Effects Of Melatonin-mediated Lipid Metabolism On The In Vitro Development Of Porcine Cumulus Oocyte Complexes

Matured oocytes with high quality play important role on clinical assisted reproductive therapy and reproductive science,by In vitro maturation(IVM).During the development of cumulus-oocyte complexes(COCs),each biological event is dependent on energy support,and lipid metabolism provides much more energy than glucose metabolism.Thus,Lipid metabolism has attracted widely interests in the field of basic research.Melatonin is a kind of indole hormone secreted by pineal cells in the brain of mammals in the dark environment,which can improve sleep,remedy neurological diseases,enhance immunity and regulate metabolism.Studies have shown that melatonin can promote in vitro maturation of oocytes in many species,and can regulate lipid metabolism in oocytes.However,the specific regulation mechanism of melatonin on lipid metabolism during the development of porcine cumulus oocyte complex is still unclear.Therefore,here the regulation of melatonin on lipid metabolism in porcine cumulus-oocyte complex was studied.The results showed that during the in vitro maturation of porcine oocytes,the addition of exogenous melatonin had no effect on the in vitro maturation rate,but it could promote the expansion of cumulus cells and increase the blastocyst rate and number of blastocysts by PA.By staining lipid droplets(LDs),fatty acids and ATP in oocytes,it was found that melatonin decreased the size of lipid droplets and increased the content of lipid droplets,fatty acids and ATP.And,the results of immunofluorescence showed that melatonin significantly increased the protein level of growth differentiation factor-9(GDF9)and bone morphogenetic protein(BMP15),and promoted the expression of lipolytic enzymes and lipogenic factors in pig oocytes.expression.We also found that after adding melatonin,the melatonin receptor in the cumulus cells was activated.The downstream of melatonin receptor 2,protein kinase A(PKA)in the cumulus cells was also activated.The PKA signaling pathway is activated,and lipolytic enzymes are activated,showing the lipid decomposition becomes more active,and a large amount of lipid droplets,fatty acids,and ATP are produced in cumulus cells and oocytes.Furthermore,when melatonin receptor antagonists are used to inhibit melatonin receptor 1 and melatonin receptor 2 in cumulus cells,the promotion of lipid metabolism by melatonin was inhibited.However,when only melatonin receptor 2 was specifically inhibited,the lipid metabolism level in oocytes and cumulus cells has no significant difference from the control group.In oocytes,the expression of the fatty acid transporter CD36 was greatly enhanced by adding melatonin,indicating that a large amount of fatty acids produced in cumulus cells may be transported to the oocytes.In summary,we demonstrate that in the porcine cumulus oocyte complex,melatonin can activate lipolytic enzymes by activating the PKA signaling pathway mediated by the melatonin receptor 2,to promote lipolysis and release a large amount of fatty acids and ATP,and improve the quality of the oocytes.The study are help to reveal the mechanisms of oocyte maturation,and provide reference for clinical assisted reproductive therapy.