Pig parthenotes were able to develop in vivo for 30 days with normal morphology. In pig, during blastocyst elongation between day 10 and 12 of gestation, estrogen production and secretion by conceptus increases, serving not only as the signal for maternal recognition of pregnancy, but also as a stimulus for the production of proteins and growth factors within the uterine environment that initiate implantation. Cloning efficiency is still very low regardless of species. To increase the productive efficiency of (transgenic, TG) clones, an advanced somatic cell nuclear transfer (SCNT) method may need. Here we report the productions of transgenic cloned pigs using cloned embryos and parthenotes simultaneously. Fibroblasts were isolated from an ear skin of a 10‐day‐old NIH miniature pig. The ear fibroblast cells were transfected with the alpha1,3‐ Galactosyltransferase knock‐out/human CD46 knock‐in (GalT KO/hCD46 KI). For SCNT, the TG somatic cells were used as donor cells. Immediately after fusion confirmation, the TG cloned embryos and parthenotes were transferred into both oviducts of surrogates. The mean number of TG cloned embryos and parthenotes was 137 (±15.2) and 123(±27.1), respectively. The pregnancy and delivery rate was (55.6%, 10/ 18) (44.4%, 8/18), respectively. Totally 19 GalT KO/hCD46 KI cloned piglets were delivered. Among them, 11 piglets were survived and 8 piglets were born stillbirth. The healthy 5 piglets are still survived.

Urokinas type plasminogen activator (uPA) has been used as a therapeutic agent for treating human diseases such as thrombosis. Attempts to transgenically overexpress the uPA in animal bioreactors have been hampered due to side effects associated with this functional protein hormone on homeostasis. Recently, chicken has been emerged as a potential candidate for use as bioreactor to produce proteins of pharmaceutical importance. Since this species has low homology uPA sequence with mammals, we hypothesized that chicken could be used as a potential bioreactor for production of human uPA. In this study, using replication‐defective Murine Leukemia Virus (MLV)‐based retrovirus vectors encapsidated with Vesicular Stomatitis Virus G Glycoprotein (VSV‐G), we attempted to make transgenic chicken expressing human uPA (huPA). The recombinant retrovirus was injected beneath the blastoderm of non‐incubated chicken embryos (stage X, at laying). After 21 days of incubation (at hatching), all of the 38 living chicks that assayed, were found to express the vector‐encoded huPA gene in various organs and tissues, which was under the control of the Rous Sarcoma Virus (RSV) or Cytomegalovirus (CMV) promoter. Using specific primer set for huPA, PCR and RTPCR analyses of gDNA isolated from these samples demonstrated these chickens were transgenic for huPA. Furthermore, successful germ line transmission of huPA transgene was confirmed and next generation whole body huPA transgenic chickens were also produced. We also assayed huPA protein titer in blood (17.1 IU/ml) and eggs (4.4 IU/ml) of whole body huPA transgenic chicken. Thus, our results demonstrated that chicken could be used as bioreactors to produce huPA.

The production of transgenic animals using somatic cell nuclear transfer (SCNT) has been widely described. A critical problem in the production of transgenic animals is the uncontrolled constitutive expression of the foreign gene which occasionally results in serious physiological disorders in the transgenic animal. In this study, we designed three different expression vectors that express the hEPO gene. hEPO is a hormone produced by the kidney that promotes the formation of red blood cells by the bone marrow. For the in vitro production of transgenic embryos, the different expression vectors were transduced into holstein ear fibroblast cells, respectively, and GFP expressed donor cells were transferred into enucleated oocytes, and then the reconstructed SCNT embryos were developed into pre-implantation stage. From three replicates, GFP expressed 112 transgenic SCNT embryos were produced. When their cleavage rate and blastocyst rate were compared with non-transgenic SCNT embryos, the results were presented into 73.2% vs. 76.9% and 26.8% vs. 30.6%, respectively, there were no differences. Also, total cell number and ICM cell numbers of day 8 blastocysts were statistically not different between the transgenic SCNT groups (120.6±7.9 and 31.4±8.2) and control SCNT group (128.3±4.8 and 35.3±4.0). The GFP expression levels were presented consecutively high during the culture of transgenic SCNT embryos. By analysis of semi-quantitative RT-PCR, the relative expression levels of hEPO mRNA and pluripotent gene were determined. These results demonstrated that the hEPO expressed transgenic bovine embryos can be efficiently produced in vitro by SCNT technique, while their potential of cloned animal production have to be examined in further study.

Despite of the absence of hyperacute rejection and acute humoral xenograft rejection, the organ graft of the a1,3-galactosyltransferase (GalT) gene knockouted (KO) and complement regulatory protein (CRP) expressing pig into a nonhuman primate is rejected by development of a thrombotic microangiopathy and/or a consumptive coagulopathy. Thus further introduction of genes to overcome the coagulation incompatibilities between pig and primate under GalT KO/CRP genetic background has been strongly suggested. CD73 (ecto-5'-nucelotidase) is an enzyme attached via a glycosyl phosphoinositol anchor to the extracellular membrane of endothelial cells, which catalyses the hydrolysis of adenosine triphosphate to adenosine. Loss of activity of CD73 results in activation and aggregation of platelets by a reduced capacity to convert nucleotides to adenosine. In previous study, we reported generation of GalT KO fibroblasts concurrently expressing membrane cofactor protein and produced cloned pigs by nuclear transfer of the fibroblast cells (1). In this study, we constructed a vector for expression of human CD73 under control of promoter of pig Icam2 gene expressed specifically at endothelial cells. This vector was introduced into porcine fibroblasts using the nucleofection technology, by which we had forty three fibroblasts clones carrying pIcam2- CD73 vector. Somatic cell nuclear transfer resulted in generation of two transgenic piglets survived.

Stem cell therapy is undoubtedly the most promising therapeutic approach for neurological disorders. Adipose tissue is ubiquitous and it can be easily harvested in large quantities under local anesthesia with little patient discomfort, making adipose tissue into the ideal large-scale source for research on clinical applications. In this study we monitored the neuronal cell differentiation potential of human adipocyte in the following condition; i) N2 medium containing 200 uM ascorbic acid (AA) and/or 10 uM flavonoid (F) and ⅱ) N2 medium containing AA and/or 10 ng/ml brain derived neurotrophic factor (BDNF) and/or, 200 ng/ml sonic hedgehog (SHH) plus 100 ng/ml fibroblast growth factor (FGF) 8. Adipose stem cells were cultured in above described differentiation condition for three weeks. RT-PCR analysis demonstrated that the mRNA levels of neuronal cell markers in differentiated adipose stem cells. Under the culture condition using N2 medium containing AA, the expression level of nestin (neural progenitor marker) m- RNA was high in all groups, while those of Neuro D, and LEP and FABP4 (adipocyte marker) mRNA were significantly decreased. Also, the addition of BDNF or SHH+FGF8 in N2 medium containing AA enhanced the neural cell differentiation from adipose stem cells, the expression level of Map2 (mature neuron) mRNA was increased, and that of TH (dopaminergic neuron marker) mRNA was high. In addition, we confirmed that the flavonoid addition has effect on the increase of Map2 expression. These results demonstrate that our designed culture condition has effect on the neural cell differentiation of adipose stem cells and this stimulatory effect may be further enhanced by transplantation.

Somatic cell nuclear transfer (SCNT) is an efficient technique which has been successfully applied to developmental biology, and resulted in the production of offspring from various species. It offers many opportunities in basic and medical research as well as endangered species preservation. On the other hand, embryonic stem (ES) cells are useful research tools for genetic engineering and developing disease models. In previous study, we established bovine IVF embryo derived ES cell line which can be grow indefinitely as undifferentiated cell state. In this study, we compared the effect of two different age cells (bovine ES cell; JNU-ibES-05 or adult ear fibroblast cell) on in vitro developmental potential of bovine SCNT embryo. To produce SCNT embryos, the ES cells or somatic cells were dissociated and transferred into enucleated MⅡ oocytes, and cleaved reconstructed embryos were cultured in CR1aa medium containing 10% FBS, 1 ug/ml epidermal growth factor (EGF) and 1 ug/ml insulin growth factor (IGF) for 8 days. In the result, blastocyst development rate was similar between ES cell treatment group and somatic cell treatment group, 27.7% (10/36) and 28.9% (11/ 38), respectively. However, there was particular difference in development speed from day 5 post SCNT, blastocyst expanding was 1 day faster in ES cell group than in somatic cell group. This difference was analyzed by semi-quantitative RT-PCR using pluripotency, growth and cell cycle gene markers. These results demonstrated that SCNT embryo using ES cell as a donor cell has better growth potential than somatic cell, and it will be a useful tool for a transgenic animal production.

It is known that oocytes can be activated without male contribution in vitro and develop to blastocysts which are used to isolate parthenogenetic embryonic stem (ES) cells. Differentiation capacity of the parthenogentic ES cells was rather lower than that of fertilized embryos derived ES cells, which might be the result of the absence of male genome. However, parthenogenetic ES cells might be useful research tool for genetic engineering and generating SCNT embryo derived ES cells. In our previous study, we reported that establishment of several bovine ES cell lines from in vitro fertilized (IVF) embryos named JNU-ibES. Based on this data, the objective of this study is to generate parthenogenetic ES cells and to examine their stem cell characteristics. Total 107 parthenogenetic embryos produced at day 8 or 9 were classified into their developmental stages (full expanded x 40, hatched x 67). For producing ES cells, ICM and trophetoderm-rich clumps were mechanically dissociated and were cultured on mitomycin- C treated mouse embryonic fibroblast feeder cell drop and covered with mineral oil in DMEM medium containing 20% FBS, 5 ng/ml basic FGF, 1% nonessential amino acids, and 0.55 mM b-mercaptoethanol. We obtained 20 primary parthenogenetic bovine ES (pbES)-like cell colonies. And pbES colony formation was higher in hatched blastocyst (25.4%, 16/67) than expanded blastocysts (10%, 4/40). Among those colonies, 5 pbES cell lines were successfully established and they were named as a series of JNU-pbES. These pbES cells were positively expresssed pluripotency markers such as Oct4, Nanog, TRA-1-81, SSEA-1 and alkaline phosphatase. This result demonstrated that the establishment efficiency and characteristics of pbES cell line was very similar to those of ibES cell line.

The event that occur in sperm during chemotaxis are only partly known. As a essential step of fertilization, sperm cells should undergo capacitation process inside female genital tracts. To understand the molecular event of calcium signals on sperm cells, Fluo 4 loaded spermatozoa was treated with follicular fluid. The motility of sperm was reduced by follicular fluids. Simultaneously, level of calcium in head and tail was also reduced for 5 10 second. The inhibition of sperm motility was believed as a reversible event, so the follicular fluid in graffiaan follicles in vivo could act as a selector on active spermatozoa that recover motility and calcium signals during ovulation. This suggested that the normal levels of calcium in sperm was also critical for active state of sperm cells and the follicular fluids during ovulation could inhibit the motility of sperm cell via calcium signaling.

The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. This study was to examine the in vitro neuron cell differentiation characteristics of our established human (h) iPS cells (IMR90-iPS-1~2) derived from human somatic cells. For the neuron differentiation, well grown hiPS colonies were recovered by collagenase treatment and then suspended cultured in a non-adherent bacteriological culture dish using human embryonic stem (hES) cell culture medium for 4 days. Embryoid bodies were plated and cultured in serum-free ITSFN (insulin/transferrin/selenium/fibronectin) medium for 8 days to select neural precursor cells. Then selected neuronal cells were dissociated, plated onto poly-L-ornithin/laminin coated dish at a concentration of 2 x 105 cells/cm2 and expanded in N2 medium containing 20 ng/ml bFGF, 200 ng/ml SHH and 100 ng/ml FGF-8 for 7 days. For the final differentiation step involved removing agents and culturing for 14 days in 20 ng/ml BDNF added N2 medium. In the neural precursor stage, >90% of nestin positive cells and >50% NCAM positive cells were obtained. Also, in final differentiation step, we confirmed the high percent (>80%) of mature neuron tubulin-β positive cells and approximately >20% of tyrosine hydroxylase positive cells. Also, these results were confirmed by RT-PCR. These results indicated that hiPS cells have potential to generate specific neuron differentiation and especially TH+ neuron was also can be obtained, and thus hiPS-derived neural cells might be an usable source for the study of neuro-degenerative disease.

There are replete numbers of reports which have apparently shown that established patterns of methylation are critical for normal mammalian development. DNA methyltransferase 1 (Dnmt1) gene contains three different isoform transcripts, Dnmt1s, Dnmt- 1o, and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1o is specific to oocytes and preimplantation embryos, whereas Dnmt1s is expressed in somatic cells. Here we determined that porcine Dnmt1o gene had differentially methylated regions (DMRs) in 5’-flanking region, while those were not found in the Dnmt1s promoter region. The methylation patterns of the porcine Dnmt1o/Dnmt1s DMRs were investigated using bisulfite sequencing and pyrosequencing analysis through all preimplantation stages from one cell to blastocyst stage in in vivo or somatic cell nuclear transfer (SCNT). The Dnmt1o DMRs contained 8 CpG sites, which located in ‒ 640 bp to ‒ 30 bp upstream region from transcription start site of the Dnmt1o gene. The methylation status of 5 CpGs within the Dnmt1o DMRs were distinctively different at each stage from one-cell to blastocyst stage in the in vivo or SCNT, respectively. 55.62% methylation degree of the Dnmt1o DMRs in the in vivo was increased up to 84.38% in the SCNT embryo, moreover, de novo methylation and demethylation occurred during development of porcine embryos from the one-cell stage to the blastocyst stage. However, the DNA methylation states at CpG sites in the Dnmt1s promoter regions were hypomethylated, and dramatically not changed through one-cell to blastocyst stage in the in vivo or SCNT embryos. In the present study, we demonstrated that the DMRs in the promoter region of the porcine Dnmt1o was well conserved, contributing to establishment and maintenance of genome-wide patterns of DNA methylation in early embryonic development.

The prediction of male fertility is of paramount importance for breeding animal herds when artificial insemination is applied. While the male fertility assays provide valuable quantitative data, they yield limited information concerning the functional competence of the spermatozoa. The objective of this study was to standardize a method for predicting in vivo fertility in bulls using the capacitation status that was assessed by chlortetracycline (CTC) staining. To optimize the capacitation process, sperm were treated with various concentrations of heparin (0, 10, 20, 50, and 100 μg/mL) and incubated for 10, 20, and 30 min each at 39℃ in 5% CO2. We found that maximum capacitation condition obtained from 10 μg /mL heparin treated sperm cells for 20 min (p<0.05). Optimized methods were used to determine the fertility of 17 batches of frozen bull semen representing a wide range of field fertility levels as indicated by non-return rates (NRR) (35.29% 93.18%). There was no significant correlation between NRR and the percentage of capacitated spermatozoa (B type) and non-capacitated spermatozoa (F type). However, acrosome reacted spermatozoa (AR type) was significantly correlated with NRR (p<0.01). To determine the normal range for the AR type, lower limits of the AR (%) were established as 23% for low fertility (NRR < 75%) using receiver operating characteristic curve. The overall accuracy of the assay was 88.24% for low fertility, sensitivity and specificity were 81.82 and 100%, respectively. These results indicate that capacitation status as measure by CTC staining is a useful predictor of male fertility. Therefore, low and high fertility bulls can be identified primarily by the functional capacitation status.

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on sperm capacitation status and sperm survival. Frozen epididymal sperm samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk- Tris-glycerol extender) were thawed in 37℃ water bath for 1 min. To rule out individual variation, 3 sperm samples were mixed after thawing. The mixed samples then were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities (300 X g and 700 X g) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Sperm capacitation status and sperm survival were evaluated using combined Hoechst 33258 and chlortertracycline fluorescence staining assay. The acrosome reacted spermatozoa (AR pattern), uncapaciated spermatozoa (F pattern) and sperm survival were significantly correlated with centrifugation time (p< 0.01). Significantly decreased F pattern observed as centrifugal time increased. As centrifugal time increased, spermatozoa with F pattern decreased and spermatozoa showing AR pattern increased. Moreover, the dead spermatozoa were significantly stimulated in time-dependent manner. However, there were no significant differences in various force of centrifugation and Percoll volume. These results suggest that only centrifugation time significantly affects sperm capacitation status and sperm survival.

Voltage-dependent anion channel (VDAC) is mitochondrial protein of all eukaryotes. It has been reported that VDAC is a large voltage-dependent channel, regulation of ion (including Ca2+), and transportation of various metabolites. Ca2+ is an important factor in sperm function. In our previous study, we found high frequency of VDAC2 expression in spermatozoa from low-fertility bulls. However, to date, there is limited information available on its effects on male fertility. Therefore this experiment was designed to evaluate the effects of VDAC and Ca2+ on sperm function in vitro. To achieve this, four treatment conditions were established with or without Ca2+ and VDAC inhibitor, namely, 4’-diisothiocyano-2,2’-disulfonic acid stilbene (DIDS). Spermatozoa from adult ICR were collected and released into modified Tyrode’s salt media. And then, they were incubated in the different media with or without Ca2+and DIDS for 90 min at 37℃ in 5% CO2. Intracellular pH ([pH]i) and Ca2+ ([Ca2+]i) were measured by their fluorescent indicators, 2,7-bicarboxyethyl-5,6-carboxy- fluorescein acetoxymethyl ester (BCECF- AM) and fura-2 AM, respectively. Western blot of extracted sperm proteins with an anti-phosphotyrosine antibody (pY20) was carried out to determine tyrosine phosphorylation after sperm incubation in different treatments. To evaluate the fertilizing ability after treatments, in vitro fertilization was performed. DIDS significantly decreased [Ca2+]i regardless of Ca2+. [pH]i was efficiently affected by the presence of Ca2+ and/or DIDS. However, the highest decrease of pH level was observed under the presence of DIDS and the absence of Ca2+ in culture condition. Tyrosine phosphorylated protein 1 was significantly different under all treatments. However, tyrosine phosphorylated protein 2 was not significantly different under the presence of DIDS. Fertilization rate was significantly decreased under the presence of DIDS. Blastocyst formation was significantly altered different to compare to control and each treatment group. Therefore it suggests that a voltage-dependent anion channel may involved paramount importance in regulation of male fertility.

Phosphorylation of proteins is a post-translational modification process which plays a significant role in a wide range of cellular processes. Addition or removal of phosphate groups result in conformational changes in proteins leading either to their activation or inactivation. Tyrosine phosphorylation of protein is associated with sperm function in several mammalian species. The control of this process may via the changes in cyclic adenosine monophosphate (cAMP); the changes in cAMP levels that occur in the spermatozoa regulate protein kinase A (PKA) activity which, in turn, leads to the tyrosine phosphorylation of protein substrates by either the activation of sperm tyrosine kinases and/or the inhibition of phosphoprotein phosphatases. Cyclic nucleotides, in particular, cAMP, are important regulators of various maturation events in sperm including capacitation and motility. Interestingly, some environmental chemicals (ECs) may exert broader endocrine disrupting effects through possible modulation of cAMP/PKA second messenger systems. Otherwise, because the mature spermatozoa are transcriptionally inactive, therefore the study of sperm proteins phosphorylation may permit more information about the agents and conditions affects on sperm function. In the present study, to examine the effect of ECs on human sperm function, human spermatozoa were incubated with a group of ECs represent a widespread chemicals in the environment bisphenol A (BPA, 100 μM), nonylphenol (NP, 10 μg/ml), 2,3,7,8-Tetrachlorodibenzo- pdioxin (TCDD, 2.5 μg/ml), genistein (Gen, 100 μM), and the following pesticides, dibromochloropropane (DBCP, 10 μg/ml), atrazine (Atraz, 500 μM), and diazinone (Diaz, 500 μM) for 6 hr at 37℃ in 5% CO2. Then, western blot analysis was carried out using extracted sperm proteins. Antiphosphorylation antibody (pY20) was used to determine sperm tyrosine phosphorylation after EDs treatment. The pY20 antibody labeled three common bands of approximately 90, 110, and 150 KDa. There were no significant differences between negative and positive control groups in regard to the tyrosine phosphorylated proteins except at the band with molecular weight 110 KDa. However, except Diaz treatment group, the other treatment groups showed decreasing (TCDD, Gen, NP, BPA, and DBCP) or increasing (Atraz) in the tyrosine phosphorylated proteins at least in one band from the three common bands studied. Therefore, it sug-gests that ECs effectively alters human sperm function and this effect may detect via their effect on tyrosine phosphorylation pattern.