Insufficient Luteinizing Hormone-Induced Intracellular Signaling Disrupts Ovulation in Preovulatory Follicles Lacking Estrogen Receptor-β

Abstract

Gonadotropin-stimulated estrogen receptor-β (ERβ)-null preovulatory follicles exhibit submaximal estradiol production, insufficient acquisition of LH receptor, and attenuated expression of essential ovulatory genes. These observations lead to low ovulatory rates compared with wild-type (WT) follicles. We hypothesize that insufficient LH receptor results in reduced cAMP production after an ovulatory stimulus. Individual preantral follicles were cultured with FSH for 4 d and then induced to ovulate with a single dose of human chorionic gonadotropin (hCG). cAMP levels 1 h after hCG were 50% lower in ERβ-null than WT follicles. To determine whether the lack of LH receptor, and resulting lack of cAMP, could be bypassed by direct activation of adenylyl cyclase, WT and ERβ-null follicles were induced to ovulate with forskolin. Ten micromolar forskolin doubled the ovulatory rate of ERβ-null follicles compared with treatment with hCG (∼50 vs. 25%, respectively). In WT follicles, 10 μm forskolin reduced the ovulation rate compared with hCG (14 vs. 83%, respectively), indicating that high doses of forskolin inhibited WT ovulation. A 10 μm concentration of forskolin induced cAMP levels in ERβ-null follicles that were comparable to levels produced in WT follicles after hCG and either partially or completely rescued the attenuated expression of LH-responsive genes. These data indicate that direct activation of adenylyl cyclase, resulting in increased production of cAMP, partially rescues the ovulatory response of ERβ-null follicles, suggesting that insufficient LH receptor and low cAMP levels contribute to their poor ovulatory rates. We also determined that ERβ-null ovaries exhibit an alteration in the activation of ERK1/2. Our evaluation of the ERβ-null ovarian phenotype indicates that ERβ plays a role in facilitating folliculogenesis. We show that expression of ERβ in preovulatory follicles is required for adequate cAMP production and propose that an optimal level of cAMP is required for hCG-stimulated ovulation.