In the present study, we investigated the effect of porcine follicular fluid (PFF) concentration (10% vs. 1%) and protein-free media (PFF 0%) on maturation of porcine oocytes in vitro and analysed difference in gene expression in resulting blastocysts following parthenogenetic activation. Three groups were tested; 1) 10% PFF: Tissue culture medium (TCM) 199+10% PFF; 2) 1% PFF: TCM 199+1% PFF; and 3) 0.1% PVA: TCM 199+0.1 PVA. Cumulus-oocyte-complexes were cultured in the respective media containing gonadotrophin (1 ug/ml), epidermal growth factor (10 ng/ml), cystein (0.57 mM), sodium pyruvate (0.91 mM), insulin (5 ug/ml), 9-cis retinoic acid (5 nM) for 20～22 h and then without hormonal supplements for an additional 20-22 h. Data was analyzed using statistical analysis system(SAS) program. There was no significant difference in oocyte maturation rate. However, significantly higher (p<0.05) proportions of embryos developed to the blastocyst stage when oocytes were matured in 10% PFF group (45%) than in the 1% PFF group (31.1%). The total cell numbers were not significantly different among groups (52 ± 1.3 vs. 54.6±3.1 vs. 54.4±2.5, respectively). The relative abundance (ratio to beta-actin mRNA) of gene transcripts related to apoptosis in blastocysts was measured by real- time PCR. The expression of anti-apoptotic gene (BclxL) was up-regulated and the expression of pro-apoptotic gene (Bax) was down-regulated in 10% PFF group than in the other groups. Therefore, it can be concluded that supplementation of 10% PFF during in vitro maturation improves embryo development by reduction of apoptosis. * This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), MKE (#10033839-2011-13), Institute for Veterinary Science, the BK21 program and TS Corporation.

The present study examined the expression of porcine sirtuin 1–3 (Sirt1–3) genes in immature (germinal vesicle; GV stage), mature (metaphase II; MII stage) oocytes, preimplantation embryos derived from parthenogenetic activation (PA), in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). We also investigated the role of sirtuins in oocyte nuclear and cytoplasmic maturation, and embryonic development of PA and IVF embryos using sirtuin inhibitor [5 mM nicotinamide (NAM) and 100 μM sirtinol]. The expression of Sirt1–3 mRNA was significantly (p<0.05) up-regulated during IVM. The expression patterns of Sirt1–3 mRNA in preimplantation embryos of PA, IVF and SCNT were gradually (p<0.05) decreased from MII stage of oocyte to blastocyst stage. Especially, the expressions of Sirt1 and Sirt3 in SCNT blastocysts were significantly lower than IVF blastocysts. Treatment with nicotinamide (NAM) during IVM resulted in significantly decreased nuclear maturation but it was restored when NAM treated with 2 μM resveratrol (RES; known as antioxidant and sirtuin activator) compared to the control (control: 88.9%, NAM: 67.9% and NAM+RES: 86.4% respectively). Intracellular reactive oxygen species (ROS) level of oocytes matured with NAM was significantly increased and with NAM+RES was significantly decreased compared to the control. Treatment with sirtuin inhibitors during IVC resulted in significantly decreased blastocyst formation and total cell number of blastocyst derived from PA (NAM: 29.4% and 29.6, sirtinol: 31.0% and 30.3, and control: 40.9% and 41.7, respectively) and IVF embryos (NAM: 10.4% and 30.9, sirtinol: 6.3% and 30.5, and control: 16.7% and 42.8, respectively). There was no significant difference in cleavage rate both PA and IVF embryos. Oocytes treated with NAM during IVM showed significantly lower expression of PCNA, Bax, Bcl-2, POU5F1 and Sirt1–3 compared to the control. Oocytes treated with NAM+RES during IVM restored gene expression except POU5F1. Similarly, PA derived blastocysts treated with NAM during IVM showed down-regulation of PCNA, Bax, Bcl–2, POU5F1 and Sirt1–2. The blastocysts derived from PA embryos treated with sirtuin inhibitors during IVC showed lower (p<0.05) expressions of POU5F1 and Cdx2 genes. Also, Sirt2 mRNA expression was significantly decreased in sirtinol treated group and Sirt3 mRNA expression was also significantly de -creased in both NAM and sirtinol treated groups compared to the control. These findings indicate that Sirt1–3 which are transcribed and stored during oocyte maturation may have physiological and important roles in porcine oocyte maturation and preimplantation embryonic development by regulating gene expressions. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

The objective of the present study was to investigate the effects of different concentrations of sorbitol supplementation for in vitro maturation medium and in vitro culture medium, on porcine cumulus oocyte complexe(COC) maturation and subsequent developmental capacity after parthenogenetic activation. Porcine COC were cultured for 44 h(0～ 22 h termed MI stage and 22～44 h termed MII stage) in TCM199 without(— ) or with(+) sorbitol (20 μM, 100 μM, 200 μM), and the resultant metaphase II oocytes cultured in PZM-3 for 7 days following activation. Our results showed that supplementation with appropriate concentrations of sorbitol (20 μM) during full term maturation culture(MI+/MII+) significantly(p<0.05) improved blastocyst formation rates and total cell number. When the concentration of sorbitol were increased to 100 μM and 200 μM during maturation culture, the maturation rate of COC were significantly reduced compared with 20 μΜ or control groups. Also blastocyst formation rates significantly(p<0.05) reduced with increasing concentration of sorbitol(200 μM). Supplementation with sorbitol(20 μM, 50 μM, 100 μM) into PZM-3 for in vitro culture significantly(p<0.05) inhibited blastocyst formation compared with control group. However, the blastocyst formation rates start to rise again when 50 μ M sorbitol was used for the first 48 hours and then cultured in PZM-3 without sorbitol. There was no significant difference in cell number between control and sorbitol treated groups. When the activated oocytes were cultured in PZM-3 for 48h and then cultured in PZM-3 with sorbitol, interestingly, the blastocyst formation rate was similar to that of PZM-3 with sorbitol for in vitro culture and significantly lower than control group. These results suggest that addition of low concentrations of sorbitol(20 μM) during oocyte maturation is beneficial for subsequent blastocyst development and improved embryo quality. However, treatment with sorbitol supplementation during in vitro culture medium is negative effect to blastocyst formation.

Epigenetic status of the genome of a donor nucleus has an important effect on the developmental potential of cloned embryos produced by somatic cell nuclear transfer (SCNT). In our previous study has results showed that the donor cells treated with 5-aza-2’- deoxyctidine (5-aza-dC, DNA methylation inhibitors) and Trichostatin A (TSA, histone deacetylase inhibitors) could improve the development of porcine nuclear transfer embryos in vitro. In this study we want to investigate why these two drugs treatment with the donor cell can improve the cloning efficiency, whether they can alter the epigenetic status of the genome of the donor nucleus. This study included 6 groups: control group, the donor cell (porcine fetal fibroblast cell) with no treatment; 2.5 nM 5-aza-dC group, the donor cells treated with 2.5 nM 5-aza-dC for 1h; 5-aza-dC group, the donor cells treated with 5 nM 5-aza-dC for 1h; TSA group, the donor cells treated with 50 nM TSA for 1h; 2.5 nM 5-aza-dC+TSA group, the donor cells treated with 2.5 nM 5-aza-dC for 1h and subsequently treated with 50 nM TSA for another 1h; 5-aza-dC+TSA group, the donor cells treated with 5 nM 5-aza-dC and 50 nM TSA together for 1h. The first experiment detected the DNA methylation status in the different groups. After treatment with these two drugs, the DNA methylation level of the donor cells decreased, however there is no significant difference among the groups. This result indicated that the donor cell treatment with 5-aza-dC and TSA can partially alter the DNA methylation status of the donor cells. The second experiment checked the histone acetylation level of the donor cells treated with these two drugs by western blot. TSA, 2.5 nM 5-aza-dC+TSA, 5 nM 5-aza-aC+TSA, these three groups can significantly improve the hisone acetylation level compared with control and 5-aza-dC groups, there is no significant difference among these three groups. The results of this study suggest that the donor cells treated with 5-aza-dC and TSA can partially decrease DNA methylation and can significantly improve the histone acetylation level of the donor cells, these alterations of the epigenetic modification maybe can improve the clonging efficiency.

An understanding of oocyte gene expression is a necessary for the study of early female gamete development. Recently, oocyte has been used in many techniques such as somatic cell nuclear transfer, intracytoplasmic sperm injection and embryonic stem cell derivation. The purpose of this study was to investigate in the proteomes of pig oocytes and identification of differential proteins between using DIGE technique. In this experiment to overcome of limitation of 2D gel method like a low reproducibility and low sensitivity for proteome analysis of very small quantities, 2D fluorescence difference gel electrophoresis (DIGE), which enables co-detection of up to three samples on the same 2DE gels with CyDyes was used for analysis of oocyte proteins. Proteins within an isoelectric point (pI) range of 3 to 10 and a molecular weight (Mw) range of 20～100 kDa were primarily analyzed in DIGE with 2 replications of each sample. Approximately 1000 spots were detected in 2-D gel. Then, image analysis of DeCyder was performed to detect variations in protein spots between mature oocyte and parthenogenesis embryo. In the comparison of mature oocyte and parthenogenesis embryo, 11 spots were identified to be up-regulated proteins and 2 spots to be down-regulated proteins in parthenogenesis embryo, among which proteins were zona pellucida glycoprotein 4, transferrin receptor, apolipoprotein B, L-3-Hydroxyacyl Coa Dehydrogenase Revisited, cytochrome P450 2C33, similar to Monocarboxylate transporter 2, 2'-5' oligoadenylate synthetase 3, interferon alpha/ beta receptor-1, Chloride channel protein 6, pyruvate carboxylase as well as2'-5' oligoadenylate synthetase 3 using MALDI-TOF-MS. These results suggested that differential proteins were present between mature oocyte and parthenogenesis embryo.

In all the studies of mammalian species, chromatin in the germinal vesicle (GV) is initially decondensed with the nucleolus not surrounded by heterochromatin (the NSN configurations). During oocyte growth, the GV chromatin condenses into perinucleolar rings (the SN configurations) or other corresponding configurations with or without the perinucleolar rings, depending on species. During oocyte maturation, the GV chromatin is synchronized in a less condensed state before germinal vesicle breakdown (GVBD) in species that has been minutely studied. As not all the species show the SN configuration and gene transcription always stops at the late stage of oocyte growth, it is suggested that a thorough condensation of GV chromatin is essential for transcriptional repression. Because the GV chromatin status is highly correlated with oocyte competence, oocytes must end the NSN configuration before they gain the full meiotic competence and they must take on the SN or corresponding configurations to stop gene transcription before they acquire the competence for early embryonic development. In this study, we firstly investigated whether the follicle size could determine chromatin configuration in porcine oocyte. For this experiment, follicles was divided into three groups (<1 mm follicle, 1～3 mm follicle and 3～6 follicle). Using DAPI staining, the GV nucleolus and chromatin of porcine oocytes was classified into SN, SN-NSN and NSN configurations. MⅠ and M Ⅱ of three groups's Mature oocytes by staining was confirmed the configuration of chromatin. The maturation rate and parthenogenetic development potential were significant different between the SN and NSN configurations oocytes. These results indicated that chromatin changes in GV oocytes affect the development potential of porcine embryos.

Cathepsins (CTSs), a family of lysosomal cysteine proteases, and their inhibitors (CSTs) play a critical role in remodeling of the uterine endometrium and placenta for the establishment and maintenance of pregnancy in many animal species including rodents, sheep, cow and pigs. It has been shown that the high rate of pregnancy failure by somatic cell nuclear transfer (SCNT) is associated with abnormal placental development. Our previous study has shown that CST6 is highly expressed in the uterine endometrium from mid to late pregnancy in pigs. In this study, to understand whether appropriate endometrial and placental tissue remodeling occurs in the uterine endometrium from gilts with conceptuses derived from SCNT during pregnancy in pigs, we investigated expression of CST6 in the uterine endometrium. Uterine endometrial tissues were obtained from gilts that carried SCNT-derived normal conceptuses (NT-No) and abnormal conceptuses (NT-Ab), and from gilts carrying conceptuses from natural mating (Non-NT) on D114 of pregnancy. Immunoblot analysis showed that CST6 protein levels in the endometrial tissues of gilts carrying NT-No were lower than those of gilts carrying Non-NT. The levels of CST6 protein in the endometrial tissues of gilts carrying NT-Ab decreased even more than those of gilts carrying NT-No. These results indicate that decreased expression of CST6 in the endometrium with NT-No and NT-Ab reflects inappropriate endometrial tissue remodeling and pregnancy failure of pigs with SCNT derived conceptuses and that CST6 plays an important role for the maintenance of pregnancy in pigs. * This work was supported by the Next Generation BioGreen 21 program (#PJ007997), RDA, Republic of Korea.

Embryo transfer (ET) is the final procedure for getting pregnancy through assisted reproductive technology such as IVF (in vitro fertilization), SCNT (somatic cell nuclear transfer). In our laboratory, the porcine cloned embryos loaded in ET medium are carried for 3 hours by portable incubator because of the great distance from the laboratory to the experimental farm. Thus, before transferring into recipient, porcine cloned embryos are exposed in vitro condition for long time. Medium which is used in this process is the TALP (Tyrode’s medium supplemented with 10 mM HEPES), but it includes little nutrients for embryo. Thus, the aim of this study is to determine whether ET media containing nutrients affect the in vitro development of embryos compared to TALP. For the experiment, porcine zygote medium (PZM)-5 which has amino acids for developing embryo was chosen as ET medium containing nutrients, added 10 mM Hepes as PZM-5 does not contain buffering system. For experiment, we carried out parthenogenesis through a chemical method using Thi/DTT. Parthenogenetic embryos were cultured in PZM-5 for 2 days, and then they were randomly divided into two group; loaded in a straw with TALP or PZM-5-Hepes, respectively. They were stored in a portable incubator for 3 hours to simulate the time consumed in ET, thereafter embryos in both TALP and PZM-5-Hepes groups were respectively cultured in PZM-5 for additional 5 days. All experiments were repeated 5 times. In result, blastocyst formation rate were 22.46%±1.47 and 23.17%± 2.13, respectively and total cell number were 32.9±2.22 and 37.09±2.18, respectively. There is no significant difference between TALP and PZM-5-Hepes groups. * Further study will investigate effect of PZM-5-Hepes on in vivo development of porcine cloned embryo. This study was supported by IPET (#311011-05-1-SB010), RNL Bio (#550-20120006), Institute for Veterinary Science, the BK21 program and TS Corporation.

The purpose of this thesis is to examine the effect of hormone treatment in blastocyst development of in vitro cultured porcine oocyte. Oocytes used in the study was matured in vitro in the presence of 10% FBS or 10% pFF, and treated with FSH, LH or FSH+ LH, and the rate of blastocyst development was assessed based on the expression of autophagic genes. There was no significant differences in blastocyst development between oocytes maturaed in 10% FBS or 10% pFF. In vitro matured oocytes treated with FSH+LH showed blastocyst development rate as high as that of untreated oocytes, while groups treated with LH only showed a decrease in blastocyst development. About the expression of cell death assosiated factors, mRNA levels of autophagy and apoptosis genes were increased in oocytes matured in 10% FBS and treated with LH. Oocytes that did not receive hormone treatment showed low expression of most cell death genes except ATG5. When oocytes were matured in 10% pFF, ATG5 expression was the highest in FSH treated group, while LC3 showed strong expression in all hormone treated groups. On the other hand, the expression level of mTOR and caspase-3 did not show significant differences between groups. We also examined the protein level of apoptotic genes in the blastocyst. The amount of caspase-3 protein was similar between groups matured in 10% FBS and 10% pFF, but was the highest when treated with LH. Blastocysts treated with FSH and FSH+LH showed similar level of caspase-3 protein, while the level was the lowest when hormone treatment was not given. Within the blastocyst, caspase-3 was mostly expressed in trophoblast cells when matured in 10% FBS, while maturation in 10% pFF caused expression of this protein in the inner cell mass (ICM). Expression of MAP1LC3A was higher in groups matured in 10% pFF than groups matured in 10% FBS in all types of hormone treatment. Among the blastocysts matured in 10% pFF, MAP1LC3A level increased in the order of untreated < FSH < FSH+ LH. Expression of MAP1LC3A within the FBS-matured blastocyst was concentrated to the trophoblast, while pFF-matured blastocyst showed expression in both trophoblast and ICM. The expression of mTOR showed a pattern opposite to that of MAP1LC3A. That is, its expression was the lowest in 10% FBS group without hormone treatment. In both FBS and pFF group, and in all three combinations of hormone treatment, mTOR expression was ovserved mostly in ICM. Together, these results indicated that hormone treatments tend to induce expression of genes associated with programmed cell death. We suggest that proper induction of programmed cell death by FSH and LH treatment would increase the rate of blastocyst development. * This work was supported by BioGreen 21 Program (No. PJ008029). Rural Development Administation, Republic of Korea.

Limited success of somatic cell nuclear transfer(SCNT) is attributed to incomplete reprogramming of transferred donor cell. Several approachs, such as histone deacetylase inhibitors and DNA methyltransferase inhibitors have been used to improve the efficiency of somatic cell nuclear transfer. Recently, it is reported that pre-treatment of somatic cells with undifferentiated cell extract, such as embryonic stem cell and mammalian oocytes is an attractive alternative ways to reprogramming control. The aim of this study was to evaluate the early development of porcine cloned embryos produced with porcine ear skin fibroblasts pre-treated with extract from porcine induced pluripotent stem cell (iPSC). For transport of porcine iPSC extract into cultured porcine ear skin fibroblasts, the ChariotTM reagent system was used. Treated cells were cultured for 3 days, and used for the analysis of histone H3K9 acetylation and SCNT The acetylation status of H3K9 was increased in cells treated with iPSC extract and cultured for 3 days compared with control. But, no significant difference was observed between the extract treated and control groups. After SCNT. no difference was observed in the rate of fusion (86.6% vs 86.2%) and embryo cleavage (86.6% vs 87.1%) between the extract treated and control groups. Also, no significant difference was noted in blastocyst rates (23.4% vs 28.4%) as well as cell numbers (43.8±10.8 vs 41.2±11.6) with extract treated group compared with control group. Overall apoptosis rate in blastocyst was not differences between the extract treated and control groups (4.6±3.5% vs 6.0± 5.8%). However, blastocyst rate with high apoptotic cells(>10% appototic cells) was significantly lower in extract treated group when compared with control group (7.1% vs 21.8%).. Our results demonstrated that pre-treatment of porcine ear skin fibroblasts using porcine iPSc extract had beneficial effect on the decreasing apoptosis in the blastocyst cultured in vitro, although there was no effect on the embryonic development.

A recent study has reported that pluripotent stem cells can be categorized according to their pluripotent state. The first is a “naïve” state, which is characterized by small, round or dome-shaped colony morphologies, LIF and BMP4 signaling pathways and two active X chromosomes in female; mouse ES cells (mESCs) represent this type. A second “primed” state has also been described and is possible in mouse epiblast stem cells (mEpiSCs) or human ES cells (hESCs). These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and Nodal/Activin signaling pathways and X chromosome inactivation in female. It has been suggested that, as a non-permissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. Meanwhile, a few studies have reported that primed pluripotent stem cell lines could be reverted to a naïve pluripotent state using various exogenous factors including GSK3β and MEK inhibitors, LIF, hypoxic conditions and up-regulation of Oct3 or klf4. Therefore, the purpose of this study was to investigate whether a LIF-dependent naïve pluripotent stem cell line could be derived from porcine embryonic fibroblasts(PEFs) via doxycycline (dox)-inducible reprogramming factors and LIF. In this study, we have been able to successfully induce PEFs into a LIF-dependent naïve pluripotent-like cell line showing a mESC-like morphology and the expression of pluripotent markers. Our results suggest the possibility of reprogramming to naive pluripotent- like stem cells from PEFs in porcine species. * This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

Acteoside acts as an anti-oxidative activity and anti-apoptosis in the cells. But, it has been not studied on maturation and development of porcine oocytes. The aims of the present study were to examine the effects of acteoside on the morphological progress of meiosis, developmental competence, and ROS in porcine oocytes. Oocytes were matured in tissue culture medium-199, supplemented with acteoside at various concentrations: 0 (control), 10, 30 and 50 μM. The oocytes maturation rates of groups supplemented with acteoside were no significantly different (81.13, 85.96, 82.95 and 83.68%, respectively). Level of ROS was significantly decreased in acteoside treated group. Furthermore, the parthenogenetic blastocyst rate was significantly improved in 10 μM acteoside treated group compared with control group (44.83 vs. 27.75%). And we investigated effect of acteoside on the oocytes condition represented by cytoplasmic maturation by homogeneous distribution and formation of cytoplasmic organelles and regulation of apoptosis-related genes. In the results. during IVM, 10 μM acteoside treated oocytes showed that the mitochondria and lipid droplet were smaller and homogeneous distribution in cytoplasm compare with control oocytes. And reverse transcription polymerase chain reaction (RTPCR) of parthenogenetic blstocysts revealed that acteoside increased the anti-apoptotic genes (Mcl-1, Bcl-2 and Bcl-xL), whereas reduced the expression of pro-apoptotic genes (Bax and Bak). In conclusion, based on the results, the effect of acteoside on IVM was not attractive. However, in acteoside treated group, cytoplasmic maturation seemed to be improved with morphologically uniform distribution of cytoplasmic organelles. Furthermore, embryonic development in acteoside treated group was significantly highly increased than that of non-treated group. Our results represents that addition of acteoside to the IVM medium has a beneficial effect in physiology of porcine oocytes, providing a improved method for porcine oocytes in vitro. * This work was supported by a grant (Code# PJ008148) from BioGreen21 Program, Rural Development Administration, Republic of Korea.

During 2008 2010, 943 swine sera were collected from 45 farms located nationwide. Antibodies against porcine epidemic diarrhea virus (PEDV) were tested via serum neutralization antibody test (SNT) using PEDV-SN, which was adapted and propagated on the Vero cell monolayer with trypsin-free culture media supplemented with more than 10% fetal bovine serum (FBS). All 45 farms were shown to have at least one or more seropositive pig. Of the 943 swine sera that were tested, 931 sera were neutralizing antibody positive against PEDV. These high seroprevalence rates seemed to be due to vaccination or natural infection of PEDV. In a plaque reduction neutralization test (PRNT) using a swine serum showing SN titer of 1:32, a greater than 50% plaque reduction was observed in up to 160 times serum dilution.

The objective of this study was to examine the effect of thymidine treatment during in vitro maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group (p<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group (p<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group (p<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups (p<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.

The objective of this study was to determine the effects of E. coli isolated from porcine semen on sperm viability, motility, and semen pH. Semen samples were prepared using commercial extender, SeminarkPro (Noahbio Tech, Korea) that did not contain antibiotics. And 4 different levels of E. coli were artificially innoculated to semen with following concentrations; 4,000 of sperms with 1 of E. coli (T1), 400 with 1 (T2), 40 with 1 (T3), and 4 with 1 (T4). Semen samples were preserved at 17℃ for 5 days in semen storage box until analyzed by flowcytometer. Aliquots were subjected to measure the sperm viability (Live/Dead® stain), motility (mitochondrial function), and semen acidity (pH) from day 0 (day of semen collection) to day 5. Sperm motility and viability were significantly decreased (p<0.05) on day 0 (4 hrs after preservation at 17℃) in T3 and T4 compared to control groups and were significantly decreased (p<0.05) in all groups from day 3. Sample pH was acidic in T3 (6.90～6.86) and T4 (6.86～6.65) from day 3 to day 5 (p<0.05). On the other hand, sample pH was maintained 7.0～7.1 in control, T1, and T2 during the experimental period. Sperm motility and viability were significantly decreased from day 0 to day 5 compared to control in samples contaminated with E. coli above a value of 40:1 (20×106 sperm cells/ml : 5×105 cfu/ml). Even on day 1 in T4 and on day 3 in T3, semen pH was acidic probably due to the acidification of dead spermatozoa. These results suggest that E. coli contamination has a concentration-dependent detrimental effect on extended porcine semen quality.

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% CO2 and 95% air at 38℃. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 0.5～10.0 mM cysteine were 34.0±3.2%, 36.0±3.5%, 48.0±3.8%, 22.0±3.2%, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.5～5.0 mM NT for 48 hrs were 24.0±4.2%, 18.0±4.9%, 8.0±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±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±4.7%, 10.0±5.3%, 6.0±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±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 cysteine (32.0±3.2%) were increased than that of BP treatment.

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.

Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cell (iPS) experiments have generally demonstrated that a differentiated cell directly converts into a undifferentiated or pluripotent state. In SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor cell nuclei to the recipient cytoplasm of matured oocytes. Although nuclear reprogramming of cells by the ex-ovo methods is not always consistent or efficient, it has been suggested that a combination of nuclear reprogramming technique may improve the efficiency or frequency of normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from GV stage sturgeon's oocytes prior to their use as nuclear donors for SCNT will improve subsequent development. We reported a reversible permeabilization protocol with digitonin to deliver sturgeon oocyte exteact (SOE) to porcine fetal fibroblast cell nuclei ex ovo. Porcine fibroblasts were permeabilized by 4 μg/ml of digitonin for 2 min at 4℃ and then incubated in SOE for 7h at 15 18℃ followed by resealing of cell membrane. As results, no difference was observed in the number of fused couplets or the number of fused couplets that cleaved between the extract treated or control group. However, there was a significantly decrease in the percentage of fused couplets that developed to the blastocyst stage in the SOE treated group (p<0.05). Histone acetylation status was determined using an antibody to acetylation at lysine 9 on histone 3 (H3K9Ac). The intensity of H3K9Ac staining in 1-cell stage NT embryos was significantly increased when treated with the SOE (p<0.05), similar to that in 1-cell stage IVF embryos. In addition, porcine NT embryos reconstructed by using donor cell exposed to SOE prior to cell fusion significantly decreased developmental competence to the blastocyst stage but increased pluripotent gene expressions (Sox2, Nanog and Oct3/4) when compared with those in normal NT embryos (p<0.05).

Porcine beta casein promoter를 이용하여 형질전환 동물에서 유선특이적으로 목적 단 백질을 발현시킬 수 있는 pPBC 벡터를 구축하였으며 이 벡터를 이용하여 보다 높은 농 도의 목적 단백질을 발현 시킬 수 있도록 pPBC 벡터 개량을 시도하였다. pPBC 벡터의 5'arm 부위를 5428 bp, 4419 bp, 3378 bp의 길이로 잘라 서로 다른 크기의 5’arm을 갖 게 하였으며 5’arm의 5’ 쪽으로 CMV enhancer를 삽입하였다. 또한, porcine beta casein promoter 조절 하에 hGH(human growth hormone) 유전자를 발현하도록 하는 벡터를 구 축한 후 세포주 및 형질전환 마우스에서 발현 양상을 확인하였다. 개량된 pPBC 벡터를 이용하여 mouse mammary gland epithelial cell line HC11에서 luciferase assay를 통해 각각의 벡터에 대한 활성을 확인한 결과, 5’arm 길이가 가장 짧고 CMV enhancer가 삽입 된 CMV-pPBC p-3378 벡터에서 활성이 가장 높게 나타났다. 이 벡터를 이용하여 hGH 유전자와 mRNA를 안정화시켜 유전자의 발현을 증가시킨다고 알려진 woodchuch hepatitis virus post-transcriptional regulator element (WPRE)를 함께 삽입하여 CMV-pPBC p-3378-hGH-WPRE 벡터를 구축하였다. 이 벡터로 transgenic mice를 생산하여 유선을 포함한 여러 조직으로부터 hGH의 발현을 RT-PCR 방법을 확인하였다. 그 결과 liver와 lung에서는 아주 약하게 hGH가 발현을 하였으나 유선 조직에서 hGH가 가장 많이 발현 하는 것을 볼 수 있었다. 형질전환 마우스 유즙 내의 hGH 발현을 western과 ELISA를 이 용하여 확인한 결과, 유즙 내에는 약 22 kDa의 hGH가 존재 하였으며 ml 당 최고 50 100 ug의 농도로 hGH를 포함하고 있었다. 이러한 결과들은 개량한 pPBC 벡터가 유선특 이적인 발현을 하며, 유선 세포주에서는 높은 활성을 나타냈지만 형질전환 마우스의 유 즙에서는 개량전의 벡터 보다 낮은 수준의 목적 단백질을 생산하여 서로 상이한 결과를 나타내었다. 따라서 앞으로도 유선특이적 발현 벡터인 pPBC 벡터의 개량 연구를 계속 진행할 것이다.

Gene targeting is a genetic technique that utilizes homologous recombination between an engineered exogenous DNA fragments with the endogenous genome of an organism. In domestic animal, gene targeting has provided an important tool for producing Knock-out pig for GGTA1 gene to use xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse ES cells. So the application of gene targeting in somatic cells has been limited by its low efficiency. Recently, knock-out rat and mouse was generated by introducing nonhomologous end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knock-out and knock-in techniques in domestic animal is very important in biomedical research. In this present study, we investigated whether homologous recombination events occurs at cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. CMAH-targeted ZFN DNA and mRNA were purchased from SIGMA-Aldrich. CMAH neo targeting vector consists of the neomycin resistance gene as a positive selectable marker gene, 789 bp 5’ arm and 763 bp 3’ arm from Exon 8 of CMAH gene. For transfection, the targeting vector and ZFN DNA or mRNA were introduced into ear fibroblasts cells of Chicago miniature pig by electroporation. After selection of G-418, PCR analysis was performed using 213 colonies transfected with ZFN DNA or mRNA. As a result, 39 positive colonies were identified in colonies transfected with ZFN DNA or mRNA. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in the production of CMAH knock-out for xenotransplantation.