• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.50-50
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• 2002

During fertilization, morphological and molecular events in male and female chromatin are precisely controlled in time. However, little information is available on onset of pronuclear formation and first S-phase entry in the pig following intracytoplasmic sperm injection. To assess species specific paternal effect on the pronuclear formation and initiation of first S-phase in the pig, we examined time of onset of male and female pronuclear formation and onset of DNA synthesis in the oocytes following pig or mouse sperm injection. (omitted)

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.103-111
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• 1993

These experiments were undertaken to establish the optimal culture systems for in vitro maturation, fertilization and subsequently embryonic development of porcine immature follicular oocytes isolated from the ovary of slaughtered pigs. Porcine ovaries were brought to the laboratory from local slaughter house within 1 hour after slaughtering and cumulus oocytes complexes were recovered from antral follicles (3~5mm) with 23 gauge needle. To maturate follicular oocytes, cumulus oocytes complexes were washed three times with TCM-199 containing 25mM HEPES and incubated (39$^{\circ}C$, 5% CO2 in air) for 42hrs. Ejaculated and liquid storaged boar spermatozoa capacitated with different sperm capacitation methods and media were prepared forfertilizaing of matured follicular oocytes in vitro. Fertilization was performed by adding 5~10${mu}ell$ of capacitated spermatozoa containing 1~5$\times$105 sperm/ml to droplets. Eighteen to twenty-eight hours after sperm insemination, fertilized eggs were washed three times with culture media and transferred to the culture media. The fertilization rates of in vitro matured follicular oocytes cultured in B. O., TCM-HEPES, m-KRB, and TALP-II media were 61.3%, 83.0%, 88.9% and 89.2%, respectively. In addition, the polyspermy rates were 60.7%, 66.5%, 53.8%, and 43.9%, respectively. These data indicated that the highest of fertilization and the lowest of polyspermy rate was shown in TALP-II medium. Spermatozoa capacitated by caffeine, heparin, and percoll density gradient treatment in the 4 different media, the fertilization rates were 33.0~57.2%, 39.9~90.2%, and 52.6~92.8%, respectively, showing the lowest rate in caffeine treatment. The development rate of follicular oocytes, fertilized with the spermatozoa capacitated by caffeine, heparin, and percoll gradient in the TALP-II medium, upto 2 to 4-cell stages were 32.6%, 74.5% and 70.9%, respectively. Finally, fertilization rates of follicular oocytes cultured with follicular fluid containing medium from 10 to 100% were 61.2~94.1% and the rates (90~94%) with 10~20% follicular fluids were significantly higher than those (85.3%) of cultured in the media without follicular fluid. In addition, the rates of pronucleus formation were also higher in follicular fluid treated group (73.1~83.0%) than those (64.7%) of oocytes cultured without follicular fluid. The highest fertilization and pronucleus formation rates was found in oocytes cultured with 10% follicular fluid. These results suggest that the addition of heparin or percoll density gradient method is better capacitation method. Furthermore, the addition of porcine follicular fluid to the fertilization medium may improve the fertilization rates and formation of pronucleus.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.56-56
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• 2001

This study was carried out to investigate on the improvement of fertilizing and developing ability of in vitro matured oocytes from individuals of bulls, sperm type, pretreatment of sperm or oocytes obtained by intracytoplasmic sperm injection(ICSI). 1. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated individual of bulls were 73.9%-87.0% and 33.3%-60.9%, respectively. 2. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated fresh and frozen sperm, tail-cutting and tail-scoring sperm were 82.0%, 78.0%, 42.2%, 51.1% and 56.0%, 42.0%, 17.8%, 22.2% respectively. and these values of fresh sperm injection were higher than that of frozen sperm, tail-cutting and tail-scoring. 3. The male pronuclear formation and developmental rates of oocytes obtained by sperm pretreated heparin, BFF(bovine follicula fluid), His, Ca Ionophore(Ⅰ) and Ⅰ ＋ caffeine methods were 66.7%-82.2% and 33.3%-60.6%, respectively. and these values of treatment of Ⅰ＋ caffeine were higher than that of other methods. 4. The male pronuclear formation and developmental rates of oocytes obtained by ICSI treated with or without zona pellucida were 80.0%, 72.0% and 46.0%, 36.0%, respectively.

• Korean Journal of Animal Reproduction
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• v.27 no.2
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• pp.103-113
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• 2003

This study was conducted to investigate the optimal condition for produce of large quantity recipient oocytes on porcine cloned embryos. In order to determined the optimum concentration and exposure time of ethanol, $Ca^{2+}$-ionophore, 6-DMAP and cycloheximide, in vitro matured oocytes were activated in TCM-199 containing various chemicals and 15% FBS. The activated oocytes were cultured in Whitten's medium containing 10% FBS at 5% $CO_2$. 1. When the porcine oocytes were activated with the ethanol, the best pronucleus formation, cleavage, and in vitro development rate were obtained in the 10% for 10 minutes, which was significantly higher than all of the other treatment(53.4%, 51.6% and 39.9%, respectively). 2. When the porcine oocytes were activated with the $Ca^{2+}$-ionophore, the pronucleus furmation, cleavage, and in vitro development rate were found significantly higher in the 25$\mu$M fur 2min. treatment than those of other concentration and exposure time(59.7%, 62.2% and 43.9%, respectively). 3. When the porcine oocytes were activated with the 6-DMAP, the best pronucleus formation, cleavage, and in vitro development rate were obtained in the 2mM for 2hr~4.5hr(57.3%, 58.4% and 29.0%, respectively). 4. When the porcine oocytes were activated with the cycloheximide, result showed that pronucleus formation, cleavage, and in vifro development rate were 52.1%, 47.7% and 31.8%, respectively, in the 5$\mu\textrm{g}$/ml for 4hr~6hr treatmrent, which was significantly higher than all of the other treatment. These results suggested that the active condition of porcine oocytes was established by optimum concentration and exposure time among different chemicals for produce of large quantity recipient oocytes.s.

• Journal of Embryo Transfer
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• v.16 no.1
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• pp.1-5
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• 2001

A total of 92 unfertilized human oocytes were treated with ethanol (EtOH), calcium ionophore A23187 (CI) or electric pulse (EP) for activating pronuclear formation and subsequent development. In Experiment 1, there was a significant (P=0.0001) treatment effect on the activation of unfertilized oocytes. No spontaneous activation was occurred in the control, but activation treatments induced PN formation with various efficacy. More unfertilized oocytes (UFOs) were activated after EtOH or EP treatment than after CI treatment. EP was as effective (63.6 %) as EtOH, but fragmentation was observed in 43% of UFOs activated by EP. Proportion of UFOs that formed presumptive haploid PN (2 PNs+1 PB or 1 PN +2 PBs) was 33.3, 0 and 28.6% after EtOH, CI and EP treatments, respectively. In Experiment 2, a significant (P=0.0362) effect of immature oocytes (IOs) status on activation was fecund. IOs at the GVBD-MI oocytes had higher potential to form PN than those at the GV stage or with abnormal morphology (25 vs. 77.8%). The results of this study clearly demonstrated that the treatment of 10% ethanol for 5 min effectively induced the activation of UFOs. IOs could form pronucleus with high efficacy by ethanol treatment, as long as they grew beyond the GVBD stage.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.229-235
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• 2018

Polo-like kinase 1 (Plk1) has been known to be a critical element in cell division including centrosome maturation, cytokinesis and spindle formation in somatic, cancer, and mammalian embryonic cells. In particular, Plk1 is highly expressed in cancer cells. Plk1 inhibitors, such as BI2536, have been widely used to prevent cell division as an anticancer drug. In this study, the fertilized murine oocytes were treated with BI2536 for 30 min after recovery from the oviduct to investigate the effect of down-regulation of Plk1 in the in vivo-fertilized murine embryos. Then, the localization and expression of Plk1 was observed by immunofluorescence staining. The sperm which had entered into the oocyte cytoplasm did not form male pronuclei in BI2536-treated oocytes. The BI2536-treated oocytes showed significantly lower expression of Plk1 than non-treated control group. In addition, alpha-tubulin and Plk1 gathered around sperm head in non-treated oocytes, while BI2536-treated oocytes did not show this phenomenon. The present study demonstrates that the Plk1 inhibitor, BI2536, hinders fertilization by inhibiting the formation of murine male pronucleus.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.281-288
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• 2000

This experiment was carried out to investigate the optimal activation condition for parthenogenetic development. In order to activate oocytes at 22 h post onset of maturation, the oocytes were subjected to 5 $\mu$M ionomycin(I) for 5 min ,10 $\mu$M calcium ionophore(Ca) for 5 min, 2 mM 6-dimethylamino-purine(DMAP) for 3 h and 10 $\mu\textrm{g}$/$m\ell$ cycloheximide(CH) for 6 h alone or in combination. The activated oocytes were cultured in modified CR$_1$aa at 5% $CO_2$, 5% $O_2$, 90% $N_2$. l. The cleavage rates after 48 h culture of oocytes treated with I, Ca, DMAP and CH were 12.7%, 14.1%, 28.9% and 22.9%, respectively. There was no blastocyst formation. 2. The cleavage rates after 48 h culture of oocytes treated with I ＋ DMAP, I ＋ CH, Ca ＋ DMAP and Ca ＋ CH were 96.9%, 82.1%, 93.1% and 34.7%, respectively. Developmental rates to blastocysts were 10.4%, 5.3%, 17.6% and 7.1 %, respectively. When oocytes were treated with Ior Ca followed by DMAP, the blastocyst formation rate was significantly higher than other groups(P <0.05). 3. According to single activation treatment, pronucleus formation rates were 5.4%, 3.6%, 28.3% and 28.8%, respectively, Whereas, all oocytes treated with the combined activation agents formed 100% pronucleus.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.69-73
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• 2003

This study was undertaken to investigate the effect of three different porcine IVM media, TCM-199, mSOF and NCSU-23 on early development of porcine spontaneous parthenogenotes. Spontaneous parthenogenotes were induced by a prolonged culture of porcine oocytes in each medium. In Experiment 1, oocytes derived from gilts were matured in three IVM media and maturation of oocytes was evaluated by the status of chromatin configuration. Oocytes matured in mSOF, NCSU-23 and TCM-199 showed no significant difference (P>0.05) in maturation. Maturation rates at 48h after IVM were 83.1$\pm$2% (mSOF), 78.0$\pm$3% (NCSU-23) and 83.5$\pm$2% (mSOF), 78.0$\pm$3% (NCSU-23) and 83.5$\pm$2% (TCM-199). In Experiment 2, pronucleus formation and development to 6~8 cell stage of pig oocytes activated spontaneously. Pronucleus formation, cleavage rate and development to 6~8 cell embryos of porcine spontaneous parthenogenotes were assessed at 55~58 h, 96 h and 168h after IVM, respectively. Pronucleus formation (5.4$\pm$2% and 3.7 $\pm$ 1% vs 1.7 $\pm$ 3%) and development to the 6$\pm$8 cell (3.2$\pm$3% and 4.0$\pm$1% vs 1.4$\pm$3%) was significantly (P<0.05) higher in mSOF or NCSU-23 than TCM-199. In conclusion, the present study showed that oocytes matured in mSOF and NCSU-23 were spontaneously activated with higher frequency.

• Molecules and Cells
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• v.40 no.8
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• pp.558-566
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• 2017

Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve ultrastructural integrity and developmental competence of oocytes following oocyte activation and embryo culture. A randomized study using mouse model was designed and in vitro development (experiment 1), ultrastructural morphology (experiment 2) and functional integrity of the oocytes (experiment 3) retrieved after PMSG/hCG injection (superovulation group) or not (natural ovulation; control group) were evaluated. In experiment 1, more oocytes were retrieved following superovulation than following natural ovulation, but natural ovulation yielded higher (p < 0.0563) maturation rate than superovulation. The capacity of mature oocytes to form pronucleus and to develop into blastocysts in vitro was similar. In experiment 2, a notable (p < 0.0186) increase in mitochondrial deformity, characterized by the formation of vacuolated mitochondria, was detected in the superovulation group. Multivesicular body formation was also increased, whereas early endosome formation was significantly decreased. No obvious changes in other microorganelles, however, were detected, which included the formation and distribution of mitochondria, cortical granules, microvilli, and smooth and rough endoplasmic reticulum. In experiment 3, significant decreases in mitochondrial activity, ATP production and dextran uptake were detected in the superovulation group. In conclusion, superovulation treatment may change both maturational status and functional and ultrastuctural integrity of oocytes. Superovulation effect on preimplantation development can be discussed.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.22-27
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• 1999

Bovine oocytes with compact and complete cumulus cells were cultured in 6 groups for up to 24h in TCM199 buffered with 25 mmol/1 HEPES and supplemented with 10% FCS, 1 mg/ml $17{\beta}$-estradiol, 20 IU/ml hCG. Half of the oocytes at each group cultured in the presence of $25{\mu}g/ml$ cycloheximide at different times during maturation (0, 6, 12, 18, 20, 22 h) were fixed at 24 h of maturation to examine the nuclear progression. The rests of them were inseminated with frozen-thawed spermatozoa in medium BO with 10 mg/ml BSA and 10 mg/ml heparin and fixed after additional 18-20 h culture to evaluate the sperm head transformation. When a protein synthesis inhibitor was added at the onset of the maturation, the oocytes were prevented to proceed GVBD. A few of the oocytes (16%) were able to be penetrated and sperm head decondensation was inhibited either. Addition of cycloheximide after 6-12 h of culture resulted in an increasing percentage of GVBCD (more than 80%), but the oocytes became arrested in M-I (69.2%). More than half of the oocytes was penetrated with a decondensing sperm head. Formation of male pronucleus was first obtained at 12 h of culture in the presence of cycloheximide. When cycloheximide was added from 18 h of culture onwards, nuclear progression to M-II was increasingly restored (80.4-85.5%). The proportion of male and female pronuclear formation increased from 17.9% to 46.2%. It is concluded that protein synthesis is necessary not only for GVBD and development from M-I to M-II, but also for sperm head decendensation and male pronuclear formation in bovine oocytes.