Optimization Of In Vitro Culture System Of Procine Preantral Follicles And Screening For Molecules Involved In Folliclar Antrum Formation

Antrum formation is a critical event of ovarian follicle development,and most follicles turn over during this process.However,molecules involving in antrum formation are still unkonwn.To optimize culture system of porcine preantral follicle and explore the mocular mechnism of follicular antrum formation not only could increase oocyte supply for resesrch and application,but also provide a reference for human preantral follicles in vitro culture.For these aims,three experiments were performed as below.Experiment 1: Aims at selecting an optimal culture method of procine PFs-OGCs in vitro growth.By porcine PFs-OGCs culture in serum adherent culture system and serum-free suspension culture system for 16.5 days,observed OGCs morphology under two culture methods and compared with PFs-OGCs antral formation rate,oocytes survival rate,found that both culture methods were capable of forming a cavity-like structure,but adherent culture was significantly better than suspension culture on survival rate and antral formation rate in procine PFs-OGCs cultured(P<0.05).Experiment 2: To selected optimal culture duration in procine PFs-OGCs serum adherent culture.By culturing procine PFs-OGCs in serum adherent culture system for16.5 d,18.5 d,20.5 d,statistics of PFs-OGCs antral formation rate,oocytes survival rate,oocytes diameter and oocytes mature rate after IVM culture.Moreover,compared oocytes cortical granules distribution,ROS level and oocytes ultrastructure with in vivo oocytes to oocyte quality.Oocytes diameters were greater when 18.5 d and 20.5 d of in vitro adherent culture compared with 16.5 d culture(P<0.05);The oocytes survival rate observed after16.5 d of in vitro culture was significantly greater than 20.5 d culture(P<0.05).The oocytes nuclear maturation rate of 18.5 d in vitro culture significantly greater than the other groups(P<0.05).Antral formation rate was no significant difference between groups(P>0.05).The results showed that 18.5 d was optimal duration in porcine PFs-OGCs cultured.In addition,oocyte cortical granules evenly distributed in the cytoplasm;ROS level;Golgi distributed near nucleus area but less with mature form;proportion of mature mitochondria were close to in vivo.However,the thickness of the zona pellucid is significantly less than in vivo oocytes(P<0.05);fat accumulate more,lipid droplet diameter significantly smaller(P<0.05).Experiment 3: Screen for molecules involved in follicular antrum formation.Separated procine PFs-OGCs and EAFs-OGCs for transcriptome sequencing(RNA-Seq).By bioinformatics analysis,found that a total of 31 genes were upregulated and 13 genesdownregulated in EAFs-OGCs group;Differentially expressed genes regulate follicle antral formation by ribosome and protein digestion and absorption signaling pathways.Moreover,selected HPGDS,STMY1,DPPA5,MCT4,FTH1,MHC,γ-TG,fibronectin of 8possible candidate genes related to follicle antral formation.Conclusion: 1.Serum adherent culture method of 18.5 d is an optimal of procine PFs-OGCs in vitro culture system;2.HPGDS,STMY1,DPPA5,MCT4,FTH1,MHC,γ-TG,fibronectin of 8 possible candidate genes related to follicle antral formation.