• Reproductive and Developmental Biology
• /
• v.33 no.2
• /
• pp.85-91
• /
• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• Journal of Life Science
• /
• v.18 no.1
• /
• pp.114-119
• /
• 2008

The molecular machinery controlling cell cycle is centered around the regulation of the activity of maturation-promoting factor (MPF), a complex composed of a catalytic Cdc2 and the cyclinB regulatory subunit. Cdc2 kinase is inactivated by phosphorylation of inhibitory kinase, Wee1. It has been known that there are three different Wee1 kinases in the mammalian cell, Wee1A, Wee1B and Myt1. To investigate the regulatory mechanism of Wee1 kinases, the phosphorylation and degradation of Wee1A and Wee1B were checked in the Xenopus oocyte cell cycle. When Wee1 kinases were injected into frog oocyte, Wee1B was more stable than Wee1A. Wee1A and Wee1B kinase were phosphorylated by many kinases such as PKA and Akt. The roles of amino or carboxyl terminal in mouse Wee1A or Wee1B kinase were investigated using chimeric constructs. The degree of protein phosphorylation, degradation and cell cycle progression were different between chimeric constructs. The amino domain of Wee1A was implicated in the protein phosphorylation and degradation while amino domain of Wee1B and carboxyl domain of Wee1A were involved in the activity regulation. These results suggested that the domains of Wee1 kinase have different and significant roles in regulating the Wee1 kinases in the cell cycle progression.

• Journal of the Korean Institute of Telematics and Electronics S
• /
• v.36S no.1
• /
• pp.70-80
• /
• 1999

In this paper, a new hierarchical image segmentation algorithm based on human visual system(HVS) characteristic is proposed which can efficiently reduce and control transmission information quantity without the degradation of the subjective and objective image quality. It consists of image segmentation based on mathematical morphology and region merging considering HVS characteristic for the pairs of two adjacent regions at each level of the hierarchy. Image segmentation is composed of 3-level hierarchical structure. In the region merging structure of each level, we extract the pairs of two adjacent regions which human vision can't discriminate, and then merge them. The proposed region merging method extracts pairs of two neighbor regions to be merged and performs region merging according to merging priority based on HVS characteristics. The merging priority for each adjacent pair is determined by the proposed merging priority function(MPF). First of all, the highest priority pair is merged. The information control factor is used to regulate the transmission information at each level. The proposed segmentation algorithm can efficiently improve bottleneck problem caused by excessive contour information at region-based very low bit rate coding. And it shows that it is more flexible structure than that of conventional method. In experimental results, though PSNR and the subjective image quality by the proposed algorithm is similar to that of conventional method, the contour information quantity to be transmitted is reduced considerably. Therefore it is an efficient image segmentation algorithm for region-based very low bit rate coding.

• Reproductive and Developmental Biology
• /
• v.30 no.3
• /
• pp.175-180
• /
• 2006

This study was conducted to examine the effect of pre activation treatment and activation time of recipient cytoplasm on the development of bovine somatic cell nuclear transfer(NT) embryos. Donor cells were transferred and electrofused to enucleated oocytes before(pre-AC) or after activation(post-AC). Activation was induced with a combination of $Ca^{2+}$-ionophore(A23187) and DMAP. NT embryos were cultured in CR1aa containing 3 mg/ml BSA for 9 days. Some NT embryos were fixed at 0.5 to 2.5 hr after fusion(for post-AC) or activation(for pre-AC) for confocal microscopy. Developmental rate to the blastocyst stage was slightly high in the post-AC group(20.6%) compared to that of pre-AC group(15.3%). However, developmental speed of embryos in the pre-AC group was faster than that of embryos in the post-AC group. Development rates to the blastocyst stage were similar among different activation time before fusion(0.5,2 and 4 hr). The result of the present study suggests that development and nuclear morphology are affected f the activation status of the recipient cytoplasm before fusion.

• Journal of Embryo Transfer
• /
• v.16 no.3
• /
• pp.223-231
• /
• 2001

The success of nuclear transplantation with mammalian oocytes depends critically on the potential of oocytes activation, which mainly caused to prevent the re-accumulation of maturation promoting factor (MPF). This study was conducted to compare the effect of combined treatment of lonomycin with a Hl-histone kinase inhibitor (dimethylaminopurine, DMAP) or cdc2 kinase inhibitor (sodium pyrophosphate, SPP) on activation of bovine oocytes. In vitro matured bovine oocytes with the first polar body (PB) and dense cytoplasm were assigned to 3 experimental groups. For activation treatment, oocytcs were exposed to 5 $\mu$M lonomycin for 5 min (Group 1), and followed by 1.9 mM dimethylaminopurine (DMAP) for 3 h (Group 2) or followed by 2 mM sodium pyrophosphate (SPP) for 3 h (Group 3). The activation effects in the three treatments and the control group (untreated) were judged by the extrusion of the second PB and formation of a pronucleus (PN). Differences among groups were analysed using one-way ANOVA after arc-sine transformation of proportional data. All three treatments led to high activation rates (90% to 95%), with significant difference from the control. However, the extrusion of the second PB and the rate of PN formation differed remarkably among treatments. In Group I and 3, about 95% of the oocytes had extruded the second polar body, but one PN had formed in a higher proportion of oocytes in Group 3 than in Group 1 (90% vs. 5%). In experiment 2, the rates of cleavage and development into blastocysts in Group 1 were significantly lower than those of Group 2 and 3 (8.7% and 0% vs. 50.5% and 11.6%, and 44.6% and 7.2%, respectively, P<0.05). In experiment 3, ~80% of parthenotes in Group 1 were developed with haploid chromosomal sets. However, when ionomycin was followed immediately by DMAP (Group 2). only 20% of parthenotes were haploid. In Group 3, combined treatment with ionomycin and SPP, the appearance of abnormal chromosomal tracts was significantly (P〈0.05) reduced and the proportion of haploid parthenotes was increased to 85% (17/20) than in Group 2. These results demonstrate that SPP acted as a cdc2 kinase inhibitor and formed the haploidy in oocyte activation. Thus, the present study suggests that cdc2 kinase inhibitor, such as sodium pyrophosphate, may have an effective role in oocyte activation for the production of cloned embryos/animals by nuclear transplantation.

• Journal of Embryo Transfer
• /
• v.20 no.1
• /
• pp.25-33
• /
• 2005

This study is a part of research that development of effective genetic resources preservation system using the in vitro spermatogenesis, in vitro insemination and culture system. We aimed for establishment of in vitro culture system with in vitro activated porcine oocytes. The porcine oocytes were matured for 48 hours in $TCM199+10\%$ FCS and activated with $7\%$ ethanol. The activated oocytes were cultured for 7 days in $TCM199+10\%$ FCS or $NCSU23+0.4\%$ BSA medium. The activated oocytes were not developed to the blastocyst stage in $TCM199+10\%$ FCS medium. However in $NCSU23+0.4\%$ medium, those were developed to blastocyst with $3\%$ of treated oocytes. We extended maturation duration of porcine follicular oocytes fur 48, 52, 56, 60, 64, 68, and 72 hours and activated with $7\%$ ethanol and cultured using $NCSU23+0.4\%$ BSA medium. The six percents of activated oocytes were developed to blastocyst in 48 hours and $10\%$ in 52 hours with comparatively low rates suggested to be not fully activated by regenerated MPF. Maturation durations from 56 hours to 68 hours supported to develop upto $11.9\~18.3\%$ of blastocysts. However the developmental rate was declined to $7.2\%$ at 72 hours of maturation duration because of cytoplasmic deterioration. The assumed time window for activation will be $56\~68$ hours of maturation duration. When the matured oocytes were activated with electric pulse of 1, 1.2, 1.4, 1.6, 1.8 and 2.0kV/cm for $80{\mu}s$, although appling the electric current once was not enough for activation, appling twice with 1.6kV/cm for $80{\mu}s$ was shown the highest developmental rate with $11.3\%$. When those were compared with activating methods, $15.7%$ of blastocyst rate was obtained in the $7\%$ ethanol. That was higher than those in electric pulse with $9.5\%$ and calcium ionophore method with $5.8\%$. In this experimental condition, the $7\%$ ethanol treatment was the most effective method for activating porcine oocytes.