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Quantitative proteomics of lipid droplets from goose hierarchical granulosa cells and pre-hierarchical granulosa cells

ABSTRACT: The lipid metabolism of granulosa cells plays a pivotal role in the development of goose follicles. However, the mechanisms by which lipid droplet-associated proteins regulate lipid droplets and influence cellular lipid metabolism remain poorly understood. To address this, our study systematically identified lipid droplet-associated proteins in goose follicular granulosa cells and analyzed these proteins at five distinct time points, in both pre-hierarchical granulosa cells and hierarchical granulosa cells, thereby obtaining relevant information on lipid droplet-associated proteins.

INSTRUMENT(S): DIA-NN

ORGANISM(S): Anser Cygnoides

TISSUE(S): Ovarian Follicle, Granulosa Cell

SUBMITTER: Xin Yuan

LAB HEAD: Xin Yuan

PROVIDER: PXD061272 | Pride | 2025-03-26

REPOSITORIES: Pride

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			Action	DRS
	Astral-1_FPEP230070151-1A.raw	Raw
	Astral-1_FPEP230070152-1A.raw	Raw
	Astral-1_FPEP230070153-1A.raw	Raw
	Astral-1_FPEP230070154-1A.raw	Raw
	Astral-1_FPEP230070155-1A.raw	Raw

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Project description:The forkhead box transcription factor FOXO1 is highly expressed in granulosa cells of growing follicles but is down-regulated by FSH in culture or by LH-induced luteinization in vivo. To analyze the function of FOXO1, we infected rat and mouse granulosa cells with adenoviral vectors expressing two FOXO1 mutants: a gain-of-function mutant FOXOA3 that has two serine residues and one threonine residue mutated to alanines rendering this protein constitutively active and nuclear, and a FOXOA3-mutant DNA-binding domain (mDBD) in which the DBD is mutated. The infected cells were then treated with vehicle or FSH for specific time intervals. Infection of the granulosa cells was highly efficient, caused only minimal apoptosis, and maintained FOXO1 protein at levels of the endogenous protein observed in cells before exposure to FSH. RNA was prepared from control and adenoviral infected cells exposed to vehicle or FSH for 12 and 24 h. Affymetrix microarray and database analyses identified, and real time RT-PCR verified, that genes within the lipid, sterol, and steroidogenic biosynthetic pathways (Hmgcs1, Hmgcr, Mvk, Sqle, Lss, Cyp51, Tm7sf2, Dhcr24 and Star, Cyp11a1, and Cyp19), including two key transcriptional regulators Srebf1 and Srebf2 of cholesterol biosynthesis and steroidogenesis (Nr5a1, Nr5a2), were major targets induced by FSH and suppressed by FOXOA3 and FOXOA3-mDBD in the cultured granulosa cells. By contrast, FOXOA3 and FOXOA3- mDBD induced expression of Cyp27a1 mRNA that encodes an enzyme involved in cholesterol catabolism to oxysterols. The genes up-regulated by FSH in cultured granulosa cells were also induced in granulosa cells of preovulatory follicles and corpora lutea collected from immature mice primed with FSH (equine choriogonadotropin) and LH (human choriogonadotropin), respectively. Conversely, Foxo1 and Cyp27a1 mRNAs were reduced by these same treatments. Collectively, these data provide novel evidence that FOXO1 may play a key role in granulosa cells to modulate lipid and sterol biosynthesis, thereby preventing elevated steroidogenesis during early stages of follicle development. Experiment Overall Design: Immature rat granulosa cells were affected with either control (GFP) or FOXO1 mutant adenovirus (FOXOA3 and FOXOA3-mDBD) for 4 hours. Then the cells were treated with either FSH or vehicle for 24 hours. The gene expression profiles for the four treatment samples (control, control +FSH, FOXOA3 + FSH, FOXOA3-mDBD + FSH) were compared using Rat Genome 230.2 Arrays with one array per sample.

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Quantitative proteomics of lipid droplets from goose hierarchical granulosa cells and pre-hierarchical granulosa cells

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