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.

Abstract
INTRODUCTION
MATERIALS AND METHODS
    Preparation of boar spermatozoa
    Collection and in vitro maturation of pig oocytes
    In vitro fertilization and culture of pig oocytes
    Evaluation of fertilization and embryonic development
    Immunofluorescence
    Western blot
    Statistical analysis
RESULTS
    Localization of high-mobility group box-1 and high-mobility group box-2 in germcells by immunofluorescence
    Identification of high-mobility group box-1 and high-mobility group box-2 in spermatozoaand oocyte extracts by Western blot analysis
    Effect of glycyrrhizic acid on porcine oocyte fertilization in vitro
    Effect of glycyrrhizic acid on the embryonic development in vitro
DISCUSSION
REFERENCES

• Sang-Myeong Lee(Laboratory of Veterinary Virology, College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Korea)
• Malavi Arachchi Gamage Nayodya Dananjanee Malavi Arachchi Gamage(Department of Agricultural Education, College of Education, Sunchon National University, Suncheon 57922, Korea)
• Young-Joo Yi(Department of Agricultural Education, College of Education, Sunchon National University, Suncheon 57922, Korea) Corresponding author
• Wijesooriya Mudhiyanselage Nadeema Dissanayake(Department of Agricultural Education, College of Education, Sunchon National University, Suncheon 57922, Korea, Department of Animal Science and Biotechnology, College of Agriculture and Life Sciences, Chungnam National University, Daejeon 34134, Korea)