• Journal of Embryo Transfer
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• v.33 no.4
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• pp.313-319
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• 2018

The purpose of this study was to analyze whether FSH and LH hormone treatment directly or indirectly affect embryo development in embryonic development. To determine this, we compared the development of embryonic cells through the expression pattern of MMPs. As a result, 33.8% of blastocysts were formed in FSH added group, 20.8% in LH added group and 10% in FSH + LH added group. In addition, the activity of MMP-9 was highly detected in the FSH-added group, and the expression of Casp-3 was much lower than that of the other groups. These results suggest that the addition of FSH seems to increase the activity of MMP-9 in embryonic cells, and that LH, on the contrary, may activate MMP-2 activity. In addition, the expression level of MMP-2 in the FSH-added group was high in the Trophoblast cell group and in the LH-added group, the hormone ideal secretion might affect the development of the embryonic cell.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.289-298
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• 2015

Edaravone (Eda) is a potent scavenger of inhibiting free radicals including hydroxyl radicals ($H_2O_2$). Reactive oxygen species (ROS) such as $H_2O_2$ can alter most kinds of cellular molecules such as lipids, proteins and nucleic acids, cellular apoptosis. In addition, oxidative stress from over-production of ROS is involved in the defective embryo development of porcine. Previous study reported that Eda has protective effects against oxidative stress-like cellular damage. However, the effect of Eda on the preimplantation porcine embryos development under oxidative stress is unclear. Therefore, in this study, the effects of Eda on blastocyst development, expression levels of ROS, and apoptotic index were first investigated in preimplantation porcine embryos. After in vitro fertilization, porcine embryos were cultured for 6 days in PZM medium with Eda ($10{\mu}M$), $H_2O_2$ ($200{\mu}M$), and Eda+$H_2O_2$ treated group, respectively. Rate of blastocyst development was significantly increased (P<0.05) in the Eda treated group compared with only $H_2O_2$ treated group. And, we measured intracellular levels of ROS by DCF-DA staining methods and investigated numbers of apoptotic nuclei by TUNEL assay analysis is in porcine blastocyst, respectively. Both intracellular ROS levels and the numbers of apoptotic nucleic were significantly decreased (P<0.05) in porcine blastocysts cultured with Eda ($10{\mu}M$). More over, the total cell number of blastocysts were significantly increased (P<0.05) in the Eda-treated group compared with untreated group and the only $H_2O_2$ treated group. Based on the results, Eda was related to regulate as antioxidant-like function according to the reducing ROS levels during preimplantation periods. Also, Eda is beneficial for developmental competence and preimplantation quality of porcine embryos. Therefore, we concluded that Eda has protective effect to ROS derived apoptotic stress in preimplantation porcine embryos.

• Journal of Embryo Transfer
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• v.7 no.1
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• pp.13-19
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• 1992

This study were carried out to investigate the effective concentration of cryoprotective agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen porcine embryos. The porcine embryos foflowing dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined by FDA test. The results are sunnnarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen4hawing in the freezing medium with a various concentration of glycerol, DMSO and propanediol added 0.25M sucrose were higher survival rate than those of sucrose concentration of 0.50M. 2. The survival rates of porcine embryos after ultrarapid ftozen4hawing in the freezing medium added 0.25M and 0.SOM sucrose were higher survival rate than those of sucrose concentration of 0.75M and 1.00M. 3. The temperature thawed at 2$0^{\circ}C$ and 3$0^{\circ}C$ resulted in a significantly higher embryos survival rate after 72 hrs in culture than did at 35$^{\circ}C$. 4. The equilibration time on the survival rate of porcine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time(10~20 min.).

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.179-183
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• 2016

Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell-derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.385-394
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• 2002

This study was constructed the correlations of the embryonic developmental rates and the frequency of chromosome aberration using ear-skin-fibroblast cell in nuclear transfer (NT) derived embryos. Karyoplast-oocyte complexes were fused and activated simultaneously, then cultured for seven days to assess development. The developmental rates of NT and in vitro fertilization (IVF) embryos were 55.4% vs 63.5%, 31.7% vs 33% and 13.4% vs 16.8% in 2 cell, 8 cell and blastocyst, respectively. Firstly, the frequency of chromosome aberrations were evaluated using fluorescent in situ hybridization (FISH) technique with porcine chromosome 1 submetacentric specific probe. Chromosome aberration was detected at day 3 on the embryo culture, the percentages of chromosomal aneuploidy in NT and IVF embryos at 4-cell stage were 40%, 31.3%, respectively. Secondly, embryonic fragmentation was evaluated at 4-cell stage embryo. Frequency of embryonic fragmentations was in 51.3% of NT, 61.3% of IVF, 28.9% of parthenogenetic activation at 4-cell stage. The proportion of fragmentation in NT embryos was higher than activation embryos. This result indicates that chromosomal abnormalities and embryonic fragments are associated with low developmental rate in porcine NT embryo. It is also suggest that abnormal porcine embryos produced by NT related with lower implantation rate, increased abortion rate and production of abnormal fetuses.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.225-231
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• 1996

This study was conducted to investigate the effects of preincubation time, concentration and exposure time of sperm on in vitro fertilization of porcine follicular oocytes rnatured in in vitro. The results obtained are as follows ; 1. Effect of preincuhation time for porcine sperm capacitation on in vitro fertilization in medium with heparin was investigated. Normal fertilization rate was highest in 15 min(26.4%). However, there were no significant differences among preincuhation times of 5~90 min, 2. Normal fertilization rates of sperm concentrations were 17.0~26.5%, and normal fertilization rate from l$\times$ l05cell /ml concentration was also higher than those of other sperm concentration. 3. Normal fertilization rates of sperm exposure time of 4, 8, 12, 16 and 20 hours were 6.1, 20.8, 27.8, 25.0 and 26.7%, respectively. Normal fertilization rate from sperm exposure time of 12 hours was also higher than that of other sperm exposure times.

• Journal of Animal Science and Technology
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• v.63 no.2
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• pp.281-294
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• 2021

Although somatic cell nuclear transfer (SCNT) is frequently employed to produce cloned animals in laboratories, this technique is expensive and inefficient. Therefore, the handmade cloning (HMC) technique has been suggested to simplify and advance the cloning process, however, HMC wastes many oocytes and leads to mitochondrial heteroplasmy. To solve these problems, we propose a modified handmade cloning (mHMC) technique that uses simple laboratory equipment, i.e., a Pasteur pipette and an alcohol lamp, applying it to porcine embryo cloning. To validate the application of mHMC to pig cloning, embryos produced through SCNT and mHMC are compared using multiple methods, such as enucleation efficiency, oxidative stress, embryo developmental competence, and gene expression. The results show no significant differences between techniques except in the enucleation efficiency. The 8-cell and 16-cell embryo developmental competence and Oct4 expression levels exhibit significant differences. However, the blastocyst rate is not significantly different between mHMC and SCNT. This study verifies that cloned embryos derived from the two techniques exhibit similar generation and developmental competence. Thus, we suggest that mHMC could replace SCNT for simpler and cheaper porcine cloning.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.319-325
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• 2015

Gangliosides exist in glycosphingolipid-enriched domains on the cell membrane and regulate various functions such as adhesion, differentiation, and receptor signaling. Ganglioside GM3 by ST3GAL5 enzyme provides an essential function in the biosynthesis of more complex ganglio-series gangliosides. However, the role of gangliosides GM3 in porcine oocytes during in vitro maturation and early embryo development stage has not yet understood clear. Therefore, we examined ganglioside GM3 expression patterns under apoptosis stress during maturation and preimplantation development of porcine oocytes and embryos. First, porcine oocytes cultured in the NCSU-23 medium for 44 h after $H_2O_2$ treated groups (0.01, 0.1, 1 mM). After completion of meiotic maturation, the proportion MII (44 h) was significantly different among control and the H2O2 treated groups ($76.8{\pm}0.3$ vs $69.1{\pm}0.4$; 0.01 mM, $55.7{\pm}1.0$; 0.1 mM, $38.2{\pm}1.6%$; 1 mM, P<0.05). The expressions of ST3GAL5 in $H_2O_2$ treated groups were gradually decreased compared with control group. Next, changes of ST3GAL5 expression patterns were detected by using immunofluorescene (IF) staining during preimplantation development until blastocyst. As a result, we confirmed that the expressions of ST3GAL5 in cleaving embryos were gradually decreased (P<0.05) according to the early embryo development progress. Based on these results, we suggest that the ganglioside GM3 was used to the marker as pro-apoptotic factor in porcine oocyte of maturation and early embryo production in vitro, respectively. Furthermore, our findings will be helpful for better understanding the basic mechanism of gangliosides GM3 regulating in oocyte maturation and early embryonic development of porcine in vitro.

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.103-110
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• 2010

The first morphogenetic event of preimplantation development, compaction, was required efficient production of porcine embryos in vitro. Compaction of the porcine embryo, which takes place at post 4-cell stage, is dependent upon the adhesion molecule E-cadherin. The E-cadherin through ${\beta}$-catenin contributes to stable cell-cell adhesion. Rho-associated kinase (ROCK) signaling was found to support the integrity of E-cadherin based cell contacts. In this study, we traced the effects of ROCK-1 on early embryonic development and structural integrity of blastocysts in pigs. Then, in order to gain new insights into the process of compaction, we also examined whether ROCK-1 signaling is involved in the regulation of the compaction mediated by E-cadherin of cellular adhesion molecules. As a result, real-time RT-PCR analysis showed that the expression of ROCK-1 mRNA was presented throughout porcine preimplantation stages, but not expressed as consistent levels. Thus, we investigated the blastocyst formation of porcine embryos treated with LPA and Y27632. Blastocysts formation and their qualities in LPA treated group increased significantly compared to those in the Y27632-treated group (p < 0.05). Then, to determine whether ROCK-1 associates embryonic compaction, we explored the effect of activator and/or inhibitor of ROCK-1 on compaction of embryos in pigs. The rate of compacted morula in LPA treated group was increased compared to that in the Y27632-treated group (39.7 vs 12.0%). Furthermore, we investigated the localization and expression pattern of E-cadherin at 4-cell stage porcine embryos in both LPA- and Y27632-treated groups by immunocytochemical analysis and Western blot analysis. The expression of E-cadherin was increased in LPA-treated group compared to that in the Y27632-treated group. The localization of E-cadherin in LPA-treated group was enriched in part of blastomere contacts compared to that Y27632-treated group. ROCK-1 as a crucial mediator of embryo compaction may plays an important role in regulating compaction through E-cadherin of the cell adhesion during the porcine preimplantation embryo. We concluded that ROCK-1 gene may affect the developmental potential of porcine blastocysts through regulating embryonic compaction.

• Journal of Embryo Transfer
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• v.26 no.4
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• pp.251-256
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• 2011

Presently, the effect of 0.5 mM dibutyryl cAMP (dbcAMP)-supplemented maturation medium during different incubation time on meiotic arrest (germinal vesicle) and resumption (metaphase II) of porcine oocytes and embryonic development of porcine oocytes following in vitro fertilization (IVF) or parthenogenetic activation (PA) was determined. Porcine cumulus oocyte complexes (COCs) were cultured in 0.5 mM dbcAMP for 17, 22, 27, or 42 h, and an additional 22 h without 0.5 mM dbcAMP. The nuclear status was examined at each time point. Oocytes cultured from 39~49 h displayed more than 80% meiotic resumption. More than 85 % of meiotic arrest was presented at 17~22 h. Oocytes were cultured for 22 h with 0.5 mM dbcAMP and additional 22 h without dbcAMP to assess developmental potential following IVF or PA. There were no significant differences in blastocyst rates among the dbcAMPIVF, IVF, dbcAMP-PA, and PA groups, although cleavage rate of IVF group was significantly higher than those of dbcAMP-PA, and PA groups. In conclusion, 0.5 mM dbcAMP influenced meiotic maturation of porcine oocytes depending on incubation time of oocyte, although embryonic development was not improved in both IVF and PA.