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Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas)
Comparative Biochemistry and Physiology Part C 128 Ž2001. 127᎐141
Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas)夽 Kathleen M. JensenU , Joseph J. Korte, Michael D. Kahl, Mumtaz S. Pasha, Gerald T. Ankley U.S. En¨ ironmental Protection Agency, Mid-Continent Ecology Di¨ ision, 6201 Congdon Boule¨ ard, Duluth, MN 55804 USA Received 12 July 2000; received in revised form 5 October 2000; accepted 16 October 2000
Abstract The fathead minnow Ž Pimephales promelas. has been proposed as a model species for assessing the adverse effects of endocrine-disrupting chemicals ŽEDCs. on reproduction and development. The purpose of these studies was to develop baseline reproductive biology and endocrinology data for this species to support interpretation of tests with potential EDCs. Pairs of reproductively-active fathead minnows Ž n s 70. were evaluated with respect to reproductive cyclicity in terms of spawning interval and fecundity. The mode and mean Ž"SE. spawning intervals for the fish in this study were 3.0 and 3.7" 0.1 days, respectively. The mean number of eggs produced per spawn was 85 " 2.8. Animals were sacrificed at periodic intervals during the established spawning cycle and measurements made of gonadal condition Žgonadosomatic index wGSIx, histopathology. and plasma concentrations of vitellogenin and sex steroids Ž-estradiol, testosterone, 11-ketotestosterone.. The GSI in females varied significantly as a function of spawning interval, with the largest values occurring day 2 post-spawn, just prior to the interval of maximum spawning activity. Plasma -estradiol concentrations in females also varied significantly relative to peak values in the GSI and spawning activity. Vitellogenin concentrations in the female, and male GSI and steroid concentrations did not vary significantly relative to position in the spawning cycle. Concentrations of -estradiol in females and 11-ketotestosterone in males were positively correlated with testosterone concentrations. Published by Elsevier Science Inc. Keywords: Endocrine disruption; Fathead minnow; Gonad; Reproduction; Secondary sex characteristics; Sex steroids; Spawning cycle; Vitellogenin
1. Introduction Recent media reports have heightened public concern for the potential effects of endocrine夽
Disclaimer: This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. EPA, and approved for publication. Mention of trade names of commercial products does not constitute endorsementrrecommendation for use. U Corresponding author. Tel.: q1218-529-5177. E-mail address: [email protected] ŽK.M. Jensen.. 1532-0456r01r$ - see front matter Published by Elsevier Science Inc. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 0 . 0 0 1 8 5 - X
disrupting chemicals ŽEDCs. on reproductive and developmental processes in both humans and wildlife ŽColborn et al., 1996.. This has led, in turn, to a legislated mandate to the US Environmental Protection Agency ŽEPA. to develop and implement standardized screening and testing methods to identify and assess potential EDCs. The EPA, in conjunction with the input of a multi-stakeholder advisory group, defined a tiered testing paradigm and associated assays for identification of chemicals that could elicit toxicity through alterations of endocrine pathways con-
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trolled by estrogens, androgens and thyroid hormones ŽEPA, 1998a.. Current recommendations for Tier 1 Žinitial. EDC screening include 3 assays with male or female rats at different life stages, a metamorphosis test with an amphibian Ž Xenopus lae¨ is. and a short-term reproduction test with the fathead minnow Ž Pimephales promelas.. The fathead minnow was chosen as a model species for EDC screening for a number of reasons ŽAnkley et al., 2000.. This species is representative of the ecologically-important and ubiquitous Cyprinidae family. It has been used extensively in chronic life-cycle and early life-stage survival and development tests in support of regulatory programs in both North America and Europe ŽEPA 1982, 1989, 1991, 1994; Organization for Economic Cooperation and Development, 1992a,b,c.. The fathead minnow is relatively easy to culture in a laboratory setting using standard methods ŽEPA, 1987. and control of its reproductive cycle is readily achieved through alterations in temperature and photoperiod. The fathead minnow is a fractional spawner and, under favorable conditions, females can produce clutches of 50᎐100 eggs every 3᎐5 days. These factors, in conjunction with a relatively rapid life-cycle Žhatch to sexual maturity in 4᎐5 months., make the fathead minnow an attractive model species with respect to short-term reproductive studies that might need to be conducted for, theoretically, thousands of chemicals ŽEPA, 1998a.. The specific regime recommended by EPA ŽEPA, 1998a. for a short-term reproduction assay with the fathead minnow was largely conceptual in nature that is, although the proposed test was reasonable in the context of detecting possible estrogen andror androgen agonistsrantagonists, methods did not exist for the assay. In response to this, a significant amount of work over the last 2᎐3 years has focused upon the effects of different EDCs on reproduction in the fathead minnow Že.g., Kramer et al., 1998; Miles-Richardson et al., 1999a,b; Nichols et al., 1999; Ankley et al., 2000; Giesy et al., 2000; Harries et al., 2000; Makynen et al., 2000. and a standard method was recently described ŽEPA, 1999; Ankley et al., 2000.. The method proposed by Ankley et al., 2000 is initiated with reproductively-active fish that are exposed to chemicals of concern for up to 21 days, following a 14᎐21-day ‘pre-exposure’ phase to document baseline reproduction. In this test, survival, behavior, secondary sexual characteristics
and fecundity are assessed daily. Fertility and early development of the F1 generation also may be evaluated. At conclusion of the test, plasma concentrations of vitellogenin ŽVtg. and sex steroids Ž  -estradiol, testosteron e, 11ketotestosterone. are measured and gonadal status assessed based on the gonadosomatic index ŽGSI. and gonad histopathology. At sufficiently high concentrations, virtually any test chemical or environmental stressor could adversely affect reproductive success in the fathead minnow. Therefore, the biochemical and histological endpoints, in addition to secondary sexual characteristics, are particularly critical to the assay in the context of assessing specific effects on endocrine systems controlled by estrogens and androgens ŽAnkley et al., 2000.. A significant uncertainty in using the fathead minnow in an assay of this type is a general lack of knowledge concerning basic aspects of reproduction. There is little systematically-collected information concerning fecundity of this species in the context of laboratory holding and testing. Similarly, gonadal status in reproductively-active fathead minnows has not been well studied and there is virtually nothing known concerning sex steroid concentrations and cycles in this species. Several recent studies have measured Vtg in the fathead minnow, but all of this work has focused upon abnormal production of the lipoprotein in male or immature fish exposed to steroidal and non-steroidal estrogen receptor agonists ŽKramer et al., 1998; Parks et al., 1999; Tyler et al., 1999; Ankley et al., 2000; Giesy et al., 2000; Korte et al., 2000., rather than documenting normal cyclical variations associated with reproduction. The goal of this study was to comprehensively document several aspects of basic reproductive biology and endocrinology in the adult fathead minnow as a basis for testing this species with potential EDCs. This work was conducted largely in the context of using adult animals in laboratory assays, but also may provide insight for possible impacts on the development of immature fish or reproductive cycles in the wild.
2. Materials and methods 2.1. Animals Data were collected from fathead minnows held
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in the on-site culture facility at the EPA laboratory in Duluth, Minnesota ŽEPA, 1987.. This culture has been maintained for approximately 30 years and was initiated using animals from the Newtown EPA facility in Cincinnati, OH. To enhance genetic diversity, fathead minnows collected from the field have routinely been introduced as supplemental breeding stock approximately every 4 years. Adult and juvenile Ž) 96 h old. fish are held in a continuous flow Ž200᎐300 mlrmin. of filtered Ž1 m., Lake Superior water treated with ultraviolet light to eliminate pathogens. General water quality characteristics are monitored at least weekly in the culture unit and include: dissolved oxygen, 5.5᎐8.0 mgrl; pH 7.5᎐8.1; alkalinity 42 mgrl as CaCO 3 ; and hardness 45 mgrl as CaCO 3 . Water temperature is maintained at 25 " 1⬚C. The photoperiod is 16:8 L:D under bulbs with a visible light intensity of approximately 800 lux. Young Ž- 30 days old. fathead minnows are fed live brine shrimp ŽBioMarine Aquafauna, Hawthorne, CA, USA. naupilii twice daily, while older fish receive thawed adult brine shrimp ŽWilson Pet Supply, Minneapolis, MN, USA., also twice daily. Adults are held as spawning pairs in partitioned glass tanks Žca. 31 = 61 = 32 cm deep. with standpipe drains adjusted to provide 20 cm of water depth. Spawning substrates are constructed of a 7᎐10 cm length of 7.6᎐10.2 cm PVC pipe that had been spilt horizontally ŽEPA, 1987.. The culture facility at Duluth typically maintains approximately 50 breeding pairs of fathead minnows at any given time. The spawning substrates are checked for eggs which are enumerated and held until hatch Žapprox. 96 h. in a flow-through tank containing aerated Lake Superior water. Approximately 100 newly-hatched fry are subsampled from these hatching tanks over the course of 1 week and placed in a mass culture containing 400 animals, with an age range of 4 weeks, for maturation and subsequent use as fresh breeding stock. At 5᎐6-months of age, fish in the mass culture tanks are randomly culled to approximately 30 animals to decrease density and promote development of secondary sexual characteristics Ždorsal pad and nuptial tubercles in males, ovipositor in females. as a basis for identifying breeding pairs. The isolated breeding pairs are maintained for no more than 3 months as active spawners. For the purpose of this study, a total of 70 individual pairs of breeding adults Žmaintained
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under the conditions described above. were intensively monitored for reproductive activity and egg output for periods of time ranging from 3 - 7 weeks on 8 different occasions over the course of approximately 2 years. At conclusion of the observation period, the adults were sampled for determination of gonadal status and measurement of plasma steroid and vitellogenin concentrations. The fish were anaesthetized with MS-222 Ž100 mgrl buffered with 200 mg NaHCO3rl., weighed, caudal peduncle partially severed and blood collected from the caudal veinrartery with heparinized microhematocrit tubes. The gonads of the fish were removed and weighed for determination of the GSI w100 = gonad wt. Žg.rbody wt. Žg.x. Not all animals were used for all analyses, e.g., steroids and Vtg were measured only in a subset of the fish for which reproductive output was monitored. However, the studies were designed so that there were representative measurements of all the endpoints at multiple times during the 3᎐4-day breeding cycle characteristic of reproductively-active fathead minnows Žsee Results.. Furthermore, care was taken to have ‘matched’ measurements in enough individual animals so that meaningful comparisons of potentially related endpoints Že.g., fecundity, GSI, steroids and Vtg. could be made. 2.2. Biochemical, histological and morphological analyses Plasma was separated from blood by centrifugation for 3 min at 15 000 = g; a proteinase inhibitor Ž0.13 units aprotinin. then was added to the plasma, which was stored at y80⬚C until Vtg andror steroid analyses. Vitellogenin measurements were made using an enzyme-linked immunosorbent assay ŽELISA. as previously described ŽParks et al., 1999; Korte et al., 2000.. Typically, plasma samples were diluted between 300- Žmales. and 120 000-fold so that the values obtained would fall within the reliable region of the standards. The diluted samples, standards of purified fathead minnow Vtg between 12 and 3000 ngrml and appropriate controls were combined with an equal volume of a 1:20 000 dilution of rabbit anti-fathead minnow Vtg antisera Žprovided by Dr Louise Parks, EPA, Research Triangle Park, NC, USA.. After incubating for 1 h at 37⬚C, two 200-l aliquots were removed and
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added to a 96-well plate that had been previously coated with purified fathead minnow Vtg at 3.5 grml. After incubating for 1 h at 37⬚C, the plate was washed and 200 l of a 1:40 000 dilution of peroxidase-conjugated secondary antibody ŽBioRad, Hercules, CA, USA. was added. The plates were again incubated for 1 h at 37⬚C, washed and allowed to react with TMB peroxidase substrate ŽKirkegaard and Perry, Gaithersburg, MD, USA. for 5᎐10 min. The color was measured with a plate reader at 450 nm after the addition of 100 l of 1 M phosphoric acid. Using the 300-fold dilution, the minimum detection limit Ždescribed below. in the plasma varied from assay to assay, but was typically less than 0.005 mgrml. The general radioimmunoassay ŽRIA. methods for measuring concentrations of -estradiol ŽE2., testosterone ŽT. and 11-ketotestosterone Ž11-KT. have been previously described ŽKagawa et al., 1981; Korte et al., 2000; Makynen et al., 2000.. Some modifications were necessary to optimize measurements for the small volume of plasma available from fathead minnows Žtypically 20᎐60 lrfish.. Thawed plasma was measured in multiples of 6 l, with 1 aliquot for each steroid measurement made. The aliquots were placed in a disposable 12 = 75-mm glass test tube with 150 l of 0.1 M phosphate buffered saline ŽPBS; 0.1 M NaPO4 , 0.15 M NaCl, pH 7.6.. Then 1.5 ml of ether was added to the tube and vortexed for 30 s. After 1 min, tubes were placed in a y80⬚C freezer for 10 min, following which the ether phase was decanted into a second tube, leaving the frozen aqueous phase behind. After thawing, the ether extraction was repeated. The combined ether phases were dried and resuspended in 20 volumes of assay buffer Ž0.01 M PBS, pH 7.4 with 1% bovine serum albumin. by vortexing for 15 s. Typically, 100 l of resuspended extract, representing 5 l of plasma, was analyzed per steroid determination. Extracts were incubated with equal volumes of appropriately diluted antisera ŽE2 and T antisera were obtained from Endocrine Sciences, Calabasas Hill, CA, USA; 11-KT antisera was provided by Dr David Kime, University of Sheffield, Sheffield, UK. and the corresponding tritiated compound Ž5000 cpm, 100 Cirmmol, Amersham Life Sciences, Arlington Heights, IL, USA., both in assay buffer, for 1.5᎐2 h at 25⬚C. In addition to the plasma samples, standards Ž0.01᎐5 ngrml. diluted in assay buffer, nonspecific binding Žno antibody. and maximum bind-
ing Žantibody and labeled compounds only. samples were analyzed. Tubes were placed in an ice bath for 15 min, followed by the addition of 0.4 ml of cold Ž0᎐4⬚C. 0.5% activated charcoal with 0.05% dextran in 0.1 M PBS, pH 7.6. After 15 min, the tubes were centrifuged at 4⬚C for 30 min at 3000 = g to separate the bound and unbound fractions. Aliquots of the supernatant Ž0.5 ml. were removed, added to 5 ml of Scintiverse 䊛 ŽFisher, Pittsburgh, PA, USA., and counted in a scintillation counter. Detection limits for the three steroids were typically 0.4 or 0.8 ngrml of plasma Žrange 0.2᎐1.6 ngrml.. Extraction efficiency, as determined by recovery of tritiated steroids from spiked plasma, was 85᎐90%. Both the inter-assay Žeffective concentration of standard at 50% response. and intra-assay Žreplicate analysis of a plasma pool. coefficients of variation were approximately 10%. Gonads from some of the fish were preserved in 1% glutaraldehyder4% formaldehyde in 0.1 M phosphate buffer for histological analyses. Tissues were embedded in JB-4 methacrylate, sectioned at 3᎐5 m in a serial-step fashion, and stained with hematoxylin and eosin. At least 2 cross-sections of ovaries from females Ž n s 12. were microscopically examined and staged according to criteria described by Selman and Wallace Ž1986., Selman et al. Ž1993. and Shimizu Ž1997.. Developing oocytes were categorized into primary growth, cortical alveolus, early vitellogenic or late vitellogenic stages and the percentage of each stage of oocyte development calculated. Corpora lutea, or post-ovulatory follicles, also were counted. Oogonia were not counted. Some ovaries were also sectioned longitudinally to confirm that oocyte distribution was similar throughout the length of the ovary. Testes from males Ž n s 3. were evaluated for the general degree of spermatogenic activity present ŽNagahama, 1983; Selman and Wallace, 1986.. A morphological characteristic in fathead minnows that appears to be indicative both of estrogens and androgens are the nuptial tubercles normally present on the head of reproductivelyactive males ŽAnkley et al., 2000.. Specifically, exposure of fathead minnows to androgen receptor agonists, such as the synthetic androgen methyltestosterone, can cause the de novo development of nuptial tubercles in females, as well as increased expression of the tubercles in males ŽSmith, 1974; Ankley et al., 2000.. Recent studies
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also have shown that estrogen receptor agonists can reduce the number andror size of nuptial tubercles in mature male fathead minnows ŽMiles-Richardson et al., 1999b; Harries et al., 2000.. There is subjectivity, however, in quantifying this particular endpoint, in part because of a general lack of baseline data. Thus, as part of these studies, we visually assessed tubercle patterns and size in actively-spawning fathead minnows. 2.3. Data manipulations and statistical analyses Minimum detection limits for Vtg and steroid analyses were the lowest standard, based on 95% confidence limits, that was significantly different from the zero standard ŽChard, 1978.. These values were converted to concentrations in the plasma to match this standard value. Any result obtained that was within the 95% confidence limit of the zero standard was treated as zero. When the result of the assay was below the detection limit and outside the 95% confidence limit of the zero standard, the actual value used for statistical comparisons was one half of the minimum detection level ŽHaas and Scheff, 1990; EPA, 1998b.. Differences between means were assessed using ANOVA followed by Tukey’s multiple comparisons test. Pearson’s correlation coefficients were applied to examine relationships between fish weight, gonad weight, GSI, E2, T, 11-KT, Vtg and fecundity. Where necessary, data were log transformed to reduce variance heterogeneity. All computations were performed with SYSTAT 7.0 for Windows 䊛 ŽSPSS, Chicago, IL, USA.. Differences were considered to be significant at PF 0.05. All data are presented as means " SE.
3. Results The spawning interval for reproductively-active fathead minnow pairs Ž n s 70. over the course of the study ranged from 1 - 15 days (n s 478 spawns., with the majority of values falling in the range of 3᎐4 days Žmodes 3 days, mean s 3.7 days, Fig. 1.. The number of eggs per spawn per female ranged from 46 to 130, with a mean value of 85 " 2.8. There was no relationship between the spawning interval and number of eggs per spawn, nor was there any obvious relationship
Fig. 1. Spawning interval frequency of 70 reproductively-active fathead minnow pairs Ž n s 478 spawns..
between female or gonad size and fecundity Ždata not shown.. In this study, the mean fecundity rate for actively spawning females was 19 " 1 eggsr femalerday. Basic reproductive data for both sexes were plotted and analyzed as a function of a 4-day spawning interval ŽFig. 2a᎐d and Fig. 3a᎐d.. In females, the mean GSI for all fish was 9.71" 0.51%. The female GSI varied significantly as a function of spawning interval, with the smallest values occurring on the same day the fish spawned and increasing by approximately 45% to the largest values observed on day 2, just prior to the peak spawning period ŽFigs. 1 and 2a.. A significant peak in plasma E2 concentration was observed on day 1 post-spawn, followed by a gradual decline in concentration of the estrogen ŽFig. 2b.. There was no obvious cyclical variation in plasma T concentrations in the female fathead minnows as a function of spawning interval ŽFig. 2c.. Mean plasma E2 and T concentrations in actively-spawning fathead minnow females were 5.97" 1.12 and 3.08" 0.34 ngrml, respectively. Concentrations of 11-KT typically were nondetectable in females; however, on occasion, comparatively small concentrations of the androgen were measured Ž0.36" 0.11 ngrml, n s 10, data not shown.. Plasma Vtg concentrations in the females were on the order of 15 mgrml and also appeared to remain relatively constant over a spawning interval of 4 days ŽFig. 2d.. A correlation matrix developed for the various reproductive and endocrinological parameters collected in the females indicated only one noteworthy significant relationship, plasma E2 and T concentra-
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tions were positively correlated with one another Ž r s 0.70; n s 49; PF 0.001; Fig. 4a.. The GSI in male fathead minnows from active spawning pairs did not vary as a function of spawning interval ŽFig. 3a.. The mean GSI in males from these studies was 1.15" 0.02%. Overall mean T and 11-KT concentrations in the male fathead minnows were 9.11" 0.92 and 33.1" 3.14 ngrml, respectively. Neither T nor 11-KT varied significantly relative to spawning interval ŽFig. 3b᎐c.; however, there was a significant positive correlation between concentrations of the two androgens Ž r s 0.53; n s 35; PF 0.01; Fig. 4b.. Plasma concentrations of E2 in the males were low, but often measurable Ž0.40" 0.13 ngrml, Fig. 3d.. Plasma Vtg also was occasionally de-
Fig. 3. Variations in the Ža. gonadosomatic index ŽGSI.; Žb. testosterone ŽT.; Žc. 11-ketotestosterone Ž11-KT.; and Žd. estradiol ŽE2. concentrations relative to a 4-day spawning interval in the male fathead minnow. Data are expressed as means " SE. Sample size is given in parentheses.
Fig. 2. Variations in the Ža. gonadosomatic index ŽGSI.; Žb. -estradiol ŽE2.; Žc. testosterone ŽT.; and Žd. vitellogenin ŽVtg. concentrations relative to a 4-day spawning interval in the female fathead minnow. Data are expressed as means " SE. Sample size is given in parentheses. Means with different letters differed significantly at PF 0.05.
tected in the fathead minnow males but, compared to the females, concentrations were very small Ž0.004" 0.001 mgrml, n s 31, data not shown.. Representative gonadal sections of reproductively-active female and male fathead minnows from this study are shown in Fig. 5a,b, respectively. The ovary of a reproductively-active female contains oocytes in various states of maturation including primary growth, cortical alveolus, early vitellogenic and the more mature, later vitellogenic stages. Though all developmental stages were present in the ovary on each day examined Ždays 0, 1 and 2 post-spawn., significant transitions in oocyte maturational stage occurred ŽFig. 6.. Primary growth stage oocytes formed a significantly higher percentage of the total oocytes
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Fig. 4. Relationship between Ža. -estradiol ŽE2. and testosterone ŽT. concentrations in female fathead minnows; and Žb. testosterone and 11-ketotestosterone Ž11-KT. concentrations in male fathead minnows. Ža: r s 0.70; n s 49; PF 0.001; b: r s 0.53; n s 35; PF 0.01..
present on days 0᎐1 post-spawn. By days 1᎐2 post-spawn, the percentage of vitellogenic oocytes significantly increased and fewer primary growth stage oocytes were present. The percentage of cortical alveolus and early vitellogenic stage oocytes appeared to remain relatively constant over the 0᎐2-day interval. Corpora lutea were present in the ovaries of females on days 0᎐1 post-spawn, providing evidence of recent ovulation. However, by day 2 post-spawn, no corpora lutea were found. Few atretic oocytes were observed in these females. In reproductively-active males, five stages of spermatogenesis were identified based on cytological morphology ŽFig. 5b.. Primary spermatogonia are large, pale-staining cells with a large central nucleus containing dense chromatin. Secondary
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spermatogonia are more numerous and smaller with large lightly basophilic nuclei and little cytoplasm. Primary spermatocytes are similar in size to secondary spermatogonia and have increasingly basophilic nuclei. Secondary spermatocytes, approximately half the size of primary spermatocytes, have still increasingly dense basophilic nuclei. The spermatozoa have condensed intensely basophilic nuclei and very little cytoplasm. Visual evaluation of secondary sexual characteristics in the fathead minnow females from this study indicated no occurrence of nuptial tubercles in females. The nuptial tubercles in males varied among individuals, however, some basic commonalities in patterns of the tubercles were noted. The tubercles usually are arranged in a bilaterally-symmetrical pattern. There may be up to eight individual tubercles around the eyes and between the nares of the males. The greatest number and largest tubercles are located in 2 parallel lines immediately below the nares and above the mouth. In many fish there are groups of tubercles below the lower jaw; those closest to the mouth generally occur as a single pair, while the more ventral set can be comprised of up to four tubercles. In this study, the mean number of tubercles in the male fathead minnow was 25 " 0.7 Ž n s 34.; seldom did any individual have more than 30 distinct tubercles Žrange, 18᎐38.. Fig. 7 indicates the variation, with respect to size, that can occur in the nuptial tubercles of an individual male fathead minnow. The predominant tubercles Žin terms of number. are present as a single, relatively round structure, with the height approximately equivalent to the radius. Most reproductively-active males also have, at least some, tubercles which are enlarged and often shaped as a star or asterisk. Occasionally, tubercles become so enlarged and pronounced that they are indistinguishable as individual structures.
4. Discussion The purpose of these studies was to comprehensively characterize aspects of reproductive biology and endocrinology in the fathead minnow. This type of information is critical as a basis for utilization of this species in laboratory assays to detect EDCs. Despite previous extensive use for aquatic toxicity testing, there has been remark-
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Fig. 5. Reproductively-active fathead minnow gonads. Ža. Female containing oocytes in various stages of maturation including Ž1. primary growth; Ž2. cortical alveolus; Ž3. early vitellogenic; and Ž4. late vitellogenic. Also shown are corpora lutea Ž5., evidence of recent ovulation. Žb. Male showing Ž1. primary spermatogonia; Ž2. secondary spermatogonia; Ž3. primary spermatocyte; Ž4. secondary spermatocyte; and Ž5. mature spermatozoa. Calibration bars: Ža. 500 m; and Žb. 50 m.
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Fig. 6. Relative percentages of the different stages of maturation oocyte in female fathead minnows during the reproductive cycle. Data are expressed as means " SE Ž n s 3᎐5 femalesrday.. Means with different letters Žprimary growth, a᎐b; and late vitellogenic, c᎐d. differed significantly at PF 0.05. No significant differences were observed between days for cortical alveolus or early vitellogenic stage oocytes.
ably little basic reproductive biology research conducted with the fathead minnow. Some of the endpoints assessed in our study Že.g., steroid titers. had not previously been evaluated in this species and other endpoints Že.g., vitellogenin, gonad histology. had not been defined in the context of the active spawning cycle. The most exhaustive reproductive physiologyrendocrinology work in fish has been conducted with synchronous annual spawners, principally salmonids ŽZohar and Billard, 1984.. Comparatively less research has been conducted with species, such as the fathead minnow,
Fig. 7. Representative pattern of nuptial tubercles in reproductively-active male fathead minnow, highlighting Ž1᎐3. differences in relative expression of the structure: Ž1. round with height equivalent to radius; Ž2. enlarged with star or asterisk shape; and Ž3. very enlarged with no distinguishable shape.
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which are multiple or fractional spawners, with group-synchronous or asynchronous gonadal development ŽWallace and Selman, 1981.. Thus, in addition to documenting aspects of reproduction specific to the fathead minnow, these studies contribute to an enhanced understanding of general comparative reproductive physiology in fish. Several aspects of the basic spawning data collected for fathead minnows in the present study are in agreement with more limited analyses reported elsewhere. For example, Gale and Buynak Ž1982. reported the spawning interval in the fathead minnow as characterized by a mode of 3 days, and a mean of 3.9 days, which is virtually identical to our respective observations of 3 and 3.7 days. Both studies are consistent with spawning interval results reported by Harries et al. Ž2000., generated from reproductive toxicity tests with the fathead minnow, as well as previous data from our laboratory testing of EDCs ŽAnkley et al., 2000.. The GSI values observed for both males and females in the present study also are in agreement with those reported elsewhere for reproductively-active fathead minnows ŽSmith, 1978; Ankley et al., 2000; Harries et al., 2000.. The number of eggsrspawn observed in the present study is similar to previous work in our laboratory ŽEPA, 1987; Ankley et al., 2000., as well as others ŽWeber, 1993; Kramer et al., 1998., but up to 50% lower than that reported by some researchers working with the fathead minnow ŽGale and Buynak, 1982; Harries et al., 2000.. Because of variations in experimental conditions Že.g., test systems, handling, fish age and size, water quality and diet, etc.. among these various studies, it is difficult to speculate as to the causeŽs. of differential fecundity. Patterns in, and concentrations of sex steroids in male and female fathead minnows were largely consistent with observations made in other nonsalmonid fish species ŽFostier et al., 1983; Dodd and Sumpter, 1984; Kobayashi et al., 1988; Hontela and Stacey, 1990.. Both E2 and T were present at elevated levels in reproductively-active female fathead minnows and concentrations of the two steroids were significantly positively correlated. Similar observations have been made in other fish species Že.g., Scott et al., 1984; Thomas et al., 1987; Greeley et al., 1988; Matsuyama et al., 1988; Jackson and Sullivan, 1995; Hobby and Pankhurst, 1997., and the correlation between the two steroids has been attributed to the role of T
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as a precursor to E2 ŽKagawa et al., 1984; Matsuyama et al., 1988.. In our study, concentrations of 11-KT were small, or non-detectable in the female fathead minnow, which also is consistent with observations made in other fish species ŽScott et al., 1984; Greeley et al., 1988.. In males, concentrations of 11-KT were greater than those of T and relative concentrations of the two steroids were positively correlated with one another. Others also have observed higher plasma concentrations of 11-KT than T in reproductively-active male fish, as well as a positive correlation in relative concentrations of the two androgens Že.g., Scott et al., 1984; Kobayashi et al., 1986; Barry et al., 1990; Jackson and Sullivan, 1995.. The latter observation has been speculated to be due to T serving as a precursor for 11-KT ŽOzon, 1972.. Compared to T and 11-KT, concentrations of E2 were small in male fathead minnows, which is similar to observations made in the goldfish Ž Carassius auratus ., another cyprinid species ŽTrudeau et al., 1993.. However, Nichols et al. Ž1999. and Giesy et al. Ž2000. reported that T and E2 concentrations in sexually-mature male fathead minnows were comparable, which is quite different from our observation that plasma E2 concentrations were as much as 10-fold lower than those of T. Differences between the two studies could be related to the measurement techniques used for the steroids; we used RIAs to measure the two steroids, while Nichols et al. Ž1999. and Giesy et al. Ž2000. utilized ELISA techniques. Alternatively, differences in sampling procedure, Že.g., stress associated with capture. could have caused differential steroid hormone profiles ŽBerlinsky and Specker, 1991.. There have been several reports concerning Vtg concentrations in the fathead minnow Že.g., Kramer et al., 1998; Panter et al., 1998; Nichols et al., 1999; Parks et al., 1999; Tyler et al., 1999; Ankley et al., 2000; Giesy et al., 2000; Harries et al., 2000; Korte et al., 2000.. However, it is difficult to compare our baseline data collected for both sexes to most of these previous studies, because their primary focus has been upon the physiologically-abnormal induction of Vtg in males, as an indicator of exposure to estrogenic chemicals. Further complicating among study comparisons, from a quantitative perspective, is that different techniques have been used to detect the protein, including determination of alkaline-labile phosphate concentrations in the plasma
ŽKramer et al., 1998. and measurement via ELISA with polyclonal antibodies specific to the fathead minnow ŽParks et al., 1999; Ankley et al., 2000; Korte et al., 2000. or other fish species ŽNichols et al., 1999; Tyler et al., 1999; Giesy et al., 2000; Harries et al., 2000.. Two general observations can be made. First, the protein is occasionally detected at relatively low concentrations in untreated males, regardless of the measurement technique used. The physiological role of andror cause of this is uncertain. Second, concentrations of Vtg, as determined via ELISA, can vary by an order of magnitude, or more, depending upon the source of the antibody used in the assay. For example, plasma concentrations of the lipoprotein in mature females was at least 10-fold higher in studies which have used a polyclonal antibody prepared from the fathead minnow ŽParks et al., 1999; Ankley et al., 2000, the present study., compared to work utilizing Žcross-reacting . antibodies for Vtg in the goldfish ŽNichols et al., 1999; Giesy et al., 2000. or the carp ŽHarries et al., 2000.. This quantitative difference among studies may still allow assessing relative changes in concentrations of Vtg associated with chemical exposures; however, if measurement of the protein is to become a routine endpoint for EDC screening assays, it would be desirable to develop a monoclonal antibody, specific to the fathead minnow, to help ensure consistency in interlaboratory testing. Although there has been some analysis of seasonal cyclicity of reproduction in the fathead minnow Že.g., Smith, 1978., there had been no previous studies describing variations in reproductive parameters in this species as a function of spawning cycle, once spawning had been initiated. Based upon studies with other fractional or multiple spawning species, variations in morphological, histological andror biochemical indicators of reproductive status might be expected ŽZohar and Billard, 1984; Greeley et al., 1988; Matsuyama et al., 1988; Rinchard et al., 1993, 1997; Cerda ´ et al., 1996.. Because serial sampling cannot be done with relatively small fish, our experimental design could not be optimal in terms of discerning reproductive cycles in the individual ŽZohar and Billard, 1984.. Nevertheless, several relevant observations were made. For example, in female fathead minnows, there was a clear influence of spawning interval on the GSI, with the smallest values occurring on the day of spawning and the
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highest values observed on day 2 post-spawning, just prior to a spawning peak on day 3. There was also a peak in plasma E2 concentration 1-day post-spawning. Our observations of an estrogen peak and subsequent elevated GSI values prior to increased spawning activity are very similar to data described by Cerda ´ et al. Ž1996. for Fundulus heteroclitus, another fractional-spawning species. Other parameters related to reproduction did not appear to vary as a function of spawning cycle in either sex. Plasma steroid concentrations in males, as well as the GSI, appeared to remain relatively constant in reproductively-active fish, which is not unexpected given that fathead minnow males can mate with multiple females in a relatively short period of time ŽHedges and Ball, 1953.. Plasma androgen and Vtg concentrations in females also did not vary significantly relative to spawning interval. Others also have noted that Vtg concentrations in fish species that are multiple spawners do not vary markedly during active reproduction Že.g., Rinchard et al., 1997.. This observation is consistent with the seemingly asynchronous nature of oocyte development in the fathead minnow ŽWallace and Selman, 1981.. Variation in parameters such as the GSI and E2 concentrations in the female fathead minnow during the 3᎐4-day reproductive cycle has implications in terms of using this species and these endpoints to test potential EDCs. Specifically, if the assay is conducted for a predetermined length of time Že.g., 21 days. it is inevitable that upon termination, fish across and within the various treatments will be at different stages in their reproductive cycle. This of course, will contribute to the ‘normal’ variability associated with a given endpoint, thereby reducing, statistical power of the test and could be particularly problematic for endpoints such as GSI or E2 concentrations where normal variation associated with the reproductive cycle Žapprox. 45 and 100%, respectively, for females in this study. might exceed effects associated with exposure to a particular test chemical ŽAnkley et al., 2000.. In test designs where paired Žmale and female. fish comprise the experimental unit of concern Že.g., Harries et al., 2000., it may be possible to minimize variability associated with reproductive cyclicity through data normalization based on the last spawning event for that given tank; in scenarios where multiple fish comprise the basic experimental unit Že.g., Ankley et al.,
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2000., this type of normalization would be more difficult. The gonad morphology observed in this study is consistent with the ovarian and testicular development described by Smith Ž1978. for breeding fathead minnows. The ovaries of reproductivelyactive females contained oocytes in various states of maturation, characteristic of asynchronous spawners. Ovaries of day 0 post-spawn females consisted primarily of smaller oocytes in the primary growth stage and to a lesser extent, the early cortical alveolus stage. By day 2 post-spawn, the relative percentage of vitellogenic oocytes doubled, which is consistent with the increased GSI observed at this time. The percentage of cortical alveolus-stage oocytes remained fairly constant confirming a fairly continual recruitment of oocytes into vitellogenesis during the reproductive cycle, possibly as a result of constant incorporation of Vtg. The levels of plasma Vtg essentially remained constant across the reproductive period. The endocrine correlates of oocyte development in the fathead minnow appear to resemble those of other fish species. On day 0 postspawn, females generally had lower levels of steroid hormones and low GSIs while the ovaries contained a higher percentage of oocytes in the primary growth stage. Plasma E2 levels were elevated on day 1 post-spawn associated with an increase in oocyte maturation and vitellogenesis on day 2 post-spawn. This is consistent with the role of E2 in stimulating vitellogenesis. A similar steroid profile associated with a relatively constant ovarian cycle has been described for other asynchronous spawning species ŽRinchard et al., 1993.. This type of reproductive physiology is in contrast to that observed with an annual spawner, such as the brook trout, where the absolute levels and fluctuations in E2 and Vtg associated with ovarian maturation are much greater ŽTam et al., 1986.. Spermatogenesis is unrestricted in the male fathead minnow meaning that actively mitotic spermatogonia are organized in lobules along the entire length of the seminiferous tubules ŽNagahama, 1983.. It appears that male fathead minnows are typical teleosts with respect to spermatogenic activity. The development of germ cells takes place within cysts formed by Sertoli cells and spermatogonia are located within the basal region of the cysts. In reproductively-active males,
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characteristic cytological changes in the testes were observed and five cellular stages of spermatogenesis differentiated: primary and secondary spermatogonia; primary and secondary spermatocytes; and spermatozoa. All of the testes examined had the appearance of active spermatogenesis, containing numerous germ cells and a large amount of mature spermatozoa. Smith Ž1974. first described studies in which exposure of female fathead minnows to the synthetic androgen methyltestosterone caused the de novo formation of nuptial tubercles and increased the intensity of expression of these structures in mature males. Studies in our laboratory with methyltestosterone have corroborated these effects on secondary sex characteristics in the fathead minnow, as well as better defining impacts of the androgen on other aspects of the reproductive cycle ŽAnkley et al., 2000.. In addition to reflecting physiological changes caused by androgenic chemicals, recent reports indicate that estrogen receptor agonists may reduce expression of nuptial tubercles in reproductively-active fathead minnow males ŽMiles-Richardson et al., 1999a; Harries et al., 2000.. The mechanism via which estrogens affect nuptial tubercles in males has not been defined but, if this endpoint is indeed sensitive to both androgens and estrogens, it would prove very useful as a general EDC screen. As for many morphological endpoints, however, there can be some degree of subjectivity with respect to interpreting responses relative to background variation. In our current studies, no tubercles were noted in sexually-active females, which means that there would be relatively little ambiguity with respect to identifying an androgen. The mean number of tubercles we observed in control males from the present study is similar to values reported elsewhere ŽSmith, 1974; Harries et al., 2000., but number alone may not be sensitive enough to detect subtle changes in expression of this endpoint in males, particularly for weak estrogens ŽMiles-Richardson et al., 1999a.. More probably, an ‘intensity’ index is required which would reflect both numbers and relative development of tubercles in males. In summary, in this paper we describe several basic aspects of reproduction in the fathead minnow which help interpret and support the use of this species in short-term reproduction assays with
potential EDCs. The data collected in these studies are largely consistent with more limited previous analyses of reproduction in the fathead minnow, as well as general aspects of comparative endocrinology in other, more extensively studied, fish species.
Acknowledgements Discussions with Dr Glen Van Der Kraak greatly aided data interpretation. Drs Jim McKim and Dave Mount provided valuable comments on an earlier version of the manuscript. Technical assistance in culturing fish was provided by Jim Jenson, Dave Nessa and Kevin Lott. Histological assistance was provided by Ann Linnum, Joe Tietge, Rich Leino and Kevin Flynn. Diane Spehar and Roger LePage assisted in manuscript preparation. References Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., Makynen, E.A., 2000. Description and evaluation of a short-term reproduction test with the fathead minnow Ž Pimephales promelas.. Environ. Toxicol. Chem. Žaccepted.. Barry, T.P., Santos, A.J.G., Furukawa, K., Aida, K., Hanyu, I., 1990. Steroid profiles during spawning in male common carp. Gen. Comp. Endocrinol. 80, 223᎐231. Berlinsky, D.L., Specker, J.L., 1991. Changes in gonadal hormones during oocyte development in the striped bass, Morone saxatilis. Fish. Phys. Biochem. 9, 51᎐62. Cerda, ´ J., Calman, B.G., LaFleur Jr., G.J., Limesand, S., 1996. Pattern of vitellogenesis and follicle maturational competence during the ovarian follicular cycle of Fundulus heteroclitus. Gen. Comp. Endocrinol. 103, 24᎐35. Chard, T., 1978. An introduction to radioimmunoassay and related techniques. In: Work, T.S., Work, E. ŽEds.., Laboratory Techniques in Biochemistry and M olecular Biology. ElsevierrNorth-Holland Biomedical Press, New York, pp. 446᎐447. Colborn, T., Dumanoski, D., Meyers, J.P., 1996. Our Stolen Future: Are We Threatening Our Fertility, Intelligence and Survival? - A Scientific Detective Story, Dutton, New York. Dodd, J.M., Sumpter, J.P., 1984. Fishes. In: Lamming, G.E. ŽEd.., Marshall’s Physiology of Reproduction,
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Reproductive Cycles of Vertebrates, 1. Churchill Livingstone, Edinburgh, pp. 1᎐126. Fostier, A., Jalabert, B., Billard, R., Breton, B., Zohar, Y., 1983. The gonadal steroids. In: Hoar, W.S., Randall, D.J., Donaldson, E.M. ŽEds.., Fish Physiology, IXA. Academic Press, New York, pp. 277᎐372. Gale, W.F., Buynak, G.L., 1982. Fecundity and spawning frequency of the fathead minnow-A fractional spawner. Trans. Am. Fish. Soc. 111, 35᎐40. Giesy, J.P., Pierens, S.L., Synder, E.M., Miles-Richardson, S., Kramer, V.J., Snyder, S.A., Nichols, K.M., Villeneuve, D.A., 2000. Effects of 4-nonylphenol on fecundity and biomarkers of estrogenicity in fathead minnows Ž Pimephales promelas.. Environ. Toxicol. Chem. 19, 1368᎐1377. Greeley Jr., M.S., MacGregor III, R., Marion, K.R., 1988. Variation in plasma oestrogens and androgens during the seasonal and semilunar spawning cycles of female gulf killifish, Fundulus grandis ŽBaird and Girard.. J. Fish. Biol. 33, 419᎐429. Harries, J.E., Runnalls, T., Hill, E., Harris, C.A., Maddix, S., Sumpter, J.P., Tyler, C.R., 2000. Development of a reproductive performance test for endocrine disrupting chemicals using pair-breeding fathead minnows Ž Pimephales promelas.. Environ. Sci. Technol. 34, 3003᎐3011. Haas, C.N., Scheff, P.A., 1990. Estimation of averages in truncated samples. Environ. Sci. Technol. 24, 912᎐919. Hedges, S.B., Ball, R.C., 1953. Production and Harvest of Bait Fishes in Michigan. Mich. Dept. Conserv. Misc. Publ. No. 6. Ann Arbor, MI, pp. 1᎐30. Hobby, A.C., Pankhurst, N.W., 1997. The relationship between plasma and ovarian levels of gonadal steroids in the repeat spawning marine fishes Pagrus auratus ŽSparidae. and Chromis dispilus ŽPomacentridae.. Fish. Physiol. Biochem. 16, 65᎐75. Hontela, A., Stacey, N.E., 1990. Cyprinidae. In: Munro, A.D., Scott, A.P., Lam, T.J. ŽEds.., Reproductive Seasonality in Teleosts: Environmental Influences. CRC Press, Boca Raton, pp. 53᎐77. Jackson, L.F., Sullivan, C.V., 1995. Reproduction of white perch: the annual gametogenic cycle. Trans. Am. Fish. Soc. 124, 563᎐577. Kagawa, H., Takano, K., Nagahama, Y., 1981. Correlation of plasma estradiol-17 and progesterone levels with ultrastructure and histochemistry of ovarian follicles in the white-spotted char, Sal¨ elinus leucomaenis. Cell Tissue Res. 218, 315᎐329. Kagawa, H., Young, G., Nagahama, Y., 1984. In vitro estradiol-17 and testosterone production by ovarian follicles of the goldfish, Carassius auratus. Gen. Comp. Endocrinol. 54, 139᎐143. Kobayashi, M., Aida, K., Hanyu, I., 1986. Annual
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changes in plasma levels of gonadotropin and steroid hormones in goldfish. Bull. Jpn. Soc. Sci. Fish. 52, 1153᎐1158. Kobayashi, M., Aida, K., Hanyu, I., 1988. Hormone changes during the ovulatory cycle in goldfish. Gen. Comp. Endocrinol. 69, 301᎐307. Korte, J.J., Kahl, M.D., Jensen, K.M., Pasha, M.S., Parks, L.G., LeBlanc, G.A., Ankley, G.T., 2000. Fathead minnow vitellogenin: complementary DNA sequence and messenger RNA and protein expression after 17᎐estradiol treatment. Environ. Toxicol. Chem. 19, 972᎐981. Kramer, V.J., Miles-Richardson, S., Pierens, S.L., Giesy, J.P., 1998. Reproductive impairment and induction of alkaline-labile phosphate, a biomarker of estrogen exposure, in fathead minnows Ž Pimephales promelas. exposed to waterborne 17-estradiol. Aquat. Toxicol. 40, 335᎐360. Makynen, E.A., Kahl, M.D., Jensen, K.M., Tietge, J.E., Wells, K.L., Van Der Kraak, G., Ankley, G.T., 2000. Effects of the mammalian antiandrogen vinclozolin on development and reproduction of the fathead minnow Ž Pimephales promelas.. Aquat. Toxicol. 48, 461᎐475. Matsuyama, M., Adachi, S., Nagahama, Y., Matsuura, S., 1988. Diurnal rhythm of oocyte development and plasma steroid hormone levels in the female red sea bream, Pagrus major, during the spawning season. Aquaculture 73, 357᎐372. Miles-Richardson, S.R., Kramer, V.J., Fitzgerald, S.D., Render, J.A., Yamini, B., Barbee, S.J., Giesy, J.P., 1999a. Effects of waterborne exposure of 17estradiol on secondary sex characteristics and gonads of fathead minnows Ž Pimephales promelas.. Aquat. Toxicol. 47, 129᎐145. Miles-Richardson, S.R., Pierens, S.L., Nichols, K.M., Kramer, V.J., Snyder, E.M., Snyder, S.A., Render, J.A., Fitzgerald, S.D., Giesy, J.P., 1999b. Effects of waterborne exposure to 4-nonylphenol and nonylphenol ethoxylate on secondary sex characteristics and gonads of fathead minnows Ž Pimephales promelas.. Environ. Res. 80, 122᎐137. Nagahama, Y., 1983. The functional morphology of teleost gonads. In: Hoar, W.S., Randall, D.J., Donaldson, E.M. ŽEds.., Fish Physiology, IXA. Academic Press, New York, pp. 223᎐275. Nichols, K.M., Miles-Richardson, S.R., Snyder, E.M., Giesy, J.P., 1999. Effects of exposure to municipal wastewater in situ on the reproductive physiology of the fathead minnow Ž Pimephales promelas.. Environ. Toxicol. Chem. 18, 2001᎐2012. Organization for Economic Cooperation and Development, 1992a. Fish acute toxicity test. OECD guidelines for testing of chemicals. Section 2. Test Guideline 203. Paris, France.
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Organization for Economic Cooperation and Development, 1992b. Fish early-life stage toxicity test. OECD guidelines for testing of chemicals. Section 2. Test Guideline 204. Paris, France. Organization for Economic Cooperation and Development, 1992c. Fish early-life stage acute toxicity test. OECD guidelines for testing of chemicals. Section 2. Test Guideline 210. Paris, France. Ozon, R., 1972. Androgens in fishes, amphibians, reptiles, and birds. In: Idler, D.R. ŽEd.., Steroids in Nonmammalian Vertebrates. Academic Press, New York, pp. 329᎐389. Panter, G.H., Thompson, R.S., Sumpter, J.P., 1998. Adverse reproductive effects in male fathead minnows Ž Pimephales promelas. exposed to environmentally relevant concentrations of the natural oestrogens, oestradiol and oestrone. Aquat. Toxicol. 42, 243᎐253. Parks, L.G., Cheek, A.O., Denslow, N.D., Heppell, S.A., McLachlan, J.A., LeBlanc, G.A., Sullivan, C.V., 1999. Fathead minnow Ž Pimephales promelas. vitellogenin: purification, characterization and quantitative immunoassay for the detection of estrogenic compounds. Comp. Biochem. Physiol. 123C, 113᎐125. Rinchard, J., Kestemont, P., Kuhn, E.R., Fostier, A., ¨ 1993. Seasonal changes in plasma levels of steroid hormones in an asynchronous fish the gudgeon Gobio gobio L. ŽTeleostei, Cyprinidae.. Gen. Comp. Endocrinol. 92, 168᎐178. Rinchard, J., Kestemont, P., Heine, R., 1997. Comparative study of reproductive biology in single and multiple-spawner cyprinid fish. II. Sex steroid and plasma protein phosphorus concentrations. J. Fish. Biol. 50, 169᎐180. Scott, A.P., MacKenzie, D.S., Stacey, N.E., 1984. Endocrine changes during natural spawning in the white sucker, Catostomus commersoni. II. Steroid hormones. Gen. Comp. Endocrinol. 56, 349᎐359. Selman, K., Wallace, R.A., 1986. Gametogenesis in Fundulus heteroclitus. Am. Zool. 26, 173᎐192. Selman, K., Wallace, R.A., Sarka, A., Qi, X., 1993. Stages of oocyte development in the zebrafish, Brachydanio rerio. J. Morphol. 218, 203᎐224. Shimizu, A., 1997. Reproductive cycles in a reared strain of the mummichog, a daily spawner. J. Fish. Biol. 51, 724᎐737. Smith, R.J.F., 1974. Effects of 17␣-methyltestosterone on the dorsal pad and tubercles of fathead minnows ŽPimephales promelas.. Can. J. Zool. 52, 1031᎐1038. Smith, R.J.F., 1978. Seasonal changes in the histology of the gonads and dorsal skin of the fathead minnow, Pimephales promelas. Can. J. Zool. 56, 2103᎐2109. Tam, W.H., Roy, R.J.J., Makaran, R., 1986. Ovarian
cycle and plasma concentrations of estrogen and vitellogenin in brook trout Ž Sal¨ elinus fontinalis, Mitchill.. Can. J. Zool. 64, 744᎐751. Thomas, P., Brown, N.J., Trant, J.M., 1987. Plasma levels of gonadal steroids during the reproductive cycle of female spotted seatrout Cynoscion nebulosus. In: Idler, D.R., Crim, L.W., Walsh, J.M. ŽEds.., Reproductive Physiology of Fish. Memorial University of Newfoundland, St. Johns, p. 219. Trudeau, V.L., Wade, M.G., Van Der Kraak, G., Peter, R.E., 1993. Effects of 17-estradiol on pituitary and testicular function in male goldfish. Can. J. Zool. 71, 1131᎐1135. Tyler, C.R., van Aerle, R., Hutchinson, T.H., Maddix, S., Trip, H., 1999. An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin. Environ. Toxicol. Chem. 18, 337᎐347. U.S. EPA, 1982. User’s guide for conducting life-cycle chronic toxicity tests with fathead minnows Ž Pimephales promelas.. EPA-600r8-81-011. Duluth, MN. U.S. EPA, 1987. Guidelines for the culture of fathead minnows Pimephales promelas for use in toxicity tests. EPAr600r3-87r001. Duluth, MN. U.S. EPA, 1989. Pesticide assessment guidelines. Subdivision E, Hazard evaluation: Wildlife and aquatic organisms. EPA-540r09-82r024. Washington, DC. U.S. EPA, 1991. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms 4th ed. In: Weber, C.I., ŽEd.., EPA-600r4-90r027. Office of Research and Development, Environmental Monitoring Systems Laboratory, Cincinnati, OH. U.S. EPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms, 3rd edition In: Lewis, P.A., Klemm, D.J., Lazorchak, J.M., Norberg-King, T.J., Peltier, W.H., Heber, M.A., ŽEds.., EPAr600r491r002. Office of Research and Development, Environmental Monitoring Systems Laboratory, Cincinnati, OH. U.S. EPA, 1998a. Endocrine Disruptor Screening and Testing Advisory Committee ŽEDSTAC. report. Office of Prevention, Pesticides, and Toxic Substances, Washington, DC. U.S. EPA, 1998b. Guidance for data quality assessment. Practical methods for data analysis. EPAr600rR-96r084. Office of Research and Development, Washington, DC. U.S. EPA, 1999. Screening level reproduction assay with the fathead minnow Ž Pimephales promelas.. Duluth, MN. Wallace, R.A., Selman, K., 1981. Cellular and dynamic
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aspects of oocyte growth in teleosts. Am. Zool. 21, 325᎐343. Weber, D.N., 1993. Exposure to sublethal levels of waterborne lead alters reproductive behavior patterns in fathead minnows Ž Pimephales promelas.. Neuro Toxicology 14, 347᎐358.
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Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas)夽 Kathleen M. JensenU , Joseph J. Korte, Michael D. Kahl, Mumtaz S. Pasha, Gerald T. Ankley U.S. En¨ ironmental Protection Agency, Mid-Continent Ecology Di¨ ision, 6201 Congdon Boule¨ ard, Duluth, MN 55804 USA Received 12 July 2000; received in revised form 5 October 2000; accepted 16 October 2000
Abstract The fathead minnow Ž Pimephales promelas. has been proposed as a model species for assessing the adverse effects of endocrine-disrupting chemicals ŽEDCs. on reproduction and development. The purpose of these studies was to develop baseline reproductive biology and endocrinology data for this species to support interpretation of tests with potential EDCs. Pairs of reproductively-active fathead minnows Ž n s 70. were evaluated with respect to reproductive cyclicity in terms of spawning interval and fecundity. The mode and mean Ž"SE. spawning intervals for the fish in this study were 3.0 and 3.7" 0.1 days, respectively. The mean number of eggs produced per spawn was 85 " 2.8. Animals were sacrificed at periodic intervals during the established spawning cycle and measurements made of gonadal condition Žgonadosomatic index wGSIx, histopathology. and plasma concentrations of vitellogenin and sex steroids Ž-estradiol, testosterone, 11-ketotestosterone.. The GSI in females varied significantly as a function of spawning interval, with the largest values occurring day 2 post-spawn, just prior to the interval of maximum spawning activity. Plasma -estradiol concentrations in females also varied significantly relative to peak values in the GSI and spawning activity. Vitellogenin concentrations in the female, and male GSI and steroid concentrations did not vary significantly relative to position in the spawning cycle. Concentrations of -estradiol in females and 11-ketotestosterone in males were positively correlated with testosterone concentrations. Published by Elsevier Science Inc. Keywords: Endocrine disruption; Fathead minnow; Gonad; Reproduction; Secondary sex characteristics; Sex steroids; Spawning cycle; Vitellogenin
1. Introduction Recent media reports have heightened public concern for the potential effects of endocrine夽
Disclaimer: This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. EPA, and approved for publication. Mention of trade names of commercial products does not constitute endorsementrrecommendation for use. U Corresponding author. Tel.: q1218-529-5177. E-mail address: [email protected] ŽK.M. Jensen.. 1532-0456r01r$ - see front matter Published by Elsevier Science Inc. PII: S 1 5 3 2 - 0 4 5 6 Ž 0 0 . 0 0 1 8 5 - X
disrupting chemicals ŽEDCs. on reproductive and developmental processes in both humans and wildlife ŽColborn et al., 1996.. This has led, in turn, to a legislated mandate to the US Environmental Protection Agency ŽEPA. to develop and implement standardized screening and testing methods to identify and assess potential EDCs. The EPA, in conjunction with the input of a multi-stakeholder advisory group, defined a tiered testing paradigm and associated assays for identification of chemicals that could elicit toxicity through alterations of endocrine pathways con-
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trolled by estrogens, androgens and thyroid hormones ŽEPA, 1998a.. Current recommendations for Tier 1 Žinitial. EDC screening include 3 assays with male or female rats at different life stages, a metamorphosis test with an amphibian Ž Xenopus lae¨ is. and a short-term reproduction test with the fathead minnow Ž Pimephales promelas.. The fathead minnow was chosen as a model species for EDC screening for a number of reasons ŽAnkley et al., 2000.. This species is representative of the ecologically-important and ubiquitous Cyprinidae family. It has been used extensively in chronic life-cycle and early life-stage survival and development tests in support of regulatory programs in both North America and Europe ŽEPA 1982, 1989, 1991, 1994; Organization for Economic Cooperation and Development, 1992a,b,c.. The fathead minnow is relatively easy to culture in a laboratory setting using standard methods ŽEPA, 1987. and control of its reproductive cycle is readily achieved through alterations in temperature and photoperiod. The fathead minnow is a fractional spawner and, under favorable conditions, females can produce clutches of 50᎐100 eggs every 3᎐5 days. These factors, in conjunction with a relatively rapid life-cycle Žhatch to sexual maturity in 4᎐5 months., make the fathead minnow an attractive model species with respect to short-term reproductive studies that might need to be conducted for, theoretically, thousands of chemicals ŽEPA, 1998a.. The specific regime recommended by EPA ŽEPA, 1998a. for a short-term reproduction assay with the fathead minnow was largely conceptual in nature that is, although the proposed test was reasonable in the context of detecting possible estrogen andror androgen agonistsrantagonists, methods did not exist for the assay. In response to this, a significant amount of work over the last 2᎐3 years has focused upon the effects of different EDCs on reproduction in the fathead minnow Že.g., Kramer et al., 1998; Miles-Richardson et al., 1999a,b; Nichols et al., 1999; Ankley et al., 2000; Giesy et al., 2000; Harries et al., 2000; Makynen et al., 2000. and a standard method was recently described ŽEPA, 1999; Ankley et al., 2000.. The method proposed by Ankley et al., 2000 is initiated with reproductively-active fish that are exposed to chemicals of concern for up to 21 days, following a 14᎐21-day ‘pre-exposure’ phase to document baseline reproduction. In this test, survival, behavior, secondary sexual characteristics
and fecundity are assessed daily. Fertility and early development of the F1 generation also may be evaluated. At conclusion of the test, plasma concentrations of vitellogenin ŽVtg. and sex steroids Ž  -estradiol, testosteron e, 11ketotestosterone. are measured and gonadal status assessed based on the gonadosomatic index ŽGSI. and gonad histopathology. At sufficiently high concentrations, virtually any test chemical or environmental stressor could adversely affect reproductive success in the fathead minnow. Therefore, the biochemical and histological endpoints, in addition to secondary sexual characteristics, are particularly critical to the assay in the context of assessing specific effects on endocrine systems controlled by estrogens and androgens ŽAnkley et al., 2000.. A significant uncertainty in using the fathead minnow in an assay of this type is a general lack of knowledge concerning basic aspects of reproduction. There is little systematically-collected information concerning fecundity of this species in the context of laboratory holding and testing. Similarly, gonadal status in reproductively-active fathead minnows has not been well studied and there is virtually nothing known concerning sex steroid concentrations and cycles in this species. Several recent studies have measured Vtg in the fathead minnow, but all of this work has focused upon abnormal production of the lipoprotein in male or immature fish exposed to steroidal and non-steroidal estrogen receptor agonists ŽKramer et al., 1998; Parks et al., 1999; Tyler et al., 1999; Ankley et al., 2000; Giesy et al., 2000; Korte et al., 2000., rather than documenting normal cyclical variations associated with reproduction. The goal of this study was to comprehensively document several aspects of basic reproductive biology and endocrinology in the adult fathead minnow as a basis for testing this species with potential EDCs. This work was conducted largely in the context of using adult animals in laboratory assays, but also may provide insight for possible impacts on the development of immature fish or reproductive cycles in the wild.
2. Materials and methods 2.1. Animals Data were collected from fathead minnows held
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in the on-site culture facility at the EPA laboratory in Duluth, Minnesota ŽEPA, 1987.. This culture has been maintained for approximately 30 years and was initiated using animals from the Newtown EPA facility in Cincinnati, OH. To enhance genetic diversity, fathead minnows collected from the field have routinely been introduced as supplemental breeding stock approximately every 4 years. Adult and juvenile Ž) 96 h old. fish are held in a continuous flow Ž200᎐300 mlrmin. of filtered Ž1 m., Lake Superior water treated with ultraviolet light to eliminate pathogens. General water quality characteristics are monitored at least weekly in the culture unit and include: dissolved oxygen, 5.5᎐8.0 mgrl; pH 7.5᎐8.1; alkalinity 42 mgrl as CaCO 3 ; and hardness 45 mgrl as CaCO 3 . Water temperature is maintained at 25 " 1⬚C. The photoperiod is 16:8 L:D under bulbs with a visible light intensity of approximately 800 lux. Young Ž- 30 days old. fathead minnows are fed live brine shrimp ŽBioMarine Aquafauna, Hawthorne, CA, USA. naupilii twice daily, while older fish receive thawed adult brine shrimp ŽWilson Pet Supply, Minneapolis, MN, USA., also twice daily. Adults are held as spawning pairs in partitioned glass tanks Žca. 31 = 61 = 32 cm deep. with standpipe drains adjusted to provide 20 cm of water depth. Spawning substrates are constructed of a 7᎐10 cm length of 7.6᎐10.2 cm PVC pipe that had been spilt horizontally ŽEPA, 1987.. The culture facility at Duluth typically maintains approximately 50 breeding pairs of fathead minnows at any given time. The spawning substrates are checked for eggs which are enumerated and held until hatch Žapprox. 96 h. in a flow-through tank containing aerated Lake Superior water. Approximately 100 newly-hatched fry are subsampled from these hatching tanks over the course of 1 week and placed in a mass culture containing 400 animals, with an age range of 4 weeks, for maturation and subsequent use as fresh breeding stock. At 5᎐6-months of age, fish in the mass culture tanks are randomly culled to approximately 30 animals to decrease density and promote development of secondary sexual characteristics Ždorsal pad and nuptial tubercles in males, ovipositor in females. as a basis for identifying breeding pairs. The isolated breeding pairs are maintained for no more than 3 months as active spawners. For the purpose of this study, a total of 70 individual pairs of breeding adults Žmaintained
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under the conditions described above. were intensively monitored for reproductive activity and egg output for periods of time ranging from 3 - 7 weeks on 8 different occasions over the course of approximately 2 years. At conclusion of the observation period, the adults were sampled for determination of gonadal status and measurement of plasma steroid and vitellogenin concentrations. The fish were anaesthetized with MS-222 Ž100 mgrl buffered with 200 mg NaHCO3rl., weighed, caudal peduncle partially severed and blood collected from the caudal veinrartery with heparinized microhematocrit tubes. The gonads of the fish were removed and weighed for determination of the GSI w100 = gonad wt. Žg.rbody wt. Žg.x. Not all animals were used for all analyses, e.g., steroids and Vtg were measured only in a subset of the fish for which reproductive output was monitored. However, the studies were designed so that there were representative measurements of all the endpoints at multiple times during the 3᎐4-day breeding cycle characteristic of reproductively-active fathead minnows Žsee Results.. Furthermore, care was taken to have ‘matched’ measurements in enough individual animals so that meaningful comparisons of potentially related endpoints Že.g., fecundity, GSI, steroids and Vtg. could be made. 2.2. Biochemical, histological and morphological analyses Plasma was separated from blood by centrifugation for 3 min at 15 000 = g; a proteinase inhibitor Ž0.13 units aprotinin. then was added to the plasma, which was stored at y80⬚C until Vtg andror steroid analyses. Vitellogenin measurements were made using an enzyme-linked immunosorbent assay ŽELISA. as previously described ŽParks et al., 1999; Korte et al., 2000.. Typically, plasma samples were diluted between 300- Žmales. and 120 000-fold so that the values obtained would fall within the reliable region of the standards. The diluted samples, standards of purified fathead minnow Vtg between 12 and 3000 ngrml and appropriate controls were combined with an equal volume of a 1:20 000 dilution of rabbit anti-fathead minnow Vtg antisera Žprovided by Dr Louise Parks, EPA, Research Triangle Park, NC, USA.. After incubating for 1 h at 37⬚C, two 200-l aliquots were removed and
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added to a 96-well plate that had been previously coated with purified fathead minnow Vtg at 3.5 grml. After incubating for 1 h at 37⬚C, the plate was washed and 200 l of a 1:40 000 dilution of peroxidase-conjugated secondary antibody ŽBioRad, Hercules, CA, USA. was added. The plates were again incubated for 1 h at 37⬚C, washed and allowed to react with TMB peroxidase substrate ŽKirkegaard and Perry, Gaithersburg, MD, USA. for 5᎐10 min. The color was measured with a plate reader at 450 nm after the addition of 100 l of 1 M phosphoric acid. Using the 300-fold dilution, the minimum detection limit Ždescribed below. in the plasma varied from assay to assay, but was typically less than 0.005 mgrml. The general radioimmunoassay ŽRIA. methods for measuring concentrations of -estradiol ŽE2., testosterone ŽT. and 11-ketotestosterone Ž11-KT. have been previously described ŽKagawa et al., 1981; Korte et al., 2000; Makynen et al., 2000.. Some modifications were necessary to optimize measurements for the small volume of plasma available from fathead minnows Žtypically 20᎐60 lrfish.. Thawed plasma was measured in multiples of 6 l, with 1 aliquot for each steroid measurement made. The aliquots were placed in a disposable 12 = 75-mm glass test tube with 150 l of 0.1 M phosphate buffered saline ŽPBS; 0.1 M NaPO4 , 0.15 M NaCl, pH 7.6.. Then 1.5 ml of ether was added to the tube and vortexed for 30 s. After 1 min, tubes were placed in a y80⬚C freezer for 10 min, following which the ether phase was decanted into a second tube, leaving the frozen aqueous phase behind. After thawing, the ether extraction was repeated. The combined ether phases were dried and resuspended in 20 volumes of assay buffer Ž0.01 M PBS, pH 7.4 with 1% bovine serum albumin. by vortexing for 15 s. Typically, 100 l of resuspended extract, representing 5 l of plasma, was analyzed per steroid determination. Extracts were incubated with equal volumes of appropriately diluted antisera ŽE2 and T antisera were obtained from Endocrine Sciences, Calabasas Hill, CA, USA; 11-KT antisera was provided by Dr David Kime, University of Sheffield, Sheffield, UK. and the corresponding tritiated compound Ž5000 cpm, 100 Cirmmol, Amersham Life Sciences, Arlington Heights, IL, USA., both in assay buffer, for 1.5᎐2 h at 25⬚C. In addition to the plasma samples, standards Ž0.01᎐5 ngrml. diluted in assay buffer, nonspecific binding Žno antibody. and maximum bind-
ing Žantibody and labeled compounds only. samples were analyzed. Tubes were placed in an ice bath for 15 min, followed by the addition of 0.4 ml of cold Ž0᎐4⬚C. 0.5% activated charcoal with 0.05% dextran in 0.1 M PBS, pH 7.6. After 15 min, the tubes were centrifuged at 4⬚C for 30 min at 3000 = g to separate the bound and unbound fractions. Aliquots of the supernatant Ž0.5 ml. were removed, added to 5 ml of Scintiverse 䊛 ŽFisher, Pittsburgh, PA, USA., and counted in a scintillation counter. Detection limits for the three steroids were typically 0.4 or 0.8 ngrml of plasma Žrange 0.2᎐1.6 ngrml.. Extraction efficiency, as determined by recovery of tritiated steroids from spiked plasma, was 85᎐90%. Both the inter-assay Žeffective concentration of standard at 50% response. and intra-assay Žreplicate analysis of a plasma pool. coefficients of variation were approximately 10%. Gonads from some of the fish were preserved in 1% glutaraldehyder4% formaldehyde in 0.1 M phosphate buffer for histological analyses. Tissues were embedded in JB-4 methacrylate, sectioned at 3᎐5 m in a serial-step fashion, and stained with hematoxylin and eosin. At least 2 cross-sections of ovaries from females Ž n s 12. were microscopically examined and staged according to criteria described by Selman and Wallace Ž1986., Selman et al. Ž1993. and Shimizu Ž1997.. Developing oocytes were categorized into primary growth, cortical alveolus, early vitellogenic or late vitellogenic stages and the percentage of each stage of oocyte development calculated. Corpora lutea, or post-ovulatory follicles, also were counted. Oogonia were not counted. Some ovaries were also sectioned longitudinally to confirm that oocyte distribution was similar throughout the length of the ovary. Testes from males Ž n s 3. were evaluated for the general degree of spermatogenic activity present ŽNagahama, 1983; Selman and Wallace, 1986.. A morphological characteristic in fathead minnows that appears to be indicative both of estrogens and androgens are the nuptial tubercles normally present on the head of reproductivelyactive males ŽAnkley et al., 2000.. Specifically, exposure of fathead minnows to androgen receptor agonists, such as the synthetic androgen methyltestosterone, can cause the de novo development of nuptial tubercles in females, as well as increased expression of the tubercles in males ŽSmith, 1974; Ankley et al., 2000.. Recent studies
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also have shown that estrogen receptor agonists can reduce the number andror size of nuptial tubercles in mature male fathead minnows ŽMiles-Richardson et al., 1999b; Harries et al., 2000.. There is subjectivity, however, in quantifying this particular endpoint, in part because of a general lack of baseline data. Thus, as part of these studies, we visually assessed tubercle patterns and size in actively-spawning fathead minnows. 2.3. Data manipulations and statistical analyses Minimum detection limits for Vtg and steroid analyses were the lowest standard, based on 95% confidence limits, that was significantly different from the zero standard ŽChard, 1978.. These values were converted to concentrations in the plasma to match this standard value. Any result obtained that was within the 95% confidence limit of the zero standard was treated as zero. When the result of the assay was below the detection limit and outside the 95% confidence limit of the zero standard, the actual value used for statistical comparisons was one half of the minimum detection level ŽHaas and Scheff, 1990; EPA, 1998b.. Differences between means were assessed using ANOVA followed by Tukey’s multiple comparisons test. Pearson’s correlation coefficients were applied to examine relationships between fish weight, gonad weight, GSI, E2, T, 11-KT, Vtg and fecundity. Where necessary, data were log transformed to reduce variance heterogeneity. All computations were performed with SYSTAT 7.0 for Windows 䊛 ŽSPSS, Chicago, IL, USA.. Differences were considered to be significant at PF 0.05. All data are presented as means " SE.
3. Results The spawning interval for reproductively-active fathead minnow pairs Ž n s 70. over the course of the study ranged from 1 - 15 days (n s 478 spawns., with the majority of values falling in the range of 3᎐4 days Žmodes 3 days, mean s 3.7 days, Fig. 1.. The number of eggs per spawn per female ranged from 46 to 130, with a mean value of 85 " 2.8. There was no relationship between the spawning interval and number of eggs per spawn, nor was there any obvious relationship
Fig. 1. Spawning interval frequency of 70 reproductively-active fathead minnow pairs Ž n s 478 spawns..
between female or gonad size and fecundity Ždata not shown.. In this study, the mean fecundity rate for actively spawning females was 19 " 1 eggsr femalerday. Basic reproductive data for both sexes were plotted and analyzed as a function of a 4-day spawning interval ŽFig. 2a᎐d and Fig. 3a᎐d.. In females, the mean GSI for all fish was 9.71" 0.51%. The female GSI varied significantly as a function of spawning interval, with the smallest values occurring on the same day the fish spawned and increasing by approximately 45% to the largest values observed on day 2, just prior to the peak spawning period ŽFigs. 1 and 2a.. A significant peak in plasma E2 concentration was observed on day 1 post-spawn, followed by a gradual decline in concentration of the estrogen ŽFig. 2b.. There was no obvious cyclical variation in plasma T concentrations in the female fathead minnows as a function of spawning interval ŽFig. 2c.. Mean plasma E2 and T concentrations in actively-spawning fathead minnow females were 5.97" 1.12 and 3.08" 0.34 ngrml, respectively. Concentrations of 11-KT typically were nondetectable in females; however, on occasion, comparatively small concentrations of the androgen were measured Ž0.36" 0.11 ngrml, n s 10, data not shown.. Plasma Vtg concentrations in the females were on the order of 15 mgrml and also appeared to remain relatively constant over a spawning interval of 4 days ŽFig. 2d.. A correlation matrix developed for the various reproductive and endocrinological parameters collected in the females indicated only one noteworthy significant relationship, plasma E2 and T concentra-
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tions were positively correlated with one another Ž r s 0.70; n s 49; PF 0.001; Fig. 4a.. The GSI in male fathead minnows from active spawning pairs did not vary as a function of spawning interval ŽFig. 3a.. The mean GSI in males from these studies was 1.15" 0.02%. Overall mean T and 11-KT concentrations in the male fathead minnows were 9.11" 0.92 and 33.1" 3.14 ngrml, respectively. Neither T nor 11-KT varied significantly relative to spawning interval ŽFig. 3b᎐c.; however, there was a significant positive correlation between concentrations of the two androgens Ž r s 0.53; n s 35; PF 0.01; Fig. 4b.. Plasma concentrations of E2 in the males were low, but often measurable Ž0.40" 0.13 ngrml, Fig. 3d.. Plasma Vtg also was occasionally de-
Fig. 3. Variations in the Ža. gonadosomatic index ŽGSI.; Žb. testosterone ŽT.; Žc. 11-ketotestosterone Ž11-KT.; and Žd. estradiol ŽE2. concentrations relative to a 4-day spawning interval in the male fathead minnow. Data are expressed as means " SE. Sample size is given in parentheses.
Fig. 2. Variations in the Ža. gonadosomatic index ŽGSI.; Žb. -estradiol ŽE2.; Žc. testosterone ŽT.; and Žd. vitellogenin ŽVtg. concentrations relative to a 4-day spawning interval in the female fathead minnow. Data are expressed as means " SE. Sample size is given in parentheses. Means with different letters differed significantly at PF 0.05.
tected in the fathead minnow males but, compared to the females, concentrations were very small Ž0.004" 0.001 mgrml, n s 31, data not shown.. Representative gonadal sections of reproductively-active female and male fathead minnows from this study are shown in Fig. 5a,b, respectively. The ovary of a reproductively-active female contains oocytes in various states of maturation including primary growth, cortical alveolus, early vitellogenic and the more mature, later vitellogenic stages. Though all developmental stages were present in the ovary on each day examined Ždays 0, 1 and 2 post-spawn., significant transitions in oocyte maturational stage occurred ŽFig. 6.. Primary growth stage oocytes formed a significantly higher percentage of the total oocytes
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Fig. 4. Relationship between Ža. -estradiol ŽE2. and testosterone ŽT. concentrations in female fathead minnows; and Žb. testosterone and 11-ketotestosterone Ž11-KT. concentrations in male fathead minnows. Ža: r s 0.70; n s 49; PF 0.001; b: r s 0.53; n s 35; PF 0.01..
present on days 0᎐1 post-spawn. By days 1᎐2 post-spawn, the percentage of vitellogenic oocytes significantly increased and fewer primary growth stage oocytes were present. The percentage of cortical alveolus and early vitellogenic stage oocytes appeared to remain relatively constant over the 0᎐2-day interval. Corpora lutea were present in the ovaries of females on days 0᎐1 post-spawn, providing evidence of recent ovulation. However, by day 2 post-spawn, no corpora lutea were found. Few atretic oocytes were observed in these females. In reproductively-active males, five stages of spermatogenesis were identified based on cytological morphology ŽFig. 5b.. Primary spermatogonia are large, pale-staining cells with a large central nucleus containing dense chromatin. Secondary
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spermatogonia are more numerous and smaller with large lightly basophilic nuclei and little cytoplasm. Primary spermatocytes are similar in size to secondary spermatogonia and have increasingly basophilic nuclei. Secondary spermatocytes, approximately half the size of primary spermatocytes, have still increasingly dense basophilic nuclei. The spermatozoa have condensed intensely basophilic nuclei and very little cytoplasm. Visual evaluation of secondary sexual characteristics in the fathead minnow females from this study indicated no occurrence of nuptial tubercles in females. The nuptial tubercles in males varied among individuals, however, some basic commonalities in patterns of the tubercles were noted. The tubercles usually are arranged in a bilaterally-symmetrical pattern. There may be up to eight individual tubercles around the eyes and between the nares of the males. The greatest number and largest tubercles are located in 2 parallel lines immediately below the nares and above the mouth. In many fish there are groups of tubercles below the lower jaw; those closest to the mouth generally occur as a single pair, while the more ventral set can be comprised of up to four tubercles. In this study, the mean number of tubercles in the male fathead minnow was 25 " 0.7 Ž n s 34.; seldom did any individual have more than 30 distinct tubercles Žrange, 18᎐38.. Fig. 7 indicates the variation, with respect to size, that can occur in the nuptial tubercles of an individual male fathead minnow. The predominant tubercles Žin terms of number. are present as a single, relatively round structure, with the height approximately equivalent to the radius. Most reproductively-active males also have, at least some, tubercles which are enlarged and often shaped as a star or asterisk. Occasionally, tubercles become so enlarged and pronounced that they are indistinguishable as individual structures.
4. Discussion The purpose of these studies was to comprehensively characterize aspects of reproductive biology and endocrinology in the fathead minnow. This type of information is critical as a basis for utilization of this species in laboratory assays to detect EDCs. Despite previous extensive use for aquatic toxicity testing, there has been remark-
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Fig. 5. Reproductively-active fathead minnow gonads. Ža. Female containing oocytes in various stages of maturation including Ž1. primary growth; Ž2. cortical alveolus; Ž3. early vitellogenic; and Ž4. late vitellogenic. Also shown are corpora lutea Ž5., evidence of recent ovulation. Žb. Male showing Ž1. primary spermatogonia; Ž2. secondary spermatogonia; Ž3. primary spermatocyte; Ž4. secondary spermatocyte; and Ž5. mature spermatozoa. Calibration bars: Ža. 500 m; and Žb. 50 m.
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Fig. 6. Relative percentages of the different stages of maturation oocyte in female fathead minnows during the reproductive cycle. Data are expressed as means " SE Ž n s 3᎐5 femalesrday.. Means with different letters Žprimary growth, a᎐b; and late vitellogenic, c᎐d. differed significantly at PF 0.05. No significant differences were observed between days for cortical alveolus or early vitellogenic stage oocytes.
ably little basic reproductive biology research conducted with the fathead minnow. Some of the endpoints assessed in our study Že.g., steroid titers. had not previously been evaluated in this species and other endpoints Že.g., vitellogenin, gonad histology. had not been defined in the context of the active spawning cycle. The most exhaustive reproductive physiologyrendocrinology work in fish has been conducted with synchronous annual spawners, principally salmonids ŽZohar and Billard, 1984.. Comparatively less research has been conducted with species, such as the fathead minnow,
Fig. 7. Representative pattern of nuptial tubercles in reproductively-active male fathead minnow, highlighting Ž1᎐3. differences in relative expression of the structure: Ž1. round with height equivalent to radius; Ž2. enlarged with star or asterisk shape; and Ž3. very enlarged with no distinguishable shape.
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which are multiple or fractional spawners, with group-synchronous or asynchronous gonadal development ŽWallace and Selman, 1981.. Thus, in addition to documenting aspects of reproduction specific to the fathead minnow, these studies contribute to an enhanced understanding of general comparative reproductive physiology in fish. Several aspects of the basic spawning data collected for fathead minnows in the present study are in agreement with more limited analyses reported elsewhere. For example, Gale and Buynak Ž1982. reported the spawning interval in the fathead minnow as characterized by a mode of 3 days, and a mean of 3.9 days, which is virtually identical to our respective observations of 3 and 3.7 days. Both studies are consistent with spawning interval results reported by Harries et al. Ž2000., generated from reproductive toxicity tests with the fathead minnow, as well as previous data from our laboratory testing of EDCs ŽAnkley et al., 2000.. The GSI values observed for both males and females in the present study also are in agreement with those reported elsewhere for reproductively-active fathead minnows ŽSmith, 1978; Ankley et al., 2000; Harries et al., 2000.. The number of eggsrspawn observed in the present study is similar to previous work in our laboratory ŽEPA, 1987; Ankley et al., 2000., as well as others ŽWeber, 1993; Kramer et al., 1998., but up to 50% lower than that reported by some researchers working with the fathead minnow ŽGale and Buynak, 1982; Harries et al., 2000.. Because of variations in experimental conditions Že.g., test systems, handling, fish age and size, water quality and diet, etc.. among these various studies, it is difficult to speculate as to the causeŽs. of differential fecundity. Patterns in, and concentrations of sex steroids in male and female fathead minnows were largely consistent with observations made in other nonsalmonid fish species ŽFostier et al., 1983; Dodd and Sumpter, 1984; Kobayashi et al., 1988; Hontela and Stacey, 1990.. Both E2 and T were present at elevated levels in reproductively-active female fathead minnows and concentrations of the two steroids were significantly positively correlated. Similar observations have been made in other fish species Že.g., Scott et al., 1984; Thomas et al., 1987; Greeley et al., 1988; Matsuyama et al., 1988; Jackson and Sullivan, 1995; Hobby and Pankhurst, 1997., and the correlation between the two steroids has been attributed to the role of T
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as a precursor to E2 ŽKagawa et al., 1984; Matsuyama et al., 1988.. In our study, concentrations of 11-KT were small, or non-detectable in the female fathead minnow, which also is consistent with observations made in other fish species ŽScott et al., 1984; Greeley et al., 1988.. In males, concentrations of 11-KT were greater than those of T and relative concentrations of the two steroids were positively correlated with one another. Others also have observed higher plasma concentrations of 11-KT than T in reproductively-active male fish, as well as a positive correlation in relative concentrations of the two androgens Že.g., Scott et al., 1984; Kobayashi et al., 1986; Barry et al., 1990; Jackson and Sullivan, 1995.. The latter observation has been speculated to be due to T serving as a precursor for 11-KT ŽOzon, 1972.. Compared to T and 11-KT, concentrations of E2 were small in male fathead minnows, which is similar to observations made in the goldfish Ž Carassius auratus ., another cyprinid species ŽTrudeau et al., 1993.. However, Nichols et al. Ž1999. and Giesy et al. Ž2000. reported that T and E2 concentrations in sexually-mature male fathead minnows were comparable, which is quite different from our observation that plasma E2 concentrations were as much as 10-fold lower than those of T. Differences between the two studies could be related to the measurement techniques used for the steroids; we used RIAs to measure the two steroids, while Nichols et al. Ž1999. and Giesy et al. Ž2000. utilized ELISA techniques. Alternatively, differences in sampling procedure, Že.g., stress associated with capture. could have caused differential steroid hormone profiles ŽBerlinsky and Specker, 1991.. There have been several reports concerning Vtg concentrations in the fathead minnow Že.g., Kramer et al., 1998; Panter et al., 1998; Nichols et al., 1999; Parks et al., 1999; Tyler et al., 1999; Ankley et al., 2000; Giesy et al., 2000; Harries et al., 2000; Korte et al., 2000.. However, it is difficult to compare our baseline data collected for both sexes to most of these previous studies, because their primary focus has been upon the physiologically-abnormal induction of Vtg in males, as an indicator of exposure to estrogenic chemicals. Further complicating among study comparisons, from a quantitative perspective, is that different techniques have been used to detect the protein, including determination of alkaline-labile phosphate concentrations in the plasma
ŽKramer et al., 1998. and measurement via ELISA with polyclonal antibodies specific to the fathead minnow ŽParks et al., 1999; Ankley et al., 2000; Korte et al., 2000. or other fish species ŽNichols et al., 1999; Tyler et al., 1999; Giesy et al., 2000; Harries et al., 2000.. Two general observations can be made. First, the protein is occasionally detected at relatively low concentrations in untreated males, regardless of the measurement technique used. The physiological role of andror cause of this is uncertain. Second, concentrations of Vtg, as determined via ELISA, can vary by an order of magnitude, or more, depending upon the source of the antibody used in the assay. For example, plasma concentrations of the lipoprotein in mature females was at least 10-fold higher in studies which have used a polyclonal antibody prepared from the fathead minnow ŽParks et al., 1999; Ankley et al., 2000, the present study., compared to work utilizing Žcross-reacting . antibodies for Vtg in the goldfish ŽNichols et al., 1999; Giesy et al., 2000. or the carp ŽHarries et al., 2000.. This quantitative difference among studies may still allow assessing relative changes in concentrations of Vtg associated with chemical exposures; however, if measurement of the protein is to become a routine endpoint for EDC screening assays, it would be desirable to develop a monoclonal antibody, specific to the fathead minnow, to help ensure consistency in interlaboratory testing. Although there has been some analysis of seasonal cyclicity of reproduction in the fathead minnow Že.g., Smith, 1978., there had been no previous studies describing variations in reproductive parameters in this species as a function of spawning cycle, once spawning had been initiated. Based upon studies with other fractional or multiple spawning species, variations in morphological, histological andror biochemical indicators of reproductive status might be expected ŽZohar and Billard, 1984; Greeley et al., 1988; Matsuyama et al., 1988; Rinchard et al., 1993, 1997; Cerda ´ et al., 1996.. Because serial sampling cannot be done with relatively small fish, our experimental design could not be optimal in terms of discerning reproductive cycles in the individual ŽZohar and Billard, 1984.. Nevertheless, several relevant observations were made. For example, in female fathead minnows, there was a clear influence of spawning interval on the GSI, with the smallest values occurring on the day of spawning and the
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highest values observed on day 2 post-spawning, just prior to a spawning peak on day 3. There was also a peak in plasma E2 concentration 1-day post-spawning. Our observations of an estrogen peak and subsequent elevated GSI values prior to increased spawning activity are very similar to data described by Cerda ´ et al. Ž1996. for Fundulus heteroclitus, another fractional-spawning species. Other parameters related to reproduction did not appear to vary as a function of spawning cycle in either sex. Plasma steroid concentrations in males, as well as the GSI, appeared to remain relatively constant in reproductively-active fish, which is not unexpected given that fathead minnow males can mate with multiple females in a relatively short period of time ŽHedges and Ball, 1953.. Plasma androgen and Vtg concentrations in females also did not vary significantly relative to spawning interval. Others also have noted that Vtg concentrations in fish species that are multiple spawners do not vary markedly during active reproduction Že.g., Rinchard et al., 1997.. This observation is consistent with the seemingly asynchronous nature of oocyte development in the fathead minnow ŽWallace and Selman, 1981.. Variation in parameters such as the GSI and E2 concentrations in the female fathead minnow during the 3᎐4-day reproductive cycle has implications in terms of using this species and these endpoints to test potential EDCs. Specifically, if the assay is conducted for a predetermined length of time Že.g., 21 days. it is inevitable that upon termination, fish across and within the various treatments will be at different stages in their reproductive cycle. This of course, will contribute to the ‘normal’ variability associated with a given endpoint, thereby reducing, statistical power of the test and could be particularly problematic for endpoints such as GSI or E2 concentrations where normal variation associated with the reproductive cycle Žapprox. 45 and 100%, respectively, for females in this study. might exceed effects associated with exposure to a particular test chemical ŽAnkley et al., 2000.. In test designs where paired Žmale and female. fish comprise the experimental unit of concern Že.g., Harries et al., 2000., it may be possible to minimize variability associated with reproductive cyclicity through data normalization based on the last spawning event for that given tank; in scenarios where multiple fish comprise the basic experimental unit Že.g., Ankley et al.,
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2000., this type of normalization would be more difficult. The gonad morphology observed in this study is consistent with the ovarian and testicular development described by Smith Ž1978. for breeding fathead minnows. The ovaries of reproductivelyactive females contained oocytes in various states of maturation, characteristic of asynchronous spawners. Ovaries of day 0 post-spawn females consisted primarily of smaller oocytes in the primary growth stage and to a lesser extent, the early cortical alveolus stage. By day 2 post-spawn, the relative percentage of vitellogenic oocytes doubled, which is consistent with the increased GSI observed at this time. The percentage of cortical alveolus-stage oocytes remained fairly constant confirming a fairly continual recruitment of oocytes into vitellogenesis during the reproductive cycle, possibly as a result of constant incorporation of Vtg. The levels of plasma Vtg essentially remained constant across the reproductive period. The endocrine correlates of oocyte development in the fathead minnow appear to resemble those of other fish species. On day 0 postspawn, females generally had lower levels of steroid hormones and low GSIs while the ovaries contained a higher percentage of oocytes in the primary growth stage. Plasma E2 levels were elevated on day 1 post-spawn associated with an increase in oocyte maturation and vitellogenesis on day 2 post-spawn. This is consistent with the role of E2 in stimulating vitellogenesis. A similar steroid profile associated with a relatively constant ovarian cycle has been described for other asynchronous spawning species ŽRinchard et al., 1993.. This type of reproductive physiology is in contrast to that observed with an annual spawner, such as the brook trout, where the absolute levels and fluctuations in E2 and Vtg associated with ovarian maturation are much greater ŽTam et al., 1986.. Spermatogenesis is unrestricted in the male fathead minnow meaning that actively mitotic spermatogonia are organized in lobules along the entire length of the seminiferous tubules ŽNagahama, 1983.. It appears that male fathead minnows are typical teleosts with respect to spermatogenic activity. The development of germ cells takes place within cysts formed by Sertoli cells and spermatogonia are located within the basal region of the cysts. In reproductively-active males,
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characteristic cytological changes in the testes were observed and five cellular stages of spermatogenesis differentiated: primary and secondary spermatogonia; primary and secondary spermatocytes; and spermatozoa. All of the testes examined had the appearance of active spermatogenesis, containing numerous germ cells and a large amount of mature spermatozoa. Smith Ž1974. first described studies in which exposure of female fathead minnows to the synthetic androgen methyltestosterone caused the de novo formation of nuptial tubercles and increased the intensity of expression of these structures in mature males. Studies in our laboratory with methyltestosterone have corroborated these effects on secondary sex characteristics in the fathead minnow, as well as better defining impacts of the androgen on other aspects of the reproductive cycle ŽAnkley et al., 2000.. In addition to reflecting physiological changes caused by androgenic chemicals, recent reports indicate that estrogen receptor agonists may reduce expression of nuptial tubercles in reproductively-active fathead minnow males ŽMiles-Richardson et al., 1999a; Harries et al., 2000.. The mechanism via which estrogens affect nuptial tubercles in males has not been defined but, if this endpoint is indeed sensitive to both androgens and estrogens, it would prove very useful as a general EDC screen. As for many morphological endpoints, however, there can be some degree of subjectivity with respect to interpreting responses relative to background variation. In our current studies, no tubercles were noted in sexually-active females, which means that there would be relatively little ambiguity with respect to identifying an androgen. The mean number of tubercles we observed in control males from the present study is similar to values reported elsewhere ŽSmith, 1974; Harries et al., 2000., but number alone may not be sensitive enough to detect subtle changes in expression of this endpoint in males, particularly for weak estrogens ŽMiles-Richardson et al., 1999a.. More probably, an ‘intensity’ index is required which would reflect both numbers and relative development of tubercles in males. In summary, in this paper we describe several basic aspects of reproduction in the fathead minnow which help interpret and support the use of this species in short-term reproduction assays with
potential EDCs. The data collected in these studies are largely consistent with more limited previous analyses of reproduction in the fathead minnow, as well as general aspects of comparative endocrinology in other, more extensively studied, fish species.
Acknowledgements Discussions with Dr Glen Van Der Kraak greatly aided data interpretation. Drs Jim McKim and Dave Mount provided valuable comments on an earlier version of the manuscript. Technical assistance in culturing fish was provided by Jim Jenson, Dave Nessa and Kevin Lott. Histological assistance was provided by Ann Linnum, Joe Tietge, Rich Leino and Kevin Flynn. Diane Spehar and Roger LePage assisted in manuscript preparation. References Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., Makynen, E.A., 2000. Description and evaluation of a short-term reproduction test with the fathead minnow Ž Pimephales promelas.. Environ. Toxicol. Chem. Žaccepted.. Barry, T.P., Santos, A.J.G., Furukawa, K., Aida, K., Hanyu, I., 1990. Steroid profiles during spawning in male common carp. Gen. Comp. Endocrinol. 80, 223᎐231. Berlinsky, D.L., Specker, J.L., 1991. Changes in gonadal hormones during oocyte development in the striped bass, Morone saxatilis. Fish. Phys. Biochem. 9, 51᎐62. Cerda, ´ J., Calman, B.G., LaFleur Jr., G.J., Limesand, S., 1996. Pattern of vitellogenesis and follicle maturational competence during the ovarian follicular cycle of Fundulus heteroclitus. Gen. Comp. Endocrinol. 103, 24᎐35. Chard, T., 1978. An introduction to radioimmunoassay and related techniques. In: Work, T.S., Work, E. ŽEds.., Laboratory Techniques in Biochemistry and M olecular Biology. ElsevierrNorth-Holland Biomedical Press, New York, pp. 446᎐447. Colborn, T., Dumanoski, D., Meyers, J.P., 1996. Our Stolen Future: Are We Threatening Our Fertility, Intelligence and Survival? - A Scientific Detective Story, Dutton, New York. Dodd, J.M., Sumpter, J.P., 1984. Fishes. In: Lamming, G.E. ŽEd.., Marshall’s Physiology of Reproduction,
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cycle and plasma concentrations of estrogen and vitellogenin in brook trout Ž Sal¨ elinus fontinalis, Mitchill.. Can. J. Zool. 64, 744᎐751. Thomas, P., Brown, N.J., Trant, J.M., 1987. Plasma levels of gonadal steroids during the reproductive cycle of female spotted seatrout Cynoscion nebulosus. In: Idler, D.R., Crim, L.W., Walsh, J.M. ŽEds.., Reproductive Physiology of Fish. Memorial University of Newfoundland, St. Johns, p. 219. Trudeau, V.L., Wade, M.G., Van Der Kraak, G., Peter, R.E., 1993. Effects of 17-estradiol on pituitary and testicular function in male goldfish. Can. J. Zool. 71, 1131᎐1135. Tyler, C.R., van Aerle, R., Hutchinson, T.H., Maddix, S., Trip, H., 1999. An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin. Environ. Toxicol. Chem. 18, 337᎐347. U.S. EPA, 1982. User’s guide for conducting life-cycle chronic toxicity tests with fathead minnows Ž Pimephales promelas.. EPA-600r8-81-011. Duluth, MN. U.S. EPA, 1987. Guidelines for the culture of fathead minnows Pimephales promelas for use in toxicity tests. EPAr600r3-87r001. Duluth, MN. U.S. EPA, 1989. Pesticide assessment guidelines. Subdivision E, Hazard evaluation: Wildlife and aquatic organisms. EPA-540r09-82r024. Washington, DC. U.S. EPA, 1991. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms 4th ed. In: Weber, C.I., ŽEd.., EPA-600r4-90r027. Office of Research and Development, Environmental Monitoring Systems Laboratory, Cincinnati, OH. U.S. EPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms, 3rd edition In: Lewis, P.A., Klemm, D.J., Lazorchak, J.M., Norberg-King, T.J., Peltier, W.H., Heber, M.A., ŽEds.., EPAr600r491r002. Office of Research and Development, Environmental Monitoring Systems Laboratory, Cincinnati, OH. U.S. EPA, 1998a. Endocrine Disruptor Screening and Testing Advisory Committee ŽEDSTAC. report. Office of Prevention, Pesticides, and Toxic Substances, Washington, DC. U.S. EPA, 1998b. Guidance for data quality assessment. Practical methods for data analysis. EPAr600rR-96r084. Office of Research and Development, Washington, DC. U.S. EPA, 1999. Screening level reproduction assay with the fathead minnow Ž Pimephales promelas.. Duluth, MN. Wallace, R.A., Selman, K., 1981. Cellular and dynamic
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