The anti-Müllerian hormone (AMH) induces forkhead box L2 (FOXL2) expression in primary culture of human granulosa cells in vitro

• Published in: Journal of assisted reproduction and genetics Vol. 34; no. 9; pp. 1131 - 1136
• Main Authors: Sacchi, Sandro, Marinaro, Federica, Xella, Susanna, Marsella, Tiziana, Tagliasacchi, Daniela, La Marca, Antonio
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.09.2017; Springer Nature B.V
• Subjects: Actin; Activation; Antibodies; Aromatase; Cell culture; Culture; Cytochrome P450; Feedback; Feedback loops; Forkhead protein; FOXL2 gene; Gene expression; Genes; Granulosa cells; Gynecology; Human Genetics; Medicine; Medicine & Public Health; Pituitary (anterior); Positive feedback; Proteins; Reproductive Medicine; Reproductive Physiology and Disease; Ribonucleic acid; Ribosomal protein S7; RNA; Spectrophotometry; Statistical analysis; Steroidogenesis; Transcription factors; FOXL2; AMH; CYP19A1; hGCs
• ISSN: 1058-0468; 1573-7330
• DOI: 10.1007/s10815-017-0980-9

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.