Journal of Fish Biology (2003) 62, 895906 doi:10.1046/j.1095-8649.2003.00074.x, available online at http://www.blackwell-synergy.

com

Morphological development of the gonads in zebrafish

G. M A A C K *
AND

H. S E G N E R

UFZ Centre for Environmental Research, Department of Chemical Ecotoxicology, Permoserstr. 15, D-04318 Leipzig, Germany (Received 15 July 2002, Accepted 18 February 2003)
Gonadogenesis of zebrafish Danio rerio was investigated by means of light microscopy to test the suitability of gonad histology as an endpoint in hazard assessment of endocrine-active compounds. At age 2 weeks post-fertilization (pf), primordial germ cells were found in a dorsocaudal position in the body cavity. At 4 weeks pf, the majority of the fish (86%) possessed paired gonads with meiotic germ cells; these gonads represented presumptive ovaries. At week 5 pf, 87% of the fish examined had ovaries with perinucleolar oocytes. Further development of the gonads in female zebrafish up to week 11 pf was characterized by an increase in gonad size as well as in the number and size of perinucleolar oocytes. Starting with week 5, some fish showed alterations of gonad morphology, including a decrease in the number and size of the oocytes, an enhanced basophilia and irregular shape of the oocytes, and finally their degeneration into residual bodies. With the decline in oocyte number, stromal cells became more numerous and they infiltrated the gonadal matrix. In several 7 week-old zebrafish with altered gonadal morphology, enhanced numbers of gonial cells arranged in cyst-like groups appeared. These gonads were interpreted as presumptive testes. In one fish out of 32 individuals examined, spermatocytes were detected, in addition to the gonial cells. During the subsequent weeks, the percentage of fish showing early testes with spermatogonia, spermatocytes and spermatids increased and reached 40% at 11 weeks pf. The sequence of gonadal alterations taking place in some of the individuals from week 5 pf onwards was interpreted to reflect the transition of protogynic ovaries into testes. The developmental pattern described identifies zebrafish to be a juvenile hermaphrodite. The results of this study are of relevance for the use of gonadal histopathology as endpoint in endocrine disruption testing, particularly in order to avoid false # 2003 The Fisheries Society of the British Isles diagnoses of intersex gonads in zebrafish. Key words: gonad morphological development; juvenile hermaphroditism; zebrafish.

INTRODUCTION Zebrafish Danio rerio (Hamilton) is recommended as test species in a number of existing ecotoxicological test protocols, e.g. different Organisation for Economic Co-operation and Development (OECD) and International Organisation for Standardisation (ISO) guidelines. The advantages of zebrafish as bioassay

*Author to whom correspondence should be addressed at present address: Department of Biological Sciences, Hatherly Laboratories, University of Exeter, Prince of Wales Road, Exeter EX4 4PS, U.K. Tel.: 44 (0) 1392 263796; fax: 44 (0) 1392 263700; email: g.maack@exeter.ac.uk Present address: Centre for Fish and Wildlife Health, University of Bern, La nggass-Str. 122, Postbox, CH-3001 Bern, Switzerland.

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organism include its small size, robustness, short life cycle and the fact that under laboratory conditions it can be induced to breed all year round. Development from the fertilized egg to full reproductive maturity takes only 34 months. This relatively short generation time makes zebrafish suitable for partial and full life cycle tests to evaluate the effects of chemicals on ontogenetic differentiation and reproduction of fishes. Since the evaluation of developmental and reproductive effects is particularly relevant in hazard assessment of endocrine-active compounds (EACs) (Arcand-Hoy & Benson, 1998; Harries et al., 2000; La nge et al., 2001), zebrafish is a candidate species for EAC testing (Fenner-Crisp et al., 2000; Huet, 2000; OECD, 2000). An important question in developing test procedures for the assessment of endocrine-mediated actions on fishes is the selection of suitable effect endpoints. Recognized consequences of exposure of fishes to EACs are disturbances of gonad morphology and gonad ontogenetic differentiation. A number of reports have demonstrated that gonad development in fishes is highly sensitive to exposure to substances with oestrogenic activity. The structural alterations induced by exposure to environmental oestrogens can include the development of ovo-testes, the manifestation of gonadal malformations (e.g. duct disruption) and histopathological alterations, or the increased frequency of atretic oocytes (Jobling et al., 1996; Gimeno et al., 1998; Flammarion et al., 2000; Kinnberg et al., 2000; Metcalfe et al., 2000; Nolan et al., 2001). For gonad histopathology to be used as an endpoint in the testing of EACs, it is necessary to know normal gonad morphology as well as ontogenetic gonad differentiation of the test species. Takahashi (1977) reported that zebrafish is a juvenile hermaphrodite, with all individuals having ovary-like gonads during early life and the bisexual differentiation taking place only later during development. The present study extends the observations of Takahashi (1977) and examines in more detail the morphological changes in gonads of juvenile zebrafish during transition from the protogynic ovary to the early testis. The results from this study will be of relevance for the interpretation of histopathological findings on gonads from EAC-treated zebrafish.

MATERIALS AND METHODS

Zebrafish were reared in the aquarium facilities of the UFZ Centre for Environmental Research at Leipzig, Germany. Fish were fed daily ad libitum with TetraMin Hauptfutter (Tetra Werke, Melle, Germany) and with Artemia sp. nauplii. Fish were kept at a rearing temperature of 26 C, range 1 C, and a 12L : 12D photoperiod. The water of the aquaria was renewed every second day. For egg and embryo production, only healthy fish without disease and overt abnormalities were used as parental fish. Spawning of zebrafish took place in the early morning when aquaria lights were switched on. Sixty fertilized eggs were transferred into one 4 l glass aquarium and kept under semi-static conditions. On day 6 post-fertilization (pf) feeding of the hatched larvae was started with the starter diet, TetraMin AZ 100 (Tetra Werke), and with freshwater rotifers (Brachionus calyciflorus). From day 14 pf fish were fed according to the standard feeding regime given above. Four weeks pf, the fish were transferred into 25 l glass aquaria. At week 6 pf, the number of fish per aquarium was adjusted to 40 individuals in each 25 l tank to reduce variability in growth that can occur as a consequence of different fish densities. A further reduction to 30 individuals per 25 l aquarium was made at week 8 pf.
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For the study of gonadogenesis, a minimum of 20 fish were sampled every week, starting with week 2 pf and continuing to week 11 pf. The fish were taken randomly from at least two independent tanks. In addition, 153 adult, reproductively active fish were sampled, to examine the normal sex ratio of adult zebrafish. Altogether the gonads of 406 zebrafish were examined histologically. Young fish sampled for histological examination were anaesthetized in ice water, decapitated and the remaining body was fixed in Bouins fluid. In older fish, the gonads were dissected and fixed in Bouins fluid. The fixed samples were dehydrated and infiltrated with Technovit 7100 (hydroxyethylmethacrylate) according the manufacturers instructions (Kulzer GmbH, Germany). Serial sections of 25 mm thickness were prepared on a Leitz microtome. Slides were stained in toluidinemethylene blue, dried overnight at 60 C and mounted with Entellan. The use of serial sections was found to be important to determine the developmental stage of the gonads.

RESULTS
ZE B R A F I S H A T 2 W E E K S P F

In zebrafish 2 weeks pf (n 12), primordial germ cells (PGCs) were found in the dorsolateral part of the body cavity. The PGCs were arranged in groups of 1020 cells. Morphologically, the round to slightly oval PGCs were distinguished from somatic cells by their relatively large diameter (c. 12 mm) and their histological features. PGCs contained a large nucleus measuring 610 mm in diameter, possessing a distinct nuclear membrane and a network of fine chromatin filaments distributed throughout the karyoplasm. Along the inner side of the nuclear membrane, deeply staining granular material occurred. The cytoplasm was weakly stained and occupied only a small fraction of the cell volume. The groups of PGCs were surrounded by flattened somatic cells.
ZE B R A F I S H A T 4 W E E K S P F

Four weeks after fertilization, the gonads of zebrafish (n 22) were comprised of two lobes that were located on the dorsal peritoneal wall on both sides of the body cavity. Three different germ cell types could be distinguished. Germ cell type 1 (type 1) had morphological features resembling oogonia, possessing a rather homogeneous, slightly basophilic karyoplasm, a large nucleus : cytoplasm ratio and one to multiple nucleoli (Fig. 1). The second morphological form of germ cells (type 2) was larger than the first one, but again the cytoplasm was restricted to a small rim around the nucleus. The large nucleus of germ cell type 2 contained thin chromatin strands, usually distributed throughout the karyoplasm (Fig. 1). The morphological characteristics of type 2 germ cells correspond to early meiotic stage primary oocytes. The third germ cell type, occurring in the gonads of 4 weeks pf zebrafish, were perinucleolar oocytes, characterized by the presence of nucleoli at the periphery of the karyoplasm and by an enhanced basophilia of the cytoplasm (Fig. 1). Somatic cells were located mainly at the gonadal periphery. The relative frequency of the different cell types within the gonad varied between individuals. The gonads of 19 out of 22 individuals of age 4 weeks contained germ cell types 1 and 2 only. These gonads are presumptive ovaries; however, since male- and female-specific gonia of fishes are difficult to
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gc2

gc1 li poc 20 m

FIG. 1. Early ovary of a 4 weeks pf zebrafish. The majority of the cells are meiotic type 2 germ cells (gc2), with thread-like chromosomes distributed throughout the karyoplasm. The more basophilic type 1 germ cells (gc1) are arranged in groups and contain one to multiple prominent nucleoli. Additionally, one single perinucleolar oocyte is seen (poc). li, Liver.

distinguish solely on the basis of cell morphological characteristics (Parmentier & Timmermans, 1985), gonads containing only germ cell types 1 and 2 were categorized to be indifferent gonads. The gonads of three out of the 22 fish examined histologically contained, in addition to germ cell types 1 and 2, perinucleolar oocytes. These gonads were classified to be ovaries.
Z E B R A F I SH A T 5 W E E K S P F

Two out of 31 fish (1015 mm total length, LT, n 31) possessed indifferent gonads, i.e. gonads containing only type 1 and type 2 germ cells (presumptive ovaries). Twenty-eight fish (90%) showed ovaries containing both type 1 and type 2 germ cells and, in addition, perinucleolar oocytes (Fig. 2). Compared to 4 week-old fish, oocytes were increased in size, mainly because of an enlargement of the ooplasm. The basophilia of the ooplasm was enhanced. Around the oocytes, a layer of flattened stromal cells developed, indicating the onset of ovarian follicular organization. Pronounced interindividual variation occurred with respect to the frequency of perinucleolar oocytes: while in some fish almost
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100 80 n = 12 n = 22 n = 31 n = 26

Gonad type (%)

n = 32

60 40 20 0

n = 33

n = 32

n = 30

n = 35

n = 153

10

11

Adult

Age (weeks pf )

FIG. 2. Percentage (%) of the morphological gonad types. , Indifferent gonads; &, ovaries; &, altered ovaries; , testes; n, number of histologically examined individuals in the respective week. The indifferent gonads in zebrafish at 2 weeks pf are characterized by the presence of primordial germ cells, whereas in zebrafish at 4 and 5 weeks pf, the indifferent gonads contain the germ cell types 1 and 2 and represent presumptive ovaries.

the complete ovary was occupied by perinucleolar oocytes (Fig. 3), in other fish perinucleolar oocytes made up <20% of the ovarian volume, and the remaining parts of the gonad were occupied by type 1 and type 2 germ cells. One individual out of the 31 fish examined in the 5 week group showed a gonadal morphology that was different to the other fish. The gonad morphology was characterized by a reduction in oocyte number, diversity of oocyte size and irregular oocyte shape. This gonad was classified as an altered ovary (Fig. 2). Congruent gonadal alterations were observed in a number of 6 weekold fish, and therefore these tissue modifications are described in more detail there.
ZE B R A F I S H A T 6 W E E K S P F

The majority (76%) of the examined (917 mm LT, n 26) fish had rather large gonads containing densely packed and well-differentiated perinucleolar oocytes. Most of the gonadal tissue was occupied by perinucleolar oocytes, and only at the cranial or caudal ends of the ovary were groups of type 1 and type 2 germ cells present. In other individuals, the relative portion of the ovary occupied by perinucleolar oocytes was smaller and, in turn, the number of type 1 and type 2 germ cells was elevated. In five individuals of the 6 weeks pf zebrafish, gonad morphology was clearly different in that both size and number of the oocytes were reduced. The tight contact between the perinucleolar oocytes was loosened and the oocytes became isolated from each other [Fig. 4(a)]. Concomitant to the decline in the number of oocytes, they also showed morphological changes indicative of a degenerative process: their shape tended to become irregular, ooplasm basophilia was enhanced, and the nuclei lost their round form and underwent disintegration [Fig. 4(b),(d)]. Finally, the degenerative oocytes seemed to give rise to strongly basophilic residual bodies. The number of somatic cells was increased in the
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poc

poc

poc

20 m
FIG. 3. Ovary of a 5 weeks pf zebrafish containing densely packed oocytes at the perinucleolar stage (poc).

altered ovaries [Fig. 4(b),(d)], particularly in the ventral part of the gonads. Phagocyte-like cells could infiltrate the gonadal tissue [Fig. 4(a)]. The extent of the degenerative alterations of the ovaries varied between individuals; in some fish, moderate reductions in oocyte number and size were evident [Fig. 4(a)], whereas in other individuals, only few oocytes of irregular form and strong basophilia were left, and the remaining part of the gonads were occupied by stromal cells [Fig. 4(b),(d)]. The morphologically altered ovaries were clearly smaller than the ovaries with densely packed perinucleolar oocytes.
Z E B R A F I SH A T 7 W E E K S P F

The features as described for the gonad morphology of 6 weeks pf zebrafish were also found in 7 weeks pf zebrafish (1322 mm LT, n 32). While 78% of the 32 fish examined possessed well-developed, large ovaries with densely packed perinucleolar oocytes, the remaining fish showed the various types of ovarian alteration as described above. In few individuals, a new development was evident in that small groups of germ cells became prominent within the stroma cell-dominated gonadal matrix [Fig. 5(a)]. The germ cells contained large, prominent nuclei surrounded by a small cytoplasm ring. Two to six of these cells were arranged in cyst-like clusters. In some cases, a slit-like lumen was visible in the centre of the germ cell groups [Fig. 5(a)]. The number of oocytes in those gonads was reduced to single, shrunken oocytes and residual bodies, or they were completely absent [Fig. 5(a)]. The gonads with few or no oocytes, but with groups of undifferentiated germ cells, sometimes arranged
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(a)

(b) pc poc

li

poc

poc

poc

20 m (c) (d) rb 20 m 20 m

50 m

FIG. 4. Alterations of ovarian morphology in 58 weeks pf zebrafish during transition from the protogynic ovary to early testis. (a) Six weeks pf zebrafish. The morphology of the perinucleolar oocytes (poc) is altered, partly they are shrunken, the nuclear shape is getting irregular and cellular basophilia increases ( ). In the gonadal matrix, spaces devoid of oocytes appear which may contain phagocyte-like cells (pc). (b) Eight weeks pf zebrafish. The number of perinucleolar oocytes (poc) has decreased, and they are no longer in contact but isolated. The spaces between the oocytes are filled by stromal cells. Oocyte morphology shows degenerative features such as shrinkage, irregular shape of cell and nucleus, increased basophilia and patchy patterns of the ooplasm. li, Liver. (c) Six weeks pf zebrafish. Most of the few perinucleolar oocytes found in this gonad show degenerative features ( ). Stromal cells occupy the majority of the gonad matrix. The degenerating oocytes may develop into residual body-like structures (rb). (d) Six weeks pf zebrafish. The majority of the cells inside the gonad are stromal cells. The few remaining oocytes show degenerative features.

around a central small lumen, were classified as presumptive testes since the germ cell groups resemble spermatogonial cysts. In one out of 32 fish of the 7 week group, a single, small group of spermatocytes was detected in the gonadal tissue.
ZE B R A F I S H A T 8 W E E K S P F

Also in 8 weeks pf zebrafish (1323 mm LT, n 33), gonad morphology varied between individual fish. A relation between gonadal development and individual size of fish could not be found. The percentage of fish containing well-differentiated ovaries was 55%, while the remaining individuals had transforming gonads or early testes (Fig. 2). In the testes, in addition to the cyst-like arranged gonial cells, spermatocytes and spermatids were found [Fig. 5(b),(c)].
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(b) (a)

* 20 m * (c) lu

* ga 20 m spt

sga

spc spd 20 m

FIG. 5. Zebrafish testes of different developmental stages. (a) Seven weeks pf zebrafish. Presumptive testis. A larger part of the gonad is occupied by stromal cells. In addition, groups of gonial cells (ga) are present and they are arranged in a cyst-like manner ; sometimes a slit-like lumen (*) can be observed in the centre of the germ cell groups. (b) Seven weeks pf zebrafish. Early testis, containing spermatocytes (spt) and few sperm cells in the lumen of the tubuli. (c) Eight weeks pf zebrafish. Early testis containing cysts with spermatogonia (sga), spermatocytes (spc) and spermatids (spd). lu, Lumen of tubulus.

Z E B R A F I SH A T 9 W E E K S P F

In 9 weeks pf zebrafish (1121 mm LT, n 32), the number of individuals with clearly differentiated testes was elevated. The occurrence of ovaries with altered structure was restricted to two out of 32 examined individuals. Ovaries were further developed, in that oocyte size was enlarged due to the increasing cytoplasm volume. All oocytes were still in the primary growth stage and had not yet entered the cortical alveolar or vitellogenic phase.
Z E B R A F I SH A T 1 0 W E E K S P F

Apart from one fish, in which the gonad showed the morphology of an altered ovary, 10 weeks pf zebrafish (1223 mm LT, n 32) possessed either a differentiated ovary or a differentiated, early stage testis. Testis development was characterized by the disappearance of stromal cells from the central parts of the gonadal tissue and a now peripheral localization of the stromal cells. At the same time, male germ cells at different developmental stages, including spermatozoa, became more frequent. In four out of the 42 examined testes,
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a few, single oocytes with degenerative morphology were found. The presence of these degenerated oocytes caused no apparent disruption to the organization of male tissue.
ZE B R A F I S H A T 11 W E E K S P F

In 11 weeks pf zebrafish (1227 mm LT, n 35), the same pattern as described for 10 weeks pf fish was observed. The data on gonadal sex-ratio of the different sampling times are summarized in Fig. 2. An association between size of individual fish and its gonadal morphology was not evident on the basis of the sample size analysed in this study. DISCUSSION Patterns of gonadal development differ among fish species. The majority of fish species are gonochoristic: PGCs differentiate directly into female or male germ cells during ontogenesis. PGCs develop next to the dorsal wall of the coelomic cavity at the site of the future gonadal ridges. During the larval period, the gonad anlage gradually arises from the PGCs and surrounding somatic cells (Yamamoto, 1969; Parmentier & Timmermans, 1985; Timmermans, 1987; Nagai et al., 2001), and grows into morphologically differentiated gonads during puberty. A sex change does not occur in gonochoristic species. In addition to gonochorism, several forms of hermaphroditism are found among fish species, including both simultaneous hermaphroditism and sequential hermaphroditism (Sadovy & Sharpiro, 1987; Helfman et al., 1997). Sequential hermaphroditism, both functional and non-functional, is not unusual in teleosts; examples are eels, hagfishes, lampreys, butterfly fishes, wrasses, parrotfishes, gobies and belontiid paradise fishes (Colombo & Grandi, 1996; Helfman et al., 1997). Protrandrous fishes develop first as males and only later do they change to females, whereas protogynous fishes such as zebrafish develop first as females and later become males. The histological examination of gonad differentiation in developing zebrafish revealed that at: (1) 5 weeks pf all fish had ovary-type gonads, while no fish contained testis-type gonads; (2) 7 weeks pf for the first time testis-like gonads were observed; (3) between 5 and 11 weeks pf, a variable percentage of the fish examined showed distinct alterations of gonadal morphology. These morphological changes included a decline in number up to complete disappearance of oocytes and degenerative alterations such as irregularities of form and shape of nucleus and oocyte, reduced cell size and enhanced basophilia of cyto- and nucleoplasm. At the same time, stromal cells became more prominent and occupied larger fractions of the gonadal tissue. While the oocytes were gradually disappearing from the altered gonads, new gonial cells developed within the stroma cell-dominated gonadal matrix, and these gonial cells eventually differentiated into spermatocytes. The alterations of gonadal morphology in some of the 511 week-old pf zebrafish are interpreted as a transformation of early ovary-type gonads into testes. This interpretation is in line with the earlier work of Takahashi (1977) who classified zebrafish to be a protogynic hermaphrodite, i.e. all individuals, irrespective of their genetic sex, develop initially an ovarian-like gonadal
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morphology, while later during ontogeny the protogynic ovaries of some fish undergo transformation into testes. The fact that the protogynic ovaries of juvenile zebrafish contain only oocytes of the primary growth stage but no cortical alveolar or vitellogenic oocytes indicates that the protogyny of zebrafish is a non-functional protogyny. As recently shown by Uchida et al. (2002), the disappearance of the early meiotic oocytes (germ cell type 2 in this study) from the protogynic ovary appears to occur by apoptosis, while the loss of the perinucleolar oocytes may be due to other cell death pathways. Once the differentiation of early ovaries and testis is completed, zebrafish shows the typical pattern of gonadal maturation as described for synchronously spawning fishes (Selman & Wallace, 1989; Takashima & Hibiya, 1995; Tyler & Sumpter, 1996), where spermatogenesis and oogenesis are continuous processes. The time period for transition from the protogynic stage into the bisexually differentiated stage may vary in relation to zebrafish strain and rearing conditions. In the study by Takahashi (1977), sexual differentiation of zebrafish gonads was more or less completed at c. day 30 pf. Also in the investigation of Uchida et al. (2002), gonad transformation occurred in 2130 days pf fish. Timing of developmental events in fishes can depend on body growth (Osse & van den Boogaart, 1995), which again depends on environmental factors such as stocking density, feeding conditions and water temperature. Therefore, betweenlaboratory differences in the timing of gonad differentiation in zebrafish may be related to inter-laboratory variations of rearing conditions and the related differences of fish growth. In the present study, however, an unequivocal relation between body size of zebrafish and gonadal differentiation status could not be revealed, but the size range of fish showing ovaries, early testes or transition gonads varied over a wide range of body length. Nevertheless, for comparison of results on developing zebrafish from different laboratories, a staging system referring to the physiological instead of the numerical age of the fish would be most valuable (Segner et al., 1994). Sexual differentiation of developing gonads in fishes is considered to be under the control of steroid hormones (Nakamura et al., 1998; Baroiller et al., 1999; Jalabert et al., 2000; Nagahama, 2000; Piferrer, 2001). Yamamoto (1969) suggested that sex steroids are the natural inducers of sex in fishes. Since then, numerous studies including recent molecular work (Baroiller et al., 1999; Nagahama, 2000) have corroborated and extended the original hypothesis of Yamamoto (1969), although contradictory observations do exist (Kawahara & Yamashita, 2000). Cytochrome P450 aromatase, an enzyme aromatizing testosterone into oestrogen, is known to be involved in the regulation of ovarian differentiation of teleosts. In zebrafish, a specific ovarian-type aromatase does exist (Chiang et al., 2001; Kishida & Callard, 2001) which shows dimorphic expression pattern in juvenile fishes (Trant et al., 2001). Modulation of aromatase activity of juvenile zebrafish is able to induce a feminization or masculinization of developing zebrafish (M. Fenske & H. Segner, unpubl. data). This observation points to a role of aromatase and steroid hormones for the transition of developing zebrafish from the protogynic gonadal stage to the bisexual stage. The protogynic development of zebrafish as described in this study can lead to problems in the assessment of endocrine effects on gonad histopathology. For instance, it will not be possible to detect a feminizing effect of an exogenous
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compound on zebrafish gonads, if samples are taken during the protogynic stage. Furthermore, histological examination of zebrafish gonads during the transition period may lead to the erroneous interpretation that a test substance induced intersex gonads.
This study was funded by the European Commission (Contract ENV4-CT97-0509). We thank J. Ha bler and C. Haiduk for assisting in the preparation of the histological sections. We are grateful to C.R. Tyler for critically reading the manuscript.

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2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 62, 895906