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.