Estrogen receptor alpha signaling pathways differentially regulate gonadotropin subunit gene expression and serum follicle-stimulating hormone in the female mouse.

Estrogen, acting via estrogen receptor (ER)alpha, regulates serum gonadotropin levels and pituitary gonadotropin subunit expression. However, the cellular pathways mediating this regulation are unknown. ERalpha signals through classical estrogen response element (ERE)-dependent genomic as well as nonclassical ERE-independent genomic and nongenomic pathways. Using targeted mutagenesis in mice to disrupt ERalpha DNA binding activity, we previously demonstrated that ERE-independent signaling is sufficient to suppress serum LH levels. In this study, we examined the relative roles of ERE-dependent and -independent estrogen signaling in estrogen regulation of LH, FSH, prolactin, and activin/inhibin subunit gene expression, pituitary LH and FSH protein content, and serum FSH levels. ERE-independent signaling was not sufficient for estrogen to induce pituitary prolactin mRNA or suppress pituitary LHbeta mRNA, LH content, or serum FSH in estrogen-treated ovariectomized mice. However, ERE-independent signaling was sufficient to reduce pituitary glycoprotein hormone alpha-subunit, FSHbeta, and activin-betaB mRNA expression. Together with previous serum LH results, these findings suggest ERE-independent ERalpha signaling suppresses serum LH via reduced secretion, not synthesis. Additionally, ERE-dependent and ERE-independent ERalpha pathways may distinctly regulate steps involved in the synthesis and secretion of FSH.

Nonclassical estrogen receptor alpha signaling mediates negative feedback in the female mouse reproductive axis.

Ovarian estrogen exerts both positive and negative feedback control over luteinizing hormone (LH) secretion during the ovulatory cycle. Estrogen receptor (ER) alpha but not ERbeta knockout mice lack estrogen feedback. Thus, estrogen feedback appears to be primarily mediated by ERalpha. However, it is now recognized that, in addition to binding to estrogen response elements (EREs) in DNA to alter target gene transcription, ERalpha signals through ERE-independent or nonclassical pathways, and the relative contributions of these pathways in conveying estrogen feedback remain unknown. Previously we created a knockin mouse model expressing a mutant form of ERalpha (AA) with ablated ERE-dependent but intact ERE-independent activity. Breeding this allele onto the ERalpha-null (-/-) background, we examine the ability of ERE-independent ERalpha signaling pathways to convey estrogen feedback regulation of the female hypothalamic-pituitary axis in vivo. ERalpha-/AA exhibited 69.9% lower serum LH levels compared with ERalpha-/- mice. Additionally, like wild type, ERalpha-/AA mice exhibited elevated LH after ovariectomy (OVX). Furthermore, the post-OVX rise in serum LH was significantly suppressed by estrogen treatment in OVX ERalpha-/AA mice. However, unlike wild type, both ERalpha-/AA and ERalpha-/- mice failed to exhibit estrous cyclicity, spontaneous ovulation, or an afternoon LH surge response to estrogen. These results indicate that ERE-independent ERalpha signaling is sufficient to convey a major portion of estrogen's negative feedback actions, whereas positive feedback and spontaneous ovulatory cyclicity require ERE-dependent ERalpha signaling.

Effect of treatment with the selective oestrogen receptor modulator LY117018-HCl on pituitary sensitivity to GnRH and subsequent ovulation.

The effects of LY117018-HCl (LY; a benzothiophene similar to raloxifene) were examined on various reproductive parameters in female rats. Four-day cyclic rats were treated (10:00 h on dioestrus) with LY (0.01, 0.1, 0.5, 1, 2, 4 or 16 mg kg(-1) p.o.) and assessed for ovulation at oestrus. LY inhibited ovulation at doses as low as 0.5 mg kg(-1), and ovulation did not occur at doses of 4 and 16 mg kg(-1). LY (16 mg kg(-1)) reduced wet uterine mass and LH concentrations at the time of the expected ovulatory surge. Ovulation induced by hCG in pentobarbital-treated rats was not altered by LY treatment, indicating normal ovarian sensitivity to gonadotrophins. LY, however, completely blocked the effects of oestradiol (under either negative or positive feedback modes) on LH secretion in ovariectomized rats. GnRH secretion into hypophyseal portal blood during pro-oestrus was not affected by treatment with LY, whereas the concentrations of serum LH remained reduced. Finally, treatment with LY markedly reduced pituitary sensitivity to GnRH during pro-oestrus, as it completely blocked GnRH-induced LH secretion. These results demonstrate that LY inhibits oestradiol action in the uterus and prevents ovulation in normal cyclic rats. LY-induced inhibition of ovulation is not caused by an alteration of the ovarian response to gonadotrophins or an impairment of GnRH secretion at the hypothalamus, but by a reduction in the sensitivity of gonadotrophs to the stimulatory effects of GnRH during pro-oestrus.

Androgen responsiveness of the pituitary gonadotrope cell line LbetaT2.

Androgens have a profound effect on the hypothalamic-pituitary axis by reducing the synthesis and release of the pituitary gonadotropin LH. The effect on LH is partly a consequence of a direct, steroid-dependent action on pituitary function. Although androgen action has been well studied in vivo, in vitro cell models of androgen action on pituitary gonadotropes have been scarce. Recently, an LH-expressing cell line, LbetaT2, was generated by tumorigenesis targeted to the LH-producing cells of the mouse pituitary. The purpose of these studies was to determine the presence of androgen receptor (AR) and establish its function in this cell line. RT-PCR analysis indicated that the LbetaT2 cell line expresses AR mRNA. Transient transfection assays, using the mouse mammary tumor virus (MMTV) promoter, showed that a functional AR is also present. Testosterone (TEST), dihydrotestosterone (DHT), 7alpha-methyl-19-nortestosterone (MENT), and fluoxymesterone (FLUOXY) increased reporter gene activity in the rank order of potencies MENT>DHT> TEST>FLUOXY. Additionally, activation of MMTV promoter activity by DHT in LbetaT2 cells was diminished by the AR antagonists casodex and 2-hydroxy-flutamide, indicating that the effects of DHT are mediated through AR. In summary, these studies showed that the LbetaT2 cell line is a useful model for the evaluation and molecular characterization of androgen action in pituitary gonadotropes.