The transcription factor RUNX1 affects the maturation of porcine oocytes via the BMP15/TGF-β signaling pathway

Abstract

Bone morphogenetic protein 15 (BMP15) is specifically expressed in oocytes in pigs at all stages from early stages to ovulation and has an important role in oocyte maturation. However, there are few reports on the molecular mechanisms by which BMP15 affects oocyte maturation. In this study, we identified the core promoter region of BMP15 using a dual luciferase activity assay and successfully predicted the DNA binding motif of the transcription factor RUNX1. The effect of BMP15 and RUNX1 on oocyte maturation was examined using the first polar body extrusion rate, a reactive oxygen species (ROS) assay and total glutathione (GSH) content at three time points of 12, 24 and 48 h of in vitro culture of porcine isolated oocytes. Subsequently, the effect of the transcription factor RUNX1 on the TGF-β signaling pathway (BMPR1B and ALK5) was further verified using RT–qPCR and Western blotting. We found that the overexpression of BMP15 significantly increased the first polar body extrusion rate (P < 0.01) and total glutathione content of oocytes cultured in vitro for 24 h and decreased reactive oxygen levels (P < 0.01), whereas interference with BMP15 decreased the first polar body extrusion rate (P < 0.01), increased reactive oxygen levels in oocytes cultured in vitro for 24 h (P < 0.01), and decreased glutathione content (P < 0.01). The dual luciferase activity assay and online software prediction showed that RUNX1 is a potential transcription factor binding to the core promoter region (−1203/−1423 bp) of BMP15. Overexpression of RUNX1 significantly increased the expression of BMP15 and oocyte maturation rate, while inhibition of RUNX1 decreased the expression of BMP15 and the oocyte maturation rate. Moreover, the expression of BMPR1B and ALK5 in the TGF-β signaling pathway increased significantly after overexpression of RUNX1, whereas their expression decreased after inhibition of RUNX1. Overall, our results suggest that the transcription factor RUNX1 positively regulates the expression of BMP15 and influences oocyte maturation through the TGF-β signaling pathway. This study provides a theoretical basis for further complementing the BMP15/TGF-β signaling pathway to regulate mammalian oocyte maturation.