FSH/LH-Dependent Upregulation of Ahr in Murine Granulosa Cells Is Controlled by PKA Signaling and Involves Epigenetic Regulation
Abstract
:1. Introduction
2. Results
2.1. The Upregulation of Ahr in GCs during Follicular Maturation Requires both FSH and LH Activity
2.2. The Effect of PMSG on The Expression Dynamics of Ahr and Follicle Maturation Marker Genes
2.3. Ahr Is Upregulated in Large Antral Follicles in Response to PMSG
2.4. Ahr Is Downregulated by Protein Kinase A Signaling Pathway
2.5. The Increased Transcription of Ahr in Response to PMSG is Regulated by Chromatin Accessibility
3. Discussion
4. Materials and Methods
4.1. Medium, Chemicals, and Antibodies
4.2. Ethics
4.3. Animals and Granulosa Cell Isolation
4.4. Western Blotting
4.5. Isolation of RNA and mRNA/hnRNA Measurement by RT-qPCR
4.6. Immunofluorescence
4.7. Plasmids, Transient Transfection and Reporter Gene Assay
4.8. Chromatin Accessibility by Real-Time PCR (CHART-PCR)
4.9. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
AHR | aryl hydrocarbon receptor |
TCDD | 2,3,7,8-tetrachlorodibenzo-p-dioxin |
FSH | follicle stimulating hormone |
LH | luteinizing hormone |
FSK | forskolin |
PKA | Protein kinase A |
hnRNA | heteronuclear RNA |
ActD | actinomycin D |
CHART-PCR | chromatin accessibility by real-time PCR |
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Application | Oligonucleotide | Sequence (5′–3′) | |
---|---|---|---|
qPCR | Ahr mRNA | Forward | GGCCAAGAGCTTCTTTGATG |
Reverse | TGCCAGTCTCTGATTTGTGC | ||
Ahr hnRNA E2-I2 | Forward | TAGGCTCAGCGTCAGCTACC | |
Reverse | GTCACCAACATTTAAAGGACCAC | ||
Fshr mRNA | Forward | GCGGCAAACCTCTGAACTTC | |
Reverse | TGATCCCCAGGCTGAGTCAT | ||
Cyp19a1 mRNA | Forward | GCCTCCTTCTCCTGATTTGGA | |
Reverse | CTGCCATGGGAAATGAGGG | ||
Lhcgr mRNA | Forward | AGTCACAGCTGCACTCTCC | |
Reverse | GTGAGAGATAGTCGGGCGAG | ||
Tbp mRNA | Forward | GGCCTCTCAGAAGCATCACT | |
Reverse | GCCAAGCCCTGAGCATAA | ||
CHART-PCR | Ahr promoter | Forward | TTTTGAGGCTGGAAAACAGGTACT |
Reverse | ACGTGATGACGCAGGACGTA | ||
Cyp19a1 promoter | Forward | CCAATCCAGCACCCTTCCAA | |
Reverse | GGGAAGAAAGCAGTGGTGGA | ||
Pax7 promoter | Forward | GTTATCGGTCCCCTCTCCCT | |
Reverse | CTCAACGGCAGGGAAGGG |
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Matvere, A.; Teino, I.; Varik, I.; Kuuse, S.; Tiido, T.; Kristjuhan, A.; Maimets, T. FSH/LH-Dependent Upregulation of Ahr in Murine Granulosa Cells Is Controlled by PKA Signaling and Involves Epigenetic Regulation. Int. J. Mol. Sci. 2019, 20, 3068. https://doi.org/10.3390/ijms20123068
Matvere A, Teino I, Varik I, Kuuse S, Tiido T, Kristjuhan A, Maimets T. FSH/LH-Dependent Upregulation of Ahr in Murine Granulosa Cells Is Controlled by PKA Signaling and Involves Epigenetic Regulation. International Journal of Molecular Sciences. 2019; 20(12):3068. https://doi.org/10.3390/ijms20123068
Chicago/Turabian StyleMatvere, Antti, Indrek Teino, Inge Varik, Sulev Kuuse, Tarmo Tiido, Arnold Kristjuhan, and Toivo Maimets. 2019. "FSH/LH-Dependent Upregulation of Ahr in Murine Granulosa Cells Is Controlled by PKA Signaling and Involves Epigenetic Regulation" International Journal of Molecular Sciences 20, no. 12: 3068. https://doi.org/10.3390/ijms20123068