GPER membrane receptor-mediated estrogen regulation of the AMH promoter

URRUTIA MARIELA ; REY RODOLFO; LOVAISA MARÍA DE LOS MILAGROS ; SCHTEINGART HELENA F

Mar del Plata

Congreso; LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica; 2018

SAIC-SAI-SAFIS

Anti-Müllerian hormone (AMH) is secreted by testicular Sertoli cells from fetal life until puberty, when it is inhibited by androgens. In conditions characterized by high estrogen levels, like Peutz-Jeghers and androgen insensitivity syndromes, AMH production is increased. The physiological effects of estrogens are mediated by classical nuclear receptors (ERα and ERβ), and also by a G protein-coupled membrane receptor (GPER). Previously, we demonstrated that estradiol (E2) mediates the increase of AMH promoter activity in Sertoli cells by ER. In this study we assessed whether E2 regulates AMH promoter activity also through GPER in the mouse pre-pubertal Sertoli cell line SMAT1.Reporter assays were performed in SMAT1 cells co-transfected with a GPER expression plasmid associated with fluorescent protein EGFP, a firefly luciferase-associated AMH promoter plasmid, and a renilla luciferase plasmid as transfection efficiency reporter. Luciferase was measured after incubation with 17β-E2 (10-9 M), GPER-specific agonist G1 (100 nM) or GPER antagonist G15 (100 nM) for 24 hours. Results, expressed as a relative luciferase unit (RLU, mean ± SEM), were compared using a paired-samples Friedman test and Dunn´s multiple comparison post-test.The expression of GPER-EGFP in SMAT1 cells was confirmed by fluorescence microscopy. Basal AMH promoter activity (0.979 ± 0.081 RLU) increased modestly but significantly after incubation with E2 (1.062 ± 0.078 RLU, n=10, P=0.048) or G1 (1.109 ± 0.085 RLU, n=10, P=0.022). The co-incubation with antagonist G15 did not produce significant changes in AMH promoter activity compared to E2 (E2: 1.062 ± 0.078 RLU, G15 + E2: 1.027 ± 0.132 RLU, n=10, P=0.227).Conclusion: Estrogen effect on AMH promoter activity is also partially exerted through GPER in SMAT1 cells. This suggests a mild modulatory action of prepubertal Sertoli cells by estrogens through GPER under physiological conditions, which could be increased in situations of hyperestrogenism.