Effects And Mechanisms Of Melatonin On Porcine Pre-antral Follicular Development In Vitro

Melatonin is an endogenous hormones that widely present in various tissuea and organs of the body's,and plays important role in modulating animal fertility.But the mechanisms of melatonin on folliculogenesis,especially on early state of folliculogenesis is still unclear.Porcine is an ideal animal model in medical research,focus on porcine pre-antral follicles research can not only compensate for the defect of animal models such as mice,but also provide supporting of methods and technology for human pre-antral follicles culture in vitro.For the complex effects of folliculogenesis in vivo,this study aimed to explore the effects and mechanisms of melatonin,a single factor on folliculogenesis by establishing pre-antral follicle culture system in vitro.We define to select smooth surface ovary which has no corpusluteum and densely distributed small antral follicles as standard by morphological index of the gilts' ovary.By accurately measure the diameter of follicle,oocyte and the antral formation of follicles,we find that the vast majority of follicles which diamater less than 300?m have no antral formation,and their oocyte diameter were all less than 90?m.We use serum contain anherent culture method culture oocyte-granulosa cells complexes derived from pre-antral follicles?PFs-OGCs?for 18.5 d and then in vitro maturation,obtained matured oocyte.Following parthenogenetic,we obtained parthenogenetic blastocysts and successfully established porcine serum contain adherent culture system.By comparing with the differences between the rate of recovery,maturation,parthenogenetic cleavage and blastocyst formation of different concentrations of melatonin on porcine PFs-OGCs culture in vitro,we find that melatonin at 10^-5 M can improve the rate of antral formation,parthenogenetic cleavage and blastocyst formation.By measuring the ROS levels and mRNA expression of anti-peroxidase related genes,we find that melatonin(10^-5 M)have no effects on ROS levels but enhance the activity of Gpx4 of PFs-OGCs culture in vitro.By co-treated PFs-OGCs with melatonin and Luzindole,a general melatonin receptor inhibitor,we find that Luzindole could neutralize the benefit effects of melatonin on PFs-OGCs culture on vitro.we also collect PFs-OGCs that were exposured to melatonin(10^-5 M)and cultured in vitro for 18.5 d for RNA-Seq analy,and finally we find that 71 genes were up-regulated and 1 gene was down-regulated q-PCR verified the trend of the genes differentially expression between control groups and melatonin treated groups,and the results were in accordance with RNA-Seq.we find that more genes were invovle in biological processes by Gene Ontology analysis.The rresults of KEGG Pathway analyse show that melatonin exert its function mainly by participating metabolic pathway,and oocyte meiosis and other pathways were also participated.At the same time,we examine the effects of melatonin on theca cells.We obtained high purity theca cells?Vimentin positive 93.8%;cytokeratin positive 6.7%and Factor ? positive 0.7%?by using microdissection-enzymic digestion and discontinuous gradient centrifugation methods.We find the existence of melatonin receptor 2?MT2?in theca cell?layers?by RT-PCR and immunohistochemical staining.Using MTT method to assay the cell viability of theca cells treated with melatonin(10^-3?10^-9M)for 48 h,we find that melatonin(10^-5?10--M)improve cell viability,but there is no significant difference between groups.Using RIA method measuring the hormone levels of theca cells treated with melatonin for 48 h,we find that melatonin(10^-5,10^-7 and 10^-9 M)could inhibit androstenedione production in a dose-dependent manner,but have no effects on progesterone productipon.q-PCR results show that melatonin significantly down-regulated the mRNA expression of steroidogenic enzymes related genes and Luzindole can only neutralize the effect of melatonin on the down-regulated mRNA expression of Hsd3(3,but not androstenedione production inhibitory action.Also,melatonin treatment can also up-regulated Gpx4 mRNA expression,but have no effects on ROS levels.In conclusion,we successfully established a stability culture system that support to obtain parthenogenetic blastocyst derived from PFs-OGCs.We evidenced that melatonin benefit PFs-OGCs development in vitro by receptors-mediated and enhance antioxidant capacity.The result of RNA-Seq suggest that the participation of melatonin on biological metabolism may be another important mechanism for melatonin function.We find that,MT2 exist in theca cells and receptor-mediated and enhance antioxidant capacity were involve in the effects of melatonin on theca cells androstenedione production inhibition.But the main cause of melatonin on theca cells androstenedione production inhibition was still unclear,need for further research.