Differential Modulation of Gonadotropin Secretion by Selective Estrogen Receptor 1 and Estrogen Receptor 2 Agonists in Ovariectomized Ewes

BIOLOGY OF REPRODUCTION 77, 320–328 (2007) Published online before print 11 April 2007. DOI 10.1095/biolreprod.107.060046 Differential Modulation of Gonadotropin Secretion by Selective Estrogen Receptor 1 and Estrogen Receptor 2 Agonists in Ovariectomized Ewes1 J. Alejandro Arreguin-Arevalo, Tracy L. Davis, and Terry M. Nett2 Department of Biomedical Science, Colorado State University, Fort Collins, Colorado 80523 INTRODUCTION The objectives of this study were to determine whether activation of estrogen receptor 1 (ESR1; also known as ERalpha), or estrogen receptor 2 (ESR2; also known as ERbeta), or both are required to: 1) acutely inhibit secretion of LH, 2) induce the preovulatory-like surge of LH, and 3) inhibit secretion of FSH in ovariectomized (OVX) ewes. OVX ewes (n = 6) were administered intramuscularly 25 micrograms estradiol (E2), 12 mg propylpyrazoletriol (PPT; a subtype-selective ESR1 agonist), 21 mg diaprylpropionitrile (DPN; a subtype-selective ESR2 agonist), or PPT + DPN. Like E2, administration of PPT, DPN, or combination of the two rapidly decreased (P , 0.05) secretion of LH. Each agonist induced a gradual, prolonged rise in secretion of LH after the initial inhibition, but neither agonist alone nor the combined agonists was able to induce a ‘‘normal’’ preovulatory-like surge of LH similar to that induced by E2. Compared with E2-treated ewes, the beginning of the increase in secretion of LH occurred earlier (P , 0.01) in DPN-treated ewes, later (P , 0.05) in PPT-treated ewes, and at a similar interval in ewes receiving the combined agonist treatment. Like E2, PPT decreased (P , 0.05) secretion of FSH, but the duration of suppression was much longer in PPT-treated ewes. DPN did not alter secretion of FSH in this study. Modulation of the number of GnRH receptors by PPT and DPN was examined in primary cultures of ovine pituitary cells. In our hands, both PPT and DPN increased the number of GnRH receptors, but the dose of DPN required to stimulate synthesis of GnRH receptors was 10 times higher than that of PPT. We conclude that in OVX ewes: 1) ESR1 and ESR2 mediate the negative feedback of E2 on secretion of LH at the level of the pituitary gland, 2) ESR1 and ESR2 do not synergize or antagonize the effects of each other; however, they do interact to synchronize the beginning of the stimulatory effect of E2 on secretion of LH, 3) ESR1 and ESR2 may mediate at least partially the positive feedback of E2 on LH secretion by increasing the number of GnRH receptors, and 4) only ESR1 appears to be involved in the negative feedback of E2 on secretion of FSH. Estradiol (E2) regulates many physiologic processes in the reproductive system [1–3] through at least two main estrogen receptors (ERs), estrogen receptor 1 (ESR1) and estrogen receptor 2 (ESR2) (also known as ERa and ERb, respectively). These receptors are widely distributed in the reproductive axis [4–8]. ESR1 is the more abundant receptor in most reproductive tissues, particularly the uterus and pituitary of human and rat [5, 6, 9–13]. The ESR2 isoform has not been reported in ovine pituitaries, but it has been localized in rat gonadotropes [9]. In brain and hypothalamus of rat, mRNAs for Esr1 and Esr2 are expressed in a partially overlapping pattern [5, 6, 11, 14]. Apparently, only mRNAs for Esr2 and its protein are localized within rodent GnRH neurons [15–17]. During recent years, indirect but increasing amounts of evidence strongly support the presence of binding proteins distinct from ESR1 and ESR2 isoforms that mediate actions of E2 in the brain [11, 18, 19]. Information generated in Esr1/ knockout (alpha ERKO), Esr2/ knockout (beta ERKO), and double Esr1//Esr2/ knockout (alpha/beta ERKO) mice has led to the general consensus that ESR1 is the primary receptor through which E2 mediates its negative feedback on gonadotropin secretion [1, 20–23]. The use of ER-selective ligands has confirmed the importance of ESR1 in the regulation of the hypothalamicpituitary axis [24–31]; however, use of these ligands has also revealed the roles of ESR2 in various systems, including the hypothalamic-pituitary axis [28, 29, 32, 33]. A selective effect of E2 acting exclusively though ESR1 [25, 31, 34, 35] or ESR2 [24, 31, 33] has been demonstrated using propylpyrazoletriol (PPT, a subtype-selective ESR1 agonist), diaprylpropionitrile (DPN, a subtype-selective ESR2 agonist), and other ERselective ligands. Moreover, some ESR1-mediated actions of E2 can be reduced [34], enhanced [34, 36], or not modified [24, 34] by activation of ESR2. The evaluation of these ERselective ligands includes their ability to mimic, in vitro, nongenomic actions of E2 [36, 37]. The ability of PPT and DPN to cross the blood-brain barrier and elicit a biologic response has also been demonstrated. Systemic administration of PPT upregulated progesterone receptor (Pgr) mRNA [38] and PGR immunoreactivity [24] in rat hypothalamus and elicited a neuroprotective effect in mouse dopaminergic neurons [35]. Similarly, systemic administration of DPN produced an anxiolytic behavior in rats [24, 31] and bound to nuclear ER in brain within 30 min of administration [24]. Our knowledge of the actions of E2 in the hypothalamicpituitary axis has grown exponentially during the last decade; however, the participation of the main ER subtypes in the regulation of gonadotropin secretion in ewes is practically unexplored. It has been well established that administration of E2 to ovariectomized (OVX) ewes rapidly (within minutes) decreases secretion of LH, followed several hours later by a preovulatory-like surge of LH [39–41]; in contrast, secretion of FSH does not decrease until several hours after anterior pituitary, estradiol, estradiol receptor subtypes, folliclestimulating hormone, luteinizing hormone 1 Supported by National Research Initiative Competitive Grant no. 2005-35203-15376 from the United States Department of Agriculture Cooperative State Research, Education, and Extension Service. 2 Correspondence: Terry M. Nett, Animal Reproduction and Biotechnology Laboratory, Department of Biomedical Science, 3801 West Rampart Rd., Fort Collins, CO 80523-1683. FAX: 970 491 3557; e-mail: Received: 9 January 2007. First decision: 7 February 2007. Accepted: 9 April 2007. Ó 2007 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org 320 ABSTRACT REGULATION OF GONADOTROPIN SECRETION BY ER AGONIST 321 FIG. 1. RBAs of PPT and DPN agonists were determined in cytosolic fraction of ovine uterus. RBAs for PPT and DPN were 1.52% and 0.52%, respectively. RBA = IC50E2/IC50 test compund 3 100. RBA for E2 is 100%. Data (mean 6 SEM) represent the average of three replicates using the same tissue preparation and different stocks of reagents. endocrine paradigm by using ER-selective ligands in OVX ewes. The specific objectives of this study were to determine whether activation of ESR1, ESR2, or both is required to: 1) acutely (...truncated)