Proteomic Analysis of Healthy and Atretic Porcine Follicular Granulosa Cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Sus Scrofa Domesticus
SUBMITTER: 
Ya Liu
PROVIDER: PXD021092 | iProX | Tue Aug 25 00:00:00 BST 2020
REPOSITORIES: iProX
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Project description:Ovarian follicular atresia is a natural physiological process, but its mechanism is not fully understood. In this study, a quantitative proteomic and phosphoproteomic of granulosa cell (GC) in the healthy (H), slightly atretic (SA), and atretic follicles (A) of porcine were performed by TMT labeling, enrichment of phosphopeptides and LC-MS/MS analysis. Altogether, 6,201 proteins were quantified, and 4,723 phosphorylation sites of 1,760 proteins were quantified. There are 24 (11 up, 13 down) and 50 (29 up, 21 down) proteins with FC>5 in H/SA and H/A, respectively. In addition, there are 20 (H/SA, up) and 39 (H/A, up) phosphosites with FC>7, which could serve as potential biomarkers to distinguish different quality categories of follicles. The results of western blotting and immunofluorescence indicated the reliability of the proteomic analysis. Further analysis of the differential expressed proteins (DEPs) and phosphorylated proteins (DEPPs) revealed some key proteins (e.g. MIF, beta catenin, integrin β2), key phosphosites (e.g. S76 of Caspase6, S22 and S636 of Lamin A/C), pathways (e.g. apoptosis, regulation of actin cytoskeleton pathway), transcription factors (e.g. STAT5A, FOXO1, BCLAF1), and kinases (e.g. PBK, CDK5, CDK12, AKT3) that involved in atresia process. Our proteomic and phosphoproteomic profiling and functional research comprehensively analyze the dynamic changes of protein expression and phosphorylation during follicular atresia, and gave a new explanation for the regulation of this process.
2021-09-09 | PXD020899 | Pride
Project description:Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state into a M-bM-^@M-^Xstem cellM-bM-^@M-^Y-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. We showed that follicular granulosa cells (GC), which have distinct functions in vivo, can dedifferentiate during culture in vitro and acquire multipotency. We investigated the dedifferentiation of GC using global gene expression analyses. Total RNA was isolated from GCs to DFOG cells at 5 time points during dedifferentiation (cultured in 0day, 1day, 2day, 4day and 7day). Each timepoint was performed in triplicate (ie, biological replicates). Using Affymetrix porcine genome array, we performed microarray time course experiments to analyze gene expression profiles during GC dedifferentiation.
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Project description:Goal:Circular RNAs (circRNAs) are thought to play important roles in multiple biological processes including apoptosis. In the ovary granulosa cell apoptosis plays an essential role in the follicular phase of the estrous cycle as it determines if the selected follicles will degenerate or escape this fate and ovulate. Whether circRNAs play a role in granulosa cell apoptosis is at present unknown. By investigating the potential role of circRNAs in the granulosa cell apoptotic cascade we hope to gain novel information regarding the regulation of antral follicular apoptosis using the porcine ovary as a model. Method: Ribosomal-depleted RNA-sequencing was performed to generate circRNA expression profiles from isolated mural porcine granulosa cells of healthy antral (HA) and atretic antral (AA) follicles, respectively. Results:In total, more than 9,632 circRNAs were identified, of which 62 circRNAs were differentially expressed (DE-circRNAs). GO and KEGG analysis of DE-circRNAs showed that apoptosis, cellular responses to stress and cell cycle pathways, were significantly enriched. Next the characteristics of DE-circRNAs, including back-splicing, RNase R resistance and stability were validated, and miRNA binding sites of DE-circRNAs were predicted. Conclusion: Based on these observations, we conclude that aberrantly expressed circRNAs and their targeted genes seem to be associated with granulosa cell apoptosis and thus antral follicular atresia.
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Project description:Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state into a M-bM-^@M-^Xstem cellM-bM-^@M-^Y-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. We showed that mature adipocytes (MA) and follicular granulosa cells (GC), which have distinct functions in vivo, can dedifferentiate during culture in vitro and acquire multipotency. We investigated the dedifferentiation mechanism of MA and GC using global gene expression analyses. Using Affymetrix porcine genome array, we compared global gene expression profiles during dedifferentiation to search for particular biological functions in genes of which expression intensities were increased or decreased by MA and GC dedifferentiation.
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Project description:Sma- and Mad-related protein 4 (SMAD4) is closely associated with the development of ovarian follicular. However, current knowledge of the genome-wide view on the role of SMAD4 gene in mammalian follicular granulosa cells (GCs) is still largely unknown. In the present study, RNA-Seq was performed to investigate the effects of SMAD4 knockdown by RNA interference (SMAD4-siRNA) in porcine follicular GCs. A total of 1025 differentially expressed genes (DEGs), including 530 upregulated genes and 495 downregulated genes, were identified in SMAD4-siRNA treated GCs compared with that treated with NC-siRNA. Furthermore, functional enrichment analysis indicated that upregulated DEGs in SMAD4-siRNA treated cells were mainly enriched in cell-cycle related processes, interferon signaling pathway, and immune system process, while downregulated DEGs were mainly involved in extracellular matrix organization/disassembly, pathogenesis, and cell adhesion. In particular, cell cycle and TGF-β signaling pathway were discovered as the canonical pathways changed under the SMAD4 silencing. Taken together, our data reveals SMAD4 knockdown alters the expression of numerous genes involved in key biological processes of the development of follicular GCs and provides a novel global clue of the role of SMAD4 gene in porcine follicular GCs.
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Project description:Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state into a ‘stem cell’-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. We showed that follicular granulosa cells (GC), which have distinct functions in vivo, can dedifferentiate during culture in vitro and acquire multipotency. We investigated the dedifferentiation of GC using global gene expression analyses.
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