Mechanism Of The SCD Gene Regulated Lipid Metabolism In Goose Follicle Granulosa Cell

There is a complex and delicate regulatory network during the avian follicle maturation and ovulation,the gradual maturation of follicle is accompanied yolk deposition,hormonal change and cell apoptosis.Among them,yolk deposition is a key step in the follicle maturation,it’s generally believed that most of yolk are synthesized by the liver and transported into the oocytes,however,previous studies have confirmed that de novo lipogenesis occurs in goose granulosa cell(GC),numerous of intracellular lipid droplet(LD)formation,and stearoyl-Co A desaturase(SCD)plays an important role in regulated the metabolic of follicular development,but the relevant research is still very limited.Therefore,in this study,the geese(from a healthy Tian Fu maternal line)used as the experiment object,to explore the characteristics of the expression pattern of SCD gene in granulosa layer and theca layer during the follicle development and expression trend of SCD gene and its enzyme activity in primary culture GC of preovulatory follicles,SCD gene overexpression and knockdown vector were constructed and transfected GC,to analyze the changes of intracellular metabolites by metabolomics,and explore the mechanism of SCD gene regulating lipid metabolism in GC elucidated by combined analysis of lipidomics and transcriptomics.The main results of this study are as follows:(1)The characteristics of the expression pattern of SCD gene in granulosa layer and theca layer during the follicle development showed that SCD was primarily expressed in the granulosa layer,the expression was highest in F1 GC.The expression pattern of SCD in cultured GC of preovulatory follicles for 7 d in vitro showed that a time-dependent oscillation pattern of SCD expression,and SCD enzyme activity showed a similar trend.(2)Overexpression and knockdown of SCD,a total of 333 intracellular metabolites were identified,and confirmed cholesterol and pantothenol as maximum fold change metabolite,35 pathways were screened to be related to lipid metabolism after pathway enrichment analysis.(3)Overexpression and knockdown of SCD,a total of 662 species of lipids were identified,pathway enrichment analysis found that the greatest impact on glycerophospholipid metabolism,this lipid-related pathway contains 6 representative lipids:PC(16:0/20:3),PC(18:1/18:4),PC(18:2/20:3),LPC(18:3/0:0),PE(22:2/12:0)and PS(20:4/20:0).The follow is the glycerolipid metabolism,which contains two representative lipids: FFA(22:2)and TG(14:0/22:0/22:2).(4)Overexpression of SCD,a total of 16 DEGs were assigned to lipid-related GO terms;knockdown of SCD,a total of 56 DEGs were assigned to lipid-related GO terms.Analysis by integrating two-omics datasets showed that lipid and DEGs were enriched in glycerolipid metabolism,glycerophospholipid metabolism,glycine,serine and threonine metabolism,and sphingolipid metabolism associated with lipid metabolism in the SCD overexpression and knockdown.However,including fatty acid degradation,fatty acid elongation,inositol phosphate metabolism,phosphatidylinositol signaling system,alpha-linolenic acid metabolism,arachidonic acid metabolism,and linoleic acid metabolism were enriched in the SCD knockdown.(5)GC contained numerous internalized LD that were generally spherical in shape and dispersed throughout the cytoplasm by confocal microscopy.SCD overexpress significantly stimulated accumulation of LD,stimulated intracellular TC synthesis,and significantly stimulated extracellular TC secretion;However,knockdown SCD significantly inhibited accumulation of LD,inhibited intracellular TC synthesis,and inhibited extracellular TC secretion.The intracellular steroidogenesis(progesterone and estrogen)and extracellular secretion were increased in overexpression of SCD,and decreased in knockdown of SCD.Cell cycle analysis by flow cytometry was showed that the cell ratio of G2/M + S phase was increased in overexpression of SCD,while the cell ratio of G2/M +S phase was decreased in knockdown of SCD.In summary,this study analyzed that SCD gene mainly regulate the key lipids and DEG of glycerophospholipid metabolism and glycerolipid metabolism,and participating in steroidogenesis mediated via the lipid droplet deposition in GC,which laid a foundation for further understanding the lipid metabolism mechanism of GC.