| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
a Department of Marine Science, Marine Science Institute, University of Texas at Austin, Port Aransas, Texas 78373
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-dihydrotestosterone had much lower affinities for the receptor, whereas no displacement of specific binding occurred with 11-ketotestosterone or any of the C21 maturation-inducing steroids. A variety of xenoestrogens, including o,p'-dichlorodiphenyltrichloroethane (DDT), chlordecone (Kepone), nonylphenol, hydroxylated polychlorinated biphenyls (PCBs), and the mycotoxin zearalenone, bound to the receptor with relatively low binding affinities, 10-3 to 10-5 that of estradiol. A comparison of the binding affinities of various ligands for the testicular ER and the hepatic ER in this species revealed that the testicular ER was saturated at a lower [3H]estradiol concentration (1 nM vs. 4 nM). The binding affinities of several compounds, including testosterone and nafoxidine, exhibited marked differences for the two ERs; and most of the estrogens and xenoestrogens tested had higher binding affinities for the testicular receptor. Minor amounts of estradiol (0.12 ng/g tissue/h) were produced by testicular tissue fragments incubated in vitro, and estradiol was detected in male Atlantic croaker plasma. The identification of a testicular ER and evidence that estradiol is produced by the testes in croaker suggest that estrogens participate in the hormonal control of testicular function in teleosts.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The ER in the testis is also a potential target for endocrine disruption by xenoestrogens. Evidence is accumulating which suggests that estrogenic compounds, including pesticides and industrial chemicals, impair male reproductive function in wildlife [11, 12]. Rainbow trout exposed to estrogenic alkylphenolic chemicals—nonionic surfactants that are often detected in sewage effluent—exhibit inhibition of testicular growth [13]. Moreover, increased exposure to environmental xenoestrogens has been suggested as a possible cause of the apparent increase in male reproductive disorders and purported decrease in sperm count in humans over the past several decades [14–16].
Although the hepatic ER has been characterized in a wide variety of teleosts [17–19], until now a testicular ER has not been identified in any teleost species. The purpose of this study, therefore, was to identify the ER in the testis of the marine teleost, Atlantic croaker (Micropogonias undulatus), in order to begin defining the role of estrogens in male reproductive physiology in teleosts. The hormone binding characteristics of the nuclear ER in the Atlantic croaker testis were examined as well as the binding affinities of several xenoestrogens. Synthesis of estradiol was also investigated to confirm testicular production of the natural ligand of the ER in this species.
Recently a second ER, ERß, has been identified and characterized in mammals [20]. There are differences in the binding affinities of various ligands for the and ß forms of the mammalian ER. Moreover, they have different tissue distributions, with ERß showing high expression levels in the testis [21]. Preliminary evidence has also been obtained for multiple forms of the ER in Atlantic croaker, one form predominating in the gonads and another in the liver [22]. Therefore, the binding affinities of a variety of estrogens and xenoestrogens for the testicular and hepatic ERs in Atlantic croaker were compared to determine whether similar differences in ERs from different tissues exist in a teleost species.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
One-year-old Atlantic croaker (~35 g) were collected by otter trawl from the bays near Port Aransas, Texas. Fish were caught in September, at the beginning of gonadal recrudescence, and were maintained in 4200-liter circular, recirculating tanks at a temperature of 22–25°C under an 11L:13D photoperiod, and fed a mixed diet of commercial pellets and shrimp (3% of BW/day). Spermiating males and mature, vitellogenic females were rapidly killed by severing the spinal cord, and testicular and liver tissues, respectively, were collected. Blood was collected from the caudal vein of spermiating males. Tissue for the ER assays and plasma for estradiol measurement were stored at -80°C and -20°C, respectively, whereas testicular estradiol production in vitro was measured immediately after tissue collection. Frozen samples showed negligible loss of receptor binding when stored at -80°C for at least 6 mo.
Chemicals
[2,4,6,7-3H]Estradiol-17ß ([3H]estradiol, 84 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Nonradioactive steroids were purchased from Sigma Chemical Company (St. Louis, MO) or Steraloids Inc. (Wilton, NH). Diethylstilbestrol (DES), tamoxifen citrate, and nafoxidine hydrochloride were purchased from ICN Biomedicals Inc. (Aurora, OH). Zearalenone was obtained from Sigma Chemical Company. Hydroxylated polychlorinated biphenyls (PCBs: 4,4'-PCB-3-OH, 4,4'-dichloro-3-biphenylol; 2',5'-PCB-3-OH, 2',5'-dichloro-3-biphenylol; 2,2',5'-PCB-4-OH, 2,2',5'-trichloro-4-biphenylol; 2',3',4',5'-PCB-4-OH, 2',3',4',5'-tetrachloro-4-biphenylol) were purchased from Ultra Scientific (North Kingston, RI). Dichlorodiphenyltrichloroethane (DDT) compounds and chlordane were purchased from Chem Service (West Chester, PA). Chlordecone (Kepone) was obtained from the the National Institute of Environmental Health Sciences Repository. 4-Nonylphenol was obtained from the Huntsman Corporation (Port Neches, TX). Aroclor 1254 was purchased from the Foxboro Co. (North Haven, CT). ICI 164384 and ICI 182780 were gifts from Dr. A.E. Wakeling at Zeneca Pharmaceuticals (Cheshire, England). All other chemicals were reagent grade and purchased from general laboratory suppliers.
ER Assay Buffers
Homogenization buffer (TEDG) consisted of Tris base (50 mM), Na2 EDTA (1.5 mM), dithiothreitol (1.0 mM), and glycerol (30% v:v), pH = 7.4. Nuclear wash buffer (TMDS) comprised Tris base (50 mM), MgCl2 (10.7 mM), sucrose (250 mM), and dithiothreitol (1.0 mM), pH = 7.4. Nuclear extraction buffer (TEDGK) consisted of TEDG buffer + KCl (600 mM). Dextran-coated charcoal (DCC) was 0.4% w:v activated charcoal, 0.1% w:v Dextran T-70 in TEDG (for saturation analysis, association and dissociation kinetics, and steroid competition curves); or 0.6% w:v activated charcoal, 0.15% w:v Dextran T-70 in TEDG (for xenobiotic competition curves).
Preparation of Tissue Extracts
Liver and testicular tissue extracts were prepared using the protocol described for the spotted seatrout hepatic ER [18]. All procedures were performed on ice or at 4°C. One gram of tissue was homogenized in 9 ml TEDG with 4 passes of a polytetrafluoroethylane (Teflon) pestle in a glass homogenizer. The homogenate was centrifuged for 20 min at 820 x g. The supernatant was centrifuged for 1 h at 103 000 x g to obtain a cytosolic extract, which was assayed for receptor content. Lipid layers were aspirated from the liver tissue extracts after each centrifugation step. The cytosolic fraction was assayed immediately or frozen at -80°C until use (binding remained constant in cytosolic and nuclear extracts for at least 1 mo).
To obtain a nuclear fraction, the pellet from the initial centrifugation was washed with 10 ml TMDS, the mixture was centrifuged at 1150 x g for 20 min, and the supernatant was discarded. The washing step was repeated 3 times, and the remaining pellet was extracted with 3 ml TEDGK for 1 h with vigorous mixing every 10–15 min, and then centrifuged at 12 800 x g for 45 min.
Measurement of Kd and Bmax
Saturation analysis was performed by incubating 100 µl of cytosolic or nuclear extract with 100 µl of varying concentrations of [3H]estradiol in TEDG, with final concentrations ranging from 0.125 to 4 nM. Nonspecific binding was determined by incubating extracts with [3H]estradiol and 100-fold excess concentrations of DES. Specific binding was defined as the fraction of total binding displaced by 100-fold excess DES. Extracts (1:20 w:v, cytosol; 1:6 w:v, nuclear) were incubated for 18 h at 4°C. Free steroid was removed by incubation with 0.5 ml DCC for 20 min followed by centrifugation at 3200 x g for 20 min. Bound [3H]estradiol was measured in a liquid scintillation counter for 5 min (LS 6000SC; Beckman Instruments Inc., Fullerton, CA). The equilibrium dissociation constant (Kd) and binding capacity (Bmax) were calculated from Scatchard analysis of the specific binding data [23]. Protein content of tissue extracts was determined using the method of Bradford [24].
Variation of Kd and Bmax during Testicular Recrudescence
Saturation analyses were performed on cytosolic fractions of testes collected throughout gonadal recrudescence from 17 individuals with gonado-somatic indexes (GSIs) ranging from 1.62% to 12.7%. GSI was calculated as (gonad weight x 100)/(total weight - gonad weight). Kd and Bmax were determined for individual fish in most cases. Testes were pooled from fish with GSIs lower than 1.95%.
Association and Dissociation Kinetics of the Testicular ER
To determine the association kinetics of receptor binding, 0.5 ml of cytosolic extract was incubated in triplicate with 1 nM [3H]estradiol in the presence or absence of 100 nM estradiol. At specific time points between 15 min and 24 h, the reaction was stopped with the addition of 0.5 ml DCC. The extracts were incubated with DCC for 20 min, and then centrifuged at 3200 x g for 20 min.
Dissociation kinetics of receptor binding were determined after a 14-h incubation of cytosolic extract with 1 nM [3H]estradiol. Dissociation began with the addition of 100 nM estradiol, and specific binding was measured at time points between 15 min and 24 h. Incubations were terminated with the addition of 0.5 ml DCC. Dissociation was expressed as a percentage of the specific binding present in control incubations at each time point.
Steroid Specificity and Xenobiotic Binding to Testis and Liver ER
Two hundred microliters of testicular cytosolic extract was incubated with 300 µl of [3H]estradiol (final concentration 1 nM) in TEDG for 18 h with or without competitors. Concentrations of competitors ranged from 10 pM to 1 mM. All competitors were dissolved in ethanol and added to the cytosolic extract for a final ethanol concentration of 1%. Free steroid was removed with the addition of 0.5 ml DCC followed by a 20-min incubation before centrifugation at 3200 x g. Maximum specific binding was defined as the fraction of total [3H]estradiol binding suppressed by 100-fold excess DES (100 nM). Competitor binding was expressed as a percentage of maximum specific binding. Five concentrations were tested for each competitor, and the assay was repeated with 3 different cytosolic extracts for each competitor.
The hepatic ER is an intermediary in the regulation of vitellogenesis by estradiol in females and is usually not present in high concentrations in males [25]. Therefore, livers from mature females were used in these investigations. The liver ER saturates between 4 and 8 nM estradiol, and 4 nM [3H]estradiol was used for all liver competition assays. Maximum specific binding was determined by incubation with 400 nM DES.
Measurement of Testicular Estradiol Production In Vitro
Testes were removed from spermiating Atlantic croaker and kept on ice. Approximately 100-mg tissue fragments were incubated in 24-well polystyrene incubation plates with 1-ml aliquots of Dulbecco's Modified Eagle's medium lacking any pH indicator dye, supplemented with sodium bicarbonate (1.2 g/L), penicillin (60 mg/L), and streptomycin (100 mg/L), at a pH of 7.4. After a 30- to 60-min preincubation, tissue was coarsely chopped, medium was removed, and 1 ml of fresh medium was added to each incubation well. Tissue fragments were incubated for 9 h in a Dubnoff metabolic shaking incubator (Precision Scientific, Chicago, IL) in a humid environment at 25°C under an atmosphere of oxygen. At the end of the incubation period, media were removed and frozen at -20°C until assayed for estradiol. Because estradiol production was expected to be low, samples were concentrated by filtering several milliliters of incubation medium through Sep-Pak C18 cartridges (Waters Chromatography Division, Millipore Corporation, Milford, MA). Media from several incubations were combined, and analysis was performed on 2 or 2.8 ml of media. Steroids were eluted from Sep-Paks with 2 ml water followed by 2 ml 10% methanol, and finally 2 ml 100% methanol, which was collected and dried down under a stream of nitrogen. Extracts were resuspended in a phosphate buffer and subsequently analyzed for estradiol by RIA according to the protocol of Smith and Thomas [25]. The overall mean steroid recovery efficiency was 85%.
Measurement of Estradiol in Male Plasma
Two hundred-microliter aliquots of plasma from 12 mature early recrudescing male Atlantic croaker (mean GSI = 5.28 ± 0.56%) were assayed for estradiol after solvent extraction (hexane/ethyl acetate, 70:30) by specific RIA [25]. Mean steroid recovery efficiency was 76%. The antiserum shows negligible cross-reaction with teleostean androgens (< 0.02%) and recognizes primarily estradiol-17ß (cross-reactivity with estrone and estriol < 0.7%) [25].
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Saturation and Scatchard analysis of binding to testicular cytosolic extracts showed a single class of high-affinity binding sites that saturated at 1–2 nM [3H]estradiol and had a mean equilibrium dissociation constant (Kd) of 0.40 ± 0.04 nM (n = 13) and a mean maximum binding capacity (Bmax) of 0.023 ± 0.004 nM, equal to 0.23 ± 0.04 pmol/g testis (n = 17, Fig. 1, A and B).
|
Specific binding of [3H]estradiol to testicular nuclear extracts was also saturated between 1 and 2 nM (Fig. 2, A and B). Scatchard analysis showed a single class of high affinity, saturable binding sites with a mean Kd of 0.33 ± 0.01 nM (n = 16). The mean Bmax for nuclear extracts was 0.028 ± 0.002 nM, equal to 0.083 ± 0.007 pmol/g testis, for individuals with a GSI greater than 3% (n = 11).
|
Variation in Kd and Bmax during Gonadal Recrudescence
The Kd of testicular cytosolic extracts varied from 0.21 to 0.73 nM, but no consistent trend was apparent during the period of testicular growth. The Bmax ranged from nondetectable to 0.043 nM, and on a per-testis basis increased during recrudescence from below the assay detection limits to 3.04 pmol per testis (r2 = 0.72, data not shown). The Kd for nuclear fractions varied little during gonadal growth, ranging from 0.25 to 0.44 nM. The receptor content of the nuclear extracts ranged from 0.013 to 0.071 nM during recrudescence, with higher values in individuals with GSIs below 3%, and fewer binding sites as GSIs increased. This trend was consistent when Bmax was calculated in terms of moles per gram of protein, but no trend was apparent when the Bmax was calculated on a per-testis basis (data not shown).
Association and Dissociation Kinetics of Testicular ER
The association and dissociation kinetics of [3H]estradiol specific binding to the testicular ER are shown in Figure 3. At 4°C, association occurred with a T1/2 of 1 h, and maximum association was reached after 5 h. Binding remained constant up to 16 h, and approximately 36 ± 21% of specific binding was lost by 24 h. Dissociation of [3H]estradiol occurred with a T1/2 of 8 h, and after 24 h 84 ± 2% of the [3H]estradiol had dissociated from its binding site.
|
Binding Specificity of Testicular ER
The synthetic estrogen DES bound to the ER with approximately 5 times higher affinity than estradiol (EC50 1.3 x 10-10 vs. 7.0 x 10-10 for estradiol, Table 1), and this compound was used for calculating relative binding affinities (RBAs). The natural estrogens estradiol, estriol, and estrone all bound with high affinity to the testicular ER (Fig. 4A). Estriol and estrone had 20 times less affinity for the receptor than estradiol on the basis of RBA values (Table 1). The antiestrogens ICI 164384 and ICI 182780 bound with 1% and 3% of the affinity for DES, respectively. The antiestrogens tamoxifen citrate and nafoxidine hydrochloride also bound, but with 100- to 1000-fold less affinity than the ICI compounds.
|
|
Among the other steroids assayed for binding affinity, including androgens, cortisol, and the two maturation-inducing steroids—17,20ß,21-trihydroxy-4-pregnen-3-one (20ß-S) and 17,20ß-dihydroxy-4-pregnen-3-one (17,20ß-P)—only two of the androgens, testosterone and 5-dihydrotestosterone, were able to displace specific binding (Fig. 4B).
Xenobiotic Binding to the Testicular ER
Zearalenone, 4-nonylphenol, and one hydroxylated PCB (2',3',4',5'-tetrachloro-4-biphenylol) were all able to displace 50% of the specific binding at concentrations around 1 µM (Fig. 4C). All o,p' congeners of the DDT compounds were able to displace 50% of the specific binding at approximately 30 µM (Fig. 4C, Table 1), whereas the p,p' congeners of DDT bound with less affinity and did not displace 50% of the specific binding. The chlorinated pesticide Kepone was able to displace more than 80% of the specific binding at a concentration of 100 µM.
Saturation Analysis of Hepatic ER
Saturation and Scatchard analysis of binding to hepatic cytosolic extracts showed a single class of binding sites with a mean Kd of 2.75 ± 0.53 nM and a mean Bmax of 0.46 ± 0.11 nM, equal to 4.6 ± 1.1 pmol/g liver (n = 5; Fig. 5). Saturation of this receptor occurred between 4 and 8 nM [3H]estradiol, a concentration four times higher than that required to saturate the testicular ER.
|
Testicular and Hepatic ER Binding Comparisons
A comparison of hepatic and testicular ER binding showed several differences. The testicular extracts had a higher affinity than the liver extracts for the majority of competitors tested, as shown by the concentrations required to displace 50% of the specific binding (EC50s, Table 1). The EC50s of DES, testosterone, nafoxidine, and several xenobiotics were more than an order of magnitude higher in the liver extracts. However, this was not the case for all compounds tested. The EC50s of ICI 182780 and estrone were similar, and those for zearalenone were nearly identical. The RBAs, which compare the affinity of a competitor with the affinity of DES at the EC50, were also different for many compounds. The RBAs of testosterone, the antiestrogens nafoxidine hydrochloride and ICI 182780, all hydroxylated PCBs tested, Kepone, zearalenone, and chlordane were all more than an order of magnitude different for the two receptors.
The competition curves for the binding of several representative compounds to ERs in the two tissues are compared in Figure 6. Estradiol, the natural ligand, caused 50% displacement at nearly an order of magnitude lower concentration in testicular extracts (Fig. 6A), whereas the affinity of estrone for the two receptors was similar (Fig. 6B). Nafoxidine displaced only 15% of the specific binding in the liver extracts, whereas it caused 75% displacement in the testicular extracts (Fig. 6C). The differences in binding affinities for the two receptors of the antiestrogen ICI 164384 and the xenobiotic o,p'-DDT were similar to those observed with estradiol (Fig. 6, A, D, and E). A variety of xenobiotics, including 2,2',5'-PCB-4-OH, did not cause 50% displacement of [3H]estradiol from the hepatic ER at the highest concentration tested (100 µM, Fig. 6F). The nonionic surfactant 4-nonylphenol also showed a distinct difference in binding affinities between tissues (Fig. 6G). Interestingly, the mycotoxin zearalenone was the only compound for which the binding curves in the two tissues overlapped (Fig. 6H).
|
Measurement of Testicular and Plasma Estradiol
Measurable amounts of estradiol were produced by testicular fragments in vitro in 2 out of 4 experiments when the incubation media were concentrated. Testicular estradiol production was 0.12 and 0.07 ng/g tissue/h in the two experiments. Estradiol was also detected in all of the plasma samples collected from mature male Atlantic croaker at a midpoint in gonadal recrudescence, with a mean concentration of 0.28 ± 0.03 ng/ml (n = 12).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We also report here that Atlantic croaker testes synthesize estradiol in vitro. Synthesis of estradiol and aromatase activity in the testis have been described previously in the rainbow trout (Oncorhynchus mykiss) [27], and expression of the aromatase mRNA has been described in the testis of the channel catfish (Ictalurus punctatus) [28]. Testicular synthesis of estradiol has been reported in a variety of other vertebrate species, including amphibians and humans [2, 29, 30], although it typically represents a minor component of testicular steroidogenesis (< 2% of testosterone production in humans). Most evidence suggests that the Leydig cell is the site of estradiol synthesis in the adult testis of both mammalian and nonmammalian vertebrates [1, 31]. Estradiol is also present in male Atlantic croaker plasma, and levels are comparable to those reported in several other teleost species (0.1–0.7 mg/ml) including tilapia (Sarotherodon melanotheron), the tropical freshwater teleost Pygocentrus cariba, and the weakly electric fish Sternopygus macrurus [32–34]. The presence of estradiol in the plasma and testes is further evidence that estrogens have a physiological role in male Atlantic croaker.
Varriale and coworkers suggested that estradiol has a role in seasonal reproduction of the male frog (Rana esculenta) from observations that estradiol peaks in both the plasma and testis in early spring, when plasma testosterone levels are decreasing and spermatogenesis is initiated [29]. Moreover, the peak in binding capacity of the testicular ER correlated with the beginning of spermatogenesis in this species [35]. In the present study, the ER was identified in both cytosolic and nuclear fractions of Atlantic croaker testis throughout the period of gonadal recrudescence. Receptor content per testis increased in cytosolic fractions as GSI increased, while in nuclear fractions receptor concentrations were higher per gram of protein during early recrudescence. Additional data on seasonal changes in plasma estradiol levels and testicular estradiol production, as well as ER localization within the testis, will be required to interpret the significance of these changes in ER content during the Atlantic croaker reproductive cycle. However, these preliminary data indicate that estradiol may be active throughout the reproductive season in male Atlantic croaker.
A number of other actions of testicular estradiol have been described in several vertebrate species. Estradiol decreases serum testosterone levels and gonadotropin-stimulated testicular testosterone production in vitro in adult male rats [36, 37]. Involvement of estradiol has also been suggested in the process of Leydig cell development in rats [38]. Estradiol also influences the spermatogonial mitotic index in the frog, Rana esculenta [39] and spermatogenic progression in the dogfish shark, Squalus acanthias [40]. In addition, estradiol has been shown to regulate the reabsorption of luminal fluid in the head of the epididymis in mice [41]. It is likely that many of these actions of estrogens are mediated by binding to a nuclear ER. ER knock-out male mice were infertile, had abnormal spermatogenesis, reduced testis size, and dismorphogenic seminiferous tubules; and the motility of their sperm was decreased [10, 42].
The recent discovery of ERß in the mouse, a second form of the ER with different tissue distribution and ligand binding from those of ER, has indicated an additional element of differential transcriptional control by estrogens at the level of the receptor [20, 21, 43]. The results of the present study suggest that tissue differences in ER binding characteristics also occur in Atlantic croaker. Differential binding affinities of several ligands to the hepatic and testicular ERs were identified. The testicular ER had a higher affinity for nearly all of the compounds tested and also saturated at a lower estradiol concentration. While these results do not prove the existence of two forms of the ER, preliminary data from our laboratory suggest that multiple forms of the ER are present in this species, with different forms dominant in the gonads and liver [22]. The relative binding affinities of several natural estrogens were similar in both tissues, whereas the relative binding affinities for two of the antiestrogens, nafoxidine hydrochloride and ICI 182780, differed markedly. In addition, the order of binding affinity of estrogens and anti-estrogens for the two ERs were different (DES > estradiol >> ICI 182780 > ICI 164384 = estrone = estriol > tamoxifen > nafoxidine, for the testis ER; DES > estradiol >> ICI 182780 > estrone > estriol = ICI 164384 > tamoxifen > nafoxidine, for the liver ER). Differences in the relative binding affinities of several synthetic estrogens (moxestrol, ICI 164384) to ER and ß have also been observed in mice [21]. Of all the compounds tested in this study, only zearalenone had overlapping binding curves for the two receptors. Interestingly, the binding curves of zearalenone for human ER and ß also overlap [43].
The ER in the testis is not only a site of estrogen action but is also a likely target for endocrine disruption by xenoestrogens. Testicular growth was impaired in male rainbow trout chronically exposed to several estrogenic alkylphenolic compounds [13]. A recent study has shown that a large proportion of the male fish collected from several European rivers in the vicinity of sewage outfalls were intersex, with ovarian as well as testicular tissue in their gonads, and also had elevated plasma vitellogenin levels, evidence of xenoestrogen exposure [12]. Rats exposed to the estrogenic chemicals DES, octylphenol, or butyl benzyl phthalate during gestation or lactation exhibited reduced testicular weight and reduced sperm production [44]. Although the site of interference by these chemicals was not determined, the testis appears to be sensitive to xenoestrogens, either directly or indirectly, during gonadal development and neonatal life.
The data presented in this study show that a variety of xenobiotics bind to the testicular ER of a teleost and therefore have the potential to interfere with male reproductive function. Many of the compounds tested in this study which bound to the ER, including o,p'-DDT, Kepone, and nonylphenol, have been shown to have estrogenic effects in mammals, birds, or fish [13, 45, 46]. Hydroxylated PCBs, which have been identified in fish-eating birds, marine mammals, and humans, also have the potential for estrogenic and antiestrogenic activity [47]. However, fish do not appear to metabolize PCBs as readily as do other vertebrates [48], although hydroxylated metabolites of PCBs have been identified in some species [49].
All of the DDT congeners tested bound to the Atlantic croaker ERs. The o,p' derivatives had higher affinities than the p,p' derivatives, in agreement with the results of previous studies [50, 51]. In general, the differences in binding affinities of the o,p' and p,p' derivatives for the Atlantic croaker ERs were smaller than those reported for other species. In rat uterine receptors, the o,p'-isomers (approximately 300 µM) caused 50–75% inhibition of [3H]estradiol binding, whereas the p,p'-isomers caused 20% or less inhibition of binding [50]. In the kelp bass hepatic ER, o,p'-1, 1-dichloro-2,2'-bis(p-chlorophenyl)ethylene (DDE) displaced more than 70% of the specific binding, whereas at similar concentrations p,p'-DDE displaced only 10% [51]. Similarly, o,p'-DDT (100 µM) caused a 60% inhibition of binding to the rabbit uterine ER, whereas p,p'-DDT and p,p'-DDE caused only a 10% inhibition of [3H]estradiol binding [52]. In contrast, our results show that the o,p'-DDT inhibited 60% of binding and p,p'-DDT inhibited almost 50% of binding in both the testicular and liver ERs at a concentration of 100 µM. Thus, it is important to test potential endocrine-disrupting chemicals on a broad range of vertebrate species in view of possible species differences in their receptor binding. The competitive binding assays with Atlantic croaker tissues show that a variety of environmental chemicals including pesticides, nonionic surfactants, and hydroxylated PCBs have the potential to interfere with the reproductive function of both the liver and testis in this species. We have observed estrogenic feedback effects of o,p'-DDT on gonadotropin secretion in croaker, with tissue concentrations in the low per-million range, similar to the EC50 of the pesticide for the testicular ER in this species [53, 54].
The differences in binding of xenobiotics to the testicular and hepatic ER observed in this study may have important implications for the pattern of endocrine disruption in different tissues. [3H]Estradiol binding is displaced from the testicular ER by xenobiotic concentrations only 10–20% of those necessary to displace it from the hepatic ER. Moreover, the number of cytosolic receptors in the liver is 20 times higher than that in the testis on a per gram of tissue basis, and the testicular ER saturates at one-fourth of the [3H]estradiol concentration required to saturate the hepatic ER. These results suggest that the testis may be more sensitive than the liver to environmental estrogens. Therefore, investigations of the effects of xenoestrogens in males should include an examination of testicular structure and function. Xenoestrogen-induced changes in the testis may be discernible at concentrations below those required to induce vitellogenin production.
In conclusion, we report the first evidence for the existence of a testicular ER in a teleost species. Differences in binding characteristics between the hepatic and testicular ER of this species were observed, suggesting that the testis may be more sensitive to estrogens than the liver. We also demonstrate that several xenobiotics can bind to both the testicular and hepatic ERs and therefore could potentially disrupt estrogen action.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
2 Correspondence: Peter Thomas, University of Texas Marine Science Institute, 750 Channel View Drive, Port Aransas, TX 78373. FAX: 512 749 6777; thomas{at}utmsi.utexas.edu
Accepted: February 5, 1999.
Received: December 3, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and ß. Endocrinology 1997; 138:863–870.This article has been cited by other articles:
|
|
 |
|
  E. Filardo, J. Quinn, Y. Pang, C. Graeber, S. Shaw, J. Dong, and P. Thomas Activation of the Novel Estrogen Receptor G Protein-Coupled Receptor 30 (GPR30) at the Plasma Membrane Endocrinology, July 1, 2007; 148(7): 3236 - 3245. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Thomas, Y. Pang, E. J. Filardo, and J. Dong Identity of an Estrogen Membrane Receptor Coupled to a G Protein in Human Breast Cancer Cells Endocrinology, February 1, 2005; 146(2): 624 - 632. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kristensen, E. Baatrup, and M. Bayley 17{alpha}-Ethinylestradiol Reduces the Competitive Reproductive Fitness of the Male Guppy (Poecilia reticulata) Biol Reprod, January 1, 2005; 72(1): 150 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. H. Rasmussen, T. K. Andreassen, S. N. Pedersen, L. T. M. Van der Ven, P. Bjerregaard, and B. Korsgaard Effects of waterborne exposure of octylphenol and oestrogen on pregnant viviparous eelpout (Zoarces viviparus) and her embryos in ovario J. Exp. Biol., December 15, 2002; 205(24): 3857 - 3876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Bouma and J. J. Nagler Estrogen Receptor-{{alpha}} Protein Localization in the Testis of the Rainbow Trout (Oncorhynchus mykiss) During Different Stages of the Reproductive Cycle Biol Reprod, July 1, 2001; 65(1): 60 - 65. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Hawkins, J. W. Thornton, D. Crews, J. K. Skipper, A. Dotte, and P. Thomas Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts PNAS, September 26, 2000; 97(20): 10751 - 10756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. K. Loomis and P. Thomas Effects of Estrogens and Xenoestrogens on Androgen Production by Atlantic Croaker Testes In Vitro: Evidence for a Nongenomic Action Mediated by an Estrogen Membrane Receptor Biol Reprod, April 1, 2000; 62(4): 995 - 1004. [Abstract] [Full Text]
|
|
|
|
|
|
K Kinnberg, B Korsgaard, P Bjerregaard, and A Jespersen Effects of nonylphenol and 17beta-estradiol on vitellogenin synthesis and testis morphology in male platyfish Xiphophorus maculatus J. Exp. Biol., January 1, 2000; 203(2): 171 - 181. [Abstract] [PDF]
|
|
|
|
|
|
T. S. Sperry and P. Thomas Identification of Two Nuclear Androgen Receptors in Kelp Bass (Paralabrax clathratus) and Their Binding Affinities for Xenobiotics: Comparison with Atlantic Croaker (Micropogonias undulatus) Androgen Receptors Biol Reprod, October 1, 1999; 61(4): 1152 - 1161. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||
