163 Treatment of Growth Differentiation Factor 8 (GDF8) During Porcine Oocyte In Vitro Maturation Changed Transcriptional Pattern and AKT Signaling

Abstract

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 was to investigate the effects of GDF8 on porcine oocytes during in vitro maturation (IVM). We investigated specific gene transcription levels in oocytes and cumulus cells (CC) after IVM by realtime PCR array, and specific protein expression and activation levels in matured CC by Western blotting. To collect IVM oocytes, the cumulus–oocyte complexes (COC) were aspirated and matured in 500 μL of M199 containing 10% porcine follicular fluid and eCG and hCG for 22 h, and then cultured in M199 without hormones for 22 h. Each concentration (0, 1, 10, and 100 ng mL⁻¹) of GDF8 was included in M199 during whole process of in vitro maturation. Data were analysed by ANOVA followed by Duncan using SPSS (IBM/SPSS, Armonk, NY, USA). Data are presented as means, and differences were considered significant at P < 0.05. After 44 h of IVM, oocytes were mechanically denuded from CC with 0.1% of hyaluronidase, and then the separated oocytes and CC were sampled following each group. To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, a realtime PCR array was performed. In CC, the 1 and 10 ng mL⁻¹ GDF8 supplement groups showed that transcription levels of transcription co-factors CBP and SP1, cell metabolic regulator MAPK1, and cumulus expansion-related genes Has2, Cox-2, Ptx3, and Areg were significantly different from control when hierarchically clustered by euclidean distance with average linkage method after IVM. In matured oocytes, the 10 and 100 ng mL⁻¹ GDF8 supplement groups 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 different compared with control. To determine the effect of GDF8 supplement during IVM, the oocyte maturational regulator AKT protein expression and phosphorylation levels analysed in CC by Western blotting. The 10 and 100 ng mL⁻¹ supplement groups showed significantly increase phosphorylated (P)-AKT per total (T)-AKT (1.22 and 1.18 times higher than control) protein levels (P < 0.05). In conclusion, supplement of GDF8 during IVM activates FOXO homologue transcription and induces cumulus cell expansion via activation of AKT signalling in CC. During the process of IVM, the changes in transcriptional landscape in CC may result in accumulation of maternal factors and mitochondrial activation in oocytes.

This work was supported, in part, by a grant from the ‘the Next-Generation BioGreen 21 Program (Project No. PJ011288, PJ011077)’ Rural Development Administration and the ‘National Research Foundation of Korea Grant funded by the Korean Government (NRF-2016R1D1A1B03933191, NRF-2017R1A2B4002546)’, Republic of Korea.