• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.239-246
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• 1992

Capacitated and acrosome~reacted spermatozoa were microinjected into the perivitelline space of bovine oocytes matured in vitro. Oocytes obtained from the ovaries of slaughtered heifers and cows were cultured in vitro in the TCM-199 supplemented with 20% FCS for 24 hr at 39$^{\circ}C$ under an atmosphere of 5% CO$_2$ 8% O$_2$. Fresh or frozen spermatozoa were incubated for 2 hr at 39°C under an atmos-phere of 5% CO$_2$, 8% O$_2$ in Ham's F-lO medium containing 0.75% BSA for capacitation, and kept for 30 min in culture medium containing 12 mM of dbcGMP and lOmM of immidazol for acrosome resction. One motile spermatozoon was injected into the perivitelline space of each oocyte. The 2nd polar body and the pronuclei were observed in 9.5% and 5.4% of oocytes, respectively. The rate of cleavage of oocyte over 2-cell stage was 4.1%(10 of 242), These results indicate that the microinjection may be a useful technique to study sperm-oocyte interaction.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.85-93
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• 2006

본 연구는 소 형질 전환 체세포 핵이식에서 용이한 탈핵을 위해 demecolcine을 이용할 시 탈핵율과 핵이식란의 발육능을 높이기 위한 최적의 조건을 알아보고자 실시되었다. 도축장 유래 미성숙 난자를 18시간 체외성숙 후 제1극체가 확인된 성숙 난자를 0.1, 0.2, 0.4 및 0.8 ug/ml의 demecolcine이 첨가된 배지에서 1시간 더 처리한 다음 세포막이 돌출되어 있는 난자를 체세포 핵이식에 공여하여 각 군간 배반포로의 발육능을 비교하였다. Demecolcine 처리 후 핵이 포함된 셰포막의 protrusion rates를 각 군간 비교한 결과 0.2, 0.4 및 0.8 ug/ml 군에서 0.1 ug/ml 군보다 유의적으로 높았으며 (82.8, 86.2, 90.4 vs. 70.1%), conventional blind 방법과 비교한 결과 demecolcine를 이용한 군에서 유의적으로 높은 탈핵율을 보였다(75.3 vs. 96.2%; p<0.05). 체세포 핵이식란의 발육능 비교에서는 0.1 및 0.2ug/ml 군에서 대조군과 함께 유의적으로 높은 분할율 및 배반포로의 발육능을 보였다(p<0.05). 결론적으로 소 형질전환 체세포 핵이식을 위한 탈핵시 높은 탈핵율과 배반포로의 발육율을 얻을 수 있는 demecolcine의 적정농도는 0.2ug/ml이라고 사료된다.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.1-14
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• 2003

This study was conducted to investigate that the immature and mature oocytes of porcine can be cryopreserved by vitrification. Oocytes were centrifuged to polarize the cytoplasmic lipid droplets. The lipids were removed from cytoplasm by micromanipulation. Delipated oocytes were centrifuged after being preincubated with cytochalasin B(CB) fer 10 min, and lipid droplets were removed. Centrifuged oocytes were treated with CB and centrifuged to polorize lipid droplets but not delipated and control oocytes is not-treatment. Oocytes of three types were vitrified in electron microscope(EM) grids. The results of survival, maturation and cleavage rates were as follows. 1 The survival rates of immature oocytes were 15.1%, 0% and 0% in the Delipated, Centrifuged and Control after vitrification, respectively, and its rate of Delipated wassignificantly higher than Centrifuged and Control(P<.01). 2. The survival rates of mature oocytes were 12.21%, 0% and 0% in the Delipated, Centrifuged and Control after vitrification, respectively, and its rate of Delipated was significantly higher than Centrifuged and Control(P<.01). 3 The maturation rates of immature oocytes were 37.5% and 68.9% for metaphase II in the Delipated after vitrification and Non-vitrification, respectively, and its rate of Non-vitrification was significantly higher than Delipated after vitrification(P<.01). 4. The cleavage rates of immature oocytes were 12.5%, 0%, 0% and 56.1% in the Delipated, Centrifuged, Control after vitrification and Non-vitrification, respectively. It's rate of Delipated was higher than Centrifuged and Control, but there were no significant difference, and its rate of Non-vitrification was significantly higher than Delipated, Centrifuged and Control(P<.05). 5 The cleavage rates of mature oocytes were 25.0%, 0%, 0% and 67.9% in the Delipated, Centrifuged, Control after vitrification and Non-vitrification, respectively. It's rate of Delipated was higher than Centrifuged and Control, but there were no significant difference, and its rate of Non-vitrification was significantly higher than Delipated, Centrifuged and Control(P<.05).

• 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.

• Reproductive and Developmental Biology
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• v.28 no.3
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• pp.173-179
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• 2004

This study was conducted to investigate the toxi-cological effects of different vitrification solution on development of immature porcine oocytes in vitro. Oocytes were exposed to EFS solution [40% ethylene glycol (EG) + 18% Ficoll + 0.3M sucrose], ES solution (5.5M EG + 1.0M sucrose) or GE solution [10% glycol (G) + 20% EG], and these oocytes were transferred to sucrose solution directly. Maturation rates were significantly (P<0.05) higher in the ES solution (44.5%) and control (57.6%) than in the EFS solution (38.8%) and GE solution (22.4%). No differences among three solution were found in fertilization rates. Cleavage rates was significantly (P<0.05) higher in the ES solution (47.1%) and control (65.9%) than in the EFS solution (21.9%) and GE solution (19.0%), but no difference among three solutions was found in the blastocyst formation rates. These results indicate that combination of EG and sucrose solutions had effects on development of immature porcine oocytes.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.331-337
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• 1994

Response of the oocytes to parthenogenetic activation is one of the indice for cytoplasmic maturation. Maturational age-dependent parthenogenetic activation was examined in bovine oocytes. Follicular oocytes recovered from the slaughter house ovaries were matured in vitro in TCM 199+15% FCS+1Oiu/ml PMSG +10 iu/ml hCG from 24 to 48 h at 6 h intervals. The in vitro matured oocytes were activated by 7% ethanol for 7 min. The nuclear maturation and the cytoplasmic maturation were analysed by the nuclear configuration and pronuclei formation stained by rapid staining method. Cumulus oophori expansion increased as the maturation time increased. Proportions of the nuclear maturation were 81, 89, 72, 60 and 60% in IVM 24, 30, 36, 42 and 48 h groups, respectively. Abnor¬mality in metaphase II chromosome increased sharply from 36 h IVM. The rates of the pronuclei formation and diploid upon ethanol activation were 67, 68, 73, 84 and 87%, and 4, 5, 10, 16 and 20% in IVM 24, 30, 36, 42 and 48 h groups, respectively. It was suggested that maturational age increased the formation of the pronuclei and diploid, and that cytoplasmic maturation require longer maturation period than normal nuclear maturation. These results should be useful for determination of an appropriate time for fertilization in mammalian eggs matured or preincubated in vitro.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.89-99
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• 2004

Stretch-activated channels (SACs) responds to membrane stress with changes in open probability (Po). They play essential roles in regulation of cell volume and differentiation, vascular tone, and in hormonal secretion. SACs highly present in Xenopus oocytes and Ascidian oocytes are suggested to be involved in the regulation of pH and fluid transport to balance the osmotic pressure, but remain unclear in mammanlian oocytes. This study was investigated to find the presence of SACs in hamster oocytes and to examine their electrophysiological properties. To infer a role of SAC in relation to the development of early stage, we followed up to the stage of two-cell zygote with patch clamp techniques. Single channels were elicited by negative pressure (lower than ­15 cm$H_2O$). Interestingly, SACs were dependent on permeable cations such as $Na^+$ or $K^+$. As permeable cation removed from both sides across the membrane, SAC activity completely disappeared. When permeable cations present only in intracellular compartment, outward currents appeared at positive potentials. In contrast to this, inward currents occurred only at the negative voltage when permeable cation absent in cell interior. These result suggests that SAC carry cations through the nonselective cation channel (NSC channel). Taken together, we found that stretch activated channels present in hamster oocyte and the channel may carry cations through NSC channels. This stretch activated-NSC channels may play physiological role(s) in oocyte growth, maturation, fertilization and embryogenesis in fertilized oocytes to two-cell zygotes of hamster.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.25-29
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• 1994

This study was carried out to investigate the in-vitro fertilization rate survival rate of rapidly frozen porcine immature embryos. The porcine embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on vitro culture or FDA test. The results are summarized as follows : 1. The in vitro fertilization rate of all frozen immature oocytes(6.7~26.7%) was very low, 35.0% of unfrozen oocytes and the rate of immature oocytes was very higher than that of mature oocytes. 2. The survival rate of all frozen immature oocytes(10.3~25.0%, 13.3~30.0%) was very low, 45% of unfrozen oocytes and the rate of immature oocytes was slightly higher than that of mature oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.3
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• pp.189-198
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• 2006

Objective: We previously described that Diva is highly expressed in matured metaphase II (MII) oocytes compared to immature germinal vesicle (GV) oocytes in mouse. We report here that the expression of Diva transcript as well as protein is oocyte-specific. To elucidate its physiological role in oocyte, the binding partner(s) of Diva has been identified by using immunoprecipitation (IP) followed by Mass Spectrometry. Methods: NIH/3T3 cells were transiently transfected for 24 h with either empty vector for control or FLAG-tagged mouse Diva construct, and IP was performed with anti-FLAG antibody. The immuno-isolated complexes were resolved by SDS-PAGE on a 12% gel followed by Coomassie Blue staining. For in-gel digestion, 15 bands of interest were excised manually and digested with trypsin. All mass spectra were acquired at a positive reflector mode by a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Proteins were identified by searching the NCBI nonredundant database using MASCOT Peptide Mass Fingerprint software (Matrixscience, London). Results: Diva-associated complexes were formed in FLAG-tagged mouse Diva-overexpressed NIH/3T3 cells via IP using anti-FLAG-conjugated beads. Among the excised 15 bands, actin and actin-binding proteins such as tropomyosin, tropomodulin 3, and ${\alpha}$-actinin were identified. Binding between Diva and actin or tropomyosin was confirmed by IP followed by Western blot analysis. Both bindings were also detected endogenously in mouse ovaries, indicating that Diva works with actin and tropomyosin. Conclusions: This is the first report that immuno-isolated Diva-associated complexes are related to actin filament of the cytoskeletal system. When we consider the association of Diva with actin and tropomyosin, oocyte-specific Diva may play a role in modulating the cytoskeletal system during oocyte maturation.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.85-92
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• 1993

In the previous study, we observed that Purine has a time dependent effect in maintaining the oocytes in meiotic arrest, and human fetal cord serum(HFCS) and human mature follicular fluid(HMFF) reverse the GVBD suppressed by purines. And it was reported that purine has a harmful effect on the development of oocytes or embryos, when they were cultured for a long time, in vitro. Therefore this study was performed to investigate the effects of purine on extrusion rates of 1st pb and viability of oocytes cultured for a long time, in vitro. Immature oocytes(GV stage) were collected from ovaries of 25~28 day old ICR mice at 48 hrs after PMSG injection. Cumulus-enclosed and denuded oocytes collected were assigned randomly to one of several culture conditions. Some of the oocytes were cultured in 4mM hypoxanthine for 24hr, and the extrusion rates of 1st pb and viability of the oocytes were assessed at every 12 hrs. In the viability, the oocytes showed granulation, pigmentation of cytoplasm or lysis of 1st pb extruded were regarded as degenerating oocytes. Also some of the oocytes were cultured in hypoxanthine for 12 hrs then the resulting oocytes were transferred to hypoxanthine-free medium and cultured for 12 hrs to determine whether the inhibitory effect of hypoxanthine on the 1st pb extrusion was reversible. The rest of the oocytes were cultured in medium containing hypoxanthine and adenosine for 18 hrs to compare the 1st pb extrusion be attendant upon hte concentration of HFCS or HMFF supplemented. Hypoxanthine suppressed the extrusion of 1st pb and viability of the oocytes significantly, when they were cultured for more than 12 hrs and the harmful effect of hypoxanthine was showed in denuded oocytes, prominently. The suppressive effect of hypoxanthine was reversed by just removal of the hypoxanthine from the cultrue medium. Also there was no difference in reverse the pb extrusion rate suppressed between HFCS and HMFF. The extrusion rate of 1st pb in medium containing adenosine and hypoxanthine was increased in line with the concentration of HFCS or HMFF supplemented. Hypoxanthine suppressed the extrusion of 1st pb and viability of the oocytes significantly, when they were cultured for more than 12 hrs and the harmful effect of hypoxanthine was showed in denuded oocytes, prominetly. The suppressive effect of hypoxanthine was reversed by just removal of the hypoxanthine fromthe culture medium. Also there was no difference in reverse the pb extrusion rate suppressed between HFCS and HMFF. The extrusion rate of 1st pb in medium containing adenosine and hypoxanthine was increased in line with the concentration of HFCS or HMFF supplemented.