• Journal of Embryo Transfer
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• v.12 no.1
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• pp.21-27
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• 1997

The studies were carried out to investigate the effects of bisection method and with and without-zona pellucida of embryos on in vitro developmental rate bisected embryos by micromanipulator, micropipette and pipetting. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TCM-199 mediurn containing 10 IU /ml의 PMSG(Sigma, USA), 10 IU /ml의 hCG, 1$\mu$g /ml의 $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$ and then, matured oocytes were again cultured for 12~ 18 hrs with motile capacitated sperm by preincubation of heparin. Bisected embryos cultured for 1~5 days in 20% FCS + TCM-199 medium. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are summarized as follows :1. The survival rates of bisected bovine embryos by micromanipulator and micropipett were 29.2% and 19.1%, respectively. The rates of non-bisection embryos(46.7%) were significantly higher than those of bisection embryos. 2. The in vitro developmental rates of bisected bovine embryos by micromanipulator, micropipett and pipetting method were 32.4%, 19.4% and 25.6%, respectively.3. The in vitro developmental rates of with and without-zona pellucida of bisected bovine embryos by raicromanipulator were 30.8% and 25.0%, respectively. The rates of nonbisection embryos(53.1%) were significantly higher than those of bisection embryos.

• 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.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.71-80
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• 1996

The envelope of the rnannnalian oocyte plays crucial roles in sperm-oocyte interactions by providing sperm receptors, inducing acrosome reaction and preventing polyspermy. Understanding of properties of the zona pellucida (ZP) is essential for the artificial control of fertility in mammals. This study was carried out to produce and characterize monoclonal antibodies(MAbs) to porcine ZP proteins. Approximately 8,000 ZPs were obtained from follicular oocytes and dissolved in 40$\mu$l of double distilled water. Following immunization through foot-pad injections of Balb /c mice with a ZP solution, the popliteal lymph nodes were recovered at 2 weeks after the last injection. Hybridoma cell lines were established by fusing lymph node cells with P3X63 myeloma cells through selection using HAT medium and screening by immunofluorescence(IF) microscopy on the isolated ZP. Secreted MAbs were found to consist k chains and different heavy chains as evidenced by isotyping. Some of the MAbs demonstrated high specificity to the ZP in IF. The Mabs also showed positive cross reactivity with hamster and mouse eggs, while negative with bovine eggs. The results implicate that the MAbs can be used not only for identification of functional regions of the ZP, but also for elucidation of mechanisms involved in fertilization of mammals. The MAbs will provide basic information on biochemical anatomy of the ZP as well as can be candidates for the future contraceptive vaccines.

• 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.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.239-250
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• 1996

The objective of the present experiments were to determine whether micromanipulative and electro-stimulation conditions for blastomere survival overlapped those for oocyte activation in porcine. Eggs selected for in vitro development potential of blastomeres isolated from 4-cell embryos and oocyte activation by electrostimulation were equilibrated for 5~10 min, in 0.3M sucrose solution containing 7.5$\mu\textrm{g}$/ml cytochalasin B, and then electrostimulated for 30$\mu$sec using one pulse of 100, 120, 150 or 180 volts DC with electrodes 0.2mm apart. Single blastomeres were inserted into empty zona pellucida prior to electrostimulaticn. Then they were cultured in 20${mu}ell$ drops of fresh BECM to observe their developmental ability in vitro in a humidified incubat or at 38.5$^{\circ}C$. The results obtained from these experiments are as follows : 1. When one pulse of 100, 120, 150 or 180 volts DC for 30$\mu$sec were applied to porcine oocytes having the slit formed on zona pellucida for activation, activation rates were 65.1, 66.7, 70.7 and 91.7%, respectively. Higher activation rate was observed in 180V. 2. Infact oocytes incubated for 30 min, in 0.3M sucrose solution after electrostimulation were significantally different from control group with increasing of voltages(p<0.05). When voltages used for electrostimulation were increased, activation rates of oocytes were improved in all treatment groups. 3. When zona punctured-oocytes were only electrostimulated, or incubated in 0.3M sucrose solution for 30 min. after electrostimulation at 180 volt DC, activation rates were 90.5 and 95.5%, respectively. And activation rates of zona punctured-oocytes were significantly different from the groups for which zona pellucida was not punctured(P<0.05). 4. When single blastomeres form 4-cell transferred into empty zona pellucida were incubated for 0, 15 and 30 min. in 0.3M sucrose solution after electrostimulation using one pulse of 180 volt DC for 30 $\mu$sec, developmental rates of electrostimulated-single blastomeres to blastocyst were 72.5, 59.0 and 51.2%, respectively, and the ratio of control group developed to blastocyst were 80.0%. 5. The average cell number in electrostimulated-blastomeres developed to blastocyst were 7.9~10.8, and reduced than the cell number in diploid control ; Also cell number decreased with increasing of voltages. The results of these experiments indicate that the optimal condition for achieving in vitro developmental ability of single 4-cell blastomeres and oocyte activatin is 1 pulse, duration 30 $\mu$sec. in 180 volt, and incubation of blastomeres and oocytes in 0.3M sucrose solution after electrostimulation was not significantally different from another treatment groups. The results also show that this condition is suitable for nuclear transplantation using porcine eggs.

• Proceedings of the Zoological Society Korea Conference
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• 1997.04a
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• pp.64.2-64
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• 1997

No Abstract, See Full Text

• Korean Journal of Veterinary Research
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• v.29 no.2
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• pp.123-128
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• 1989

Demi-embryos were successfully produced by bisection of ICR mouse embryos at preimplantation stages. They were microsurgically bisected using a microsurgical blade attached to a micromanipulator after pretreatment with 0.5% pronase in PBS for two minutes or not. Embryos with softened zona pellucida were more easily bisected and less damaged than intact embryos. The highest success rate in bisection has been achieved by selecting blastocysts(94.1% in success rate with intact blastocysts and 100% in success rate with zona softened blastocysts). Demi-embryos without zona pellucida were cultured in D-PBS or M-16 medium at $37^{\circ}C$, 5% $CO_2$ in air for 72 hours for 2-cell stage embryos, 48 hours for 4-to 8-cell stage embryos, 24 hours for morula stage embryos and 6~12 hours for blastocyst stage embryos. For the in vitro culture of 2-cell stage embryos, $100{\mu}M$ 2Na-EDTA was added to the media. M-16 medium was better for the in vitro development of mouse embryos than PBS, and PBS is not considered to be suitable for long-term culture of embryos, especially at early stage of cleavage. In M-16 medium, developing rate of demi-embryos of which pair underwent development to form eublastocysts was 15.8% at 2-cell stage, 16.8% at 4-cell stage, 38% at 8-cell stage, 89.6% at morula stage and 94.4% at blastocyst stage, respectively. The more rapid and efficient production of demi-embryos and higher viability after bisection can be expected by softening zona pellucida with pronase and by selecting morulae or blastocysts rather than embryos at early stage of cleavage.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.7
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• pp.962-968
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• 2009

The aim of this study was to compare the effects of oviductal fluid, porcine zygote medium (PZM)-3, PZM-4 and PZM-5, and modified PZM-5 culture media, and determine the effects of zona pellucida (ZP) removal on the development of nuclear transfer (NT) embryos. There were no significant differences in the rates of fusion and cleavage among the five different oviductal fluid concentrations. However, the rates of blastocyst formation and the cell numbers per blastocyst were high in the embryos at the 14 and 28 $\mu{g}$/ml concentrations of oviductal fluid compared to the 0, 56 and 100 $\mu{g}$/ml concentrations. The rates of cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the PZM-3, PZM-5 and modified PZM-5 media than in the PZM-4 medium. However, there were no significant differences in the fusion rates of oocytes among the four culture media. The cell numbers per blastocyst in the embryos without ZP were significantly greater than those with ZP. However, there were no significant differences in the rates of fusion, cleavage and blastocyst formation between the embryos with and without ZP. In conclusion, we improved blastocyst development and the quality of NT embryos by replacing PVA with 3 mg/ml of BSA in PZM-5 medium and supplementing the PZM-5 medium with 14 $\mu{g}$/ml oviductal fluid. The NT embryos produced by the zona-free NT method had a high rate of blastocyst formation in the modified PZM-5 medium.

• Korean Journal of Animal Reproduction
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• v.15 no.3
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• pp.225-231
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• 1991

These experiments were carried out ot detect autoantiboies to zona pellucida in sera from infertile women using indirect ELISA and IFA and to investigate their effect on in vitro fertilization of mouse ova. In inidirect ELISA test, 12 of 116(10.3%) serum samples form infertile women gave positive reaction whereas all of 16 samples from fertile women and men were negative. Furthermore, in indirect IFA test, 17 of 116 (14.7%) serum samples from infertile women gave positive fluorescence whereas all of control sera were negative fluorescence. The fertilization rates(15.9%) of mouse eggs treated with positive sera were significantly lower than those(51.9%＋71.2%) autoantibodies to zonapellucida are responsible for infertility in unexplained infertile women, presumably by perventing sperm attachment and penetration.

• Proceedings of the Zoological Society Korea Conference
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• 1998.04a
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• pp.49.1-49
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• 1998

No Abstract, See Full Text