The nature of molecular mechanisms governing embryo development is largely unknown, but recent reports have demonstrated that proper execution of programmed cell death is crucial for this process. The main objective of this study is to examine the mode of programmed cell death during nuclear transfer embryos development in porcine. In particular, the relative employment of two major pathways in programmed cell death; e.g. apoptosis (type I) and autophagy (type II) was compared. Oocytes use in the study was matured in vitro in the presence of 10% FBS maturation medium. After nuclear transfer embryos were cultured for each programmed cell death control factor [Cysteamine(Cyst : 0.4mM), 3-methyladenine(3MA : 2.5mM) and Rapamycin(RP : 100nM)] in TCM-199 medium supplemented with 0.1% BSA. In this study results of among the blastocysts development in 3MA; PCNA, MAP1LC3A and ATG5 RNA gene expression level increased in the order of IVF<Cyst < 3MA < RP. However Casp-3 and TNF-r RNA gene expression level decreased in the order of IVF < 3MA and RP< Cyst. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in Cyst group. And next experiments analysis of MMP expression patterns. Analysed this MMPs enzyme activation to evaluate the effectiveness of high quality brastocyst culture in porcine. In this results of the enzymatic activity of MMP-2 and MMP-9 was assessed in culture, the level of active MMP-9 was higher expression in the medium of each 3MA and RP treatment group, with the level of another treatment group being relatively higher. These results suggest that the autophagy activation culture medium is more effective for stable and innovative nuclear transfer embryos development.

Although assisted reproductive technology (ART) has been developed in many mammalian species including cows, the only embryo preservation technology that is available is cryopreservation. In the present study, small molecules were used to preserve embryos at room temperature. The basic medium for embryo preservation consisted of 1% BSA non-cryopreservation medium (BNC) instead of fetal bovine serum (FBS). To maintain survival and prevent damage during embryo storage, three candidate small molecules were selected—CHIR99021, Y-27632 and Thiazovivin—and their concentrations were optimized. Then, the embryos in the small molecule supplemented preservation medium were stored at room temperature. The viability and hatching rate of embryos stored at 10°C were greater for Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. However, the rate was lower for Thiazovivin-BNC compared to BNC. Although there were no surviving embryos after storage at 20°C, the viability and hatching rate of embryos significantly increased in Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. The mechanism by which small molecules enhance survival of embryos during storage was investigated, and expression of heat shock protein 70 was observed to increase. The findings of this work may be useful in improving ART in the agricultural field.

This study was conducted to investigate the effect of activation method on the endoplasmic reticulum (ER) stress induction, apoptosis and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by four activation methods; 1) electric stimulus (ES) (E), 2) ES+10 μM Ca-ionophore (A23187) treatment (EC), 3) ES+2 mM 6-dimethylaminopurine (6-DMAP) treatment (ED), or 4) ES+A23187 and 6-DMAP treatments (ECD). Parthenogenetic embryos were sampled to analyze x-box binding protein 1 (Xbp1) mRNA, ER stress-associated genes and apoptosis genes at 3 h after ES and the 1-cell and blastocyst stages. In the EC group, the band intensity of spliced Xbp1 (Xbp1s) mRNA was higher than those of the other groups at the 3 h and 1-cell stage, and higher than that of the E group at the blastocyst stage. Four ER stress-associated genes were expressed at the highest level in the EC group and weakly expressed in the ED group at 3 h after activation. However, most of the genes were highly expressed at the 1-cell and blastocyst stages with some variation in the EC and ECD groups. Expression of Bcl-2-associated X protein (Bax) and caspase-3 mRNA was significantly higher in the EC group than in the other groups at all development stages. The developmental rates to the blastocyst stage were higher in the ED and ECD groups than in the E and EC groups. These results suggest that the intracellular ER stress of parthenogenetic porcine embryos is affected by the activation method and subsequently lead to the apoptosis of embryos.

CD26, also known as Dipeptidyl peptidase IV (DPP-4), is a cell surface glycoprotein that belongs to the serine protease family and has wide spread organ distribution throughout the body. CD26 was previously characterized in immune cells but also has important metabolic functions which are not yet fully understood. Thus, we investigated the effect of CD26 in porcine parthenogenetic embryos. We attempted CD26 downregulation of porcine embryos by siRNA, and evaluated CD26 suppression of developmental competencies. Although the porcine embryos injected with CD26 siRNA were able to develop to the early stage, these embryos were decreased to form blastocysts. Our results indicated that CD26 is one of factors for the regulation of development of porcine embryos.

It is well known that the production of transgenic bovine embryos is more difficult than the production of non-transgenic bovine embryos. We performed whether the quality of transgenic bovine embryos expressing the enhanced green fluorescent protein (EGFP) can be improved when cultured for 4 days from day 4 until day 8 after activation with epidermal growth factor (EGF), insulin-like growth factor (IGF), and flavonoid (F) supplements. The EGFP gene was introduced into bovine IVF embryos using microinjector. In experiment, transgenic bovine embryos were cultured in modified CR1aa medium containing 10% FBS at 38.8℃ in an incubator (5% CO2, 5% O2, and 90% N2) for 8 days and embryos were equally divided into four groups [non-treated group (control), 1 μM IGF+EGF (IE), 10 μM F and 1 μM IGF+EGF+10 μ M F (IEF)] at day 4 embryos. In the result, development rate of F treatment group (41.8%) was higher than that of control (33.3%), IE (23.9%) and IEF groups (28.0%). However, hatching rate was significantly high in IE (53.0%) and IEF (65.0%) groups than in control (42.9%) and F group (42.8%) (p<0.05). The EGFP expression rate was not different among all groups (30.0~33.3%) at blastocyst stage. In comparison of total cell number, IE group (145.2±10.4) was significantly higher than control group (101.4±14.3). Apoptotic index of IE group (1.9%) was the lowest compared with that of control (3.3%), F (3.5%) and IEF groups (4.6%). This result demonstrate that the combination of IGF, EGF and flavonoid can be helpful to improve the development potential and the quality of transgenic bovine embryos.

Abnormal epigenetic reprogramming of donor nuclei is supposed to be one of the factors that causes low development efficiency of mammalian somatic cell nuclear transfer (SCNT). Trichostatin A (TSA) is an inhibitor of histone acetylase, and so development of SCNT embryos could be increased by treatment with TSA. In the present study, we examined the effect of TSA on in vitro development of porcine embryos derived from NT (nuclear transfer) by investigating the status of histone acetylation in TSA-treated and control NT embryos and the expression of developmental related genes. In this study, we found that incubating　NT embryos with 40nM TSA for 24h after activation could improved the blastocyst formation rate from 13.7% to 32.5%. Thechange in histone acetylation level as a reslut of TSA treatment were validated using immunofluorescence and confocal microscopy. Immunofluorescence results indicated that the level of aetylation at histone 3 lysine 18 (AcH3K18) was increased at early embryo development stage after TSA treatment. furthermore, we compared the expression patterns of several genes (developmental related genes; Oct4, Sox2, Nanog, Cdx2, the imprinting genes; igf2r). TSA treatment improved the expression of development related genes such as Oct4, Cdx2, Nanog as well as the imprinted genes like igf2r. In conclusion, our results demonstrated that TSA treatment improves the in vitro development of porcine NT embryos, increased the global histone acetylation (AcH3K18) and enhances the expression of some developmentally important genes (Oct4, Cdx2, Nanog) at blastocyst stages.

Jeju Black Cattle (JBC) is an indigenous species of Korea and their mass production and industrialization are required for this high quality indigenous species. For production of elite JBC zygotes, selection of high quality sperm is necessary for in vitro fertilizatioin. In this study, we compared the sperm fertility and developmental capacity of IVF embryos using various JBC sperm (Bull A, B and C). The frozen semen was thawed and confirmed sperm viability and motility. In addition, frozen-thawed sperm was used for a chlorotetracycline(CTC) staining assay and in vitro fertilization. Sperm were classified into three staining patterns. The F pattern is indicative of uncapacitated sperm, the B pattern is indicative of capacitating and capacitated sperm and the AR pattern is indicative of acrosome-reacting sperm or acrosome-reacted sperm, respectively. Several kinds of JBC sperm was inseminated in 44 ㎕ IVF drop contained 10 oocytes with sperm concentration of 1 × 106 cells/ml, and then 2 ㎕ heparin and 2 ㎕ PHE (20 μM penicillamine, 10 μM hypotaurine, 2 μM epinephrine) were added. The sperm viability and motility were higher in sperm 3 species (n=8). When we confirmed sperm capacitation, F pattern and B pattern rate were higher than AR pattern in sperm A group. After IVF, the rates of cleavage and blastocyst development were higher in sperm C group compared to other sperm group. However, the cell number of blastocyst was higher in sperm E group. These results demonstrate that the use of sperm C was effective in production of elite JBC IVF embryos. Additional experimental data are required for more accurate analysis.

Mitochondrial dysfunction is found in oocytes and transmitted to the offspring due to maternal obesity. This is curable by endoplasmic reticulum (ER) stress inhibitors such as salubrinal (SAL). Recently pigs are considered as a model animal for biomedical research due to its physiological similarity with human. Pig oocytes have shown ER stress mostly in metaphase II stage. ER stress is hindering the in vitro embryo production (IVP). This study investigated the effect of ER stress inhibition by using SAL during 44 h of in vitro maturation (IVM) of oocytes at 1, 10, 50 and 100 nM concentrations. Firstly, we defined the concentration of SAL during IVM of pig oocytes. SAL at 10 nM showed higher (44.2 to 55.6%, P<P0.05) development competence to the blastocyst state than control and other concentrations after parthenogenetic activation (PA). Secondly, we sorted out the time-dependent treatment at 10 nM of SAL for IVM of oocytes. It revealed that treatment with SAL during 22 to 44 h and 0 to 44 h of IVM improved PA embryonic development significantly (40.5, 51.7 and 60.2% for control, 22 to 44 h and 0 to 44 h of IVM, respectively, P<0.05). Glutathione (GSH) level is an indicator of cytoplasmic maturation of oocytes. Reactive oxygen species (ROS) have a harmful effect on development competence of oocytes. For this, we determined the intraoocyte levels of GSH and ROS after 44 h of IVM. It was found that SAL increased intraoocyte GSH level and also decrease ROS level (P<0.05). Finally, we performed somatic cell nuclear transfer (SCNT) after treating oocytes with 10 nM SAL during IVM. SAL treatment significantly improved blastocyst formation of SCNT embryos compared to control (24.7 vs. 39.6%, P<0.05). Our results indicate that treatment of pig oocytes with ER stress inhibitor SAL during IVM improves preimplantation development cloned pig embryos by influencing cytoplasmic maturation in terms of increased GSH content and decreased ROS level in IVM pig oocytes.

Lysophosphatidic acid (LPA) is a member of the phospholipid autacoid family and has growth factor and hormone-like activities on various animal cells. In this study, we investigated the effect of LPA on porcine embryo development. Porcine parthenogenetic embryos were treated into various concentrations of 0 (control), 0.1, 1 and 10 μM LPA (0 LPA, 0.1 LPA, 1 LPA and 10 LPA) during in vitro culture for 7 days or cultured in basic culture medium until day 4 and treated LPA from day 4 to day 7. In the LPA treatment for culturing from day 0 to day 7, there was no significant difference on cleavage and blastocyst formation rate. In addition, the blastocyst development proportion which was classified as expanded, hatching, or hatched blastocystshas was no significant difference among all groups. In the LPA treatment for culturing from day 4 to day 7, 0.1 and 1 LPA groups were presented increased blastocyst formation compared to other groups, but cleavage rate and over-expanded blastocyst formation rate were not significantly different among all LPA treated groups. The total cell number was not different but apoptosis was reduced when 1 LPA treated from day 4 to day 7. The relative mRNA expression level of anti-apoptosis gene, BCL2L1 was higher and pro-apoptosis gene, BAK was lower in the 1 LPA treated group than the control. In comparison with the control and the 1 LPA treated group using time-lapse monitoring system, 1 LPA treated embryo was accelerated developmental speed via morula compaction and expanded blastocyst. The 1 LPA treated group significantly increased the relative expression levels of gap junction and tight junction related genes, GJD1, CDH1 and ZO-1 compared to the control. These results indicated that 1 μM LPA supplementation for culturing from day 4 to day 7 post activation is efficient in blastocyst formation and LPA may be helpful for embryo developmental capacity.

This study was conducted to examine the effects of activation methods on the ER stress induction and subsequent apoptosis and in vitro development of porcine parthenogenetic embryos. Porcine in vitro matured oocytes were activated by four activation methods; 1) electric stimulus(ES) with two DC pulses of 1.25 kV/cm, for 30 ㎲ (E), 2) ES + 10 μM Ca-ionophore (A23187) treatment for 5 min (EC), 3) ES + 2 mM 6-dimethylaminopurine treatment for 3 h (ED), or 4) ES + A23187 + 6-DMAP (ECD). After activation, parthenogenetic embryos were in vitro cultured in PZM-3 medium and sampled to analyze the x-box binding protein 1 (Xbp1) mRNA, ER stress-associated genes and apoptotic genes at 3 h post ES and the 1-cell and blastocyst stages. The un-spliced and spliced x-box binding protein 1 (Xbp1) mRNA were confirmed by RT-PCR. Also ER stress-associated genes, such as the C/EBP homologous protein (CHOP), binding protein (BiP), activating transcription factor 4 (ATF4) and glucose-regulated protein 94 (GRP94), and apoptotic genes were analyzed by real-time quantitative RT-PCR (RT-qPCR). The band intensities of spliced Xbp1 (Xbp1s) mRNA was higher in the EC group than other three groups at 3 h and the 1-cell stage, while it was higher in the ED groups compared with E group at the blastocyst stage. Four ER stress-associated genes were showed the highest expression in the EC group and weakly expressed in the ED group at 3 h. However, most of those genes were highly expressed in EC and ECD groups at the 1-cell and blastocyst stages with some variation. The expressions of Bcl-2-associated X protein (Bax) and caspase-3 mRNAs were significantly higher in EC group than other three groups at all stages. The developmental rate to the blastocyst stage was higher (p<0.05) in ED and ECD groups (32.1±3.8 to 34.6±2.2%) than that of E group (26.1±3.9%). These results suggest that the intracellular ER stress of parthenogenetic porcine embryos is affected by activation method and subsequently lead to the apoptosis of embryos.

Somatic cell nuclear transfer (SCNT) technique is a key point of producing transgenic animal disease models. During in vitro production of SCNT embryo, the quality of matured oocytes are one of the important factors that regulate embryo developmental capacity. In preliminary test, we confirmed the effect of fibroblast growth factor 10 (FGF10) on porcine oocyte maturation. In this study, we investigated the developmental potential of SCNT embryos treated with the 10 ng/ml FGF10 (10 F) during in vitro maturation of recipient oocytes. The polar body emission rate was significantly higher in the 10 F treated group than control group. After SCNT, although the rate of fusion was no significant difference, the rate of cleavage and blastocyst formation was significantly increased in the 10 F treated group (p<0.05). In 10 F treated group, the total cell number was increased and the percentage of apoptotic cell was decreased in the blastocyst stage at day 7 (p<0.1). The transcription level of apoptosis relative gene, Casp3 was significantly decreased, while anti-apoptosis gene BCL2l1 was increased in the 10 F treated group compared to control group. The 10 F treated group was highly expressed the reprogramming related genes, Sox2 and POU5f1. Also, the first cleaving time was more faster and the percentage of cell block was significantly lower in 10 F treated group than in control group. In this study, we confirmed that 10 ng/ml FGF10 has effect on enhance the oocyte maturation and developmental capacity. These results demonstrate that FGF10 treatment can be used for in vitro development of porcine SCNT embryos and subsequent production of transgenic animal model.

Increase of bovine embryos produced by in vitro fertilization (IVF) has been seen. The main reason for producing in vitro fertilized embryos in Korea has been to utilize the genetics of cows with higher carcass grade. Ovaries are collected from the cows in the slaughter house and the information on the carcass grade of the cow can be traced. Embryos produced from cows with higher carcass grade have been favored by the farmers. PCR has been one of the main techniques for sex determination of embryos targeting various genes. Bovine sex determining region Y (SRY) is specific to Y chromosome. However, it requires a control gene for PCR, if the embryo is female. In comparison to SRY, amelogenin can be amplified from male or female embryos with different fragment sizes due to differential splicing in all bovidae. The goal of this study was to determine whether there are any differences in the sex ratio of embryos produced in vitro and to compare the efficiency of sex determination using PCR. Ovaries of Hanwoo were collected and transported to the laboratory in thermal bottles. For in vitro maturation, oocytes were collected from the follicles with less than 8 mm of diameter and placed in either the Brackett & Oliphant media (BO), Tissue culture medium-199 (TCM-199), or IVMD101 media, containing 3% fetal bovine serum (FBS), 0.5 mg/ml FSH, 0.5 mg/ml LH, and 1 mg/ml estradiol-17β. For IVF, frozen sperm from Hanwoo bulls were used. After 22-24h IVF, embryos were transferred and cultured either in BO or TCM-199 with 10% FBS until the embryos were hatched. Hatched blastocysts were stored in PBS frozen, and later thawed and treated with embryo lysis buffer. After isolating genomic DNA, it was used for PCR using primers for casein beta (CSN2), as PCR control, or for male specific SRY primers. Alternatively, primers for amelogenin were used. Sex of embryos was determined and the sex ratio was analyzed. Out of 94 embryos, sex of 83 embryos (88.3%) was determined and there were 40 male embryos (48.2%) and 43 female embryos (51.8%). Sex of 31 embryos was determined using both SRY and amelogenin. Among those, 17 embryos were determined as having identical sex, while 1 embryo was determined as having different sex, and the sex of 11 and 2 embryos were determined only by amelogenin or SRY primers, respectively. In conclusion, the success of determining the sex of embryos by PCR was relatively high. Using amelogenin primer for PCR tends to be more efficient than SRY primer in determining the sex. Slightly higher ratio of female embryos was different from previous years and the cause for the difference may require further investigation.

In vitro production of mammalian embryos has been achieved with the oocytes derived from middle-size follicles (MF, mainly 3-6 mm in diameter) in many species including domestic animals. In the ovaries, however, there are more small-size follicles with less than 3 mm in diameter (SF). If we can develop an efficient system to produce embryos in vitro from the oocytes from SF. In this presentation, I would like to review about embryo production in vitro from the oocytes derived from SF. As well as the diameter of oocytes, the number of cumulus cells surrounding the oocyte derived from SF is significantly smaller those of oocytes from MF. The comparative analysis in electrophoresis about secretions of cumulus-oocyte complexes derived from SF and MF demonstrated a significant difference in the proteins with a molecular weight. Proteins secreted from cumulus cells, vascular endothelial growth factor (VEGF), are 34- to 42-kDa proteins, including seven family members. The molecular weight of VEGF was similar with the secretion we observed. Supplementation of medium for in vitro maturation with VEGF significantly improved the oocytes competence not only to complete the meiosis in vitro but also to develop to the blastocyst stage following parthenogenetical activation. Removing cumulus cells 20 h after the start of culture for in vitro maturation also significantly improved the competence of oocytes derived from SF to achieve the meiosis. A combination of these new techniques may improve more the meiotic and developmental competences.

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.

The study aims to assess the embryo development and survivability of bovine embryos cultured in vitro by addition of cysteine. The rates of metaphase II formation are not significantly different among the three groups(73.8% for TCM199, 76.9% for TCM199 with 0.3mM cysteine and 83.8% for TCM199 with 0.5mM cysteine). No differences on cleavage rate(70.6~74.6%) was observed among three culture medium(70.6% for TCM199, 71.3% for CR1aa, and 74.6% for SOF) with 0.5mM cysteine. However, significantly(P<0.05) higher development rate was obtained in the blastocyst stage by adding 0.5mM cysteine in SOF medium(35.6%) than in TCM199(27.6%) or CR1aa(26.6%). No significant differences in the cleavage rates were observed among the three culture. After freezing the blastocysts cultured with 0.5M cysteine, the re-expansion rates ranged from 61.3% to 86.4% among groups, and hatching rates are from 26.3% to 46.9% among groups. The rates of re-expansion and hatching are significantly(P<0.05) higher in SOF medium(86.4% and 46.9%, respectively) than those in TCM199(61.3% and 26.3%) and CR1aa medium(87.1 and 44.4%). After thawing, the blastocyst re-expansion rate become significantly(P<0.05) higher in in vivo (87.1%) and in vitro (70.3%) embryos. In conclusion, our results demonstrate that supplementation of IVM and IVC media with 0.5mM cysteine improved the quality of in vitro production of embryo and post-thawed embryo. Future studies comparing these culture systems in well-designed trials should be performed.

Historically, Korea old cattle had been consisted with various lines of coat color brindle, black and white-brown breeds or more. The two rare lines of black and white coat color are maintained for animal resources and preserved critically. The present study was carried out to evaluate potential usage of cysteamine supplementation during in vitro matration (IVM) and in vitro culture/production of embryo (IVP) by transvaginal ultrasound-guided follicle aspiration (Ovum Pick-Up: OPU) for the establishment of cryo-banking system. Immature slaughterhouse-derived cumulus-oocyte complexes (SL-COCs) were matured in IVM medium supplemented with 0, 0.1, 0.3 or 0.9 mM cysteamine, and then cultured in mSOF-BAS for 8 days after in vitro fertilization. The treatment of 0.1 mM cysteamine on SL-COCs showed higher rate of blastocyst, so OPU-derived COCs from rare breeds were matured in TCM media supplemented with or without 0.1 mM cysteamine, FSH and 5% FBS. The embryos were evaluated their developmental stages on day 8. During IVM, cysteamine treatment significantly increased the embryo production rate of slaughterhouse-derived COCs (19.6% vs. 30.5%). The presence of cysteamine during IVM of OPU-derived COCs from rare Korean cattle breeds (albino white and black line) also increased embryo production rates than those from SL-COCs (27.4% vs. 41.9% and 36.4%). With these results, cysteamine treatment during IVM is one of key factors IVP of blastocysts to establish banking system of endangered rare Koarean cattle with OPU derived transferable blastocysts.

국내에 서식하는 두꺼비의 배아를 이용하여 화학물질의 독성평가에 대한 가능성을 파악하기 위해 FETAX(Frog Embryo Teratogenesis Assay-Xenopus) 기법에 따라 두꺼비(Bufo gargarizans)의 배아를 배양하면서 Zn과 Benomyl의 효과를 probit 분석법으로 조사하였다. 그 결과, Zn과 Benomyl의 농도에 의존하여 유생의 체장 길이는 감소하고 치사율 과 기형율은 증가하였으며 Zn과 Benomyl의 teratogenic concentration(EC50)은 각각 2.3, 1.0㎎/ℓ을 나타내었고 embryo lethal concentrations(LC50)은 10.3, 6.9㎎/ℓ을 나타내었다. Teratogenic index(TI=LC50/EC50)는 Zn의 경우 4.4, Benomyl의 경우 6.7을 나타내어 Zn과 Benomyl은 두꺼비 배아 발달에 최기형성 물질로 작용함을 알 수 있었다. 이상의 결과들로 보아 Zn과 Benomyl 모두 낮은 농도에서 두꺼비 배아발달에 민감하게 반응하였다. 이는 다량의 배아 확보가 가능하며 배양이 용이하고 치사율, 기형율, 성장률, 기형양상 등을 기존의 연구들과 비교하였을 때 유사한 결과를 나타내어 두꺼비 배아를 활용한 시험기법은 화학물질 및 환경오염물질의 독성검정에 활용할 수 있을 것으로 판단된다.

This study is performed to evaluate the effect of insulin in the porcine parthenogenetic embryo development. In porcine embryo culture, insulin is helpful factor in the process of embryo development. To identify this, insulin is used in pig embryos development. Therefore, this study was performed to investigate the effect of insulin on early embryonic development in pigs. For that, insulin positive or negative (0, 10 ug/mL) was supplemented in the porcine IVM media and then compared two groups divided by the cytoplasm of the black groups and white ring groups based on the distribution of lipid material of the cell cytoplasm in microscope. In maturation rates of porcine oocytes, significant higher black group rates were shown in the insulin positive groups compared with other groups (56.0±2.1 vs 46.2±0.3). In the embryo culture, black groups were showed the significant higher cleavage rates (82.1±0.8, 78.3±0.1 vs 63.2±0.3, 63.4±0.0), and blastocyst formation rates (15.5±3.6, 16.6±0.4 vs 11.7±1.3, 7.4±0.2) regardless of whether the addition of insulin. Also, black groups were showed higher cell number of blastocyst (33.2±2.5, 35.5±2.6 vs 31.2±2.1, 31.3±2.2). In conclusion, supplement of insulin producing black group in vitro maturation, it was effective in vitro maturation and embryonic development of pig embryos.

Transcription factor called activating enhancer binding protein 2C (AP2-gamma) is found in a variety of species and expressed from oocyte stage onwards, particularly restricted to the trophectoderm. Recent studies demonstrated that ablation of Tfap2c led to failure of tight junction biogenesis, particularly the knock-down embryos of Tfap2c did not form cavity from morula to blastocyst in mouse and pig. We speculated that the Tfa2pc may also be involved in desmosome biogenesis because blastocoel formation is coincident with the establishment of desmosome. To determine this, we depleted Tfap2c injecting siRNA into one-cell zygote and analysed the expression levels of genes that are required for desmosome complex such as PkP2, Pkp3, Dsc2, and Dsg2. We found only Pkp3 was up-regulated in the knockdowned morula embryos. Interestingly, upstream region of Pkp3 had putative Tfap2c binding sites. In conclusion, our results suggest that Tfap2c is not a crucial factor but somehow it might be involved in desmosome biogenesis directly or indirectly via Pkp3.