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FSH Promotes Progesterone Synthesis by Enhancing Autophagy to Accelerate Lipid Droplet Degradation in Porcine Granulosa Cells.

ABSTRACT: Little is known about the molecular relationships among follicle stimulating hormone (FSH), lipid droplet (LD) degradation, and autophagy. In this study, we aimed to investigate the pathway by which FSH regulates autophagy and the potential role of autophagy in progesterone production. Our results revealed that FSH stimulated progesterone production in mammalian follicular granulosa cells (GCs) through a non-canonical pathway. In porcine secondary follicles cultured in vitro, FSH treatment increased the level of the autophagic marker, LC3-II, as well as increased the number of autophagic vacuoles in GCs. The underlying molecular mechanism and biological functions were then investigated in porcine GCs. Our results demonstrated that FSH could upregulate Beclin1 levels in porcine GCs; however, this effect was blocked by LY294002 (a PI3K/AKT inhibitor) and SP600125 (SAPK/JNK inhibitor). Further research confirmed that the transcriptional factor, c-Jun, was phosphorylated by FSH, then translocated into the nucleus from the cytoplasm and bound to the BECLIN1 promoter region, and that LY294002, SP600125, or c-Jun knockdown prevented the increase in Beclin1 levels induced by FSH. Interestingly, inhibition of autophagy using chloroquine or SP600125 decreased progesterone production in porcine GCs treated with FSH, although the expression of StAR and P450scc was not disturbed. Moreover, FSH treatment reduced the average number and size of LDs in porcine GCs, but these effects were eliminated by knocking down the key autophagy genes, ATG5 and BECLIN1; in addition, the effect of FSH on promoting progesterone secretion by the cells was also reduced significantly. Based on the above results, we concluded that FSH promoted progesterone production by enhancing autophagy through upregulation of Beclin1 via the PI3K/JNK/c-Jun pathway to accelerate LD degradation in porcine GCs, independent of the classical steroidogenic pathway.

SUBMITTER: Liu Q

PROVIDER: S-EPMC7921800 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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FSH Promotes Progesterone Synthesis by Enhancing Autophagy to Accelerate Lipid Droplet Degradation in Porcine Granulosa Cells.

Liu Qiang Q Gao Hui H Yang Feng F Zhang Hanxue H Zeng Shenming S

Frontiers in cell and developmental biology 20210216

Little is known about the molecular relationships among follicle stimulating hormone (FSH), lipid droplet (LD) degradation, and autophagy. In this study, we aimed to investigate the pathway by which FSH regulates autophagy and the potential role of autophagy in progesterone production. Our results revealed that FSH stimulated progesterone production in mammalian follicular granulosa cells (GCs) through a non-canonical pathway. In porcine secondary follicles cultured <i>in vitro</i>, FSH treatmen ...[more]

PMID: 33665189

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Project description:BACKGROUND:Ion channels occur as large families of related genes with cell-specific expression patterns. Granulosa cells have been shown to express voltage-gated potassium channels from more than one family. The purpose of this study was to determine the effects of 4-aminopyridine (4-AP), an antagonist of KCNA but not KCNQ channels. METHODS:Granulosa cells were isolated from pig follicles and cultured with 4-AP, alone or in combination with FSH, 8-CPT-cAMP, estradiol 17beta, and DIDS. Complimentary experiments determined the effects of 4-AP on the spontaneously established pig granulosa cell line PGC-2. Granulosa cell or PGC-2 function was assessed by radio-immunoassay of media progesterone accumulation. Cell viability was assessed by trypan blue exclusion. Drug-induced changes in cell membrane potential and intracellular potassium concentration were documented by spectrophotometric determination of DiBAC4(3) and PBFI fluorescence, respectively. Expression of proliferating cell nuclear antigen (PCNA) and steroidogenic acute regulatory protein (StAR) was assessed by immunoblotting. Flow cytometry was also used to examine granulosa cell viability and size. RESULTS:4-AP (2 mM) decreased progesterone accumulation in the media of serum-supplemented and serum-free granulosa cultures, but inhibited cell proliferation only under serum-free conditions. 4-AP decreased the expression of StAR, the production of cAMP and the synthesis of estradiol by PGC-2. Addition of either 8-CPT-cAMP or estradiol 17beta to serum-supplemented primary cultures reduced the inhibitory effects of 4-AP. 4-AP treatment was also associated with increased cell size, increased intracellular potassium concentration, and hyperpolarization of resting membrane potential. The drug-induced hyperpolarization of resting membrane potential was prevented either by decreasing extracellular chloride or by adding DIDS to the media. DIDS also prevented 4-AP inhibition of progesterone production. CONCLUSION:4-AP inhibits basal and FSH-stimulated progesterone production by pig granulosa cells via drug action at multiple interacting steps in the steroidogenic pathway. These inhibitory effects of 4-AP on steroidogenesis may reflect drug-induced changes in intracellular concentrations of K+and Cl- as well as granulosa cell resting membrane potential.

Project description:BackgroundSecreted frizzled-related proteins (SFRPs) comprise a family of WNT signaling antagonists whose roles in the ovary are poorly understood. Sfrp4-null mice were previously found to be hyperfertile due to an enhanced granulosa cell response to gonadotropins, leading to decreased antral follicle atresia and enhanced ovulation rates. The present study aimed to elucidate the mechanisms whereby SFRP4 antagonizes FSH action.MethodsPrimary cultures of granulosa cells from wild-type mice were treated with FSH and/or SFRP4, and effects of treatment on gene expression were evaluated by RT-qPCR and RNAseq. Bioinformatic analyses were conducted to analyse the effects of SFRP4 on the transcriptome, and compare them to those of FSH or a constitutively active mutant of FOXO1. Additional granulosa cell cultures from wild-type or Sfrp4-null mice, some pretreated with pharmacologic inhibitors of specific signaling effectors, were used to examine the effects of FSH and/or SFRP4 on signaling pathways, autophagy and apoptosis by western blotting and TUNEL.ResultsTreatment of cultured granulosa cells with recombinant SFRP4 was found to decrease basal and FSH-stimulated mRNA levels of FSH target genes. Unexpectedly, this effect was found to occur neither via a canonical (CTNNB1-dependent) nor non-canonical WNT signaling mechanism, but was found to be GSK3β-dependent. Rather, SFRP4 was found to antognize AKT activity via a mechanism involving AMPK. This lead to the hypophosphorylation of FOXO1 and a decrease in the expression of a portion of the FSH and FOXO1 transcriptomes. Conversely, FSH-stimulated AMPK, AKT and FOXO1 phosphorylation levels were found to be increased in the granulosa cells of Sfrp4-null mice relative to wild-type controls. SFRP4 treatement of granulosa cells also induced autophagy by signaling via AKT-mTORC1-ULK1, as well as apoptosis.ConclusionsThis study identifies a novel GSK3β-AMPK-AKT signaling mechanism through which SFPR4 antagonizes FSH action, and further identifies SFRP4 as a novel regulator of granulosa cell autophagy. These findings provide a mechanistic basis for the phenotypic changes previously observed in Sfrp4-null mice, and broaden our understanding of the physiological roles of WNT signaling processes in the ovary.

Dataset Information

FSH Promotes Progesterone Synthesis by Enhancing Autophagy to Accelerate Lipid Droplet Degradation in Porcine Granulosa Cells.

Publications

FSH Promotes Progesterone Synthesis by Enhancing Autophagy to Accelerate Lipid Droplet Degradation in Porcine Granulosa Cells.

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