Journal of Fish Biology (2015) doi:10.1111/jfb.12626, available online at wileyonlinelibrary.com

BRIEF COMMUNICATION Exogenous 17𝜷-oestradiol (E2) modifies thymus growth and regionalization in European sea bass Dicentrarchus labrax F. Seemann*, T. Knigge†, S. Olivier† and T. Monsinjon†‡ *State Key Laboratory of Marine Pollution, Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Hong Kong, Hong Kong and †UMR-I 02 Unité Stress Environnementaux et Biosurveillance des milieux aquatiques (SEBIO), 4116 Université du Havre, 25 Rue Philippe Lebon, 76058 Le Havre Cedex, France (Received 9 June 2014, Accepted 16 December 2014) The effect of 17𝛽-oestradiol (E2) on the growth of the thymus and its regionalization into cortex and medulla was investigated in juvenile European sea bass Dicentrarchus labrax as they find themselves close to sources of oestrogenic pollution whilst residing in their estuarine nursery areas. While the exposure to 2, 20 and 200 ng l−1 in 60 days post-hatch (dph) fish tended to cause a non-monotonous dose–response curve with a significant difference of the cortex size between lowest and highest exposures, the exposure to 20 ng l−1 E2 from 90 dph onwards resulted in a distinct enlargement of the cortex. It is probable that the alteration of the cortex size also affects the T-cell differentiation and proliferation. © 2015 The Fisheries Society of the British Isles

Key words: development; endocrine disruption; immunocompetence; oestrogen; T-lymphocytes.

In all vertebrates, the thymus is the primary immune organ that provides the microenvironment necessary for T-lymphocyte development, differentiation and proliferation. T-cells are indispensable for (1) the co-ordination of other immune cells through the release of cytokines as mediators and (2) for the secretion of cytotoxic factors to eliminate infected or abnormal cells (Rauta et al., 2012). The teleost thymus is situated in the dorso-lateral region of the gill chamber and is separated from the gill cavity by a single layer of epithelial cells (Boehm et al., 2003). As the organ matures, a clear zonation into cortex and medulla is observed in several teleosts (Fig. 1; Bowden et al., 2005). These two compartments, as well as the corticular/medullary border (BCM), provide the micro-environment for T-cell maturation before the mature T-cells are released into the periphery (Romano et al., 2007). Earlier research has described thymus and T-cell development in the European sea bass Dicentrarchus labrax (L. 1758) (Table I). The organ develops from 21 to 27 days post-hatch (dph) and increases in size within the first year post-hatching (Abelli et al., 1996a, b; Breuil et al., 1997). Presumably, double-negative (CD4− / CD8− ) T-cell ‡Author to whom correspondence should be addressed. Tel.: +33 2 32 74 43 24; email: [email protected]

1 © 2015 The Fisheries Society of the British Isles

2

F. S E E M A N N E T A L.

EL

M GC C

Fig. 1. Transversal cut representing a normally developed thymus of Dicentrarchus labrax at 60 days post-hatch (control group), differentiated into cortical (C) and medullary (M) region; GC, gill chamber; EL, epithelial cell layer; haematoxylin–eosin staining; magnification ×50.

precursors develop into double-positive CD3+ /T-cell receptor (TCR)+ /CD4+ /CD8+ pre-T-cells in the corticular region (Boschi et al., 2011). In D. labrax, this step is likely to occur during two stages. At first, TCR+ -cells are detected from 34 dph onwards, indicating the presence of CD3+ /TCR+ /CD4− /CD8− thymocytes, while CD8𝛼-chain+ T-cells, and concomitant CD4 and CD8 transcripts, are mostly detected from 51 dph onwards, and tend to increase up to 92 dph (Picchietti et al., 2008, 2009; Romano et al., 2011). This marks the period of early CD3+ /TCR+ /CD4+ /CD8+ T-cell development. Secondly, enhanced apoptotic activity in the cortex and near the BCM between 61 and 72 dph suggests the onset of the positive and subsequent negative selection process of double-positive pre-T-cells. In this stage, the cells are sorted due to their ability to express either CD4 or CD8 and their affinity to antigens presented by the major histocompatibility complex. Then, they migrate as single-positive naive CD4+ or CD8+ T-cells into the medulla, where smaller numbers of larger T-lymphocytes are generally observed (Boschi et al., 2011). As assumed by Romano et al. (2007), the BCM is responsible for T-cell release into the periphery and appears to fulfil its functions at the latest from 86 dph onwards. The T-cell subtypes are indispensable for an effective vertebrate adaptive immune response (Laing et al., 2006; Boschi et al., 2011). So far, studies concerning thymotoxic pollutants in teleosts have been only conducted by Wester & Canton (1987), Wester et al. (1990) and Grinwis et al. (1998, 2000, 2009), demonstrating changes of morphometric variables of the thymus, such as thymus size and volume. Endogenous oestrogens are known to play a pivotal role in both thymic and T-cell development, as well as in mammalian thymic atrophy (Erlandsson et al., 2001; Zoller & Kersh, 2006). Furthermore, the migration of immature T-cells from the outer to the inner cortex and further into the medulla is highly dependent on the cytokine environment. One of the priority substances with respect to water quality is 17𝛽-oestradiol (E2) (European Commission, 2012). It is the natural ligand of oestrogen receptors (ER) and

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

E2 AFFECTS THYMUS DEVELOPMENT IN DICENTRARCHUS LABRAX

3

Table I. T-lymphocyte and thymus maturation in Dicentrarchus labrax Fish age (dph)

Thymic development

21–27

Appearance

34 38

First TCR+ cells observed Increased organ growth

40–90

Organ regionalization into cortex and medulla Appearance of double-positive cells in the cortex, period of increased CD8 and CD4 transcripts Increased apoptotic activity in the cortex and BCM Development of a distinct BCM Adult size

51–92

61–74 86 365

Reference Abelli et al. (1996b), Breuil et al. (1997) Romano et al. (2011) Abelli et al. (1996a), Breuil et al. (1997) Romano et al. (2011) Picchietti et al. (2009, 2008)

Abelli et al. (1998) Picchietti et al. (2008) Abelli et al. (1996a, b), Breuil et al. (1997)

BCM, corticular–medullary border; dph, days post-hatching; TCR, T-cell receptor.

was therefore used as model oestrogen in this study. Concentrations of E2 in surface waters have been reported to range between 1 and 50 ng l−1 (Desbrow et al., 1998; Zhang et al., 2012). In estuaries, E2 appears to be quickly degraded into oestrone so that it is present only at very low ng l−1 ranges or even below detection limits (Langston et al., 2005; Noppe et al., 2007; Shi et al., 2014). Riverine or direct discharges of sewage and industrial waste into the estuaries, however, produce complex mixtures of substances with oestrogenic activity, which are known to induce vitellogenesis (Allen et al., 1999; Langston et al., 2005). Fish species, such as D. labrax, that spend juvenile stages within the estuaries are likely to experience exposure to biologically significant concentrations of oestrogenic compounds during highly sensitive periods of their life cycle (Matthiessen & Sumpter, 1998). Although vitellogenin induction has been repeatedly detected in offshore fishes (Allen et al., 1999; Scott et al., 2006), concentrations of compounds with oestrogenic activity generally decline from the estuaries towards the open sea (Noppe et al., 2007; Shi et al., 2014). Therefore, the question whether exogenous oestrogens may interfere with the development of the teleost thymus in species that have their nursery grounds in estuaries is of particular interest. In a previous study investigating the effects of oestrogen on the developing immune system of D. labrax, E2-induced changes in cytokine levels in juveniles were revealed (Seemann et al., 2013). As T-cell differentiation is highly dependent on correct thymus structure (Zapata et al., 2006; Vadstein et al., 2013), changes of the endogenous E2 levels through waterborne E2 exposure may induce long-lasting modifications of thymus structure, notably when the exposure occurs during sensitive developmental stages. Possible effects on the thymus during development may even have persistent consequences for the immune function of adults. Hatchery-reared D. labrax (L’écloserie marine de Gravelines, Gravelines, France) were acclimated to experimental conditions as described in the study of Seemann et al. (2013). The animals were randomly distributed into 120 l (60, 90 and

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

4

F. S E E M A N N E T A L.

120 dph) and 30 l (30 dph) tanks and fed once daily ad libitum on formulated feed (by courtesy of L’écloserie marine de Gravelines; 60, 90, 120 dph) or fresh Artemia sp. nauplii (30 dph). The water temperature was maintained at 18⋅0∘ C, range ± 0⋅5∘ C. Subsequently, two independent experiments were conducted that investigated (1) concentration-dependent changes and (2) age-dependent modifications of the thymus. The animals were treated in accordance to the European convention for protection of vertebrate animals used for experimental and other scientific purposes (Council of Europe, 1986). In the first experiment, juvenile D. labrax [60 dph, mean ± s.d. total length (LT ) = 4⋅03 ± 0⋅67 cm; n = 40] were randomly distributed into aquaria equipped with independent filter systems and exposed to 2, 20, 200 ng l−1 of waterborne E2 for 56 days as described by Seemann et al. (2013), using two tank replicates per exposure. In the second experiment, juveniles of 30 dph (1⋅78 ± 0⋅13 cm; n = 150), 90 dph (6⋅37 ± 0⋅65 cm; n = 60) and 120 dph (7⋅00 ± 0⋅71 cm; n = 45) were exposed in two independent tank replicates each to 20 ng l−1 waterborne E2 for 56 days. After 56 days of E2 exposure, fish from the 90 and 120 dph groups were allowed to depurate for another 56 days to investigate whether changes were persistent. Animal exposures were conducted in semi-static conditions following the methods described by Seemann et al. (2013). The E2 concentration was renewed every day after the exchange of 70% of the water mass by adding E2–methanol solution, the volume of which was adjusted to obtain the chosen nominal concentrations (maximum volume = 100 μl). Water oestrogenicity, which was monitored every week over the entire experimental period using the yeast oestrogen screen assay (Routledge & Sumpter, 1996), revealed the following effective E2 concentrations (mean ± s.d.): 0⋅42 ± 0⋅05 ng l−1 E2 for the control, 2⋅34 ± 0⋅5 ng l−1 for the 2 ng l−1 , 14⋅48 ± 0⋅73 ng l−1 and 11⋅30 ± 1⋅19 ng l−1 for the 20 ng l−1 in experiment 1 and 2, respectively. A mean ± s.d. of 126⋅96 ± 6⋅09 ng l−1 was measured for the highest nominal concentration of 200 ng l−1 . For the ease of presentation, however, the nominal value was used. At each sampling, two individuals per tank replicate, i.e. four fish per treatment, were anaesthetized with tricaine methanesulphonate (MS-222, Sigma; www.sigmaaldrich.com), weighed and measured. Due to the tiny organ size, slices of the head ranging from the middle of the gill chamber to the beginning of the peritoneal cavity containing the thymus were fixed in 4% formaldehyde (Sigma). Before histological processing, the tissues were decalcified for 5 days with 22% formic acid solution to eliminate bony matter. Subsequently, the tissues were dehydrated in a tissue processor (Leica; www.leicabiosystems.com) and embedded in paraffin. Sections of 5 μm were cut with a microtome (Leica) and stained with haematoxylin–eosin–safran (COT20; www.medite.de). For the first experiment, the sections were examined with a Leica DMLB 100 microscope and images were taken with a microscope-mounted camera (Leica DC 300 V2.0). The thymus surface areas and regions were measured with the programme Leica QWin 2.5 (Leica). In the second experiment, the sections of the thymus were observed with a Nikon TE 2000 microscope (Nikon; www.nikoninstruments.com) and images were taken with a microscope-mounted camera (Nikon D3S, www.nikon.com). These images were further processed with the open-source programme ImageJ (imagej.nih.gov/ij/index.html) to obtain measurements of organ areas and regions.

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

E2 AFFECTS THYMUS DEVELOPMENT IN DICENTRARCHUS LABRAX

5

2·5 c

Volume (mm3)

2·0 b c

1·5 1·0

a b

b

a b

0·5 a 0 –0·5 0

100

200

300

Days post hatch Fig. 2. Volumes of cortex ( ), medulla ( ) and whole thymus ( ) in Dicentrarchus labrax at different ages. Values are means ± s.d. from two experimental and four observational units; different lower-case letters indicate statistically significant differences at an 𝛼-level of 5% (P ≤ 0⋅05).

In both cases, surface areas of the thymus were measured in sequential sections of 100 μm from the beginning to the end of the organ (c. 20 sections per individual). In reference to Abelli et al. (1996a), Grinwis et al. (2009) and Seemann et al. (2013, unpubl. data), it is sufficient to measure only one of the paired thymus glands, due to the consistency in size. The volume of the organ ∑ and its regions were obtained by indirect measurement using the Cavalieri method: (a1 , a2 , a3 , … , an ) 100 [μm3 ], with a1 , a2 , a3 , … , an = surface area of organ or region (Casteleyn et al., 2007). All data were tested for homogeneity and normal distribution (Bartlett’s test, Kolmogorov–Smirnoff). To account for the nested experimental design with two sub-groups (tank replicates, i.e. experimental units) and four observational units (i.e. individual fish) within each treatment group, a two-level hierarchical analysis of variance (ANOVA) was applied followed by a Fisher’s LSD post hoc test (STATISTICA 9.0; www.statsoft.com). The data were considered significantly different at P ≤ 0⋅05. The observation of the controls provides fundamental information on the normal development of the thymus and its regionalization in D. labrax. The organ increased in mean ± s.d. volume from 0⋅024 ± 0⋅018 mm3 (93 dph) to 1⋅347 ± 0⋅730 mm3 at 183 dph, thereafter it decreased to 0⋅502 ± 0⋅562 mm3 in 239 dph fish (Fig. 2). Furthermore, the ratio of cortex and medulla changed from 3⋅15 (93 dph) to 1⋅31 (239 dph) over this period. The sensitivity of the thymus and its compartments to different exogenous E2 concentrations was examined in the first experiment (Fig. 3). No statistically significant differences between the exposed fish (irrespective of the E2 concentration) and the control group could be obtained for the entire thymus volume or the cortex and medulla volumes. There appeared, however, to be a non-significant trend to lower thymus volumes in the groups exposed to 20 and 200 ng l−1 E2. Interestingly, and probably as a result of these trends, the volume of the cortex was significantly decreased at the highest oestrogen concentration (200 ng l−1 ) compared with the individuals exposed to the lowest oestrogen concentration (2 ng l−1 ) (P < 0⋅05). © 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

6

F. S E E M A N N E T A L.

0·8

0·8

Cortex volume (mm3)

(b) 1·0

Thymus volume (mm3)

(a) 1·0

0·6

0·4

0·2

a

0·6

b 0·4

0·2

0

0

–0·2

–0·2 0

(c) 1·0

2

20

200

E2 concentration (ng l−1)

Medulla volume (mm3)

0·8

0·6

0·4

0·2

0

–0·2 0

2

20

200

E2 concentration (ng l−1)

Fig. 3. Volumes of (a) thymus, (b) cortex and (c) medulla in Dicentrarchus labrax (60 days post-hatch) exposed to 0, 2, 20 and 200 ng l−1 17𝛽-oestradiol (E2) for 56 days; , means; , s.d.; , ±95% c.i. for two experimental and four observational units; different lower-case letters indicate significant differences at an 𝛼-level of 5% (P ≤ 0⋅05).

The susceptibility to the medium concentration of 20 ng l−1 E2 in the experiment one at different fish stages was investigated in the second experiment. At this concentration, no differences were observed in thymus volume of D. labrax exposed from 30 dph onwards (Fig. 4). Likewise, the thymus growth was not changed in 120 dph fish after 56 days of E2 exposure. In contrast, animals exposed from 90 dph onwards showed a statistically significant (P < 0⋅05) two-fold increase of the cortex volume compared with the control group. This effect was no longer observed after the depuration period. On the contrary, as a trend, the exposure to 20 ng l−1 E2 appeared to intensify the normal age-related decrease of cortex volume in the exposed fish after the depuration. In rodent models, exposure to oestrogens modulates thymic development and causes massive atrophy and reduced cellularity of the thymus (Gould et al., 2000). Oestrogen treatment reduced thymocyte proliferation (Novotny et al., 1983; Gould et al., 2000; Hareramadas & Rai, 2006), modulated the ratios of T-cell subpopulations

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

7

E2 AFFECTS THYMUS DEVELOPMENT IN DICENTRARCHUS LABRAX

3

0·030 0·025 0·020 0·015 0·010 0·005

1·5 1·0 0·5 0·0 –0·5 –1·0 0 20 E2 concentration (ng l−1)

0·006 0·004 0·002

1·2 3

Medulla volume (mm )

b

a

1·0 0·8 Direct exposure

0·6 0·4 0·2 0·0

1·2

0·7

1·0

0·6

3

0·8 0·6 0·4 0·2 0·0

0·5 0·4 Depuration 0·3 0·2 0·1 0·0

1·4

1·4

1·2

1·2

3

Medulla volume (mm )

–0·4

1·0 0·8 0·6 0·4 0·2

1·0 0·8 Direct exposure

0·6 0·4 0·2 0·0

0·0

–0·2

1·2 1·0 0·8 0·6 0·4 0·2 0·0 –0·2 –0·4 –0·6

1·0 0·8

3

3

Cortex volume (mm )

2·0

0·008

0·000

–0·2

2·5 3 Thymus volume (mm )

1·8 1·6 1·4 1·2 1·0 0·8 0·6 0·4 0·2 0·0

0·010

Medulla volume (mm )

3

Cortex volume (mm )

0·000

2·8 2·6 2·4 2·2 2·0 1·8 1·6 1·4 1·2 1·0 0·8 0·6 0·4 0·2 0·0

–1·5

Medulla volume (mm3)

0·012

Medulla volume (mm )

Thymus volume (mm3)

(c)

0·014

0·035

3

1·8 1·6 1·4 1·2 1·0 0·8 0·6 0·4 0·2 0·0 –0·2

0·040

Cortex volume (mm )

Thymus volume (mm3) Thymus volume (mm3)

(b)

2·8 2·6 2·4 2·2 2·0 1·8 1·6 1·4 1·2 1·0 0·8 0·6 0·4 0·2 0·0

Cortex volume (mm )

0·050 0·045 0·040 0·035 0·030 0·025 0·020 0·015 0·010 0·005 0·000

Cortex volume (mm3)

3 Thymus volume (mm )

(a)

0 20 E2 concentration (ng l−1)

0·6 0·4 Depuration

0·2 0·0 –0·2 –0·4 –0·6

0 20 E2 concentration (ng l−1)

Fig. 4. Volumes of thymus, cortex and medulla for (a) 30, (b) 90 and (c) 120 days post-hatch (dph) Dicentrarchus labrax exposed for 56 days to 0 and 20 ng l−1 17𝛽-oestradiol (E2) and (b, c) after the depuration period; , means; , s.d.; , ±95% c.i. for two experimental and four observational units; different lower-case letters indicate significant differences at an 𝛼-level of 5% (P ≤ 0⋅05).

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

8

F. S E E M A N N E T A L.

(Rijhsinghani et al., 1996; Okasha et al., 2001; Zoller & Kersh, 2006; Chapman et al., 2009) and inhibited T lymphopoiesis, which eventually resulted in suppressed humoral and cell-mediated immunity (Cooke et al., 2006). It is therefore conceivable that the observed changes in thymic volumes entail negative effects on the immune response in fishes and may subsequently impair the animals’ health. Although the cellular and molecular mechanisms through which oestrogens mediate these effects remain to be elucidated completely, the importance of ERs in thymus and thymocyte development has been demonstrated (Erlandsson et al., 2001; Islander et al., 2003; Yonezawa et al., 2008). It has been shown that the endogenous E2–ER signalling pathways are fundamental to regulate normal thymus development in male and female mice Mus musculus (Staples et al., 1999; Yellayi et al., 2000). Despite the fact that the presence of ERs in the teleost thymus is still not fully established (Nakayama et al., 2009), the observed effects of oestrogens on thymus growth and, in particular, regionalization in fishes suggest an implication of these receptors in thymus development (Grinwis et al., 2009; Casanova-Nakayama et al., 2011; Shelley et al., 2013). Intriguingly, in this study, the thymus volume transiently increased at 183 dph, being reduced again during later development. These observations underscore the highly dynamic nature of the thymus (Dooley & Liston, 2012). Similar transitions in the thymus volume during development have also been reported in rainbow trout Oncorhynchus mykiss (Walbaum, 1792) (Grace & Manning, 2010). The reduction in organ volume towards 239 dph is possibly explained by the release of mature T-cells that leave the thymus at the end of the first maturation wave. This hypothesis is emphasized by a decreasing cell density in the cortex (Abelli et al., 1998) and a reduction of apoptotic cells (Romano et al., 2013) in 225 dph D. labrax, as well as a shift towards larger medulla volume in the cortex:medulla ratio. The concentration-dependent effects of E2 on thymus volume revealed a nonmonotonous dose–response curve, indicating the divergent effects of lower and higher concentrations of E2 (Straub, 2007; Vandenberg et al., 2012). Likewise, when exposing juvenile D. labrax (60 dph) to concentrations of E2 ranging from 2 to 200 ng l−1 for 1 to 5 to 8 weeks, the expression of selected cytokines in the head kidney displayed non-monotonous response curves over time and across concentrations, resulting in transient upregulation of cytokines (Seemann et al., 2013). In contrast to the head kidney cytokine expression, where most changes were observed at 20 ng l−1 E2 after 35 days of exposure, 20 ng l−1 appears to represent a concentration at which 60 dph D. labrax continue to maintain normal thymus development. Inasmuch as the cytokine microenvironment is highly organ specific and depends on the cell types present, an extrapolation of the changed gene expression in the head kidney on the thymus remains difficult. A distinct effect of the E2 concentrations was only observed on the cortex volume, suggesting that this compartment is the most sensitive at 60 dph (Leceta et al., 1988). It appears that 2 ng l−1 enhances the cortex volume, which may be explained by a higher quantity of double-negative T-cells at relatively low E2 levels (Okasha et al., 2001), due to either possible perturbations of the positive selection, blocking the migration of single-positive T-cells into the medulla, or an increased immigration of T progenitor cells into the cortex. In contrast, higher concentrations of E2, e.g. 200 ng l−1 , are likely to enhance the apoptosis of T-lymphocytes or result in a depletion of early thymic progenitors (Okasha et al., 2001; Zoller & Kersh, 2006). In combination with a reduction

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

E2 AFFECTS THYMUS DEVELOPMENT IN DICENTRARCHUS LABRAX

9

of the stromal microenvironment, these effects caused a decrease of the cortex volume. Both E2 exposure scenarios may have a possible effect on the amount of T-cells released and may, therefore, weaken the immune system caused by either auto-immune reacting T-cells, as a result of a higher cell through-put, or the release of insufficient numbers of T-cells into the periphery. A similar drastic enlargement of the cortex in response to E2 exposure, as observed in the 90 dph animals, has so far been only described by Leceta et al. (1988) and Forsberg (1996) in rodents. The 1⋅5-fold increase reported for pre-pubertally exposed mice (Forsberg, 1996, 2000) may be comparable with the observed increase in D. labrax, if the different exposure durations are taken into account. Interestingly, Forsberg (2000) observed no differences in thymus markers or changes in the double-positive and the single-positive T-lymphocytes population between exposed and non-exposed mice. An increased immigration of double-negative T-lymphocyte progenitors and a disturbed maturation within the cortex, however, may impede the emigration of mature T-cells from the thymus (Forsberg, 1996), which could explain the temporary increase of the cortex volume. The results from Forsberg (1996) also suggest that the cortex, during the final phase of regionalization or shortly thereafter, is especially sensitive to exogenous oestrogens. The exposure for fishes (this study) and for mice (Forsberg, 1996) were both conducted during the pre-pubertal period of maturation to immunocompetence, identified as potentially critical window for immunotoxicity in humans Homo sapiens by DeWitt et al. (2012). In contrast to the present results and the studies of Forsberg (1996, 2000), E2 is generally believed to result in thymus atrophy after oestrogen exposure (Erlandsson et al., 2001; Okasha et al., 2001; Islander et al., 2003), which is thought to be due to an increased apoptosis of T-cells, even if this increase in apoptotic activity has not been yet proven in vivo (Zoller & Kersh, 2006). Furthermore, E2 is supposed to inhibit thymopoiesis and therefore may create a lack of double-positive thymocytes which, consequently, would result in a smaller thymus volume (Zoller & Kersh, 2006). Oestrogen-induced changes in the interplay of ER 𝛼, ER 𝛽1 and ER 𝛽2 may be responsible for the controversial observation of an initial thymic atrophy in 60 dph animals and the thymic enlargement in the subsequent developmental stage (90 dph) in response to 20 ng l−1 E2 exposure (Leceta et al., 1988; Ascenzi et al., 2006). The depuration period, which followed oestrogen-exposure in the experiment, underscores the reversible nature of these alterations in cortex volume reported as well in mice by Forsberg (1996). This suggests that the E2-induced changes in juvenile D. labrax may in fact be reversible, particularly as the thymus volume during these developmental periods appears to be extremely dynamic. Nevertheless, since the thymus provides the microenvironment for the correct differentiation of T-cells, an effect on the T-cell development due to E2-induced thymus alteration should exist, which may have life-long consequences as stated by Chapman et al. (2009). Hence, there is potential for health problems and reduced immunity in adult fishes, if oestrogen exposures result in persistent modifications of the thymic environment and the maturation of thymocytes. The present results are the first to show thymic involution in a teleost after daily exposure to low concentrations of E2 for 2 months. Similar to the changes in head kidney cytokine expression, which in D. labrax suggests particularly sensitive periods to oestrogen exposure during the development of the immune system (Seemann et al., 2013), the thymus development in D. labrax appears to be temporarily sensitive to oestrogen exposure. This comprises notably the period between 90 and 153 dph, when

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

10

F. S E E M A N N E T A L.

exposure to oestrogenic substances appears to affect the normal development. This critical window is likely to match the first wave of maturation of thymocytes in D. labrax (Picchietti et al., 2009). The extent to which the E2-induced disruption of the thymus development is compromising the juveniles’ immune defence and possibly affecting the functionality of the adult humoral immunity remains subject to further investigations. It is hoped that the results of this study inspire future research to show the effect of E2 on the different developmental stages of thymocytes and T-cell sub-populations. The authors thank the staff of the Laboratoire d’anatomie et pathologie de l’hôpital Jacques Monod, Montivilliers, France, for their kind support and J. Humble and D. Peterson for proofreading the manuscript. The research was funded by the European Union (Interreg IV, DIESE #4040).

References Abelli, L., Picchietti, S., Romano, N., Mastrolia, L. & Scapigliati, G. (1996a). Immunocytochemical detection of thymocyte antigenic determinants in developing lymphoid organs of sea bass Dicentrarchus labrax (L). Fish and Shellfish Immunology 6, 493–505. Abelli, L., Picchietti, S., Romano, N., Mastrolia, L. & Scapigliati, G. (1996b). Ontogeny of thymocytes in sea bass Dicentrarchus labrax: studies with monoclonal antibodies. Italian Journal of Zoology 63, 329–331. Abelli, L., Baldassini, M. R., Meschini, R. N. & Mastrolia, L. (1998). Apoptosis of thymocytes in the developing sea bass Dicentrarchus labrax. Fish and Shellfish Immunology 8, 12–24. Allen, Y., Matthiessen, P., Scott, A., Haworth, S., Feist, S. & Thain, J. (1999). The extent of oestrogenic contamination in the UK estuarine and marine environments – further surveys of flounder. Science of the Total Environment 233, 5–20. Ascenzi, P., Bocedi, A. & Marino, M. (2006). Structure-function relationship of estrogen receptor alpha and beta: impact on human health. Molecular Aspects of Medicine 27, 299–402. Boehm, T., Bleul, C. C. & Schorpp, M. (2003). Genetic dissection of thymus development in mouse and zebrafish. Immunological Reviews 195, 15–27. Boschi, I., Randelli, E., Buonocore, F., Casani, D., Bernini, C., Fausto, A. M. & Scapigliati, G. (2011). Transcription of T cell-related genes in teleost fish, and the European sea bass (Dicentrarchus labrax) as a model. Fish and Shellfish Immunology 31, 655–662. Bowden, T. J., Cook, P. & Rombout, J. H. (2005). Development and function of the thymus in teleosts. Fish and Shellfish Immunology 19, 413–427. Breuil, G., Vassiloglou, B., Pepin, J. F. & Romestand, B. (1997). Ontogeny of IgM-bearing cells and changes in the immunoglobulin M-like protein level (IgM) during larval stages in sea bass (Dicentrarchus labrax). Fish and Shellfish Immunology 7, 29–43. Casanova-Nakayama, A., Wenger, M., Burki, R., Eppler, E., Krasnov, A. & Segner, H. (2011). Endocrine disrupting compounds: can they target the immune system of fish? Marine Pollution Bulletin 63, 412–416. Casteleyn, C., Van den Broeck, W. & Simoens, P. (2007). Histological characteristics and stereological volume assessment of the ovine tonsils. Veterinary Immunology and Immunopathology 120, 124–135. Chapman, J. C., Min, S. H., Freeh, S. M. & Michael, S. D. (2009). The estrogen-injected female mouse: new insight into the etiology of PCOS. Reproductive Biology and Endocrinology 7, 47. Cooke, P. S., Selvaraj, V. & Yellayi, S. (2006). Genistein, estrogen receptors, and the acquired immune response. Journal of Nutrition 136, 704–708. Desbrow, C., Routledge, E. J., Brighty, G. C., Sumpter, J. P. & Waldock, M. (1998). Identification of estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening. Environmental Science and Technology 32, 1549–1558. DeWitt, J. C., Peden-Adams, M. M., Keil, D. E. & Dietert, R. R. (2012). Current status of developmental immunotoxicity: early-life patterns and testing. Toxicologic Pathology 40, 230–236.

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

E2 AFFECTS THYMUS DEVELOPMENT IN DICENTRARCHUS LABRAX

11

Dooley, J. & Liston, A. (2012). Molecular control over thymic involution: from cytokines and microRNA to aging and adipose tissue. European Journal of Immunology 42, 1073–1079. Erlandsson, M. C., Ohlsson, C., Gustafsson, J. A. & Carlsten, H. (2001). Role of oestrogen receptors alpha and beta in immune organ development and in oestrogen-mediated effects on thymus. Immunology 103, 17–25. Forsberg, J. G. (1996). The different responses of the female mouse thymus to estrogen after treatment of neonatal, prepubertal, and adult animals. Acta Anatomica 157, 275–290. Forsberg, J. G. (2000). Neonatal estrogen treatment and its consequences for thymus development, serum level of autoantibodies to cardiolipin, and the delayed-type hypersensitivity response. Journal of Toxicology and Environmental Health Perspectives A 60, 185–213. Gould, K. A., Shull, J. D. & Gorski, J. (2000). DES action in the thymus: inhibition of cell proliferation and genetic variation. Molecular and Cellular Endocrinology 170, 31–39. Grace, M. F. T.n. & Manning, M. J. (2010). Growth of the lymphoid organs in Rainbow trout, Salmo gairdneri from one to fifteen months of age. Journal of Zoology 199, 503–520. Grinwis, G. C. M., Boonstra, A., van den Brandhof, E. J., Dormans, J. A. M. A., Engelsma, M., Kuiper, R. V., van Loveren, H., Wester, P. W., Vaal, M. A., Vethaak, A. D. & Vos, J. G. (1998). Short-term toxicity of bis(tri-n-butyltin)oxide in flounder (Platichthys flesus): pathology and immune function. Aquatic Toxicology 42, 15–36. Grinwis, G. C., Vethaak, A. D., Wester, P. W. & Vos, J. G. (2000). Toxicology of environmental chemicals in the flounder (Platichthys flesus) with emphasis on the immune system: field, semi-field (mesocosm) and laboratory studies. Toxicology Letters 112–113, 289–301. Grinwis, G. C., Wester, P. W. & Vethaak, A. D. (2009). Histopathological effects of chronic aqueous exposure to bis(tri-n-butyltin)oxide (TBTO) to environmentally relevant concentrations reveal thymus atrophy in European flounder (Platichthys flesus). Environmental Pollution 157, 2587–2593. Hareramadas, B. & Rai, U. (2006). Cellular mechanism of estrogen-induced thymic involution in wall lizard: caspase-dependent action. Journal of Experimental Zoology A 305, 396–409. Islander, U., Erlandsson, M. C., Hasseus, B., Jonsson, C. A., Ohlsson, C., Gustafsson, J. A., Dahlgren, U. & Carlsten, H. (2003). Influence of oestrogen receptor alpha and beta on the immune system in aged female mice. Immunology 110, 149–157. Laing, K. J., Zou, J. J., Purcell, M. K., Phillips, R., Secombes, C. J. & Hansen, J. D. (2006). Evolution of the CD4 family: teleost fish possess two divergent forms of CD4 in addition to lymphocyte activation gene-3. Journal of Immunology 177, 3939–3951. Langston, W., Burt, G., Chesman, B. & Vane, C. (2005). Partitioning, bioavailability and effects of oestrogens and xeno-oestrogens in the aquatic environment. Journal of the Marine Biological Association of the United Kingdom 85, 1–31. Leceta, J., Fernandez-Galaz, C., Sanchez, C., Navarro, R., Gomariz, R., Zapata, A. & Tejero, A. (1988). Effects of neonatal treatment with estrogens on the development of the thymus in rats. Developmental and Comparative Immunology 12, 375–383. Matthiessen, P. & Sumpter, J. P. (1998). Effects of estrogenic substances in the aquatic environment. In Fish Ecotoxicology (Braunbeck, T., Hinton, D. E. & Streit, B. eds), pp. 319–335. Basel: Springer-Verlag. Nakayama, A., Segner, H. & Kawai, S. i. (2009). Immunotoxic effects of organotin compounds in teleost fish. In Ecotoxicology of Antifouling Biocides (Arai, T., Harino, M. & Langston, W. J. eds), pp. 207–218. Tokyo: Springer-Verlag. Noppe, H., Verslycke, T., De Wulf, E., Verheyden, K., Monteyne, E., Van Caeter, P. & Janssen, C. R. (2007). Occurrence of estrogens in the Scheldt Estuary: a 2-year survey. Ecotoxicology and Environmental Safety 66, 1–8. Novotny, E., Raveche, E., Sharrow, S., Ottinger, M. & Steinberg, A. (1983). Analysis of thymocyte subpopulations following treatment with sex hormones. Clinical Immunology and Immunopathology 28, 205–217. Okasha, S. A., Ryu, S., Do, Y., McKallip, R. J., Nagarkatti, M. & Nagarkatti, P. S. (2001). Evidence for estradiol-induced apoptosis and dysregulated T cell maturation in the thymus. Toxicology 163, 49–62. Picchietti, S., Guerra, L., Selleri, L., Buonocore, F., Abelli, L., Scapigliati, G., Mazzini, M. & Fausto, A. M. (2008). Compartmentalisation of T cells expressing CD8 alpha and TCR

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

12

F. S E E M A N N E T A L.

beta in developing thymus of sea bass Dicentrarchus labrax (L.). Developmental and Comparative Immunology 32, 92–99. Picchietti, S., Guerra, L., Buonocore, F., Randelli, E., Fausto, A. M. & Abelli, L. (2009). Lymphocyte differentiation in sea bass thymus: CD4 and CD8-alpha gene expression studies. Fish and Shellfish Immunology 27, 50–56. Rauta, P. R., Nayak, B. & Das, S. (2012). Immune system and immune responses in fish and their role in comparative immunity study: a model for higher organisms. Immunology Letters 148, 23–33. Rijhsinghani, A. G., Thompson, K., Bhatia, S. K. & Waldschmidt, T. J. (1996). Estrogen blocks early T cell development in the thymus. American Journal of Reproductive Immunology 36, 269–277. Romano, N., Rossi, F., Abelli, L., Caccia, E., Piergentili, R., Mastrolia, L., Randelli, E. & Buonocore, F. (2007). Majority of TcRbeta + T-lymphocytes located in thymus and midgut of the bony fish, Dicentrarchus labrax (L.). Cell and Tissue Research 329, 479–489. Romano, N., Caccia, E., Piergentili, R., Rossi, F., Ficca, A. G., Ceccariglia, S. & Mastrolia, L. (2011). Antigen-dependent T lymphocytes (TcRbeta+) are primarily differentiated in the thymus rather than in other lymphoid tissues in sea bass (Dicentrarchus labrax, L.). Fish and Shellfish Immunology 30, 773–782. Romano, N., Ceccarelli, G., Caprera, C., Caccia, E., Baldassini, M. R. & Marino, G. (2013). Apoptosis in thymus of teleost fish. Fish and Shellfish Immunology 35, 589–594. Routledge, E. J. & Sumpter, J. P. (1996). Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environmental Toxicology and Chemistry 15, 241–248. Scott, A. P., Katsiadaki, I., Witthames, P. R., Hylland, K., Davies, I. M., McIntosh, A. D. & Thain, J. (2006). Vitellogenin in the blood plasma of male cod (Gadus morhua): a sign of oestrogenic endocrine disruption in the open sea? Marine Environmental Research 61, 149–170. Seemann, F., Knigge, T., Rocher, B., Minier, C. & Monsinjon, T. (2013). 17 beta-Estradiol induces changes in cytokine levels in head kidney and blood of juvenile sea bass (Dicentrarchus labrax, L., 1758). Marine Environmental Research 87–88, 44–51. Shelley, L. K., Osachoff, H. L., van Aggelen, G. C., Ross, P. S. & Kennedy, C. J. (2013). Alteration of immune function endpoints and differential expression of estrogen receptor isoforms in leukocytes from 17 beta-estradiol exposed rainbow trout (Oncorhynchus mykiss). General and Comparative Endocrinology 180, 24–32. Shi, J., Liu, X., Chen, Q. & Zhang, H. (2014). Spatial and seasonal distributions of estrogens and bisphenol A in the Yangtze River Estuary and the adjacent East China Sea. Chemosphere 111, 336–343. Staples, J. E., Gasiewicz, T. A., Fiore, N. C., Lubahn, D. B., Korach, K. S. & Silverstone, A. E. (1999). Estrogen receptor alpha is necessary in thymic development and estradiol-induced thymic alterations. Journal of Immunology 163, 4168–4174. Straub, R. H. (2007). The complex role of estrogens in inflammation. Endocrine Reviews 28, 521–574. Vadstein, O., Bergh, Ø., Gatesoupe, F.-J., Galindo-Villegas, J., Mulero, V., Picchietti, S., Scapigliati, G., Makridis, P., Olsen, Y., Dierckens, K., Defoirdt, T., Boon, N., De Schryver, P. & Bossier, P. (2013). Microbiology and immunology of fish larvae. Reviews in Aquaculture 5, S1–S25. Vandenberg, L. N., Colborn, T., Hayes, T. B., Heindel, J. J., Jacobs, D. R. Jr., Lee, D. H., Shioda, T., Soto, A. M., vom Saal, F. S., Welshons, W. V., Zoeller, R. T. & Myers, J. P. (2012). Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses. Endocrine Reviews 33, 378–455. Wester, P. W. & Canton, J. H. (1987). Histopathological study of Poecilia reticulata (guppy) after long-term exposure to bis(tri-n-butyltin)oxide (TBTO) and di-n-butyltindichloride (DBTC). Aquatic Toxicology 10, 143–165. Wester, P. W., Canton, J. H., Van Iersel, A. A. J., Krajnc, E. I. & Vaessen, H. A. M. G. (1990). The toxicity of bis(tri-n-butyltin)oxide (TBTO) and di-n-butyltindichloride (DBTC) in the small fish species Oryzias latipes (medaka) and Poecilia reticulata (guppy). Aquatic Toxicology 16, 53–72.

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626

E2 AFFECTS THYMUS DEVELOPMENT IN DICENTRARCHUS LABRAX

13

Yellayi, S., Teuscher, C., Woods, J. A., Welsh, T. H. Jr., Tung, K. S., Nakai, M., Rosenfeld, C. S., Lubahn, D. B. & Cooke, P. S. (2000). Normal development of thymus in male and female mice requires estrogen/estrogen receptor-alpha signaling pathway. Endocrine 12, 207–213. Yonezawa, Y., Shin, Y. H., Abe, A. & Kondo, Y. (2008). Estrogen receptor alpha expression in the thymus of chick embryos during late stages of embryogenesis. Animal Science Journal 79, 270–273. Zapata, A., Diez, B., Cejalvo, T., Gutierrez-de Frias, C. & Cortes, A. (2006). Ontogeny of the immune system of fish. Fish and Shellfish Immunology 20, 126–136. Zhang, X., Zhang, D., Zhang, H., Luo, Z. & Yan, C. (2012). Occurrence, distribution, and seasonal variation of estrogenic compounds and antibiotic residues in Jiulongjiang River, South China. Environmental Science and Pollution Research 19, 1392–1404. Zoller, A. L. & Kersh, G. J. (2006). Estrogen induces thymic atrophy by eliminating early thymic progenitors and inhibiting proliferation of beta-selected thymocytes. Journal of Immunology 176, 7371–7378.

Electronic References Council of Europe (1986). European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. Available at http://www.conventions.coe. int/Treaty/en/Treaties/Html/123.html/ (last accessed 16 December 2014). European Commission (2012). Memo/12/59. Proposal for a Revised Directive of the European Parliament and of the Council on Priority Substances in the Field of Water Quality. Available at http://europa.eu/rapid/press-release_MEMO-1417 2-59_en.html/ (last accessed 16 September 2014).

© 2015 The Fisheries Society of the British Isles, Journal of Fish Biology 2015, doi:10.1111/jfb.12626