inhibitory effect of hypoxanthine. The detrimental effect of hypoxanthine on viability of the oocytes be cused by hydrogen peroxide produced during the metabolism of hypoxanthine.

• Clinical and Experimental Reproductive Medicine
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• v.51 no.1
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• pp.57-62
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• 2024

Objective: The purpose of this study was to use a mouse model to investigate the blastocyst formation rate in vitrified-warmed embryos derived from vitrified-warmed oocytes. Methods: Metaphase II oocytes obtained from BDF1 mice were vitrified and warmed, followed by fertilization with epididymal sperm. On day 3, a total of 176 embryos, at either the eight-cell or the morula stage, were vitrified-warmed (representing group 1). For group 2, 155 embryos at the same developmental stages were not vitrified, but rather were directly cultured until day 5. Finally, group 3 included day-5 blastocysts derived from fresh oocytes, which served as fresh controls. The primary outcome measured was the rate of blastocyst formation per day-3 embryo at the eight-cell or morula stage. Results: The rates of blastocyst formation per day-3 embryo were comparable between groups 1 and 2, at 64.5% and 69.7%, respectively (p>0.05). The formation rates of good-quality blastocysts (expanded, hatching, or hatched) were also similar for groups 1 and 2, at 35.5% and 43.2%, respectively (p>0.05). For the fresh oocytes (group 3), the blastocyst formation rate was 75.5%, which was similar to groups 1 and 2. However, the rate of good-quality blastocyst formation in group 3 was 57.3%, significantly exceeding those of group 1 (p=0.001) and group 2 (p=0.023). Conclusion: Regarding developmental potential to the blastocyst stage, vitrified-warmed day-3 embryos originating from vitrified-warmed oocytes demonstrated comparable results to non-vitrified embryos from similar oocytes. These findings indicate that day-3 embryos derived from vitrified-warmed oocytes can be effectively cryopreserved without incurring cellular damage.

• Development and Reproduction
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• v.11 no.3
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• pp.263-272
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• 2007

Contrast to mouse where its in vitro maturation rates are high without specific supplements or presence of the cumulus cells, there are some species, such as porcine, where its in vitro oocyte maturation rates are still very low. This comparative study was conducted to investigate the role of malate dehydrogenase(Mor2) during oocyte maturation by RNAi in the mouse and porcine. The Mor2 double-stranded RNA(dsRNA) was prepared speciesspecifically and microinjected into the cytoplasm of denuded germinal vesicle(GV) oocytes. Oocytes were cultured for 48 h(porcine) and 16 h(mouse) in M199 with 10% porcine follicular fluid, pyruvate, p-FSH, EGF, cystein, and estradiol-$17{\beta}$. We measured changes in oocyte morphology, maturation rates and mRNA levels after Mor2 RNAi. We confirmed gene sequence-specific knock down of Mor2 mRNA in both species after Mor2 RNAi. In contrast to our previous finding that mMor2 RNAi resulted in GV arrest in the mouse, we found that pMor2 RNAi resulted in MI arrest in denuded porcine oocytes(58%), but developed to MII(84.4%) in COCs. To determine whether this difference between mouse and porcine RNAi is due to differences in culture media, we cultured mouse oocytes in the M199 media for 16 h after mMor2 RNAi. Mouse oocytes were developed to MII stage(62%) and there was no statistical difference compared to that of non-injected(76.8%) and buffer-injected(73.3%) control groups. Therefore, we concluded that the mouse and porcine oocytes are having different metabolic systems in relation to malate dehydrogenase for oocyte maturation. This could be a basis for differences in maturation rates in vitro in two species. Further scrutinized studies on the metabolic pathways would led us in finding better culture system to improve oocyte maturation rates in vitro, especially in more challenging species like the porcine.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.71-71
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• 2002

Our previous study has suggested that muscarinic receptor present in the mouse oocytes, and Ca/sup 2+/ waves elicited by acetylcholine (ACh) are similar to those induced by sperm. A numerous study reported that ACh could cause early activation events in mouse oocytes overexpressing the Ml muscarinic receptor (Williams et al., 1992; Moore et al., 1993; Kim et al., 1998). However, the physiological role of ACh during mouse embryonic development is poorly understood. (omitted)

• Clinical and Experimental Reproductive Medicine
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• v.18 no.1
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• pp.49-53
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• 1991

The development of 2-cell mouse embryos to the blastocyst stage in vitro has been used as a quality control for the media empolyed for human in vitro fertilization. There was a comparison between the quality control data of the culture medium as ascertained by 2-cell mouse embryos development and sperm motility and the data from fertilization and cleavage of human oocytes. However, there was no obvious association between fertilization and cleavage of human oocytes and the quality of the medium ascertained by mouse embryo development and sperm motility.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.243-250
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• 1995

Frozen storage of the oocytes has been used in a few mammalian species including mouse, hamster, human and cattle. However, frozen4hawed oocvtes show different sperm penetration on the levels of the zona pellucida and the plasma memhrane when compared with fresh oocytes. To elucidate biological changes occurring during freezing and thawing, we examined the kinetics of sperm penetration into frozen-thawed hamster oocytes. Oocytes obtained from superovulated female golden hamsters were frozen-thawed in an autofreezer according to an established method. Fresh and frozen4hawed oocytes were fertilized in vitro with capacitated hamster spermatozoa after removing the zona pellucida. The oocytes were examined at 1, 2, 3 and 6 h postinsemination. Sperm penetration found to be 1 h delayed in frozen-thawed oocytes. Other parameters such as degree of polyspermy and decondensing sperm heads were not affected by freezing and thawing. The results suggest that freezing and thawing may cause changes in the egg membrane surface and subsequently which leads to delay in the sperm-egg fusion.