Effects of Verbascoside, a bioactive compound from olive oil mill wastewater, on in vitro developmental potential and bioenergetic/oxidative parameters of prepubertal lamb oocytes

Juvenile in vitro embryo production from oocytes of prepubertal subjects is a promising assisted reproductive technology (ART). In farm animals, it reduces the generation gap and in human reproductive medicine, it helps to overcome premature ovarian failure. Oxidative stress in germ cell in vitro culture and cryopreservation procedures is an emerging problem in ART and studies aimed to evaluate the potentially antioxidant activity of natural bioactive compounds, such as phenolic compounds and polyphenols, present in plant-derived by-products would be beneficial to improve cryopreservation and in vitro culture protocols of prepubertal oocytes. Verbascoside (VB) or acteoside is a phenolic bioactive compound with known antioxidant activity, which is present in a good amount in olive oil mill wastewater (1). The aim of this study was to test the effects of VB on the developmental competence of ovine prepubertal oocytes and the bioenergetic/oxidative stress status of fresh and vitrified oocytes. Previously published methods were used for: VB extraction, purification, HPLC analysis and uptake by the cumulus-oocyte complex (1); in vitro oocyte maturation (IVM) and assessment of bioenergy redox biomarkers, such as mitochondrial distribution pattern and activity; intracellular reactive oxygen species (ROS) levels; mitochondria/ROS colocalization; catalase and total superoxide dismutase activities (2); oocyte vitrification (3); in vitro fertilization and embryo development (4). VB effects were tested at microM concentrations (1.03, 2.06 and 4.11). In fresh oocytes, 4.11 microM VB exerted pro-oxidant short-term effects, i.e. catalase activity increase and uncoupled increases of ooplasmic mitochondria and reactive oxygen species (ROS) specific fluorescence signals. It also induced pro-oxidant long-term effects, i.e reduced blastocyst formation rate, thus indicating an affected developmental competence of exposed oocytes. In vitrified oocytes, 1.03 microM VB increased ROS levels. Pro-oxidant VB effects in ovine prepubertal oocytes could be related to higher VB accumulation, which was found as being almost one thousand times higher than that reported in other cell systems in previous studies. Also, long exposure times of oocytes to VB, throughout the duration of IVM culture, may have contributed to significantly increase oocyte oxidation. In conclusion, VB, added at microM concentrations in a continuative 24 hours IVM exposure protocol, acts as a pro-oxidant molecule by impairing bioenergetic potential, oxidative status and embryo developmental competence of prepubertal lamb oocytes. Further studies are ongoing aimed to identify suitable conditions, lower concentrations and/or shorter exposure times, to figure out VB antioxidant effects in juvenile ARTs.