• Asian-Australasian Journal of Animal Sciences
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• v.8 no.6
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• pp.641-645
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• 1995

This study was conducted to develop a method for production of nuclear transplant bovine embryos using in vitro-matured (IVM) oocytes and to examine the effect of different conditions of electrofusion on fusion rate and developmental capacity of donor nucleus transplanted to enucleated oocytes. Eight- to sixteen-cell embryos derived from oocytes matured and fertilized in vitro used as donor blastomeres and IVM oocytes were used as recipient oocytes. Oocytes were enucleated immediately after 23-24 h IVM and then reconstituted with a donor blastomere in two different micromanipulation media. Fusion rate and subsequent development of the reconstituted oocytes was compared under the different electric stimuli and recipient oocyte ages. Success rate of enucleation was significantly higher in TCM-199 medium containing FCS than in DPBS. The high fusion rate(75-94%) and development (6.4-14.8%) to morulae and blastocyst (M + B) were obtained from 0.6-0.75 kV/cm DC voltage, although total cleavage was not different among the electric pulses. Most optimal condition of electric stimulation for fusion and development was 1 DC voltage of 0.75 kV/cm, in which 80.5% of oocytes were fused, 80.0% and 31.7% of which was cleaved and developed to M + B, respectively. No M + B was obtained from 1.2 kV/cm DC voltage regardless of pulse frequency. Recipint oocyte age at electrofusion greatly affected the cleavage and subsequent development to M + B, showing high rate at 40-41 h oocyte maturation. These results suggest that a suitable condition of electrofusion for donor nuclei derived from IVF may be 1-2 DC pulses of 0.7 kV/cm for $70{\mu}sec$ and that processing of a transplanted nucleus in IVM oocytes may be affected by maturation age of recipient oocytes.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.475-478
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• 2011

In the present study, the effect of cysteine and NT or bisphenol A(BP) on in vitro aturation(IVM) of porcine oocytes were examined. COCs was cultured in NCSU-23 medium supplement with 10% FCS which had previously been covered with mineral oil and equilibrated in a humidified atmosphere of 5% $CO_2$ and 95% air at $38^{\circ}C$. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 0.5~10.0 mM cysteine were $34.0{\pm}3.2%$, $36.0{\pm}3.5%$, $48.0{\pm}3.8%$, $22.0{\pm}3.2%$, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.5~5.0mM NT for 48 hrs were $24.0{\pm}4.2%$, $18.0{\pm}4.9%$, $8.0{\pm}2.2%$, respectively. NT affects oocyte in vitro maturation rate in a dose-dependent. This result were significantly lower than the control group. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM NT+5.0 mM cysteine($38.0{\pm}4.3%$) were significantly higher than that of NT treatment. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.05~5.0 mM BP for 48 hrs were $20.0{\pm}4.7%$, $10.0{\pm}5.3%$, $6.0{\pm}3.2%$, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with BP was significantly lower cultured non supplement of BP ($44.0{\pm}3.5%$). BP affects porcine oocyte maturation rate in a dose-dependent manner. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM BP+5.0 mM cycteine ($32.0{\pm}3.2%$) were increased than that of BP treatment.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.34 no.6
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• pp.584-587
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• 2001

The effects of tributyltin (TBT), triphenyltin (TPhT) and Aroclor 1254 on germinal vesicle breakdown (GVBD) and ovulation of olive flounder (Paralichthys olivaceus) were investigated in in vitro bioassay. TBT, TPhT and Aroclor 1254 showed the inhibition effects on GVBD and ovulation in response to HCG. The oocyte response appeared to be more sensitive to TBT than Aroclor 1254. TBT was more effective in inhibition GVBD at concentrations of 0.1 and 1 ppm. However, no significant inhibition was obseued in concentrations tested ($0.0001\~1\;ppm$). Significant inhibition of ovulation in response to HCG occurred at TBT (0.01, 0.1, 1 ppm), TPhT (0.01, 0.1, 1 ppm) and Aroclor 1254 (0.01, 1 ppm, except 0,1 ppm), compared to HCG control, The lowest ovulation rate was measured at 1 ppm TBT, These data suggest that TBT (or TPhT) could possibly interfere the actions of progestogens to induce GVBD and ovulation in in vitro bioassay system.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.2
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• pp.203-210
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• 1997

The aim of this study was to determine the effect of steroids and human chorionic gonadotropin (HCG) on in vitro maturation and ovulation of oocyte in Pseudobagrus fulvidraco. Oocytes were incubated in the media Leibovitz L15 supplemented with the various concentration of $17\alpha,\;20\beta-dihydroxy-4-pregnen-3-one(17\alpha20{\beta}OHP),\;17\alpha-hydroxyprogesterone(17{\alpha}OHP),\;progesterone(P_4),\;estradiol-17\beta(E_2)and\;HCG$. After 60 hours incubation, the maturation ability of oocyte was assessed by the appearance of germinal vesicle breakdown (GVBD). GVBD was significantly enhanced by the addition of $17\alpha20{\beta}OHP,\;17{\alpha}OHP,\;P_4\;and\;HCD(P<0.05)$. The highest CVBD was observed when $17\alpha20{\beta}OHP$ and HCG were supplemented to media. When oocytes were cultured for 16 hours in media containing $10\~1,000\;ng/ml\;17\alpha20{\beta}OHP,\;17{\alpha}OHP\;and\;P_4$, the rate of GVBD in oocytes cultured in the medium supplemented with 100 ng/ml $17\alpha20{\beta}OHP(65\%)$ was significantly higher than that with $17{\alpha}OHP\;(40\%)\;and\;P_4(35\%)$. The efforts of $17\alpha20{\beta}OHP$ and HCG on GVBD were assessed by various concentration of these hormones. When oocytes were cultured for 60 hours in various media containing $1\~1,000\;ng/ml\;17{\alpha}20{\beta}OHP\;or\;5\~1,000\;IU/ml$ HCG, the GVBD of oocytes was significantly increased in the medium with $10\~100\;ng/ml\;17\alpha20{\beta}OHP$ and 500 IU/ml HCT. When oocytes were cultured in the various media supplemented with $1\~1,000\;ng/ml\;17\alpha20{\beta}OHP\;or\;5\~1,000\;IU/ml$ HCG for 60 hours, the media with $1\~100\;ng/ml\;17\alpha20{\beta}OHP\;or\;50\~1,000IU/ml$ HCG significantly increased in the rate of ovulation. However supplementation with $1,000\;ng/ml\;17\alpha20{\beta}OHP$or 5 IU/ml HCG did not improve the rate of ovulation compared to controls. This results indicate that supplementation of steroid and HCG except $E_2$ can improve the in vitro maturation and ovulation of oocyte in P. fulvidrac; HCG and $17\alpha20{\beta}OHP$ may be more effective than other steroids on oocyte maturation and ovulation in P. fulvidraco.

• Development and Reproduction
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• v.2 no.1
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• pp.9-20
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• 1998

It has been wel known that phospholipase C(PLC) plays an important role in the intracellular signaling in a variety of cell types. However, involvement of PLC in mouse oocyte maturation and preimplantation embryo development remains unknown. The present study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturatio and preimplantation embryo development study examined the expression patterns of the mouse PLC \beta 1 and \gamma 1 during oocyte maturation and preimplantation embryo development by the competitive reverse transcription-polymerase chain reaction (RT-PCR method). PLC \gamma 1 mRNA (0.1 fg) was readily detected in germinal vesicle (GV)-stage oocyte and its level was reduced as meiotic resumption proceeded. PLC-\beta 1 mRNA (<0.1 fg) as detected at low level at GV-stage oocytes and scarcely detected at germinal vescle breakdown (GVBD)-stage oocytes. After fertilization, both PLC \beta 1 and \gamma 1 mRNA levels began to increase at morula-stage embryos (0.2 fg) and were more prominent in blastocyst-stage embryos(1 fg). to elucidate the possible involvement of PLC via protein kinase C(PKC) pathway during oocyte maturation and preimplantation embryo development , the effects of sphingosine (PKC inhibitor), sn-$diC_{8}$(PKC activator) anc U73122 (PLC ingibitor) were examined. Treatment of GV-stage oocytes with sphingosine (20 \mu M) facilitated the meiotic resuption by 10-20 over the control within 1 h as judged by GVBD, whereas U73122 failed to show any significant effect. U73122 (10 \mu M) effectively blocked the compaction of morula, while sn-$diC_{8}$(50 \mu M). In summary, the present study shows that the mouse PLC \beta 1 and \gamma 1 are expressed in a developmental stage-specific manner and PLC-PKC pathway may be involved in early preimplantation embryo development.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.4
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• pp.495-499
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• 2006

In our previous studies, we demonstrated that Vascular Endothelial Growth Factor (VEGF) enhances bovine oocyte maturation and early embryonic development in serum supplemented media. In this experiment, to determine the synergistic effect of VEGF with serum components on early embryonic development in vitro in cattle, 1 mg/ml polyvinyl-alcohol (PVA) was replaced with foetal bovine serum (FBS) in maturation and culture media. Bovine oocytes were matured in Synthetic Oviduct Fluid (SOF) supplemented with PVA, PVA+5 ng/ml of VEGF, FBS, or FBS+VEGF. Fertilized oocytes were cultured in the same conditions for 8 days. The development of embryos was examined at 48 h post- insemination and on days 6, 7 and 8. The results were analyzed using repeated measures two- factor ANOVA, in which the effects of VEGF and serum were assigned as two factors. The development rate to 4- to 8-cell embryos at 48 h was significantly higher in the PVA+VEGF group than in the PVA group (44.7% and 31.5%, respectively). However, the highest development rate to 4- to 8-cell embryos was obtained from the FBS+VEGF group (58.8%). On day 8, the blastocyst rates were higher in the PVA+VEGF (22.8%), FBS (32.1%, p<0.05) and FBS+VEGF (42.1%, p<0.05) groups than in the PVA group (17.1%). Two- factor ANOVA of the development rates indicates that VEGF had a significant effect, but had no synergistic effect with serum components on early embryonic development. The results of the present study demonstrate that VEGF improves the in vitro developmental competence of bovine oocytes and/or embryos independent of the effect of serum components.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.200-200
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• 2004

The purpose of this study was carried out to determine the possibility of in vitro maturation of tiger oocytes. Immature oocytes were recovered from a pairs of ovaries. A total of 78 oocytes were collected, of which forty threes were identified as compact cumulus cells and uniform cytoplasm. 43 COCs were in vitro matured at 39℃, 5% CO₂ in air atmosphere for 48 h in a IVM medium (TCM-199 supplement with 10% FBS, 0.6 mM cysteine, 0.2 mM pyruvic acid and 10 IU/㎖ HMG). (omitted)

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.61-61
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• 2003

We have investigated the novel function of tissue transglutaminase (tTG) in the germinal vesicle breakdown (GVBD) of mouse oocyte. tTG was identified in ooplasm and germinal vesicle by immunostaining assay. Spontaneous maturation of the oocytes elevated in situ activity of tTG by over 2.5 fold at 3 hr, which was determined by a confocal microscopic assay. However, incubation with monodansylcadaverine (MDC), a tTG inhibitor, blocked the activation of tTG. The possible role of tTG in GVBD was investigated by the use of two tTG inhibitors, MDC and cystamine. MDC largely inhibited the GVBD by a concentration dependent manner. GV-stage oocytes were matured to the GVBD stage by 78% at 3 hr in BWW culture medium. However, in the oocytes incubated with MDC for 3 hr, the GVBD rates were 43 and 11% by 50 and 100 mM, respectively. MDC also blocked the entry of 70 kDa TRITC-dextran from the ooplasm to the compartment of germinal vesicle, indicating a possible inhibition of nuclear pore disassembly by MDC. The role of tTG in GVBD was further investigated by microinjection with cystamine. The control oocytes, injected with DPBS, showed about 80 % of GVBD at 3 hr. But the oocytes injected with cystamine showed 15% of GVBD at 3 hr and a little higher rate at 6 hr. In addition, the inhibition of GVBD maturation by MDC was reversible by washing. These results suggested that tTG was involved in the early event of mouse oocyte maturation

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.127-132
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• 2013

The objective of this study was to examine the effect of in vitro maturation (IVM) medium, cytochalasin B (CB) treatment during intracytoplasmic sperm injection (ICSI), and electric activation on in vitro development ICSI-derived embryos in pigs. Immature pig oocytes were matured in vitro in medium 199 (M199) or porcine zygote medium (PZM)-3 that were supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 21~22 h. ICSI embryos were produced by injecting single sperm directly into the cytoplasm of IVM oocytes. The oocytes matured in PZM-3 with 61.6 mM NaCl (low-NaCl PZM-3) tended to decrease (0.05

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.111-120
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• 2001

Objective: To verify the expression of leptin receptor (OB-R) in oocytes and preimplantation embryos, the involvement of mitogen activated protein kinase (MAPK or Erk1/2) in the leptin signaling, and effect of leptin on the oocyte maturation in mice. Method: RT-PCR analysis of OB-R was conducted in germinal vesicle (GV)-intact and MII stage oocytes, and 1, 2, 8-cell embryos and blastocysts. Germinal vesicle breakdown (GVB), polar body extrusion, monitored in the presence or absence of leptin ($1{\mu}M$). Following the leptin treatment, temporal changes in MAPK activity were verified by immunoprecipitation and in vitro kinase assay in MII oocytes. Results: The expression of OB-R mRNA was found in GV and MII oocyte but not in the embryos. MAPK activity of the MII oocytes was significantly increased by brief incubation in the HTF supplemented with leptin ($1{\mu}M$). Priming of PD098059, a MEK inhibitor to leptin treatment attenuated the activation of MAPK by leptin in MII oocytes. Following 24 hrs of culture of the GV oocytes, leptin significant increased the GVB and 1 st polar body extrusion. Conclusion: This result suggested that functional interaction between leptin and OB-R resulted in potentiation of MAPK (Erk1/2) activity in MII oocytes through MEK activation and that leptin might be a local regulator of meiotic maturation of the mouse oocytes.