• Journal of Embryo Transfer
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• v.9 no.1
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• pp.117-125
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• 1994

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 $^{\circ}C$ 5% $CO_2$. The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto $\leq$ 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.52-52
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• 2002

The objective of this study was to determine the developmental competence of in vitro matured bovine oocytes after intracytoplasmic sperm injection(ICSI) with frozen-thawed epididymal spermatozoa. The ovaries were obtained from slaughtered Korean native cows. Oocytes matured in vitro for 24 hrs were fertilized by ICSI with frozen-thawed epididymal spermatozoa. After ICSI, one group of oocytes was activated with 7% ethanol for 5 min, and second group was not activated. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24-30 hrs in a incubator with 5% CO₂ in air at 38.5℃. (omitted)

• 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.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.18-18
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• 2001

Despite of importance of integrated events of nucleus and microtubule remodeling in nuclear transferred embryos with somatic cells, little information is available on this subject. In this study we configured chromatin and microtubule organization following somatic cell nuclear transfer in pre- and non-activated bovine oocytes in order to clearify nuclear remodeling process and to demonstrate centrosome inheritance during nuclear transfer. The cumulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 supplemented hormone. Matured bovine oocytes were enucleated by aspirating the frist polar body and metaphase chromatin using a beveled pipette. Bovine fibroblast cells were fused into enucleated oocyte by electrical stimulation. Reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurin, and then cultured in CRlaa medium. The organization of nuclear and microtubules were observed using laser-scanning confocal microscopy. At 1 hour after fusion, microtubule aster was seen near the transferred nucleus in most oocytes regardless activation condition. While most of fibroblast nuclei remodeled to premature chromosome condensation (PCC) and to the two masses of chromosome in non-activated oocytes, a few number of fibloblasts went to PCC and multiple pronuclear like structures in activated oocytes. Microtubular spindle was seen around condensed chromosome. Gamma-tubulin was detected in the vicinity of condensed chromosome, suggesting this is a transient spindle. The spindle seperated nucleus into two masses of chromatin which developed to the pronuclear like structures. Two pronuclear like structures were than apposed by microtubular aster and formed one syngamy like nuclear structure at 15 h following nuclear transfer. At 17 to 18 h after fusion, two centrosomes were seen near the nucleus, which nucleates micrtubules for two cell cleavage. While 31% of reconstructed oocytes in non-activated condition developed to morulae and blastocysts, a few reconstructed oocytes in pre-activated condition developed to the blastocyst. These results suggested introduction of foreign centrosome during nuclear transfer, which appeared to give an important role for somatic cell nuclear reprogramming.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.71-76
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• 2007

Previous studies have shown that BMI-1026 is a potent inhibitor of the cyclin-dependent kinases (cdk). In cell culture, the compound also arrests G2/M strongly and G1/S and S weakly. Two key kinases, cdk1 (p34cdc2 kinase) and mitogen-activated protein (MAP) kinase (erk1 and 2), perform crucial roles during oocyte maturation and, later, metaphase II (MII) arrest. In mammalian oocytes, both kinases are activated gradually around the time of germinal vesicle breakdown (GVBD) and maintain high activity in eggs arrested at metaphase II. In this study, we examined the effects of BMI-1026 on GVBD and MII arrest in mouse oocytes. BMI-1026 inhibited GVBD of immature oocytes and activated MII-arrested oocytes in a concentration-dependent manner, with more than 90% of oocytes exhibiting GVBD inhibition and MII activation at 100 nM This is approximately 500$\sim$1,000 times more potent than the activity reported for the cdk inhibitors roscovitine (${\sim}50{\mu}M$) and butyrolactone (${\sim}100{\mu}M$). Based on the results of previous in vitro kinase assays, we expected BMI-1026 to inhibit only cdk1 activation in oocytes and eggs, not MAP kinase. However, in our cell-based system, it inhibited the activity of both kinases. We also found that the effect of BMI-1026 is reversible. Our results suggest that BMI-1026 inhibits GVBD and activates MII-arrested oocytes efficiently and reversibly and that it also inhibits both cdk1/histone HI kinase and MAP kinase in mouse oocytes.

• Journal of Ginseng Research
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• v.29 no.4
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• pp.167-175
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• 2005

The $Ca^{2+}-activated$ chloride channel (CLCA) was activated by ginseng total saponin (GTS) in Xenopus oocytes. The reverse transcription PCR (RT-PCR) method was performed with gene specific primers on oocytes. The gene specific primers were deduced from spleen cDNA in expressed sequence tags (EST) database showing high homology to the mouse CLCA. Full length of cDNA sequence was completed by linkage of several 5' and 3'-half cDNA fragments have been sequenced. We named the full cDNA to oCLCA transiently. The oCLCA gene encodes a protein of 911 amino acids with $48.9\%$ identity overall to that of mouse CLCA (mCLCA4). A predicted oCLCA amino acids sequence shows the molecular weight of 108 kDa and has four or more transmembrane domains, and also the one hydrophobic C­terminal domain. oCLCA gene was expressed ubiquitously in various tissues included oocytes, also interfered in oocytes by siRNA for oCLCA. Here, we suggest that oCLCA is a endogenous chloride channel gene in oocytes. We are studying for the identification of oCLCA gene and further physiological research.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.133-139
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• 1997

Large scale production of cloned embryos requires the technology of multiple generational nuclear transfer(NT) by using NT embryos itself as the subsequent donor nuclei. In this work we investigated comparatively the effects of enucleated oocytes treated with ionomycin and 6-DMAP on the electrofusion rate and in vitro developmental potential in the first and second NT embryos. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 15 hours after hCG injection. The enucleated oocytes were pre-activated by 5 min incubation in 5$\mu$M ionomycin and 2 hours incubation in 2 mM 6-DMAP at 19~20 hours post-hCG before microinjection. In the first and second generation NT, the unsynchronized 16-cell stage embryos were used as nuclear donor. The separated donor blastomeres were injected into the enucleated activated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of single pulse for 60 $\mu$sec at 1.25kV/cm in $Ca^2$+, $Mg^2$+ - free 0.28 M mannitol solution. In the non-preactivation group, the electrofusion and electrical stimulation was given 3 pulses for 60 $\mu$sec at 1.25 kV/cm in 100$\mu$M $Ca^2$+, $Mg^2$+ 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in TCM-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The results obtained were summarized as follows: 1. In the first generational NT embryos, the electrofusion rate of preactivated and non-activated oocytes(80.4 and 87.8%) was not significantly different, but in the second generational NT embryos, the electrofusion rate was significantly(P<0.05) higher in the non-activated oocytes(85.7%) than in the preactivated oocytes(70.1%). 2) In the first and second generational NT embryos, the developmental potential to biastocyst stage was significantly(P<0.05) higher in the preactivated oocytes(39.3 and35.7%) than in the non-preactivated oocytes(16.0 and 13.3%). No significant difference in the developmental potential was shown between the first and second generational NT embryos derived from the preactivated oocytes. In conclusion, it may be efficient to use the oocytes preactivated with ionomycin and 6-DMAP for the multiple production of cloned embryos by recycling nuclear transfer.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.499-502
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• 2011

Aurora A kinase is a mitotic serine/threonine kinase whose proposed functions include the maturation of centrosomes, G2/M transition, alignment of chromosomes at metaphase, and cytokinesis. In this study, we investigated the effect of MLN8237, an aurora A kinase inhibitor, on the postovulatory aging of oocytes based on the frequency of oocyte fragmentation, cdk1 kinase activity, and cyclin B degradation. The fragmentation of ovulated oocytes during prolonged culture was inhibited by treatment with MLN8237 in a concentration-dependent manner. The frequency of fragmented oocytes was significantly lower in oocytes treated with 2 ${\mu}M$ MLN8237 (13%) than in control oocytes (64%) after two days of culture. Most of the control (non-fragmented) oocytes (91%) were activated after two days of culture. In comparison, only 22% of the MLN8237-treated oocytes were activated; the rest of the oocytes (78%) were still in metaphase with an abnormal spindle and dispersed chromosomes. Next, cdk1 activity and the level of cyclin B were examined. The level of cyclin B and cdk1 activity in MLN8237-treated oocytes were nearly equal to those in control oocytes. Our results indicate that MLN8237 inhibited the fragmentation of ovulated oocytes during prolonged culture, although it blocked the spontaneous decrease in activity of cdk1 and degradation of cyclin B. This mechanism of inhibition is different from that in oocytes treated with nocodazole, which have high levels of cdk1 activity and cyclin B.

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.221-227
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• 1998

This study was carried out to determine the effects of ionomycin and 6-dimethylaminopurine (6-DMAP) treatments on parthenogenesis of rabbit oocytes. The oocytes were randomly assigned to the activation treatments with either ionomycin plus 6-DMAP or electric stimulation. The oocytes were colected from the oviducts of superovulated rabbits at 13~14 hours and 19~20 hours post hCG injection and were activated with 5$\mu$M ionomycin for 5 min and 2 hours incubation in 2mM 6-DMAP. The other oocytes were stimulated by three pulses of 3.6kV/cm for 60 $\mu$sec each 30 min apart, starting 19 hours post in hCG in 0.28M mannitol solution with 100$\mu$M Ca2+ and Mg2+. The results obtained were summarized as follows: 1. Following treatment of the oocytes with ionomycin plus 6-DMAP, the cleavage rate and in vitro developmental rate to blastocyst were significantly(P<0.01) higher in the oocytes collected bet ween 19~20 hours than between 13~14 hours after hCG injection. 2. When the oocytes were treated with ionomycin plus 6-DMAP, 85(98.8%) of 86 treated oocytes extruded the second polar bodies, with the entire chromatin complements outside ooplasm. However when the oocytes were restored during subsequent incubation in the drug-free medium, the cytoplasts regain their full capacity for parthenogenetic activation and nuclear remodelling. 3. The cleavage rate and the in vitro developmental rate to blastocyst were not significantly different in the oocytes activated by ionomycin plus 6-DMAP treatment(91.2 and 45.6%) or electrical stimulation(89.6 and 34.3%).

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.79-85
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• 2002

We investigated the optimal concentration and exposure time of cycloheximide(CHX) on development of activated porcine oocytes following electrical pulse(EP). After 42~44 h maturation, oocytes were treated with 0.1% hyaluronidase, and denuded cumulus cells by pipetting. Oocytes were stimulated by electric pulse (1.2 kV/cm, 30 $\mu$sec, 1 pulse) or incubated for 3, 5 and 7 h in cycloheximide (1, 5 and 10 $\mu\textrm{g}$/$m\ell$, respectively) following electric pulse, and cultured for 8 days. Cleavage rate of oocytes activated with 10 $\mu\textrm{g}$/$m\ell$ CHX following EP was significantly (P＜0.05) higher than those of 1 $\mu\textrm{g}$/$m\ell$ (86.8% vs. 74.4%). The developmental competence of oocytes incubated to 5 $\mu\textrm{g}$/$m\ell$ of CHX was significantly (P＜0.05) higher development to blastocysts (13.3%), compared with 10 $\mu\textrm{g}$/$m\ell$ of CHX (5.6%). When the oocytes were activated with 5$\mu\textrm{g}$/$m\ell$ CHX for 3, 5, and 7 h following EP, the cleavage rate of oocytes in 5 h group(86.6%) was significantly (P＜0.05) higher than that in 3 h group(73.2%). The developmental rate of oocytes to morula in 5 and 7 h groups(26.7% and 16.4%) were significantly (P＜0.05) high than that in 3 h group(14.5%). Matured oocytes were activated with electric pulse (EP) or electric pulse combined with cycloheximide (EP + CHX) and cultured for 8 days. The rate of cleavage and development to blastocyst (80.1% and 11.6%) of activated with EP group were similar to EP combined with CHX group. When activated with EP or EP combined with CHX, the mean cell number of blastocysts were less in the activated with EP (18.67$\pm$5.53) than in the activated EP combined CHX (20.71$\pm$6.16), but not significantly different. This results suggest that, when the porcine oocytes were activated with CHX following EP, the developmental rate of activated oocytes can be improved by treated with a concentration of 5 $\mu\textrm{g}$/$m\ell$ CHX for 5 h exposure time.