170 SUPPLEMENT OF GROWTH DIFFERENTIATION FACTOR 8 ON PORCINE OOCYTE DURING IN VITRO MATURATION ACTIVATES SMAD2 AND cAMP RESPONSIVE ELEMENT BINDING PROTEIN SIGNALING

Abstract

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β family 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 reactive oxygen species (ROS) levels, specific gene transcription levels in oocytes and cumulus cells (CC) after IVM, and specific protein expression and activation levels in matured CC by Western blotting. Each concentration (0, 1, 10, and 100 ng mL⁻¹) of GDF8 was added in maturation medium (TCM199) during process of IVM. Data were analysed by ANOVA followed by Duncan using SPSS (IBM Corp., Armonk, NY). Data are presented as the mean (replicate numbers) and differences were considered significant at P < 0.05. After 44 h of IVM, oocytes are mechanically denuded from CC with 0.1% hyaluronidase and only metaphase II stage oocytes are counted as nuclear matured oocytes. Each group of matured oocytes are stained by 2′,7′-dichlorodihydrofluorescein diacetate and the fluorescence was captured as graphic files under an epifluorescence microscope. The fluorescence intensities of oocytes were measured using Image J software (National Institutes for Health, Bethesda, MD). The groups treated with 10 and 100 ng mL⁻¹ of GDF8 showed significantly more than 10% decrease in intracellular ROS levels compared with other groups (5 times). To assess the effect of GDF8 on specific gene transcription level changes as a dose response during IVM, real-time PCR was performed. In matured oocytes, the developmental competence marker POU5F1, antioxidant enzymes regulator NRF2, and antiapoptosis gene BCL-2 mRNA transcription levels were significantly increased in the 10 ng mL⁻¹ treatment group compared with control (4 times). In CC, the 10 ng mL⁻¹ treatment groups showed significantly higher PCNA and NRF2 mRNA levels, and the 1 and 10 ng mL⁻¹ treatment groups observed significantly increased cumulus expansion-related genes HAS2, PTX3, and TNFAIP6 mRNA expression levels after IVM (4 times). To determine effect of GDF8 treatment during IVM, GDF8 downstream effector and oocyte ovulation-related protein expression and activation levels were analysed in CC after IVM by Western blotting. The 1 and 10 ng mL⁻¹ treatment groups showed significantly increased phosphorylated (P)-SMAD2 (1.25 and 1.23 times increased compared with the control) and cyclic adenosine monophosphate responsive element binding protein (CREB; 1.31 and 1.32 times increased compared with the control) activation levels (4 times). In conclusion, supplementation of 10 ng mL⁻¹ of GDF8 during IVM effectively increased the oocytes cytoplasmic maturation by reducing of intracellular ROS, and it seems correlated with significantly increased P-SMAD2, which is possibly related with induction of the cumulus cell expansion related genes expression and P-CREB while process of IVM.

This work was supported by a grant from the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ011288), Rural Development Administration, Republic of Korea.