Abstract
Purpose
Anti-Müllerian hormone (AMH) and forkhead box L2 (FOXL2) are two pivotal genes expressed in human granulosa cells (hGCs) where both genes share similar inhibitory functions on activation and follicular growth in order to preserve the ovarian follicle reserve. Furthermore, AMH and FOXL2 contribute to inhibit steroidogenesis, decreasing or preventing the activation of gonadotrophin-dependent aromatase CYP19A1 cytochrome P450 family 19 subfamily A member 1 (CYP19A1). The purpose of this study is to evaluate the role of AMH in regulating the expression of FOXL2.
Methods
Primary cultures of hGCs were treated with increasing concentrations of recombinant human AMH (rhAMH; range 10–100 ng/ml) for 3 h. Negative controls were performed using corresponding amounts of AMH vehicle. Total RNA or proteins were purified and quantified by spectrophotometry. FOXL2 and CYP19A1 gene expression, normalized by reference gene ribosomal protein S7 (RpS7), was evaluated by RT-qPCR. Each reaction was repeated in triplicate. Statistical analysis was performed. Extracted proteins were analyzed by immunoblot using anti-FOXL2 and anti-β-actin as primary antibodies.
Results
rhAMH treatments tested did not modulate the basal expression of aromatase CYP19A1 gene. rhAMH (50 ng/ml) was able to increase FOXL2 gene expression and its intracellular content.
Conclusions
This study demonstrated the existence of an AMH-FOXL2 relationship in hGCs. AMH is capable of increasing both gene and protein expression of FOXL2. Because FOXL2 induces AMH transcription, these ovarian factors could be finely regulated by a positive feedback loop mechanism to preserve the ovarian follicle reserve.
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Acknowledgments
We are sincerely grateful to Fiorcari Stefania, Martinelli Silvia, Benatti Stefania (Hematology Division, Department of Medical and Surgical Sciences of the University of Modena and Reggio Emilia), and Riccetti Laura (Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, NOCSAE) for their patience and assistance with immunoblot.
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Informed consent was obtained from all individual participants included in the study. (Stated also in “Patients’ selection” section of Materials and Methods in the manuscript)
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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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Sacchi, S., Marinaro, F., Xella, S. et al. The anti-Müllerian hormone (AMH) induces forkhead box L2 (FOXL2) expression in primary culture of human granulosa cells in vitro. J Assist Reprod Genet 34, 1131–1136 (2017). https://doi.org/10.1007/s10815-017-0980-9
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DOI: https://doi.org/10.1007/s10815-017-0980-9