Growth differentiation factor8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. Overall of the current studies, the GDF8 is detected in oviduct fluid and uterus which led us to suggest that the GDF8 may effect on preimplantation embryonic development and act paracrine role to correlate with successful late-blastocyst implantation in in vivo. The purpose of this study is the effects of GDF8 on porcine parthenogenesis (PA) embryo development during in vitro culture (IVC). We were investigated the effect of GDF8 supplement during PA embryo IVC by cleavage and blastocyst formation rate and patterning analysis. Data were analyzed by on way ANOVA, followed by Tukey’s range test. Respectively 0.2, 2 and 20 ng/mL of GDF8 were added during IVC followed experiment design as control, 0.2, 2, and 20 GDF8 supplement groups. After 48h of embryo culture time, no significant difference was observed on cleavage rate from the different concentration (0, 0.2, 2, and 20 ng/ml) of GDF8 supplement groups (65.7%, 66.0%, 66.3%, and 65.8%, respectively). After 120h of embryo culture time, the 0.2 and 2 group showed significantly (p<0.05) higher blastocyst formation rate than control (40.4% and 36.4% VS 40.4%, respectively). In embryo developmental pattern analysis, the 0.2 ng/ml GDF8 supplement groups showed significantly higher (p<0.05) 2-3 cell cleavage- and early blastocyst pattern compared with control (12.0% and 10.4% VS 6.6% and 6.2%, respectively). However there are no significantly different pattern was observed in other groups. In conclusion, the 0.2 ng/ml of GDF8 supplementation during porcine PA embryo IVC significantly changed embryonic developmental patterns. However there are further studies are required such as analysis of blastocyst total number, specific gene transcription pattern, and ICM/TE rate to make clarify and support the conclusion.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated a specific gene transcription levels in oocytes and cumulus cells (CC) after IVM by realtime PCR arry, and specific protein expression and activation levels in matured CCs by western blotting. Each concentration (0, 1, 10, and 100 ng/ml) of GDF8 was added in maturation medium (TCM199) during process of IVM. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science). Data are presented as the mean and Differences were considered significant at P < 0.05. After 44 h of IVM, oocytes are mechanically denuded from CCs with 0.1% of hyaluronidase, and then the separated oocytes and CCs were sampled following each group. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, the realtime PCR array was performed. In CCs the 1- and 10 ng/ml of GDF8 supplement group showed the transcription co-factors CBP and SP1, cell metabolic regulator MAPK1, and cumulus expansion related genes Has2, Cox-2, Ptx3 and Areg transcription levels were significantly distinguished with control when hierarchically clustered by Euclidean distance with average linkage method after IVM. In matured oocytes the 10- and 100 ng/ml of GDF8 supplement group showed the maternal factors JMJD3 and Zar1, transcriptional regulator FOXO1, Sirt1 and Sirt2, mitochondrial activity factor Sirt3, ACSL3 and ACADL, anti-apoptosis gene BCL-2, and oocyte secrete factor BMP15 mRNA transcription levels were significantly distinguished compared with control. To determine effect of GDF8 supplement during IVM, the GDF8 down steam canonical regulator SMAD2/3 protein phosphorylation levels analyzed in CCs by western blotting. The 10- and 100 ng/ml supplement groups showed significantly increase phosphorylated (P)-SMAD3 (1.56 and 1.34 times higher than control) protein levels (P < 0.05). In conclusion, supplement of GDF8 during IVM activates FOXO homolog transcription and induced cumulus cells expansion via activation of SMAD3 signaling in CCs. While process of IVM, the transcriptional landscape changes in CCs may consequently result maternal factors accumulation and mitochondrial activation in oocytes.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β that has been identified as a strong physiological regulator. The purpose of this study is to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated a specific gene transcription levels in oocytes and cumulus cells (CC) after IVM, and protein kinase B (PKB) expression and activation levels in matured CCs by western blotting. Each concentration (0, 1, 10, and 100 ng/ml) of GDF8 was treated in maturation medium (TCM199) while process of IVM. Data were analyzed by ANOVA followed by Duncan using SPSS (Statistical Package for Social Science). Data are presented as the mean and differences were considered significant at P < 0.05. After 44 h of IVM, oocytes are mechanically denuded from CCs with 0.1% of hyaluronidase, and then the separated each group of oocytes and CCs were sampled. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, the realtime PCR was performed. In CCs, all of GDF8 treatment groups showed significantly higher CREB transcription regulator cbp mRNA and the 1- and 10 ng/ml treatment groups observed significantly increased cumulus expansion related genes areg, cox-2, has2, ptx3 and tnfaip6 transcription levels after IVM. In matured oocytes, the maternal factors jmjd3 and zar1, transcriptional regulator foxo1 and sirt1, mitochondrial activity factor sirt3 and acadl, and anti-apoptosis gene bcl-2 mRNA transcription levels were significantly increased in 1- and10 ng/mL of GDF8 treatment groups compared with control. To determine effect of GDF8 treatment during IVM, translation regulator PKB protein expression and phosphorylation levels were analyzed in CCs by western blotting. The 10 ng/ml treatment group showed significantly increased phosphorylated PKB (1.4 times higher than control) protein levels (P < 0.05). In conclusion, treatment 10 ng/ml of GDF8 during IVM activates CREB related transcription and induced cumulus cells expansion via activation of PKB signaling in CCs. The transcriptional landscape changes in CCs result maternal factors accumulation and mitochondrial activation in oocytes during IVM.

Recent 2 decades, including in vitro maturation (IVM), assisted reproductive technologies (ARTs) achieved noteworthy development. However the efficiency of ARTs with in vitro matured oocytes is still lower than that with in vivo oocytes. To overcome those limitations, many researchers attempted to adapt co-culture system during IVM and consequently maturation efficiency has been increased. The beneficial effects of applying co-culture system is contemplated base on communication and interaction between various somatic cells and oocytes, achievement of paracrine factors, and spatial effects of extracellular matrix (ECM) from somatic cell surface. The understanding of co-culture system can provide some information to narrow the gap between in vitro and in vivo. Here we will review current studies about issues for understanding cu-culture system with various somatic cells to improve in vitro maturation microenvironment and provide bird view and strategies for further studies.

A lot of works have been dedicated to clarify the reasons why the establishment of embryonic stem cells (ESCs) from pig is more difficult than that from mouse and human. Several concomitant factors such as culture condition including feeder layer, sensitivity of cell to cell contact, definitive markers of pluripotency for evaluation of the validity and optimal timing of derivation have been suggested as the disturbing factors in the establishment of porcine ESCs. Traditionally, attempts to derive stem cells from porcine embryos have depend on protocols established for mouse ESCs using inner cell mass (ICM) for the isolation and culture. And more recently, protocols used for primate ESCs were also applied. However, there is no report for the establishment of porcine ESCs. Indeed, ungulate species including pigs have crucial developmental differences unlike rodents and primates. Here we will review recent studies about issues for establishment of porcine ESCs and discuss the promise and strategies focusing on the timing for derivation and pluripotent state of porcine ESCs.

The influenza viruses can be spread from birds to people. In this process, the pig is the intermediate host, and this virus is amplified and produces many mutations in pigs. Therefore, we attempted to develop the influenza-resistant pigs for the study of the virulence test and the transgenic (TG) animal model for translational research. At interferon- α, γ treated cells, the porcine Mx2 protein has been observed near the nuclear envelope and inhibits influenza virus proliferation, but not in common cells. So, we tried to produce the Mx2 gene over-expressed pig by somatic cell nuclear transfer(SCNT).First, we establish the Mx2 gene over expressed cells for the preparation of the TG donor cells. Porcine fetal fibroblasts were transfected with cytomegalo virus vector which include the porcine Mx2 gene. The established transgenic cell was injected into the enucleated ooplasm for the production of the Mx2-TG cloned embryos. Total, 511 female TG porcine SCNT embryos (TG-SCNTembryos) were made. The 511 female TG-SCNT embryos were transferred to five surrogates. On 25 days after embryo transfer, two of female embryos’ surrogates were diagnosed as pregnant (pregnancy rate, 40%). On day 114, we obtained six cloned piglets and four mummies from two of female embryos’ surrogate. Being analyzed by PCR, all female piglets were not integrated with Mx2 gene. Hereby, we again established newly male MX-TG cell line for donor cell of SCNT. 427 male TG-SCNT embryos were made. From these, 38 of male TG-SCNT embryos were cultured in in vitro to confirm the developmental capacity of TG-SCNT embryos. Among these porcine SCNT-TG embryos, 26 embryos (68.4%) were cleaved. Finally, 5 transgenic porcine SCNT embryos (13.2%) developed to the blastocyst stage. All male TGSCNT blastocysts were proved to be integrated with Mx2 gene as PCR analysis. Therefore, we expect that newly birth male piglets will be targeted with MX2 gene. The remaining 389 male embryos were transferred to four surrogates. On 25 days after embryo transfer, one of male embryos’ surrogates was diagnosed as pregnant (pregnancy rate, 25%). Now, pregnant surrogate have maintained at 88 days after embryo transfer and shown more than eight embryonic sacs. This study has presented new possibilities of production of influenza virus resistant pig by SCNT for translational research. * This work was supported by a grant from Next-Generation BioGreen 21 program (# PJ008121), Rural Development Administration, Republic of Korea.

Porcine blastocyst’s quality derived from in vitro is inferior to in vivo derived blastocysts. In this study, to improve in vitro derived blastocyst’s quality and then establish porcine ESCs (pESCs), we treated in vitro fertilized (IVF) embryos and parthenogenetic activated (PA) embryos with three chemicals: porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), resveratrol (RES) and β-mercaptoethanol (β-ME). The control group was produced using M199 media in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The treatment group is produced using M199 with 2 μM RES in IVM and PZM5 with 10 ng/mL pGM-CSF, 2 μM RES and 10 μM β-ME in IVC. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. In total, 1210 embryos in PA and 612 embryos in IVF evaluated. As results, we observed overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (p<0.05) in the treatment groups (54.5%) compared to the control group (43.4%) in PA and hatched blastocysts rates in day 6 and 7 were also increased significantly. Total cell numbers of blastocyst were significantly higher (p<0.05) in the treatment group (55.1) compared to the control group (45.6). In IVF, hatched blastocysts rates in day 7 were increased significantly, too. After seeding porcine blastocyst, the attachment rates were higher in the treatment group (36.2% in IVF and 32.2% in PA) than the control group (26.6% in IVF and 19.5% in PA). Also, colonization rates and cell line derivation rates were higher in treatment group than control group. Colonization rates of control group were 10.8% in IVF and 2.4% in PA, but treatment group were 17.75% in IVF, and 13.1% in PA. And we investigated the correlation between state of blastocysts and attachment rate. The highest attachment rate is in hatched blastocyst (78.35±15.74 %). So, the novel system increased quality of porcine blastocysts produced from in vitro, subsequently increased attachment rates. The cell line derivation rates were 4.2% (IVF) and 2.4% (PA) in control group. In treatment group, they were 10.0% (IVF) and 7.2% (PA). We established 3 cell lines from PA blastocysts (1 cell line in control group and 2 cell lines in treatment group). All cell line has alkaline phosphatase activity and express pluri-potent markers. In conclusion, the novel system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of porcine blastocysts produced from in vitro, subsequently increased derivation rates of porcine putative ESCs.

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.