• Journal of Embryo Transfer
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• v.32 no.1
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• pp.17-24
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• 2017

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 ${\beta}$-galactoside ${\alpha}$-2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a ($10{\mu}M$) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.229-237
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• 2004

The main objective of this study was to examine the effects of serum and gonadotropins supplement during in vitro maturation(IVM) of bovine oocytes on nuclear maturation and embryo development, and we also examine the cell number. 1 . The first polar body(PB) extrusion rates of Korean native cow(KNC) oocytes matured in medium with FBS or gonadotropins were similar among treatment groups. The development rate to the blastocyst stage was significantly higher in the group of both supplement FBS and gonadotropins(26.0％) than in the group of non-supplement(9.9％) and gonadotropins (12.0％). The numbers of inner cell mass (ICM) and trophectoderm (TE) cells and total cell numbers of blastocysts were highest in the group of both supplement FBS and gonadotropins, and the number of ICM cells was increased by FBS supplementation (p<0.05). 2. The PB extrusion rates of KNC oocytes matured in medium with FBS in the different duration of IVM was significantly higher in the 0-18hr(63.1％) and in the 9-18hr(63.4％) group than in the 0-9hr.(37.4％) group (p<0.05). The embryo development rates did not differ among treatment groups. The numbers of TE cells and total cell numbers of blastocysts were similar among treatment groups, but the number of ICM cells of the 0-18h. group were significantly higher than the other treatment groups (p<0.05). The results indicate that although TCM199 alone can support bovine oocyte maturation and development to the blastocyst stage, a high quality of blastocysts can be produced from oocytes matured in medium containing serum and gonadotropins.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.86-92
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• 2019

The aim of this study was to investigate effects of hyaluronidase during IVM on oocyte maturation, oxidative stress status, expression of cumulus expansion-related (PTX, pentraxin; GJA1, gap junction protein alpha 1; PTGS2, prostaglandin-endoperoxide synthase 2) and fatty acid metabolism-related (FADS1, delta-6 desaturase; FADS2, delta-5 desaturase; PPARα, peroxisome proliferator-activated receptor-alpha) mRNA, and embryonic development of porcine oocytes. The cumulus-oocyte complexes (COCs) were incubated with 0.1 mg/mL hyaluronidase for 44 h. Cumulus expansion was measured at 22 h after maturation. At 44 h after maturation, nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels were measured. Gene expression in cumulus cells was analyzed using real time PCR. The cleavage rate and blastocyst formation were evaluated at Day 2 and 7 after insemination. In results, expansion of cumulus cells was suppressed by treatment of hyaluronidase at 22 h after maturation. Intracellular GSH level was reduced by hyaluronidase treatment (p < 0.05). On the other hand, hyaluronidase increased ROS levels in oocytes (p < 0.05). Only PTGS2 mRNA was enhanced in COCs by hyaluronidase (p < 0.05). Population of oocytes reached at metaphase II stage was higher in control group than hyaluronidase treated group (p < 0.05). Both of cleavage rate and blastocyst formation were higher in control group than hyaluronidase group (p < 0.05). Our present results showed that developmental competence of porcine oocytes could be reduce by hyaluronidase via inducing oxidative stress during maturation process and it might be associated with prostaglandin synthesis. Therefore, we suggest that suppression of cumulus expansion of COCs could induce oxidative stress and decrease nuclear maturation via reduction of GSH synthesis and it caused to decrease developmental competence of mammalian oocytes.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.123-123
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• 2003

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.173-183
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• 2004

This study was carried out to examine the effects of epidermal growth factor (EGF) and the number of cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). COCs were collected from antral follicles of porcine ovaries collected from abattoir, and were maturated in modified NCSU-23 (mNCSU-23) with 10% pFF, 0.6 mM cysteine, 50 ${\mu}mM{\beta}-mercaptoethanol$, 1 mM dbcAMP, 10 IU/mL PMSG and 10 IU/mL hCG, which was supplemented with or without 10 ng/mL EGF and into which 50 or 15 COCs per droplet was put. Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium (mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU- 23. The results are as follows. 1.In the result of IVM, 10 ng/mL EGF supplement duplicated the percentage of C4 group of COCs(41% vs 81%). But the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, and there was not a significant interaction between the two factors, either. 2. In the result of IVF, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, or was not a significant interaction between the two factors, in the rate of sperm penetration, in the percentage of oocytes with male pronucleus (MPN), and in the rate of polyspermy. 3. In the result of IVD, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet in the percentage of cleaved oocytes. There was not significantly different between the number of COCs per culture droplet, but between control and EGF supplemented (p<0.01) in the percentage of blastocysts, the number of inner cell mass (ICM), trophectoderm (TC) and total cells. There was no significant interaction between the two factors anywhere. These results suggested that 10 ng/mL EGF supplement into mNCSU-23 for IVM was effective in the production of more as well as better blastocysts during IVD through increasing the number of cells in those.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.121-128
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• 2006

In this study, we examined the effects of molecular weight, concentrations and treat the duration of polyvinylpyrrolidone (PVP) in vitro maturation (IVM) medium (Experiment 1), and the effect of PVP in IVM, in vitro fertilization (IVF) and in vitro culture (IVC) medium on the development and cell number of porcine embryos (Experiment 2). The base mediums were NCSU 23 solution for IVM, mTBM solution for IVF and PZM3 solution for IVC. In experiment 1, the development rates to 2 cell and blastocyst stage were not differ from the different molecular weight (MW), concentration and duration of PVP in IVM medium. However, the hatching rate of blastocyst was significantly higher in the group of MW 40,000, 0.5% and $0{\sim}44hr$ than in the other groups (p<0.05). In experiment 2, the results of IVM, IVF and IVC medium with (W) or without (W/O) 0.5% MW 40,000 PVP are follows. The development rate to 2 cell stage was highest in the group of W-W/O-W (p<0.05). The development rate to blastocyst and hatching rate was higher in the group of W-W/O-W and W-W/O-W/O than that of other treatments (p<0.05).

• Journal of Embryo Transfer
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• v.29 no.2
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• pp.133-139
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• 2014

$K^+$ channels are involved in the regulation of a variety of physiological functions, including proliferation, apoptosis and differentiation, in mammalian cells. Our previous study demonstrated that the blockage of $K^+$ channels inhibits mouse early embryonic development. This study was designed to identify the effect of $K^+$ channels during bovine embryonic development. $K^+$ channel blockers (tetraethylammonium (TEA), $BaCl_2$, quinine, ruthenium red and fluoxetine) were added to the culture medium during in vitro fertilization (IVF) for 6 h to first identify the short-term effect of these chemicals. Among $K^+$ channel blockers, fluoxetine, which is used as a selective serotonin reuptake inhibitor, significantly increased the blastocyst formation rate by approximately 6% when compared to control. During the in vitro maturation (IVM) of immature oocytes and the in vitro culture (IVC) of embryos, the oocytes and embryos were exposed to fluoxetine for either a short-term (6 h) or a long-term (24 h) to compare the embryonic development in response to exposure time. The 6 h exposure to fluoxetine during IVM did not affect the blastocyst formation rate, but the rate of blastocyst formation was reduced after the 24 h exposure. On the other hand, embryonic development increased approximately 10% in both groups of embryos exposed to fluoxetine for 6 and 24 h during IVC. Taken together, fluoxetine treatment during IVF and IVC, but not IVM, enhances bovine embryonic development. These results suggest that fluoxetine-modulated signals in oocytes and embryos could be an important factor towards enhancing bovine embryonic development.

• Korean Journal of Animal Reproduction
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• v.17 no.4
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• pp.287-294
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• 1994

This study was to investigate the effects of electrofusion, activation and developmental stage of donor embryos on in vitro development of nuclear transplant bovine embryos. A single blastomere nucleus from 8-cell to morula stage embryos produced by in vitro fertilization(IVF) was transferred into a recipient oocyte enucleated at 23∼25 h after in vitro maturation(IVM) or into a recipient oocyte enucleated and cultured for 14∼15 h. In one experiment the nuclear transplant embryos were subjected to additional activation treatments. Fusion rate of nuclear transplant eggs was high at direct current(D.C) voltages of 1.0 and 1.5 kV/cm 991.5 and 93.3%, respectively), but decreased at 2.0kV/cm (81.8%). Additional activation treatments by electric pulases or 7% ethanol did not affect the cleavage and development of nuclear transplant embryos. Development of nuclear transplant embryos slightly increased by delayed nuclear transfer and fusion (42∼43 h after IVM). With this system, blastocysts were obtained from transfer of 8-cell to morula stage donor nuclei (9.6%∼2.4%). The result of this study suggests that nucleo-cytoplasmic interactins, expecially activation of ooplast are very important for the development of nuclear transplant embryos, and donor cell stage does not affect the development of nuclear transplant embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.7
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• pp.956-965
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• 2019

Objective: The main goal of this study was to provide a morphological indicator that could be used to select high-quality oocytes of appropriate meiotic and developmental capabilities in pig. The higher quality of immature oocytes, the higher success rates of in vitro maturation (IVM) and in vitro fertilization (IVF). Thus, prior to the IVM culture, it is important to characterize oocytes morphologically and biochemically in order to assess their quality. Two of the largest indicators of oocyte quality are the presence of cumulus cells and status of chromatin. To investigate the effects of porcine oocyte chromatin configurations on the developmental capacity of blastocysts, we assessed oocyte chromatin status according to follicle size and measured the developmental potency of blastocysts. Methods: To sort by follicle size, we divided the oocytes into three groups (less than 1 mm, 1 to 3 mm, and more than 3 mm in diameter). To assess chromatin configuration, the oocytes were assessed for their stages (surrounded nucleolus [SN] germinal vesicle [GV], non-surrounded nucleolus [NSN] GV, GV breakdown, metaphase I [MI], pro-metaphase II [proMII], and metaphase II [MII]) at different maturation times (22, 44, and 66 h). To assess the development rate, oocytes of each follicle size were subjected to parthenogenetic activation for further development. Finally, GV oocytes were grouped by their chromatin configuration (SN, SN/NSN, and NSN) and their global transcriptional levels were measured. Results: SN GV oocytes were more suitable for IVF than NSN GV oocytes. Moreover, oocytes collected from the larger follicles had a greater distribution of SN GV oocytes and a higher developmental capacity during IVM, reaching MII more quickly and developing more often to blastocysts. Conclusion: Porcine oocytes with high-level meiotic and developmental capacity were identified by analyzing the relationship between follicle size and chromatin configuration. The porcine oocytes from large follicles had a significantly higher SN status in which the transcription level was low and could be better in the degree of meiotic progression and developmental capacity.

• Journal of Embryo Transfer
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• v.32 no.4
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• pp.257-265
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• 2017

Ferulic Acid (FA) is a metabolite of phenylalanine and tyrosine, a phenolic compound commonly found in fruits and vegetables. Several studies have shown that FA has various functions such as antioxidant effect, prevention of cell damage from irradiation, protection from cell damage caused by oxygen deficiency, anti-inflammatory action, anti-aging action, liver protective effect and anti-cancer action. In this study, we investigated the maturation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) of porcine oocytes by adding FA to the in vitro maturation (IVM) medium and examined subsequent embryonic developmental competence at 5% oxygen through parthenogenesis. There is no significant difference between the control group ($0{\mu}M$) and treatment groups ($5{\mu}M$, $10{\mu}M$, $20{\mu}M$) on maturation rates. Intracellular GSH levels in oocyte treated with $5{\mu}M$ of FA significantly increased (P < 0.05), and $20{\mu}M$ of FA revealed significant decrease (P < 0.05) in intracellular ROS levels compared with the control group. Oocytes treated with FA exhibited significantly higher cleavage rates (79.01% vs 89.19%, 92.20%, 90.89%, respectively) than the control group. Oocytes treated with $10{\mu}M$ showed significantly higher blastocyst formation rates (28.3% vs 40.3%, respectively) after PA than the control group. Total cell numbers in blastocyst of $10{\mu}M$ FA displayed significantly higher (39.4 vs 51.9, respectively) than the control group. In conclusion, these results suggested that treatment with FA during IVM improved the developmental potential of porcine embryos by increasing intracellular GSH synthesis and reducing ROS levels. Also, there was an improvement of cleavage rate, blastocyst formation and total cell numbers in blastocysts. It might be associated with Keap1-Nrf2 pathway as an antioxidant regulate pathway that plays a crucial role in determining the sensitivity of cells to oxidative damages by regulating the basal and inducible expression of enzymes which is related to detoxification and anti-oxidative effects, stress response enzymes and/or proteins and ABC transporters.