Effect of Phytoestrogens on Basal and Gnrh-Induced Gonadotropin Secretion
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S A ARISPE and others Effect of E2 and PEs on 219:3 243–250 Research gonadotrope activity Effect of phytoestrogens on basal and GnRH-induced gonadotropin secretion Correspondence Sergio A Arispe, Betty Adams and Thomas E Adams should be addressed to T E Adams Department of Animal Science, University of California, Davis, One Shields Avenue, Davis, California 95616, USA Email [email protected] Abstract Plant-derived estrogens (phytoestrogens, PEs), like endogenous estrogens, affect a diverse Key Words array of tissues, including the bone, uterus, mammary gland, and components of the neural " LH and cardiovascular systems. We hypothesized that PEs act directly at pituitary loci to attenuate " FSH basal FSH secretion and increase gonadotrope sensitivity to GnRH. To examine the effect of PEs " cell culture on basal secretion and total production of FSH, ovine pituitary cells were incubated with PEs " phytoestrogens for 48 h. Conditioned media and cell extract were collected and assayed for FSH. Estradiol (E2) " estradiol and some PEs significantly decreased basal secretion of FSH. The most potent PEs in this regard " estrogen receptor were coumestrol (CM), zearalenone (ZR), and genistein (GN). The specificity of PE-induced " ovine pituitary cells suppression of basal FSH was indicated by the absence of suppression in cells coincubated with PEs and an estrogen receptor (ER) blocker (ICI 182 780; ICI). Secretion of LH during stimulation by a GnRH agonist (GnRH-A) was used as a measure of gonadotrope responsiveness. Journal of Endocrinology Incubation of cells for 12 h with E2, CM, ZR, GN, or daidzein (DZ) enhanced the magnitude and sensitivity of LH secretion during subsequent exposure to graded levels of a GnRH-A. The E2- and PE-dependent augmentation of gonadotrope responsiveness was nearly fully blocked during coincubation with ICI. Collectively, these data demonstrate that selected PEs (CM, ZR, and GN), like E2, decrease basal secretion of FSH, reduce total FSH production, and enhance GnRH-A-induced LH secretion in a manner that is dependent on the ER. Journal of Endocrinology (2013) 219, 243–250 Introduction Phytoestrogens (PEs) are nonsteroidal, organic hormone (LH), and follicle-stimulating hormone (FSH), compounds that are bound by one or both of the estrogen which have important roles in reproduction. Specifically, receptor (ER) isoforms, ERa and ERb (Kuiper et al. 1998). in females FSH stimulates follicular growth and maturation, Although nonsteroidal, the structure of these plant- whereas LH promotes steroidogenesis and ovulation. derived compounds generally includes a phenolic ring Although estrogens can affect gonadotropin synthesis and resembling the aromatic ring of ovarian-derived estrogens, secretion by influencing gonadotropin-releasing hormone such as 17b-estradiol (E2). (GnRH) release from hypothalamic loci, direct action of Endogenous estrogens affect a diverse array of tissues estrogens on gonadotrope cells is suggested by experiments including the gonadotrope cells of the pituitary. These cells using in vivo and in vitro models in which pituitary tissue synthesize the gonadotropic hormones, luteinizing is physically separated from hypothalamic inputs http://joe.endocrinology-journals.org Ñ 2013 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-13-0158 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 08:22:59PM via free access Research S A ARISPE and others Effect of E2 and PEs on 219:3 244 gonadotrope activity (Clarke & Cummins 1984). Indeed, effects of estrogens on and E2 (United States Biochemical Corp., Cleveland, OH, basal and GnRH-stimulated gonadotropin secretion are USA) were obtained from established vendors. evident in cell culture using ovine or rodent pituitary cells (Huang & Miller 1980). Although the effect of Cell culture endogenous estrogens on gonadotrope function is well recognized, much less is known regarding the effect of PEs Pituitary tissue was collected from castrated male yearling on the synthesis and secretion of FSH. This may be a sheep euthanized at a local abattoir by electrical particularly important concern in animal production stunning and exsanguination. Tissue was collected within systems, because several common forages, including alfalfa, 10 min of euthanasia and immersed in low calcium, clover, lupin, and some grains, contain high concentrations magnesium-free (LCMF) HBSS. LCMF HBSS was prepared of PEs (Dixon 2004). In addition, the rumen and intestinal by supplementing CMF HBSS with CaCl2.H2O, 1% BSA, microbiota of domestic livestock species may enhance the 25 mM HEPES, 0.035% NaHCO3, and penicillin/ estrogenic potency of feedstuffs by transforming plant streptomycin (100 units/ml). The tissue was dissociated precursors into active PEs (Zhou et al.2009). into component cells using a modification of the The potential effects of PEs on the reproductive procedure developed by Huang & Miller (1980). Briefly, efficiency of livestock is suggested by reports that female anterior pituitary tissue was dissected free of connective sheep and cattle grazing on subterranean clover (Trifolium tissue and the posterior pituitary and sliced into 0.5 mm subterran) or alfalfa (Medicago sativa) became infertile sections using a Stadie–Riggs tissue slicer. The pituitary (Bennetts et al. 1946, Moule et al. 1963). These effects of slices were minced into roughly 2 mm2 with scissors. The PEs on reproduction are likely to reflect PE-dependent minced tissue from five pituitary glands was combined change in the ER function. Indeed, Mathieson & Kutts and washed five times with LCMF HBSS before resuspen- (1980) report that genistein (GN) and coumestrol (CM) sion into 66 ml LCMF HBSS containing 200 mg collagen- compete with E2 for binding sites in hypothalamic and ase (290 U/mg). The tissue suspension was gently agitated pituitary tissue of sheep. in a rotary shaker (90 r.p.m.) at 37 8C for 90 min. After In the studies reported here, we examine the incubation with collagenase, the partially digested tissue physiological effect of PEs commonly present in feedstuffs was pelleted by centrifugation (400 g for 4 min). The provided to domestic animals. Our particular objective collagenase containing supernatant was decanted and was to determine the effect of PEs on basal secretion of tissue fragments were resuspended in 60 ml CMF HBSS Journal of Endocrinology FSH. In addition, we assessed the effect of PEs on GnRH- containing 0.25% pancreatin. Tissue fragments were induced LH secretion from ovine pituitary cells. incubated with pancreatin for 35 min at 37 8C with gentle agitation. At the conclusion of enzyme treatment, undigested tissue was removed by passage of the cell Materials and methods suspension through a nylon mesh filter. Dispersed cells were pelleted by centrifugation (400 g for 4 min) and Reagents and supplies washed four times with CMF HBSS. Cell yield was assessed Calcium and magnesium-free (CMF) Hanks balanced salt using a hemocytometer and viability was determined by solution (HBSS), and medium 199 (without phenol red, dye exclusion. Average yield was 200!106 cells/g initial L-glutamine, or NaHCO3; M199) were obtained from tissue (nZ5), with a viability O90% (meanZ95.5%). After 6 Sigma–Aldrich. Bovine insulin, the GnRH agonist (D-Ala , the final wash with CMF HBSS, the cell pellet was 10 des-Gly GnRH ethylamide; GnRH-A), pancreatin, the ER suspended using M199 containing NaHCO3 (2.2 g/l), antagonist (ICI 780 182; fulvestrant), and most PEs were L-glutamine (200 mM), insulin (5 mg/ml), gentamycin also purchased from Sigma–Aldrich. The PE, daidzein (DZ), sulfate (60 mg/ml), penicillin/streptomycin (100 units/ml), was purchased from Tocris Bioscience (Ellisville, MO, USA). fungizone (500 ng/ml), HEPES sodium salt (25 mM), and The antifungal drug, fungizone, and the two antibiotics, 10% serum collected from castrated male sheep (M199C gentamycin sulfate and penicillin/streptomycin, 10% wether serum (WS)). One milliliter aliquot of the cell were obtained from HyClone (Logan, UT, USA), suspension (0.5!106 cells/ml) was used to seed the Mediatech (Manassas, VA, USA), and Gibco respectively. 24-well culture plates. The seeded plates were placed in a Collagenase (type-I; Worthington Biochemical Corpo- water-jacketed incubator and cells were allowed to attach ration, Lakewood, NJ, USA), L-glutamine (Invitrogen), during 36–48 h culture at 37 8Cinahumidified BSA (fraction V; EMD Chemicals, Gibbstown, NJ, USA), atmosphere of 5% CO2 and air. http://joe.endocrinology-journals.org Ñ 2013 Society for Endocrinology Published by Bioscientifica Ltd. DOI: 10.1530/JOE-13-0158 Printed in Great Britain Downloaded from Bioscientifica.com at 09/25/2021 08:22:59PM via free access Research S A ARISPE and others Effect of E2 and PEs on 219:3 245 gonadotrope activity Experimental procedure effects included treatment, experiment, and treatment– experiment interaction, while plate was included as a random The effect of E and PEs on the basal secretion of FSH from 2 effect. In contrast, GnRH-A-induced LH secretion was attached pituitary cells was assessed by removing con- analyzed using a model that included the fixed effects of ditioned culture media, washing each well with 1 ml treatment, GnRH, and treatment–GnRH-A interaction; M199C10% WS and, finally adding 1 ml M199C10% WS experiment and experiment–GnRH-A, while plate and the appropriate concentration of test compound (E with 2 plate–GnRH-A were included as random effects. The PE). The PEs (CM, zearalenone (ZR), GN, DZ, resveratrol likelihood-ratio-test was used to determine the significance (RV), biochanin A (BA), and enterolactone (EL)) were of the fixed effects (P!0.05) and competing models where diluted in DMSO to working concentrations (0.001–1.0 mM) fitted using maximum likelihood. Contrasts were setup to using M199C10% WS (final DMSO concentration was determine the mean comparisons against the negative control 0.2%). Vehicle also contained DMSO. We found no (vehicle) and positive controls (0.05 and 5 nM E2). The evidence that DMSO at the concentration (0.2%) used in Bonferroni correction was used to control the type 2 error rate.