Granulosa cell expression of Fos is critical for regulating ovulatory gene expressions in the mouse ovary
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ABSTRACT: A previous study showed that female Fos null mice fail to ovulate even when given gonadotropins, suggesting that ovarian expression of Fos is critical for successful ovulation. However, the expression of FOS and function of FOS have not been determined in the mouse ovary. FOS, a member of the Fos family (Fos, Fosb, Fosl1, and Fosl2), functions as a transcription factor by forming a heterodimer complex with a member of Jun family (Jun, Junb, and Jund). The present study demonstrated rapid increases in Fos, along with other Fos and Jun family members, after hCG administration in the ovary of immature PMSG-primed mice and after the LH surge of naturally cycling animals. ChIP-seq analysis identified 1965 FOS-binding genes in granulosa cells collected at 3h post-hCG, including Pgr, Ptgs2, Tnfiap6, and Edn2, genes known to be involved in the ovulatory process. When super-ovulation was induced, the number of oocytes released was significantly reduced in Esr2cre/+ - driven granulosa cell-specific Fos knockout (gcFosKO) mice. This reduction was accompanied by lower expression of Pgr, Ptgs2, Ptgs1, and Edn2 in preovulatory follicles of gcFosKO mice compared to those in control litter mates. In addition, gcFosKO mice showed a trend of decreasing average litter size. Together, this study provided a comprehensive characterization of the tightly controlled up-regulation of all Fos and Jun family members in mouse preovulatory follicles after the LH surge or ovulatory hCG administration and revealed FOS’s role in regulating the expression of key ovulatory genes in the mouse ovary.
Project description:Ovulation is triggered by the gonadotropin surge that induces the expression of two key genes, progesterone receptor (Pgr) and prostaglandin-endoperoxide synthase 2 (Ptgs2) in the granulosa cells of preovulatory follicles. Their gene products PGR and PTGS2 activate two separate pathways that are both essential for successful ovulation. Here we show that the PGR plays an additional essential role; attenuate ovulatory inflammation by diminishing the gonadotropin surge-induced Ptgs2 expression. PGR indirectly terminates Ptgs2 expression and PGE2 synthesis in granulosa cells by inhibiting the NF-κB, a transcription factor required for Ptgs2 expression. When the expression of PGR was ablated in the granulosa cells, the ovary undergoes hyperinflammatory condition manifested by excessive PGE2 synthesis, immune cell infiltration, oxidative damage, and neoplastic transformation of ovarian cells. Despite the ovary undergoes ovulations dozens or hundreds of times in one’s lifetime, the repetitive ovulatory inflammations do not leave significant tissue damage in the ovary. The PGR-driven termination of PTGS2 expression may protect ovary from the ovulatory inflammation.
Project description:FOS, a subunit of the activator protein-1 (AP-1) transcription factor, has been implicated in various cellular changes. In the human ovary, the expression of FOS and its heterodimeric binding partners JUN, JUNB, and JUND increases in periovulatory follicles. However, the specific role of the FOS/AP-1 remains elusive. The present study determined the regulatory mechanisms driving the expression of FOS and its partners and functions of the FOS/AP-1 using primary human granulosa/lutein cells (hGLC). hCG induced a biphasic increase in the expression of FOS, peaking at 1-3h and 12h. The levels of JUN proteins were also increased by hCG, with varying expression patterns. Co-immunoprecipitation analyses revealed that FOS is present as heterodimers with all JUN proteins. hCG immediately activated PKA and p42/44MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of FOS, JUN, and JUNB. To identify the genes regulated by FOS, high throughput RNA sequencing was performed using hGLC treated with hCG ± T-5224 (FOS inhibitor). Sequencing data analysis revealed that FOS inhibition affects the expression of numerous genes, including a cluster of genes involved in the periovulatory process such as matrix remodeling, prostaglandin synthesis, glycolysis/gluconeogenesis, and cholesterol biosynthesis. qPCR analysis verified hCG-induced, T-5224-regulated expression of a selection of these genes. Consistently, T-5224 attenuated hCG-induced increases in metabolic activities and cholesterol levels. This study unveiled potential downstream target genes of and a role for the FOS/AP-1 in granulosa cell metabolic changes and cholesterol biosynthesis in human periovulatory follicles.
Project description:Ovulation is induced by the preovulatory surge of luteinizing hormone (LH) that acts on the ovary and triggers the rupture of the preovulatory ovarian follicle by stimulating proteolysis and apoptosis in the follicle wall, causing the release of the mature oocyte. In mammals, the pro-inflammatory cytokine tumor necrosis factor α (TNFα) and prostaglandin F2α (PGF2α) are known to be involved in the control of ovulation but their role mediating the pro-ovulatory actions of LH has not been established. Here we show that Lh induces PGF2α synthesis through its stimulation of Tnfα production in trout, a primitive teleost fish. Importantly, trout recombinant Tnfα (rTnfα) and PGF2α recapitulate the stimulatory in vitro effects of salmon Lh (sLh) on contraction, proteolysis and loss of cell viability in the preovulatory follicle wall and, finally, ovulation. Furthermore, all pro-ovulatory actions of sLh are blocked by inhibition of Tnfα secretion or PG synthesis and those of rTnfα are blocked by PG synthesis inhibitors. Therefore, we provide evidence that the Tnfαâdependent increase in PGF2α production is necessary for the pro-ovulatory actions of Lh in a teleost fish. The results from this study shed light onto the mechanisms underlying the pro-ovulatory actions of LH in vertebrates and may prove important in clinical assessments of female infertility. Preovulatory follicles from brook trout from four different females (n = 4) were isolated and incubated in the absence or presence of salmon LH. Total RNA of control and LH-treated preovulatory follicles from each of the four females was analyzed.
Project description:Ovulation is induced by the preovulatory surge of luteinizing hormone (LH) that acts on the ovary and triggers the rupture of the preovulatory ovarian follicle by stimulating proteolysis and apoptosis in the follicle wall, causing the release of the mature oocyte. In mammals, the pro-inflammatory cytokine tumor necrosis factor α (TNFα) and prostaglandin F2α (PGF2α) are known to be involved in the control of ovulation but their role mediating the pro-ovulatory actions of LH has not been established. Here we show that Lh induces PGF2α synthesis through its stimulation of Tnfα production in trout, a primitive teleost fish. Importantly, trout recombinant Tnfα (rTnfα) and PGF2α recapitulate the stimulatory in vitro effects of salmon Lh (sLh) on contraction, proteolysis and loss of cell viability in the preovulatory follicle wall and, finally, ovulation. Furthermore, all pro-ovulatory actions of sLh are blocked by inhibition of Tnfα secretion or PG synthesis and those of rTnfα are blocked by PG synthesis inhibitors. Therefore, we provide evidence that the Tnfα–dependent increase in PGF2α production is necessary for the pro-ovulatory actions of Lh in a teleost fish. The results from this study shed light onto the mechanisms underlying the pro-ovulatory actions of LH in vertebrates and may prove important in clinical assessments of female infertility.
Project description: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.
Project description:The LH surge triggers dramatic transcriptional changes in genes associated with ovulation and luteinization. The present study investigated the spatiotemporal expression of nuclear factor interleukin-3 (NFIL3), a transcriptional regulator of the bZIP transcription factor superfamily, and its potential role in the ovary during the periovulatory period. NFIL3, also known as E4-binding protein 4 or NFIL3/E4BP4, was originally identified as a transcriptional repressor based on its DNA-binding activity at the promoter of the gene encoding the adenovirus E4 protein. Immature female rats were injected with PMSG, treated with hCG, and ovaries or granulosa cells were collected at various times after hCG. Nfil3 mRNA was highly induced both in intact ovaries and granulosa cells after hCG treatment. In situ hybridization demonstrated that Nfil3 mRNA was highly induced in theca-interstitial cells at 4-8 h after hCG, localized to granulosa cells at 12 h, and decreased at 24 h. Over-expression of NFIL3 in granulosa cells inhibited the induction of prostaglandin-endoperoxide synthase 2 (Ptgs2), progesterone receptor (Pgr), epiregulin (Ereg), and amphiregulin (Areg) and down regulated levels of prostaglandin E2. The inhibitory effect on Ptgs2 induction was reversed by NFIL3 siRNA treatment. In theca-interstitial cells the expression of hydroxyprostaglandin dehydrogenase 15-(NAD) (Hpgd) was also inhibited by NFIL3 over-expression. Data from luciferase assays demonstrated that NFIL3 over-expression decreased the induction of the Ptgs2 and Areg promoter activity. EMSA and ChIP analyses indicated that NFIL3 binds to the promoter region containing the DNA binding sites of CREB and C/EBP?. In summary, hCG induction of NFIL3 expression may modulate the process of ovulation and theca-interstitial and granulosa cell differentiation by regulating expression of PTGS2, PGR, AREG, EREG, and HPGD, potentially through interactions with CREB and C/EBP? on their target gene promoters.
Project description:RUNX1 is induced by the LH surge in the ovary and plays an important role in granulosa cells in the peri-ovulatory window through the induction of key gene expression. The ablation of the canonical heterodimer partner of RUNX1 (CBFβ) leads to ovulatory failure and subfertility in female mice. A possible interaction between RUNX1 and PGR on the chromatin level has also been indicated. However, the properties of RUNX1 chromatin binding in granulosa cells in response to the LH surge remains unknown. This study aims to characterise the RUNX1 cistrome in pre- and peri-ovulatory granulosa cells and explore the potential interaction between RUNX1 and the key ovulatory factor PGR.
Project description:Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status (such as lactatino) are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function RNAseq profiling was conducted on non-lactating Holstein-Friesian heifers (n=16) and lactating Holstein-Friesian cows (n=17) at three stages of preovulatory follicle development: A) newly selected dominant follicle in the luteal phase (Selection); B) follicular phase before the LH surge (Differentiation) and C) pre-ovulatory phase after the LH surge (Luteinization). Based on a combination of RNA sequencing, ingenuity pathway analysis and Q-RT-PCR validation several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, were identified to be affected (downregulated) by the catabolic state. We propose that the adverse metabolic environment caused by lactation decreases preovulatory follicle function by affecting cholesterol transport into the mitochondria to initiate steroidogenesis. Granulosa and Theca samples from the dominant follicle were taken from cows and heifers at stages: selection, differentiation and luteinization.
Project description:Cellular mechanisms that contribute to low estradiol concentrations produced by the preovulatory ovarian follicle in cattle with a compromised metabolic status (such as lactatino) are largely unknown. To gain insight into the main metabolic mechanisms affecting preovulatory follicle function RNAseq profiling was conducted on non-lactating Holstein-Friesian heifers (n=16) and lactating Holstein-Friesian cows (n=17) at three stages of preovulatory follicle development: A) newly selected dominant follicle in the luteal phase (Selection); B) follicular phase before the LH surge (Differentiation) and C) pre-ovulatory phase after the LH surge (Luteinization). Based on a combination of RNA sequencing, ingenuity pathway analysis and Q-RT-PCR validation several important molecular markers involved in steroid biosynthesis, such as the expression of steroidogenic acute regulatory protein (STAR) within developing dominant follicles, were identified to be affected (downregulated) by the catabolic state. We propose that the adverse metabolic environment caused by lactation decreases preovulatory follicle function by affecting cholesterol transport into the mitochondria to initiate steroidogenesis.
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).