Binding Characteristics of Estrogen Receptor (ER) in Atlantic Croaker
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BIOLOGY OF REPRODUCTION 61, 51±60 (1999) Binding Characteristics of Estrogen Receptor (ER) in Atlantic Croaker (Micropogonias undulatus) Testis: Different Af®nity for Estrogens and Xenobiotics from that of Hepatic ER 1 Anna Katrina Loomis and Peter Thomas2 Department of Marine Science, Marine Science Institute, University of Texas at Austin, Port Aransas, Texas 78373 ABSTRACT spermatogenesis and fertility [10]. However, although the existence of estrogens and ERs in males is well recognized An estrogen receptor (ER) was identi®ed in cytosolic and nu- throughout the vertebrates, their precise physiological roles Downloaded from https://academic.oup.com/biolreprod/article/61/1/51/2734627 by guest on 31 December 2020 clear fractions of the testis in a marine teleost, Atlantic croaker in male reproduction remain unclear. (Micropogonias undulatus). A single class of high af®nity, low capacity, and displaceable binding sites was identi®ed by satu- The ER in the testis is also a potential target for endo- ration analysis, with a K of 0.40 nM in cytosolic extracts and a crine disruption by xenoestrogens. Evidence is accumulat- d ing which suggests that estrogenic compounds, including Kd of 0.33 nM in nuclear extracts. Competition studies demon- strated that the receptor was highly speci®c for estrogens (di- pesticides and industrial chemicals, impair male reproduc- ethylstilbestrol . estradiol k estriol 5 estrone) and also bound tive function in wildlife [11, 12]. Rainbow trout exposed to several antiestrogens. Testosterone and 5a-dihydrotestosterone estrogenic alkylphenolic chemicalsÐnonionic surfactants had much lower af®nities for the receptor, whereas no displace- that are often detected in sewage ef¯uentÐexhibit inhibi- ment of speci®c binding occurred with 11-ketotestosterone or tion of testicular growth [13]. Moreover, increased exposure any of the C21 maturation-inducing steroids. A variety of xen- to environmental xenoestrogens has been suggested as a oestrogens, including o,p9-dichlorodiphenyltrichloroethane (DDT), possible cause of the apparent increase in male reproductive chlordecone (Kepone), nonylphenol, hydroxylated polychlori- disorders and purported decrease in sperm count in humans nated biphenyls (PCBs), and the mycotoxin zearalenone, bound over the past several decades [14±16]. to the receptor with relatively low binding af®nities, 1023 to Although the hepatic ER has been characterized in a 1025 that of estradiol. A comparison of the binding af®nities of wide variety of teleosts [17±19], until now a testicular ER various ligands for the testicular ER and the hepatic ER in this has not been identi®ed in any teleost species. The purpose species revealed that the testicular ER was saturated at a lower of this study, therefore, was to identify the ER in the testis [3H]estradiol concentration (1 nM vs. 4 nM). The binding af®n- of the marine teleost, Atlantic croaker (Micropogonias un- ities of several compounds, including testosterone and nafoxi- dulatus), in order to begin de®ning the role of estrogens in dine, exhibited marked differences for the two ERs; and most of male reproductive physiology in teleosts. The hormone the estrogens and xenoestrogens tested had higher binding af- binding characteristics of the nuclear ER in the Atlantic ®nities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments croaker testis were examined as well as the binding af®n- incubated in vitro, and estradiol was detected in male Atlantic ities of several xenoestrogens. Synthesis of estradiol was croaker plasma. The identi®cation of a testicular ER and evi- also investigated to con®rm testicular production of the nat- dence that estradiol is produced by the testes in croaker suggest ural ligand of the ER in this species. that estrogens participate in the hormonal control of testicular Recently a second ER, ERb, has been identi®ed and function in teleosts. characterized in mammals [20]. There are differences in the binding af®nities of various ligands for the a and b forms INTRODUCTION of the mammalian ER. Moreover, they have different tissue distributions, with ERb showing high expression levels in Although estrogens are considered to be primarily fe- the testis [21]. Preliminary evidence has also been obtained male reproductive hormones with numerous targets and ac- for multiple forms of the ER in Atlantic croaker, one form tions in the female reproductive system, recent evidence predominating in the gonads and another in the liver [22]. suggests that they may also have important roles in the Therefore, the binding af®nities of a variety of estrogens regulation of gonadal function in males. Estrogens have and xenoestrogens for the testicular and hepatic ERs in At- been detected in the testes and male plasma of several ver- lantic croaker were compared to determine whether similar tebrate species [1, 2]. In addition, classical nuclear estrogen differences in ERs from different tissues exist in a teleost receptors (ERs) have been identi®ed in the male reproduc- species. tive systems of both mammalian and nonmammalian ver- tebrates, including the rat testis, mouse epididymis and ef- MATERIALS AND METHODS ferent ductules, human epididymis and seminal vesicle, and testes of the Urodele amphibian, Necturus maculosus, the Fish and Tissue Collection freshwater turtle, Chrysemys picta, and the elasmobranch, One-year-old Atlantic croaker (;35 g) were collected by Squalus acanthias [3±9]. Studies on ER knock-out mice otter trawl from the bays near Port Aransas, Texas. Fish demonstrate that a functional ER is required for normal were caught in September, at the beginning of gonadal re- crudescence, and were maintained in 4200-liter circular, re- Accepted February 5, 1999. circulating tanks at a temperature of 22±258C under an 11L: Received December 3, 1998. 13D photoperiod, and fed a mixed diet of commercial pel- 1This study was funded by Texas Sea Grant College Program, no. R/ lets and shrimp (3% of BW/day). Spermiating males and MBT-3 and EPA STAR grant no. R826125. 2Correspondence: Peter Thomas, University of Texas Marine Science mature, vitellogenic females were rapidly killed by severing Institute, 750 Channel View Drive, Port Aransas, TX 78373. FAX: 512 749 the spinal cord, and testicular and liver tissues, respectively, 6777; e-mail: [email protected] were collected. Blood was collected from the caudal vein 51 52 LOOMIS AND THOMAS of spermiating males. Tissue for the ER assays and plasma tant was discarded. The washing step was repeated 3 times, for estradiol measurement were stored at 2808C and and the remaining pellet was extracted with 3 ml TEDGK 2208C, respectively, whereas testicular estradiol production for 1 h with vigorous mixing every 10±15 min, and then in vitro was measured immediately after tissue collection. centrifuged at 12 800 3 g for 45 min. Frozen samples showed negligible loss of receptor binding when stored at 2808C for at least 6 mo. Measurement of Kd and Bmax Saturation analysis was performed by incubating 100 ml Chemicals of cytosolic or nuclear extract with 100 ml of varying con- [2,4,6,7-3H]Estradiol-17b ([3H]estradiol, 84 Ci/mmol) centrations of [3H]estradiol in TEDG, with ®nal concentra- was purchased from New England Nuclear (Boston, MA). tions ranging from 0.125 to 4 nM. Nonspeci®c binding was Nonradioactive steroids were purchased from Sigma Chem- determined by incubating extracts with [3H]estradiol and ical Company (St. Louis, MO) or Steraloids Inc. (Wilton, 100-fold excess concentrations of DES. Speci®c binding was de®ned as the fraction of total binding displaced by NH). Diethylstilbestrol (DES), tamoxifen citrate, and naf- Downloaded from https://academic.oup.com/biolreprod/article/61/1/51/2734627 by guest on 31 December 2020 oxidine hydrochloride were purchased from ICN Biomed- 100-fold excess DES. Extracts (1:20 w:v, cytosol; 1:6 w:v, icals Inc. (Aurora, OH). Zearalenone was obtained from nuclear) were incubated for 18 h at 48C. Free steroid was Sigma Chemical Company. Hydroxylated polychlorinated removed by incubation with 0.5 ml DCC for 20 min fol- biphenyls (PCBs: 4,49-PCB-3-OH, 4,49-dichloro-3-biphen- lowed by centrifugation at 3200 3 g for 20 min. Bound ylol; 29,59-PCB-3-OH, 29,59-dichloro-3-biphenylol; 2,29,59- [3H]estradiol was measured in a liquid scintillation counter PCB-4-OH, 2,29,59-trichloro-4-biphenylol; 29,39,49,59-PCB- for 5 min (LS 6000SC; Beckman Instruments Inc., Fuller- 4-OH, 29,39,49,59-tetrachloro-4-biphenylol) were purchased ton, CA). The equilibrium dissociation constant (Kd) and from Ultra Scienti®c (North Kingston, RI). Dichlorodi- binding capacity (Bmax) were calculated from Scatchard phenyltrichloroethane (DDT) compounds and chlordane analysis of the speci®c binding data [23]. Protein content were purchased from Chem Service (West Chester, PA). of tissue extracts was determined using the method of Brad- Chlordecone (Kepone) was obtained from the the National ford [24]. Institute of Environmental Health Sciences Repository. 4- Nonylphenol was obtained from the Huntsman Corporation Variation of Kd and Bmax during Testicular Recrudescence (Port Neches, TX). Aroclor 1254 was purchased from the Saturation analyses were performed on cytosolic frac- Foxboro Co. (North Haven, CT). ICI 164384 and ICI tions of testes collected throughout gonadal recrudescence 182780 were gifts from Dr. A.E. Wakeling at Zeneca Phar- from 17 individuals with gonado-somatic indexes (GSIs) maceuticals (Cheshire, England). All other chemicals were ranging from 1.62% to 12.7%. GSI was calculated as (go- reagent grade and purchased from general laboratory sup- nad weight 3 100)/(total weight 2 gonad weight). K and pliers. d Bmax were determined for individual ®sh in most cases. Tes- tes were pooled from ®sh with GSIs lower than 1.95%. ER Assay Buffers Homogenization buffer (TEDG) consisted of Tris base Association and Dissociation Kinetics of the Testicular ER (50 mM), Na2 EDTA (1.5 mM), dithiothreitol (1.0 mM), To determine the association kinetics of receptor bind- and glycerol (30% v:v), pH 5 7.4.