Insufficient luteinizing hormone-induced intracellular signaling disrupts ovulation in preovulatory follicles lacking estrogen receptor-{beta}

Abstract

Gonadotropin-stimulated estrogen receptor-beta (ERbeta)-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 ERbeta-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 ERbeta-null follicles were induced to ovulate with forskolin. Ten micromolar forskolin doubled the ovulatory rate of ERbeta-null follicles compared with treatment with hCG ( approximately 50 vs. 25%, respectively). In WT follicles, 10 microm forskolin reduced the ovulation rate compared with hCG (14 vs. 83%, respectively), indicating that high doses of forskolin inhibited WT ovulation. A 10 microm concentration of forskolin induced cAMP levels in ERbeta-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 ERbeta-null follicles, suggesting that insufficient LH receptor and low cAMP levels contribute to their poor ovulatory rates. We also determined that ERbeta-null ovaries exhibit an alteration in the activation of ERK1/2. Our evaluation of the ERbeta-null ovarian phenotype indicates that ERbeta plays a role in facilitating folliculogenesis. We show that expression of ERbeta in preovulatory follicles is required for adequate cAMP production and propose that an optimal level of cAMP is required for hCG-stimulated ovulation.

Figure 1

Ovulation rates after hCG treatments (A) and estradiol production by preovulatory follicles (B). A, Follicles that reached at least 340 μm in diameter were induced to ovulate by treatment with (4 IU) hCG. Ovulation rates were significantly lower in ERβ-null follicles compared with WT follicles (*, P < 0.05; n = 14–16 per genotype). B, Estradiol was measured in the spent medium after a 48-h accumulation period (before hCG treatment). WT follicles produce significantly higher amounts of estradiol during culture compared with ERβ-null follicles (*, P < 0.001; n = 8 per genotype).

Figure 2

Gene expression in preovulatory follicles. Preantral follicles from WT (light bars) and ERβ-null (dark bars) mice were isolated and cultured in the presence of FSH until they reached 340 μm in diameter. Follicles were frozen, and gene expression was assessed by quantitative PCR. Expression of Lhcgr, Cyp19a, and Cyp17 was significantly lower in ERβ-null follicles compared with WT follicles (*, P < 0.05; n = 19 pooled from three experiments). Expression of ERα was found to be significantly increased in ERβ-null preovulatory follicles (*, P < 0.001; n = 7 and n = 14 follicles for WT and ERβ-null, respectively).

Figure 3

Time course of cAMP levels produced by individual follicles after hCG treatment. Preantral follicles were cultured in the presence of FSH. Follicles that reached 340 μm in diameter were treated with hCG and frozen at either 0, 30, 60, 120, and 180 min after treatment. Individual follicles were lysed, and cAMP content was measured. WT follicles produced significantly higher levels of cAMP compared with ERβ-null follicles at 60 min, and levels of cAMP remained high in WT follicles 120 min after hCG (*, P < 0.05; n = 6–8 follicles per time point).

Figure 4

Ovulation after forskolin treatment. Preantral follicles were collected and cultured with FSH as described. WT and ERβ-null preovulatory follicles were treated with vehicle (0.01% DMSO), hCG (4 IU), or 5, 10, or 15 μm forskolin. Ovulation was assessed 18 h after treatment. Asterisks represent differences between groups as shown. ERβ-null follicles treated with 10 μm forskolin exhibited a significant increase (P = 0.013) in ovulatory rates compared with hCG treatment. WT follicles treated with either concentrations of forskolin exhibited a significantly (P < 0.001) decreased ovulatory rate compared with follicles treated with hCG. This experiment was repeated eight times, and the total number of follicles per group is listed.

Figure 5

cAMP levels after forskolin treatment. Preovulatory WT and ERβ-null follicles (collected and cultured as previously described) were treated with vehicle (0.01% DMSO), hCG (4 IU), or 5 or 10 μm forskolin. Medium was collected 1 h after treatment for measurement of cAMP. *, Significant difference between genotypes within treatments (P < 0.05); **, significant difference between treatment within genotype (P < 0.05). The experiment was repeated two times and included a total of eight to 15 follicles per treatment. The shaded box highlights the optimal window of cAMP levels.

Figure 6

Gene expression 4 h after hCG or forskolin treatment. Preovulatory WT (light bars) and ERβ-null (dark bars) follicles were treated with vehicle (0.01% DMSO), hCG (4 IU), or 10 μm forskolin (FSK). Follicles were frozen 4 h after treatment, and gene expression was assessed by quantitative PCR. Gene expression was calculated based on WT vehicle expression for all genes except for Sult1e1 where expression was not detected (nd) in the vehicle group, and expression was calculated based on WT treated with hCG. The experiment was repeated three times and included a total of 12–18 follicles per treatment. ERβ-null follicles exhibited lower gene expression after hCG treatment compared with WT follicles (*, P < 0.05). Forskolin treatment resulted in an increase in gene expression in ERβ-null follicles that was comparable to the level observed in WT follicles treated with hCG.

Figure 7

Phosphorylation of ERK1/2 in vivo and expression of Areg and Ereg. Prepubertal mice, WT or ERβ-null, were treated with PMSG, and 48 h later, ovaries were collected (0 min), or they were treated with hCG and their ovaries collected 60 or 120 min after hCG. The experiment was repeated twice with three mice per experiment per time point. Expression of Areg and Ereg was measured in six to eight follicles per genotype (WT, light bars; ERβ-null, dark bars) per treatment and were treated as described. FSK, Forskolin.