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Neurotensin expressed in the granulosa cells regulates selective genes that are potentially involved in the ovulatory process

ABSTRACT: Neurotensin (NTS) is a small 13 amino acid neuropeptide. In the mouse ovary, the expression of Nts ovary increases 250-fold 4h after hCG. Similarly, in granulosa cells, the mRNA levels of Nts increased rapidly at 4h after hCG stimulation. Interestingly, the Nts mRNA levels in granulosa cells were approximately 8-fold higher at 4 h after hCG than in whole ovaries. However, the potential mechanisms of NTS action in the ovulatory process are unknown. The present study determined the regulatory mechanisms driving the expression of Nts and functions of the NTS using primary mouse granulosa cells. hCG activated PKA and p38MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of Nts. To identify the genes regulated by NTS, high throughput RNA sequencing was performed using Nts silenced mouse granulosa cells treated with or without hCG. Sequencing data analysis revealed that Nts knockdown affects the expression of several genes that were not previously identified in the ovary. qPCR analysis verified hCG-induced, Nts-regulated expression of a selection of these genes. This study revealed novel genes regulated by NTS, thereby providing a function for NTS in the ovulatory process.

ORGANISM(S): Mus musculus

PROVIDER: GSE210811 | GEO | 2022/10/03

REPOSITORIES: GEO

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Project description:Luteinizing hormone (LH) activation of the EGF receptor/RAS/ERK1/2 pathway is essential for ovulation and luteinization because granulosa cell depletion of Erk1/2 (Erk1/2gc-/- mice) renders mice completely infertile. Because of their potential as mediators of ERK1/2-dependent granulosa cell differentiation, we disrupted genes encoding the CCAAT/enhancer-binding proteins, (C/EBP) a and C/EBPb. Female Cebpbgc-/- mutant mice but not Cebpagc-/- mice were subfertile whereas Cebpa/bgc-/- double mutant females were sterile. Follicles failed to ovulate, ovaries were devoid of corpora lutea, luteal cell marker genes (Lhcgr, Prlr, Ptgfr, Cyp11a1 and Star) were absent and serum progesterone levels were low. Microarray analyses identified numerous C/EBPa/b target genes in eCG-hCG treated mice. At 4h post-hCG, a subset (19%) of genes altered in the Cebpa/b depleted cells was also altered in Erk1/2 depleted cells; hence they are common effectors of ERK1/2. Additional genes down-regulated in the Cebpa/b depleted cells at 8 and 24h post-hCG include known (Akr1b7, Runx2, Star, Saa3) and novel (Abcb1b, Apln, Igfbp4, Prlr, Ptgfr Timp4) C/EBP target genes including effectors of vascular cell development. Bhmt, a gene controlling methionine metabolism and expressed exclusively in liver and kidney, was high in WT luteal cells but totally absent in Cebpa/b mutant cells. Because numerous genes potentially associated with vascular development were suppressed in the mutant cells, C/EBPa/b appear to dictate the luteinization process by also controlling genes that regulate the formation of the extensive vascular network required to sustain luteal cells. Thus, C/EBPa/b mediate several aspects of granulosa cell differentiation as well as the complex process of luteinization. Five granulosa cell treatments were included: CEBPM-NM-1/M-NM-2 conditional KO ovary, eCG-treated 48h, hCG 4h wild type ovary, eCG-treated 48h, hCG 8h CEBPM-NM-1/M-NM-2 conditional KO ovary, eCG-treated 48h, hCG 8h wild type ovary, eCG-treated 48h, hCG 24h CEBPM-NM-1/M-NM-2 conditional KO ovary, eCG-treated 48h, hCG 24h

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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Neurotensin expressed in the granulosa cells regulates selective genes that are potentially involved in the ovulatory process

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