Short-Term Exposure to 17α-Ethynylestrodiol 
Decreases the Fertility of Sexually Maturing 
Male Rainbow Trout (oncorhynchus mykiss) 

Environmental Toxicology and Chemistry, v.22, n.6 Jun03

IRVIN R. SCHULTZ,*† ANN SKILLMAN,† JEAN-MARC NICOLAS,‡ DANIEL G. CYR,‡ and JAMES J. NAGLER§ †Battelle MSL-PNNL, 1529 West Sequim Bay Road, Sequim, Washington 98382, USA ‡INRS-Institut Armand-Frappier, University of Quebec, 245 Hymus Boulevard, Pointe-Claire, Quebec H9R 1G6, Canada §Department of Biological Sciences, Center for Reproductive Biology, University of Idaho, Moscow, Idaho 83844-3051, USA

* To whom correspondence may be addressed (irpschultz@pnl.gov). Presented at the 21st Annual Meeting of the Society of Environmental Toxicology and Chemistry, November 12–16, 2000, Nashville, Tennessee, USA.

(Received 12 June 2002; Accepted 7 November 2002)

Abstract—The synthetic estrogen 17α-ethynylestradiol (EE2) is a commonly used oral contraceptive that has been increasingly detected in sewage effluents. This study determined whether EE2 exposure adversely affected reproduction in sexually maturing male rainbow trout (Oncorhynchus mykiss). We exposed male trout to graded water concentrations of EE2 (10, 100, and 1,000 ng/ L) for 62 d leading up to the time of spawning. Semen and blood plasma samples were removed from each fish. Semen was used to fertilize groups of eggs from one nonexposed female. As a measure of fertility, eggs were incubated for 28 d after fertilization to determine the proportion that attained the eyed stage of embryonic development. Additional endpoints also measured included sperm motility, spermatocrit, gonadosomatic and hepatosomatic indices, testis histology, and circulating plasma levels of the sex steroids 17α, 20ß-dihydroxyprogesterone (17,20-DHP) and 11-ketotestosterone (11-KT). Exposure to 1,000 ng/L of EE2 caused complete mortality of the treatment group by day 57. Exposure to lower EE2 water concentrations (10 and 100 ng/L) caused an increase in sperm density, while a significant reduction in testis mass was observed only in the 100-ng/L exposure group. Most significantly, semen harvested from fish exposed to 10 and 100 ng/L EE2 caused an approximately 50% reduction in the number of eggs attaining the eyed stage of embryonic development. Plasma levels of 17,20-DHP in exposed fish were roughly twice the level of the controls, while levels of 11-KT were significantly reduced in fish exposed to 100 ng/L EE2. These results suggest that sexually maturing male rainbow trout are susceptible to detrimental reproductive effects of short-term exposures to environmentally relevant levels of EE2.

Keywords—Embryonic development Sperm motility Endocrine disruption Environmental estrogens

The consequence of xenoestrogen exposure in fish has received increasing scrutiny with the awareness that exogenous inputs of estrogens into surface waters may disturb sexual development and reproduction [1]. A number of contaminants have been demonstrated to bind to estrogen receptors from several fish species although typically at several orders of magnitude below that of 17ß-estradiol (E2) and synthetic analogues [2–4]. Ethynylestradiol (EE2) is a synthetic estrogen used in oral contraceptives, and its occurrence in surface waters is the result of municipal sewage discharges [5,6]. Recently, both E2 and EE2 have been implicated as the primary contaminants contributing to the estrogenic activity in surface waters from both the United Kingdom and United States [7,8]. Worldwide environmental monitoring of sewage effluents and surface waters near municipal sewage outfalls typically report only intermittently detectable levels of EE2. Advancements in analytical methods have improved accuracy and detection limits for EE2, and recent values for EE2 concentrations in effluents and surface waters range from less than 0.05 ng/L up to 9 ng/L [9–11]. One exception to these lower values for EE2 concentrations is a recent survey of U.S. waters that reported EE2 concentrations ranging from 73 to 831 ng/L [12]. Despite the frequently low environmental concentrations of EE2, laboratory studies have demonstrated induction of the estrogen biomarker protein, vitellogenin, in male rainbow trout after exposure to EE2 concentrations as low as 0.3 ng/L [13]. This raises the concern that exposure to environmental levels of EE2 (and E2) may lead to impaired reproduction in fish.

The toxicology of EE2 in fish has only recently been investigated but appears to be similar to E2. A partial life-cycle study in sheepshead minnows (Cyprinodon variegatus) using exposure concentrations ranging from 0.2 to 3,200 ng/L reported that exposure levels of 1,600 ng/L or higher caused severe tissue damage to kidneys, liver and gills, and overall high mortality [14]. At exposure concentrations down to 2 ng/ L, effects on the testis were observed, such as testis-ova and fibrosis formation [14]. Both reproductive and hatching success were decreased at exposure concentrations of 200 ng/L or higher [14]. In other fishes, such as the medaka (Oryzias latipes) and fathead minnow (Pimephales promelas), exposure to lower concentrations of EE2 reduces growth and fecundity and in males causes formation of ovo-testes or a complete lack of testicular development [15,16]. These studies are typical of many reproductive studies of estrogens, such as EE2, in that partial or full life cycle exposures are employed. Early life history stages are considered to be particularly vulnerable to xenoestrogens, as exposure during morphological sex differentiation may impact normal development of the phenotype [17]. This has caused a tendency to focus on intersexuality as a critical endpoint of toxicological evaluations of xenoestrogens. However, this endpoint may not always be the most sensitive to estrogen exposure.

Chronic or full life-cycle exposure studies to xenoestrogens are an important aspect of toxicity evaluations for potential endocrine active pollutants. However, this type of exposure scenario may be unrealistic for some contaminants, such as EE2, that are more likely to occur over a wide range of concentrations because of variable removal by sewage treatment and seasonal hydrologic fluctuations in surface waters. Short-term exposure (e.g., ,90 d) to contaminants such as EE2 appear to represent a more likely type of exposure duration. Short-term exposure of sexually mature fish to estrogens has received much less study compared to early life history stages. Sexually maturing male fish may represent a vulnerable life stage to xenoestrogen exposure, as sensitivity to estrogens can increase at the end of the spermatogenic cycle [18], impacting normal maturation of spermatozoa. Further evidence for adult sensitivity comes from a study using adult male transgenic zebrafish (Danio rerio) possessing an estrogen-responsive reporter gene [19]. When transgenic zebrafish were exposed to E2 for 48 h, the testes exhibited the highest induction of the reporter gene compared to other tissues examined [19]. These findings suggest that sexually mature fish that have not been previously exposed to xenoestrogens may still suffer impaired reproduction after short-term E2 or EE2 exposure. To test this possibility, we investigated the effect of a 62-d exposure to EE2 in sexually maturing male rainbow trout (Oncorhynchus mykiss). We determined reproductive success by comparing the fertilization rate of semen harvested from EE2-exposed trout and control trout. We also measured several semen quality parameters, circulating plasma levels of two sex steroids, 17a, 20b-dihydroxyprogesterone (17,20-DHP) and 11-ketotestosterone (11-KT), and testis histology. These additional measures were made to gain some insight into the mode of action of EE2.

MATERIALS AND METHODS

Chemicals

The 17α-ethynylestradiol was obtained from Sigma Chemical (St. Louis, MO, USA). The 17,20-DHP was purchased from Steraloids (Newport, RI, USA). The 17a-hydroxy [1,2,6,7-H3] progesterone was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). An antiserum to 17,20-DHP was kindly provided by A.P. Scott (Center for Environment, Fisheries and Aquaculture, Lowestoft, UK). The 11-KT enzyme-linked immunosorbent assay (EIA) kit was purchased from Cayman Chemical (Ann Arbor, MI, USA). All other chemicals were reagent grade.

Fish

Adult male rainbow trout (O. mykiss) 1+ age (mean body wt 6 standard deviation [SD]: 740 ± 220 g; n = 80) were obtained November 3 from Nisqually trout farm (Lacey, WA, USA). The spawning season for trout from this hatchery is mid-December to early January. Trout were maintained outdoors under natural lighting to allow for continued sexual maturation in synchrony with female trout that remained at the hatchery. All trout were held in 1,400-L circular tanks at a holding density not exceeding 10 kg/m3 with a minimum in- flow rate of 20 L/min at 12°C. Source water for the holding tanks was obtained from an artesian deep-well (400-ft depth) groundwater reservoir. Chemical characteristics of the inflow water were as follows (mean 6 SD): dissolved oxygen: 9.6 ± 0.21 mg/L; pH: 8.10 ± 0.03; total alkalinity: 200 ± 8.1 mg/ L (as CaCO3); ammonia: ,0.05 mg/L; and nitrate–nitrite: ,0.01 mg/L (as total N). Trout were fed a maintenance ration of 1 to 2% body mass 4 to 5 d per week with a soft, moist pelleted feed (Moore Clark Silver Cup, Vancouver, BC, Canada).

17 α-Ethynylestradiol exposures

Rainbow trout were continuously exposed to EE2 using a flow-through exposure system. Three nominal exposure levels of EE2 were tested: 10, 100, and 1,000 ng/L. A concentrated stock solution of EE2 was prepared in methanol and slowly added to the exposure tanks using a peristaltic pump at a flow rate of 0.10 ml/min (equals 0.0005% methanol in tanks). Control tanks had only methanol added (solvent control). All exposure tanks were allowed to equilibrate with the EE2 dosing system for 14 d prior to the addition of trout. Ethynylestradiol concentrations were monitored every 7 to 14 d during the exposures to quantify actual levels. On November 14, 1998, 20 male trout were placed in each exposure tank and held until time of semen collection, which occurred 62 d later.

Determination of 17 α-ethynylestradiol in exposure water

Water concentrations of EE2 were determined using gas chromatography/mass spectrometry (Hewlett-Packard, Palo Alto, CA, USA, model 5973) and methods previously described in Schultz et al. [20]. Briefly, water samples (1 L) were fortified with 100 ml of saturated NaCl solution and then passed through a C18 solid-phase extraction (SPE) cartridge (Baker bond elute, 500 mg packing material, Phillipsburg, NJ, USA). The EE2 was eluted from the SPE cartridge using 5 ml methylene chloride, evaporated to dryness, and then derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide as previously described [20]. Ethynylestradiol fortified water standards were prepared to encompass the observed concentration ranges and were processed as described for the exposure water samples. Recovery of EE2 from fortified water standards was always >95%.

Collection of gametes and determination of sperm density and in vitro fertilization rate assay

On day 62 of the exposures, trout were evaluated for spawning readiness after induction of light anesthesia using MS-222 (50 mg/L) followed by gentle massage of the abdominal region. Five trout from each exposure tank exhibiting expressible semen were selected and gametes obtained. Caution was used when obtaining the semen to prevent contamination with water and urine. Semen samples were stored at 4°C in polyethylene tubes under normal atmosphere for a maximum of 4 h prior to use.

Spermatocrit (packed cell volume/total semen volume) was immediately measured by placing aliquots of the fresh semen into microcapillary tubes and centrifuging in a hematocrit centrifuge for 5 min at 1,000 g. Sperm concentration was measured by counting spermatozoa in a hemacytometer at 3400 magnification. Semen samples were diluted 1:10,000 using a motility- inhibiting buffer that contained 103 mM NaCl, 40 mM KCl, 1 mM CaCl2, and 0.8 mM MgSO4 in a 20-mM N-2- hydroxyethylpiperazine-N-2-ethane-sulfonic acid buffer (pH 7.8). The samples were diluted to bring the number of spermatozoa in the field of view to approximately 100 per chamber. Sperm concentration was calculated from the average of three separate counts of the diluted semen samples from each trout, multiplied by the dilution factor.

The eggs used in fertilization trials were obtained from a single female donor that was brought to the laboratory from the Nisqually Trout Farm on the day semen was obtained from male trout. Eggs were obtained from the donor trout after induction of light anesthesia followed by massaging of the abdominal region. Harvested eggs were initially placed in a sieve for 3 min, and the ovarian fluid was allowed to drip away. The eggs were then divided into aliquots of 200 and placed in polyethylene cups containing 100 ml of ovarian fluid substitute: 60 mM NaHCO3 in a 50-mM tris buffer (pH 9.0). The appropriate volumes of semen were added to the cups and gently swirled for 1 min and then allowed to stand for 5 min to allow fertilization to occur. Three separate sperm:egg ratios were used to test the fertilization capacity of harvested sperm. The sperm:egg ratios were 300,000, 50,000, and 10,000 to 1. These ratios were selected to provide optimal, suboptimal, and low sperm numbers in the fertilization trials. Afterward, clean tank water was added to the cups and left to sit for 30 min and then transferred to incubation trays and placed in incubation chambers supplied with clean tank water. The incubation chamber was kept in total darkness during the initial 14- d postfertilization period and then allowed a 12:12 artificial photoperiod for an additional 14 d. As an indicator of reproductive success, embryological development was assessed on day 28 by measuring the percentage of eyed eggs in each incubation tray. All fertilization trials were done in triplicate for each sperm sample and sperm:egg ratio.

Blood sampling and determination of tissue-somatic indices

Analysis of plasma steroid levels was performed on blood samples removed from trout used in the fertilization trials. Blood samples were removed from the dorsal aorta using a heparinized 5-ml syringe and a 23-G needle and briefly stored on ice. Shortly afterward, blood samples were centrifuged, and plasma was stored at 2208C for subsequent steroid analysis.

Tissue-somatic indices were calculated from 10 additional trout removed from each exposure tank. These trout were also euthanized on day 62 of the EE2 exposure by anesthetic overdose. After determining the total mass of the trout, the liver and testes were removed and weighed. The tissue-somatic indices (either hepatosomatic [HSI] and gonadosomatic [GSI]) were calculated as the percentage of tissue mass to the total mass of the trout.

Histology of testes

Midportions of the testes of fish sampled previously for the determination of GSI were fixed in Bouin’s fluid and then stored in 70% ethanol. These tissues were subsequently dehydrated through a graded series of ethanol, cleared in xylene, and embedded in paraffin. For histological analysis, two serial sections (5–6 mm thick) were cut and stained with hematoxylin and eosin. The histology of the testis of each fish was examined using a light microscope (Zeiss Axioskop, Carl Zeiss, Göttingen, Germany).

Assessment of sperm motility

Sperm motility was assessed using computer-assisted motion analysis of video recordings of each semen sample. Initially, semen samples were diluted with the motility-inhibiting buffer as described previously. Next, an aliquot of the diluted semen was mixed with a 20-fold excess volume of activator buffer (60 mM NaHCO3 in a 50-mM tris buffer; pH 9.0) on a clean, concave glass slide and immediately placed on the microscope. For each sample, the microscope field was adjusted to ensure that a minimum of 100 spermatozoa were visible in the field. Sperm movement was recorded using a digital video camera attached to a Nikon Optiphot microscope (Nikon, Princeton, NJ, USA). Motility was recorded until no further sperm movement was observed (;180 s). Video recordings were obtained from three replicates of each semen sample.

The recordings of sperm motility were analyzed using an Integrated Visual Optical System, Ver 10 (Hamilton Thorne Research, Beverly, MA, USA). The system was adapted to use the video feed as input. Motility analysis of each sample was initiated 12 s after the start of the recording and was carried out for 15 s. This allowed for the analysis of a consistently clear recording in all samples. Within the frame of acquisition, each sperm is followed individually, and several variables were analyzed by the Integrated Visual Optical system. These included sperm concentration, percentage motile sperm, percentage progressive sperm, sperm velocity, beat frequency, lateral displacement, straightness, and linearity of the trajectory. Each variable is divided into 10 classes, and events falling within the range of each class are cumulated over the acquisition period.

Sex steroid measurements

The blood plasma levels of the progestin, 17,20-DHP, were measured using a previously validated radioimmunoassay according to the procedure described for rainbow trout in Scott et al. [21]. The only modification was that plasma samples were extracted twice with diethyl ether and dissolved in assay buffer before aliquots were used in the radioimmunoassay. Blood plasma levels of the androgen, 11-KT, were measured with an 11-KT EIA kit according to the manufacturer’s instructions. It was determined that prior solvent extraction with ethyl acetate was necessary to achieve parallelism of serial dilutions of plasma with the standard curve in the 11-KT EIA.

Statistics

Analysis of variance and a multiple comparisons test (Tukey’s test) was used to test for significant differences between experimental endpoints from control and EE2-treated trout. A probability level of p , 0.05 was taken as indicating statistically significant differences.

RESULTS

EE2 exposure concentrations and overt toxicity

The measured water concentration of EE2 in the exposure tanks during the 62-d exposures is summarized in Figure 1. At the two lower test concentrations, measured EE2 levels were slightly higher than nominal and averaged 15.6 ± 6.7 and 131 ± 23 ng/L (mean 6 SD), respectively. These lower test concentrations were well tolerated by the trout, and no obvious signs of distress (unusual swimming or feeding behavior) or mortality occurred in these exposures. The highest attempted exposure concentration of 1,000 ng/L proved difficult to maintain, and the average concentration was 750 ± 116 ng/L. This exposure level was found to be lethal to trout with initial mortalities occurring on day 34 and complete loss of all 20 trout in this exposure group occurring by day 57. Gross necropsy of selected individuals indicated extensive hemorrhaging and necrosis of the liver and kidney.

Fig. 1. Water concentrations of 17 α-ethynylestradiol (EE2) sampled during the 62-d exposure period. The EE2 was measured using gas chromatography/mass spectrometry as described in the Materials and Methods section.

Fig. 2. Tissue somatic indices in control (white bar) and 17 α-ethynylestradiol (EE2)-exposed trout. (A) hepatosomatic index (HIS); (B) gonadosomatic index (GSI). Tissue somatic indices were determined in 10 trout removed from each exposure tank on day 62. Each column is the mean ± standard deviation. * p < 0.05.

HSI, GSI, and sperm density parameters

A statistically significant difference in liver and testes mass was observed only in the 100 ng/L exposure group (Fig. 2A and B). In this treatment group, the liver was enlarged with a HSI approximately three times greater than trout from the control or 10-ng/L group (Fig. 2A). In contrast, the GSI was slightly reduced in the 10-ng/L group (although not significantly less than controls) and more than three times lower in the 100-ng/L group (Fig. 2B).

Exposure to EE2 at both the 10- and the 100-ng/L treatment level caused an increase in sperm density in the semen. Both sperm concentration and spermatocrit were significantly increased in semen from EE2-treated trout (Fig. 3A and B). The increase in both parameters was dose dependent, with semen from trout exposed to 100 ng/L being notably more viscous because of a spermatocrit approaching 70% of the total semen volume (Fig. 3B).

Embryological development

The results of in vitro fertilization assays using semen diluted to produce sperm:egg ratios of 10,000, 50,000, and 300,000 to 1 are shown in Figure 4. Increasing the sperm:egg ratio increased the number of embryos reaching the eyed stage of development in all treatment groups. However, viability of embryos from EE2-exposed trout was significantly decreased compared to control trout (Fig. 4). The decrease in viability was most pronounced at sperm:egg ratios of 50,000 and 300,000 to 1, where a reduction of almost 50% was observed (Fig. 4). An important observation is that both the 10- and the 100-ng/L treatment caused a similar reduction in viability, suggesting that a maximal response had already been attained with the 10-ng/L treatment.

Plasma sex steroids

The concentration of circulating 17,20-DHP and 11-KT in blood plasma sampled at the time of spawning is shown in Figure 5A and B. Treatment with either 10 or 100 ng/L EE2 caused a significant increase in the 17,20-DHP concentrations that was more than twice the concentration observed in control trout (Fig. 5A). The plasma levels of both steroids exhibited high interfish variation. Also, plasma levels of 11-KT appeared to display a complex dose–response relationship with the EE2 treatments (Fig. 5B). The 11-KT concentration was elevated in the 10-ng/L treatment group (although not significantly different compared to the control trout) in contrast to the 100- ng/L treatment group, where 11-KT concentrations were significantly reduced by fivefold relative to controls (Fig. 5B).

Sperm motility

No significant differences in the swimming velocity of spermatozoa were observed in semen samples from control and EE2-treated trout (data not shown). Other motility parameters (beat frequency, lateral displacement, straightness, and linearity of the trajectory) were also not significantly different between the treatment groups (data not shown). In all semen samples obtained from the EE2-treated trout, motility varied less than 10% from values observed using control semen. Overall, an average of 85 to 91% of sperm from the semen samples were motile.

Fig. 3. Sperm density in semen samples obtained on day 62 of the exposures. (A) sperm concentration; (B) spermatocrit. Each column is the mean 6 standard deviation (n 5 5). * p , 0.05.

Fig. 4. Embryological development evaluated on day 28 postfertilization as the percentage of eyed eggs. Eggs were fertilized using semen collected from 17α-ethynylestradiol-exposed and control trout (white bar). Semen was collected from five individual trout from each exposure tank on day 62 and used to fertilize eggs collected from a single nonexposed female trout. Three separate ratios of sperm:egg concentrations were tested as indicated. Each column is expressed as the mean ± standard deviation (n = 5). * Denotes treatments significantly lower than controls ( p , 0.05).

Histopathology

No observable differences were noted between the histology of the testes from control fish and those from either EE2 treatments (Fig. 6A and B). All fish examined had mature testes with tubules almost exclusively containing spermatozoa.

DISCUSSION

The results of this study indicate that short-term exposure of EE2 to sexually maturing male rainbow trout significantly decreases reproductive performance. To our knowledge, this is the first documented report of the effects of EE2 exposure on the number of viable embryos produced in controlled in vitro fertilization trials. Previously, E2 and EE2 have been reported to decrease the fertility of male fish in natural breeding experiments. For example, a 14-d exposure to E2 reduced the fertility of male medaka allowed to breed naturally with unexposed unexposed females [22]. In a similarly designed study, the reproductive performance of pair-breeding zebrafish was evaluated after 21-d exposures to measured EE2 concentrations of 5, 10, and 25 ng/L [23]. This study reported that in exposed male zebrafish mated to control females, all EE2 treatments reduced fertilization success below 70%, which is the mean value for nonexposed zebrafish reported by the authors [23]. A previous study of EE2 exposure in zebrafish also observed decreased reproduction (measured from hatching success) after a 12-d exposure to a nominal concentration of 5 ng/L [24].

In the present study, a decrease in the number of viable embryos occurred at an EE2 exposure concentration that did not cause obvious signs of toxicity or significant changes in the HSI and GSI (10 ng/L; Fig. 2). Exposure to EE2 did increase the sperm density of semen in a dose-dependent manner (Fig. 3). The average sperm concentration in semen from control trout was 7.3 3 109 cells/ml, which is similar to historical values reported by other researchers for rainbow trout [25– 27]. Sperm concentration of semen in EE2-treated trout increased to 21 and 33 3 109 cells/ml, respectively (Fig. 3). These elevated values of sperm concentration are comparable to levels observed when semen is directly obtained from the testes from either normal or intentionally sex-reversed female rainbow trout [28]. The increased sperm concentration of testicular semen has been attributed to low seminal fluid volume or, perhaps, increased quantities of immature spermatozoa [28]. In the current study, a reduction in seminal fluid volume is more likely to have occurred, as all semen samples contained a high percentage of motile sperm, implying that a high percentage of mature spermatozoa were present in the semen samples [29]. Histological analysis of testes from control and treated trout also indicated that mature spermatozoa were present (Fig. 6). These observations would imply that increased sperm density is probably due to a water-sparing effect of the EE2 treatments on seminal fluid production.

When the results of the in vitro fertilization trials are compared against the different sperm:egg ratios tested, it is clear that ratios of 50,000:1 and 300,000:1 were the most sensitive for assessing the effects of the EE2 treatments (Fig. 4). The greatest number of viable embryos from control trout semen was 60.5% at the 300,000:1 sperm:egg ratio. This number is slightly lower than typical for rainbow trout, which normally is greater than 70% [30]. However, a ratio of 300,000:1 is at the lower end of the range for optimum fertilization conditions [30], and maximal fertilization rates in rainbow trout are frequently achieved at sperm:egg ratios approaching 1 X 106 sperm per egg [31]. We chose to test graded sperm:egg ratios descending from 300,000:1 in our fertilization trials because excessively high sperm:egg ratios can overcome poor semen quality. For example, cryopreservation of rainbow trout semen causes a variety of morphological alterations and a reduction in sperm motility [32]. However, fertilization rates comparable to fresh semen can be obtained with cryopreserved semen if sperm:egg ratios exceeding 3 X 106 are used [32]. A similar finding has also been obtained when semen from the African catfish (Clarias gariepinus) was treated in vitro with mercuric chloride [33]. In this study, decreased fertilization rates using sperm treated with mercury were most apparent at sperm:egg ratios considered 50% of that needed to achieve maximal fertilization rates [33]. Our results indicate that in rainbow trout, sperm:egg ratios as low as 50,000:1 are effective at identifying decreased fertilization rates. At lower sperm:egg ratios, sensitivity for detecting toxicant effects is largely lost because of the low fertilization success observed in control semen (Fig. 4).

In addition to describing an effect on embryological development after xenoestrogen exposure, it is also important to determine the physiological mechanism(s) by which this pollutant is acting. Understanding the mechanism of action would be particularly valuable for interpreting the significance of laboratory results using controlled reproductive trials and extending this to natural breeding situations. To gain some insight into this area, we measured the additional endpoints of circulating levels of 17,20-DHP and 11-KT in blood plasma and sperm motility. These measures were chosen because it is well established that decreased sperm motility can lower fertilization rate [34] resulting in fewer viable embryos, and both 17,20-DHP and 11-KT are important hormones for proper development and function of male germ cells in fishes [21,35– 38]. In male salmonids, 17,20-DHP is the primary hormone involved with final maturation of spermatozoa and acquisition of sperm motility [37] and is produced largely by developing germ cells [39]. This led us to initially hypothesize that short-term exposure of EE2 to sexually maturing rainbow trout would decrease production of 17,20-DHP and/or 11-KT by the testes, impacting the final stages of sperm maturation and reducing sperm numbers or motility. However, the analysis of sperm motility and the results shown in Figure 5 do not support this hypothesis. Plasma concentrations of 17,20-DHP in the EE2-treated trout were significantly increased compared to control trout (Fig. 5A), and treatment with EE2 did not affect sperm motility as measured by percentage motile sperm or average velocity. These findings would indicate that testicular secretion of 17,20-DHP was stimulated by EE2, in contrast to a previous report showing the native steroid, E2, to decrease 17,20-DHP production in isolated trout testes [18]. At present, it is difficult to explain the discrepancy between the in vitro results reported in Vizziano et al. [18] and in vivo effects of EE2 on 17,20-DHP secretion, as a variety of factors could affect plasma levels, including endocrine perturbations in extratesticular tissues, altered binding of 17,20-DHP to plasma proteins, and/or decreased elimination of 17,20-DHP. In contrast to 17,20-DHP, levels of 11-KT were significantly lower in the 100-ng/L EE2 treatment group, though not statistically different at 10 ng/L (Fig. 5B). One explanation for the decrease in 11-KT could be a reflection of the increased production of 17,20-DHP in the testes of EE2-treated fish and a shunt of precursors within the steroidogenic pathway from androgens to progestins. However, the group treated with 10 ng/L EE2 had the highest levels of 17,20-DHP yet did not show decreased 11-KT levels. It is known that estrogen receptors are located in Leydig cells within the rainbow trout testis [40], which is the site for 11-KT production [41]. Exposure to levels of EE2 higher than 10 ng/L could inhibit 11-KT production within Leydig cells. However, it is not obvious that the 100- ng/L EE2 exposure, which resulted in lowered 11-KT blood plasma levels, seriously affected the number of germ cells since sperm cell counts were elevated in semen from treated fish and testis histology was unaffected. The mechanism by which EE2 affects 17,20-DHP and 11-KT blood levels and the link between reduced numbers of embryos surviving from these exposed male rainbow trout needs to be further explored. Because sperm motility was not affected, we can suggest that a decrease in fertilization rate is probably not responsible for the decrease in the number of eyed eggs found in the EE2 treatments but rather is a failure during early development in the embryo. It is apparent that additional experiments will be needed to understand how exposure to EE2 can affect sperm, resulting in fewer embryos reaching the eyed stage of development. In this regard, the rainbow trout would appear to be a useful fish model for future studies.

Fig. 6. Histological sections of sexually mature rainbow trout testis from a representative individual of either the control (A) and 100-ng/L 17aethynylestradiol treatment (B) groups. The white arrowhead indicates spermatozoa filling the enlarged tubules. The interstitial space that surrounds the spermatogenic cyst is indicated by the white arrows. Magnification is 3400.

It is also important to emphasize the significance of the EE2-induced decrease in fertility without concomitant signs of toxicity. A recent large-scale biomonitoring effort of carp (Cyprinus carpio) in the United States has observed that several biomarkers of endocrine disruption, such as unusual blood plasma concentrations of 17ß-estradiol, 11-KT, and vitellogenin, are not associated with abnormal gonad histopathology [42]. These findings suggest that any adverse effects occurring from exposure to xenoestrogens at low environmental concentrations will be manifested as more subtle changes in reproductive physiology and performance. Our results support the concept that gamete viability as opposed to gamete production or development may be a more sensitive indicator of toxicity from endocrine disruptors [24]. It also should be noted that the decrease in fertility observed in the present study was still maximal at the lowest test concentration of 10 ng/L, implying that fertility would have been impacted at lower EE2 exposure levels.

Regulatory approaches to evaluating potential endocrineactive compounds rely on short-term screening assays using asynchronous fractional spawning fishes, such as the fathead minnow, medaka, and zebrafish. Typically, high test concentrations are employed with measured endpoints, such as gonadosomatic index, plasma sex steroid, and vitellogenin levels, in addition to various measures of reproductive performance [43]. Where appropriate, short-term tests are followed by full life cycle tests or multigenerational studies [14,16]. The results of the present study would suggest that salmonids are at least as sensitive as fractional spawning fishes with regard to the effects of xenoestrogens on male fertility. Also, anadromous fishes, such as salmon, are unlikely to receive continuous lifetime exposure to EE2, a pollutant found primarily in municipal sewage effluents. A more likely exposure scenario is during the downstream ocean migration or as returning adults prior to spawning. Our results suggest that upstream migrating salmonids that may otherwise have developed normally are still susceptible to the reproductive effects of exposures to EE2 at levels that may not produce overt changes in gonad morphology. A reduction in male fertility may not represent a problem for hatchery-reared salmon where high sperm:egg ratios can be used to compensate. However, this is not possible for wild salmon and raises the concern that xenoestrogen-induced reduction in male fertility may impact reproductive success of naturally spawning salmon.

Acknowledgement—We thank Rhonda Karls, Tim Fortman, and Paul Farley. This research was supported by the U.S. Department of Energy under contract DE-AC06-76RLO 1830 (to I. Schultz and A. Skillman) and the National Science Foundation Idaho Experimental Program to Stimulate Competitive Research Program Cooperative Agreement EPS-9720634 (to J. Nagler).

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