Effect Of Phlorizin On Porcine Oocyte In Vitro Maturation And Development Of Parthenogenetic Embryos

Pig is a common animal in agricultural production.Pigs have been used as an animal models for studying organ xenotransplantation and human disease due to their physiological similarities to humans.IVP embryos plays an important role in studies of embryonic development and reproductive technologies.However,many negative environmental factors resulted in the quality of oocytes is lower in vitro than in vivo.The main reason may be that compared to in vivo matured oocytes,IVM oocytes are susceptible to effect of light,oxygen stress,and culture medium composition,which changed the metabolism in oocytes and leading to an imbalance in the production and degradation of ROS.Porcine oocytes contain a large amount of lipids,which are particularly sensitive to ROS.Excessive ROS might contribute to lipid peroxidation,mitochondrial dysfunction,DNA fragmentation and oxidative modification of proteins,which impairs oocyte quality and developmental competence.The dihydrochalcone phlorizin is a natural product and dietary constituent found in a number of fruit trees.It has been used as a pharmaceutical and tool for physiology research for over 150 years.Phlorizin's principal pharmacological action is to produce renal glycosuria and block intestinal glucose absorption through inhibition of the sodium–glucose symporters located in the proximal renal tubule and mucosa of the small intestine.Flavonoids,including chalcones such as phlorizin,are metabolites manufactured by all plant families.These polyphenolic compounds have antioxidant properties and scavenge superoxide anions,singlet oxygen,lipid peroxy radicals,and sequester metal ions through liganding.Apples,tea,red wine,and onions are the major dietary sources of flavonoids.Epidemiological observations suggest that flavonoid consumption varies inversely with the prevalence of coronary artery disease,and a number of studies suggest that phlorizin may partially account for these observations.Phlorizin has been shown to be a more potent inhibitor of lipid peroxidation than 17-? estradiol.Apple juice and apple extracts,containing phlorizin as 11 to 36% of total phenolic concentration,inhibit low-density lipoprotein oxidation.Additionally,phloretin(the aglucon of phlorizin)produces endothelium-independent relaxation of isolated coronary artery rings in organ baths.The aim of this work is to assess the effect of phlorizin on maturation in vitro and culture in vitro.In this study we used different concentrations of phlorizin(0?M,50?M,100?M,150?M)to treat oocytes and embryos during IVM and IVC.And detect the antioxidant capacity,totipotency and apoptosis of oocytes and embryos.Through experiments,we found that adding 100 ?M phlorizin during in vitro maturation can effectively increase the rate of parthenogenetic blastocyst activation in oocytes,increase the level of intracellular GSH,and reduce the level of intracellular ROS.Moreover,the relative expression levels of oxidative stress genes SOD,CAT and GPx were also significantly increased.At the same time,the relative expression levels of mitochondrial functional genes TFAM and PGC-1? also increased significantly.The addition of phlorizin decreased the relative expression of apoptosis-related genes CASPES3 and BAX,and increased the relative expression of anti-apoptosis-related genes BCL2.In the subsequent activation culture,the blastocysts obtained by phlorizintreated oocytes had higher expression of totipotent related genes,and the average total cell number of blastocysts was higher.Adding 100 ?M phlorizin in the embryo culture stage did not significantly increase the blastocyst rate,and the levels of intracellular GSH and ROS in the four-cell period were similar.However,the relative expression of blastocyst cell apoptosis-related genes CASPES3 and BAX was significantly reduced,and the expression of anti-apoptotic gene BCL2 was significantly increased.The blastocyst cells in the phlorizin treatment group had less apoptosis and more total cells.In summary,adding 100 ?M phlorizin to the medium during the in vitro maturation stage can improve the quality of oocytes and obtain more and better quality embryos.