Acute effects of estradiol on gonadotropin-releasing hormone-induced signal cascade in gonadotropes

Abstract

The importance of estrogens in numerous physiological systems, including reproduction, cardiovascular, skeletal and immune systems requires a better understanding of the subcellular mechanisms affected by exongenous and endogenous estrogens. Environmental or nutritional estrogenic endocrine disrupting chemicals are thought to mediate developmental and carcinogenic pathologies. Regarding the reproductive system, estrogens affect many target tissues in the male and female, including the mammary glands, uterus, ovaries, testes, prostate, hypothalamus and anterior pituitary gland. It has been demonstrated that estrogens exert both positive and negative effects on the hypothalamus and the anterior pituitary gland to regulate gonadotropin secretion. Classically, effects of estradiol (E2) are mediated through the nuclear estrogen receptor (ER), which modulates gene expression. In general, this genomic effect occurs over a period of several hours and involves repression or activation of synthesis of new proteins, i.e. gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) and follicle-stimulating hormone (FSH). It has been increasingly appreciated in the past decade that steroid hormones, such as E2, mediate cell functions not only through stimulation or inhibition of transcription, but also via non-genomic mechanisms. The genomic effects of estrogens and other steroid hormones are now relatively well understood, yet we lack understanding of the non-genomic mechanisms. In the present study we replicated previous work on cultured ovine pituitary cells and observed that E2 decreased GnRH-induced secretion of LH (P < 0.05). To evaluate the effects of E2 on the subcellular mechanisms controlling release of LH, we turned to the homogenous gonadotrope cell line, LβT2. Unlike cultured ovine pituitary cells, treatment of LβT2 cells with E2 failed to inhibit GnRH-induced release of LH. Interestingly, LβT2 cells express very low levels of estrogen receptor-α (ERα) in contrast to observations on normal anterior pituitary cells. Based on these data, we postulated that increasing expression of ERα in LβT2 cells would restore the negative non-genomic effects of E2 on GnRH-induced release of LH observed previously in ovine pituitary cells. LβT2 cells were stably transfected with murine ERα (LβT2-ER). Treatment of these cells with E2 resulted in decreased release of LH in response to GnRH (P < 0.01). Furthermore, membrane impermeable estrogen, E2 conjugated to bovine serum albumin (E-BSA), inhibited GnRH-induced secretion of LH in this cell line. We concluded that the non-genomic effects of E2 were likely due to a membrane-associated ERα and were not the result of a non-specific steroid effect. This conclusion is based on the fact that E2 had no effect on release of LH in the parent LβT2 cells yet decreased GnRH-induced secretion of LH in LβT2-ER cells and the effect could be mimicked using membrane impermeable E-BSA. This was further corroborated by showing that PPT, an ERα specific agonist, but not DPN, an estrogen receptor-β specific agonist, inhibited GnRH- mediated release of LH. We investigated the effects of other steroid hormones in an effort to delineate further the non-genomic mechanisms affecting LH secretion. Non-genomic effects for progesterone, cortisol and testosterone have been demonstrated in other tissues. Acute effects of 17α-estradiol depends on the tissue type. In this study using LβT2-ER cells, we demonstrated negative effects of 17α-estradiol, progesterone, testosterone and cortisol on GnRH-induced secretion of LH. Additional studies are required to fully characterize these effects. Numerous investigators have demonstrated non-genomic effects of E2 on subcellular mechanisms. Currently, the data on the acute effects of estradiol on GnRH- stimulated signal transduction is limited. In the present study, both LβT2 and LβT2-ER cells responded to GnRH with increased accumulation of IP3, and E2 had no effect on the GnRH-stimulated formation of IP3 in either cell line. We observed a GnRH-evoked spike in intracellular calcium (Ca2+i) in both LβT2and LβT2-ER cells without the characteristic large-amplitude Ca2+i oscillations observed in normal gonadotropes. Interestingly, LβT2 cells seem to have retarded re-uptake of Ca2+i with the inclusion of E2 in the GnRH treatment. In contrast, in LβT2-ER cells there is a reduction of the increase in intracellular calcium when E2 is included with the GnRH treatment compared to cells treated only with GnRH. These initial experiments cannot distinguish the Ca2+ source, i.e. intracellular or extracellular, responsible for the alteration in Ca2+i levels. Since the effects of E2 on GnRH-induced increase in Ca2+ are immediate, it is presumed that the effects are mediated non-genomically. In summary, these results 1) clearly demonstrate the utility of LβT2-ER cells for examination of the non-genomic effects of E2 on GnRH-evoked secretion of LH; 2) reveal that E2 has a negative effect on the GnRH-induced increase in Ca2+i; and 3) establish that manifestation of the negative effect of E2 on LH secretion depends upon expression of ERα. Based on the results obtained in the present study, we have concluded that the new cell line, LβT2-ER, exhibits characteristics consistent with those of normal gonadotropes, such as rapid inhibition of GnRH-induced secretion of LH. In the future, it will be possible to use the LβT2-ER cell line not only as a representation of normal gonadotropes to evaluate cellular and molecular events acutely affected by steroid hormones, but also as a model to evaluate both the physiological and pathological non- genomic effects of estrogens.