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Accumulating evidence shows that granulosa cells within both mammalian and avian ovaries have the ability to synthesize fatty acids through de novo lipogenesis and to accumulate triglycerides essential for oocyte and ovarian development. However, very little is known about the exact roles of key genes involved in the lipid metabolic pathway in granulosa cells. The goal of this study was to investigate the differential actions of diacylglycerol acyltransferase (DGAT) 1 and 2, which are recognized as the rate-limiting enzymes catalyzing the last step of triglyceride biosynthesis, in regulating lipid metabolism and steroidogenesis in granulosa cells of goose follicles at different developmental stages. It was observed that the mRNAs encoding DGAT1 and DGAT2 were ubiquitous in all examined granulosa cell layers but exhibited distinct expression profiles during follicle development. Notably, the mRNA levels of DGAT1, DGAT2, FSHR, LHR, STAR, CYP11A1, and 3βHSD remained almost constant in all except for 1-2 follicles within the 8-10 mm cohort, followed by an acute increase/decrease in the F5 follicles. At the cellular level, siRNA-mediated downregulation of DGAT1 or DGAT2 did not change the amount of lipids accumulated in both undifferentiated- and differentiated granulosa cells, while overexpression of DGAT2 promoted lipid accumulation and expression of lipogenic-related genes in these cells. Meanwhile, we found that interfering DGAT2 had no effect but interfering DGAT1 or overexpressing DGAT2 stimulated progesterone secretion in undifferentiated granulosa cells; in contrast, interference or overexpression of DGAT1/2 failed to change progesterone levels in differentiated granulosa cells but differently modulated expression of steroidogenic-related genes. Therefore, it could be concluded that DGAT1 is less efficient than DGAT2 in promoting lipid accumulation in both undifferentiated- and differentiated granulosa cells and that DGAT1 negatively while DGAT2 positively regulates progesterone production in undifferentiated granulosa cells., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

FASN plays a critical role in lipid metabolism, which is involved in regulating ovarian follicular development. However, the molecular mechanisms of how FASN regulate the function of ovarian follicular cells still remain elusive. In this study, by overexpression or interference of FASN in pre-hierarchical follicle granulosa cells (phGCs) and hierarchical follicle granulosa cells (hGCs), we analyzed their effects on the granulosa cell transcriptome and metabolome profiles using RNA-Seq and LC-MS/MS, respectively. The results showed that overexpression of FASN promoted proinflammatory factors expression by activating TLR3/IRF7 and TLR3/NF-κB pathways in phGCs, but only by activating TLR3/IRF7 pathways in hGCs. Then, necroptosis and apoptosis were triggered through the JAK/STAT1 pathway (induced by inflammatory factors) and BAK/caspase-7 pathway, respectively. The combined analysis of the metabolome and transcriptome revealed that FASN affected the demand of GCs for 5-hydroxytryptamine (5-HT) by activating the neuroactive ligand-receptor interaction pathway in two categorized GCs and only altering the metabolic pathway of tryptophan in phGCs, and ultimately participated in regulating the physiological function of geese GCs. Taken together, this study showed that the mechanisms of FASN regulating the physiological function of geese phGCs and hGCs were similar, but they also had some different characteristics. [ABSTRACT FROM AUTHOR]

Stearoyl-CoA desaturase (SCD) is a key enzyme catalyzing the rate-limiting step in monounsaturated fatty acids (MUFAs) production. There may be a mechanism by which SCD is involved in lipid metabolism, which is assumed to be essential for goose follicular development. For this reason, a cellular model of SCD function in goose granulosa cells (GCs) via SCD overexpression and knockdown was used to determine the role of SCD in GC proliferation using flow cytometry. We found that SCD overexpression induced and SCD knockdown inhibited GCs proliferation. Furthermore, ELISA analysis showed that SCD overexpression increased the total cholesterol (TC), progesterone, and estrogen levels in GCs, while SCD knockdown decreased TC, progesterone, and estrogen levels (p < 0.05). Combining these results with those of related multi-omics reports, we proposed a mechanism of SCD regulating the key lipids and differentially expressed gene (DEGs) in glycerophospholipid and glycerolipid metabolism, which participate in steroidogenesis mediated by the lipid droplet deposition in goose GCs. These results add further insights into understanding the lipid metabolism mechanism of goose GCs. [ABSTRACT FROM AUTHOR]

Background: Despite their important functions and nearly ubiquitous presence in cells, an understanding of the biology of intracellular lipid droplets (LDs) in goose follicle development remains limited. An integrated study of lipidomic and transcriptomic analyses was performed in a cellular model of stearoyl-CoA desaturase (SCD) function, to determine the effects of intracellular LDs on follicle development in geese. Results: Numerous internalized LDs, which were generally spherical in shape, were dispersed throughout the cytoplasm of granulosa cells (GCs), as determined using confocal microscopy analysis, with altered SCD expression affecting LD content. GC lipidomic profiling showed that the majority of the differentially abundant lipid classes were glycerophospholipids, including PA, PC, PE, PG, PI, and PS, and glycerolipids, including DG and TG, which enriched glycerophospholipid, sphingolipid, and glycerolipid metabolisms. Furthermore, transcriptomics identified differentially expressed genes (DEGs), some of which were assigned to lipid-related Gene Ontology slim terms. More DEGs were assigned in the SCD-knockdown group than in the SCD-overexpression group. Integration of the significant differentially expressed genes and lipids based on pathway enrichment analysis identified potentially targetable pathways related to glycerolipid/glycerophospholipid metabolism. Conclusions: This study demonstrated the importance of lipids in understanding follicle development, thus providing a potential foundation to decipher the underlying mechanisms of lipid-mediated follicle development. [ABSTRACT FROM AUTHOR]

Lipid metabolism participates in regulating the functions of granulosa cells (GCs), which is important for follicular development. In this experiment, goose GCs from pre-hierarchical follicles and hierarchical follicles were selected to be the model for studying the putative regulatory role of lipid metabolism in apoptosis and steroidogenesis, through overexpression and interference with fatty acid synthase (FASN). When FASN was overexpressed, the lipid accumulation was increased in hierarchical GCs (hGCs) and it was increased in the two categorized GCs when FASN was interfered. In addition, the apoptosis of the two categorized GCs was increased when FASN was overexpressed, and their progesterone production was decreased when FASN was interfered. The results of qRT-PCR showed that, when FASN was overexpressed, the expression level of CYP11A1 was decreased in pre-hierarchical GCs (phGCs), while the expression levels of SCD1 , DGAT2 , APOB , and StAR were increased in hGCs. When FASN was interfered, the expression levels of CPT-1 , DGAT2 , and StAR were decreased whereas the expression level of CYP11A1 was increased in phGCs, and the expression levels of CPT-1 , SCD1 , and StAR were decreased in hGCs. These results not only identify the different effects of manipulated FASN expression on lipid metabolism of goose phGCs and hGCs but also demonstrate that FASN -mediated lipid metabolism plays an important role in regulating apoptosis and steroidogenesis of in vitro cultured goose GCs. [ABSTRACT FROM AUTHOR]

The proliferation and steroid hormone synthesis of granulosa cells (GCs) are essential for ovarian follicle growth and ovulation, which are necessary to support the normal function of the follicle. Numerous studies suggest that miRNAs play key roles in this process. In this study, we report a novel role for miR-10a-5p that inhibits ovarian GCs proliferation and progesterone (P4) synthesis in chicken. Specifically, we found that miR-10a-5p significantly decreased the P4 secretion by quantitative real-time PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot. Moreover, we observed that miR-10a-5p can inhibit the proliferation of chicken GCs through the investigation of cell proliferation gene expression, cell counting kit 8 (CCK-8), cell cycle progression, and 5-ethynyl-2′-deoxyuridine (EdU) assay. Then we screened a target gene MAPRE1 of miR-10a-5p, which can promote P4 synthesis and proliferation of GCs. To explore how miR-10a-5p affects cell cycle by MAPRE1, we investigated the interaction between MAPRE1 and cyclin-dependent kinase 2 (CDK2) by Co-Immunoprecipitation (Co-IP), and then we found that MAPRE1 can form a complex with CDK2. In addition, miR-10a-5p was found to inhibit CDK2 expression by repressing the expression of MAPRE1. Overall, our results indicate that miR-10a-5p regulates the proliferation and P4 synthesis of chicken GCs by targeting MAPRE1 to suppress CDK2. • miR-10a-5p targets MAPRE1 in chicken. • miR-10a-5p regulates chicken granulosa cell proliferation and progesterone synthesis. • MAPRE1 regulates chicken granulosa cell proliferation and progesterone synthesis. • MAPRE1 interacts with CDK2 in chicken. • miR-10a-5p inhibits chicken granulosa cell proliferation and progesterone synthesis by targeting MAPRE1 to suppress CDK2. [ABSTRACT FROM AUTHOR]

The normal development of follicles determines the reproductive performance of females. Granulosa cells (GC) play crucial roles in follicular maturation. Numerous studies have shown that miRNAs are involved in the regulation of GC. According to our previous sequencing data, gga-miR-146b-3p was differentially expressed in normal and atretic chicken follicles. In this study, we verified that gga-miR-146b-3p attenuated proliferation and autophagy but promoted apoptosis in chicken GC. Threonine kinase1 (AKT1), a key member of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, was predicted to be a target gene of gga-miR-146b-3p via bioinformatic analysis. Dual-luciferase reporter gene assays were used to determine target relationships. Moreover, knockout of AKT1 decelerated proliferation and autophagy while accelerating the apoptosis of GC. However, overexpression of AKT1 reversed these results. In summary, our results demonstrated that gga-miR-146b-3p repressed the proliferation and autophagy of chicken GC while up-regulating apoptosis by targeting AKT1 through the PI3K/AKT signaling pathway. These findings may provide great insights for further exploration of the molecular regulation of gga-miR-146b-3p and AKT1 on the functions of GC during folliculogenesis. • miR-146b-3p targets AKT1 in chickens. • miR-146b-3p regulates chicken granulosa cell proliferation, apoptosis and autophagy. • AKT1 regulates chicken granulosa cell proliferation, apoptosis and autophagy. • miR-146b-3p regulates granulosa cell proliferation, apoptosis and autophagy via PI3K/AKT signaling pathway. [ABSTRACT FROM AUTHOR]