Glycyrrhizic acid improves fertilization and embryonic development of porcine oocytes fertilized in vitro: focus on HMGB proteins in embryo development

High-mobility group box-1 (HMGB1) is a conserved nuclear protein that stabilizes the nucleosome and regulates gene transcription. Recent studies have reported that glycyrrhizic acid (GA), a HMGB1 inhibitor, blocks extracellular HMGB1 cytokine activity and has a protective effect in various diseases. This study was performed to investigate the effect of GA on in vitro fertilization (IVF) and embryo culture of pig oocytes. HMGB1 was detected by immunofluorescence in the boar sperm post-acrosomal sheath and sperm tail mid-piece, as well as in the nucleus of immature, germinal vesicle stage porcine oocytes, the cytoplasm of mature metaphase II oocytes, and in the embryonic nuclei. A related protein, HMGB2, was also observed in spermatozoa and oocytes, co-localizing with HMGB1. Both HMGB1 and HMGB2 were detected in the protein extracts of spermatozoa and oocytes by Western blotting. Total fertilization rates (mono and polyspermic) increased, and more spermatozoa were bound to the zona pellucida of the oocytes when the IVF medium was supplemented with 20 µM GA compared to the control (p<0.05). In the presence of 20 µM GA, there was a significant increase in the percentage of cleaved embryos, blastocyst formation, and the mean cell numbers per blastocyst (p<0.05). GA treatment increased porcine fertilization rates and improved embryo development in vitro, possibly by blocking the cell-survival-limiting activities of HMGB proteins. Thus, GA could be a suitable therapeutic candidate in assisted reproductive technologies.