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NR5A1 and FOXL2 Induce Human Fibroblasts into Steroidogenic Ovarian Granulosa-Like Cells

ABSTRACT: Granulosa cells provide essential niche for ovarian folliculogenesis by communicating with oocytes and secreting steroids at different follicular stages, but current cellular models for studying human granulosa cells mainly rely on immortal cancer cell lines. Here, we established a reprogramming method to collect early and late granulosa cells with different steroidogenic abilities. We applied AMH-fluorescence-reporter system to screen six candidate factors for cellular reprogramming and generated human induced granulosa like cells (hiGC) by overexpressing FOXL2 and NR5A1. AMH-EGFP+ hiGC resembled human cumulus cells in transcriptome profiling and secreted high levels of estrogen as well as progesterone, similar to late-stage granulosa cells at antral or preovulatory stage. Moreover, we identified a cell surface marker CD55 which can be used to isolate early-stage granulosa cells. CD55+AMH-EGFP- hiGC secreted high levels of estrogen but low levels of progesterone, and the transcriptome profiles were more similar to early-stage granulosa cells. Both types of the hiGC promoted oocyte maturation when cocultured with immature oocytes. More importantly, when CD55+ hiGC were transplanted to mouse model with Polycystic ovary syndrome (PCOS), the number of cystic follicles was significantly reduced, showing alleviation of PCOS. Hence, hiGC provides a cellular model to study developmental program of human granulosa cells and has potential to treat PCOS.

ORGANISM(S): Homo sapiens

PROVIDER: GSE203070 | GEO | 2023/12/31

REPOSITORIES: GEO

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Project description:Primordial follicle assembly is the process by which ovarian primordial follicles are formed. During follicle assembly oocyte nests break down and a layer of pre-granulosa cells surrounds individual oocytes to form primordial follicles. The pool of primordial follicles formed is the source of oocytes for ovulation during a femaleM-bM-^@M-^Ys reproductive life. Complex networks of cellular signaling and gene expression are essential for any biological process. A systems biology experimental approach provides a global view of these gene relationships in a particular developmental process. The current study utilized a systems approach to detect all genes that are differentially expressed in response to seven different growth factor and hormone treatments known to influence primordial follicle assembly in a neonatal rat ovary culture system. One novel growth factor, basic fibroblast growth factor (FGF2), was experimentally determined to inhibit follicle assembly. The different growth factor and hormone treatments were all found to affect the same physiological pathways, but each treatment affected a unique set of differentially expressed genes (signature gene set). A gene bionetwork analysis identified gene modules of coordinately expressed interconnected genes and it was found that different gene modules appear to accomplish distinct tasks during primordial follicle assembly. Unique gene networks were identified for a number of the modules and signature gene sets. Predictions of physiological pathways important to follicle assembly were validated using ovary culture experiments in which ERK1/2 (MAPK1) activity was increased. A number of the highly interconnected genes in these gene networks have previously been linked to primary ovarian insufficiency (POI) and polycystic ovarian disease syndrome (PCOS). Observations have identified novel factors and gene networks that regulate primordial follicle assembly. This systems approach has helped elucidate the molecular control of primordial follicle assembly and provided potential therapeutic targets for the treatment of ovarian disease. We used microarrays to determine genes expressed differentially between control and P0 ovaries treated with 7 growth factors: AMH, CTGF, estradiol (E2), Activin-a, FGF2, progesterone (P4), and TNFa. RNA samples from 7 control samples are compared to 3 growth factor treated ovary samples for each AMH (human Anti-MM-CM-<lerian hormone), CTGF (connective tissue growth factor ), estradiol (E2), TNF (tumor necrosis factor ), FGF2 (fibroblast growth factor 2), Inhba (inhibin, beta A), and 2 samples for progesterone (P4).

Project description:Determining the spatial and temporal expression of genes involved in the ovulatory pathway is critical for the understanding of the role of each estrogen receptor in the modulation of folliculogenesis and ovulation. Estrogen receptor (ER) b is highly expressed in ovarian granulosa cells and mice lacking ERb (bERKO) are subfertile due to inefficient ovulation. Previous work has focused on isolated granulosa cells or cultured follicles and while informative, provides confounding results due to the heterogeneous cell types present including granulosa, theca and oocytes and exposure to in vitro conditions. Herein, we isolated preovulatory granulosa cells from WT and ERb-null mice using laser capture microdissection to examine the genomic transcriptional response downstream of PMSG (mimicking FSH) and PMSG/hCG (mimicking LH) stimulation. This allows for a direct comparison of in vivo granulosa cells at the same stage of development from both WT and ERb-null ovaries. ERb-null granulosa cells showed altered expression of genes known to be regulated by FSH (Akap12 and Runx2) as well as not previously reported (Arnt2 and Pou5f1) in WT granulosa cells. Our analysis also identified 304 genes not previously associated with ERb in granulosa cells. LH responsive genes including Abcb1b and Fam110c show reduced expression in ERb-null granulosa cells; however novel genes including Rassf2 and Megf10 were also identified as being downstream of LH signaling in granulosa cells. Collectively, our data suggests that granulosa cells from ERb-null ovaries may not be appropriately differentiated and are unable to respond properly to gonadotropin stimulation We used microarray to compare the gene expression profiles of wiltype (WT) and Erb-null (bERKO) preovulatory granulosa cells as they respond to either PMSG or PMSG+hCG treatments. Laser microdissection was used to collect a purified population of granulosa cells only from preovulatory follicles. We chose to compre the response to PMSG or PMSG+hCG of granulosa cells collected from either WT and bERKO preovulatory follicles. We chose to collect cells 48h after mice were treated with PMSG to compare the gene expression profile ot preovulatory granulosa cells. We also studied the response of these cells to LH (or hCG) as we collected cells 4h after mice were treated with hCG (peak of transcriptional response to hCG).

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NR5A1 and FOXL2 Induce Human Fibroblasts into Steroidogenic Ovarian Granulosa-Like Cells

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