Sylvain Milla

The effects of estrogenic and androgenic endocrine disruptors on the immune system of fish: a review

During the last decade, a number of studies have shown that, in addition to their classically described reproductive function, estrogens and androgens also regulate the immune system in teleosts. Today, several molecules are known to interfere with the sex-steroid signaling. These chemicals are often referred to as endocrine disrupting contaminants (EDCs). We review the growing evidence that these compounds interfere with the fish immune system. These studies encompass a broad range of approaches from field studies to those at the molecular level. This integrative overview improves our understanding of the various endocrine-disrupting processes triggered by these chemicals. Furthermore, the research also explains why fish that have been exposed to EDCs are more sensitive to pathogens during gametogenesis. In this review, we first discuss the primary actions of sex-steroid-like endocrine disruptors in fish and the specificity of the fish immune system in comparison to mammals. Then, we review the known interactions between the immune system and EDCs and interpret the primary effects of sex steroids (estrogens and androgens) and their related endocrine disruptors on immune modulation. The recent literature suggests that immune parameters may be used as biomarkers of contamination by EDCs. However, caution should be used in the assessment of such immunotoxicity. In particular, more attention should be paid to the specificity of these biomarkers, the external/internal factors influencing the response, and the transduction pathways induced by these molecules in fish. The use of the well-known mammalian models provides a useful guide for future research in fish.

Corticosteroids: Friends or foes of teleost fish reproduction?

Reproduction in vertebrates is controlled by the Hypothalamus-Pituitary-Gonad axis and the main hormone actions have been extensively described. Still, despite the scattered information in fish, accumulating evidence strongly indicates that corticosteroids play essential roles in reproductive mechanisms. An integrative approach is important for understanding these implications. Animal husbandry and physiological studies at molecular to organismal levels have revealed that these corticosteroids are regulators of fish reproductive processes. But their involvements appear strongly contrasted. Indeed, for both sexes, corticosteroids present either deleterious or positive effects on fish reproduction. In this review, the authors will attempt to gather and clarify the available information about these physiological involvements. The authors will also suggest future ways to prospect corticosteroid roles in fish reproduction.

Hydration of rainbow trout oocyte during meiotic maturation and in vitro regulation by 17,20{beta}-dihydroxy-4-pregnen-3-one and cortisol.

SUMMARY Although oocytes of many teleost fish, especially marine species, are subjected to a hydration process during meiotic maturation, which leads to an important volume increase, no noticeable hydration of the preovulatory oocyte has ever been reported in rainbow trout (Oncorhynchus mykiss). In the present study, oocyte water content and dry mass were monitored using consecutive samples taken in vivo from the same female rainbow trout, from 4–5 days prior to ovulation to up to 7 days post-ovulation. In addition, yolk protein electrophoretic patterns were compared between oocytes sampled prior to germinal vesicle breakdown (GVBD) and unfertilized eggs. Furthermore, the effect of the maturation-inducing steroid (17,20β-dihydroxy-4-pregnen-3-one, 17,20β-P), cortisol and 11-deoxycorticosterone (DOC) on oocyte dry and wet masses, as well as GVBD occurrence was assessed in vitro. Finally, mRNA expression profiles of glucocorticoid and mineralocorticoid receptors as well as 11β-hydroxysteroid dehydrogenase (11β-HSD) were monitored in the periovulatory ovary by real-time PCR. Both in vivo and in vitro data showed, for the first time in rainbow trout, that a significant oocyte hydration occurs during oocyte maturation. In addition, an intra-oocyte dry matter increase was reported in vivo during the periovulatory period. However, yolk protein migration patterns were similar in preGVBD oocytes and unfertilized eggs, suggesting that no or little yolk proteolysis occurs during oocyte maturation. We also showed that oocyte hydration can be induced in vitro by 17,20β-P and cortisol but not by DOC. In contrast, GVBD was only observed after 17,20β-P stimulation. Finally, real-time PCR analysis showed an up-regulation of 11β-HSD and glucocorticoid receptor 2 transcripts in the ovary at the time of oocyte maturation. Together, these results suggest that cortisol could participate in the control of oocyte hydration and possibly in other periovulatory ovarian functions.

Plasma 11-deoxycorticosterone (DOC) and mineralocorticoid receptor testicular expression during rainbow trout Oncorhynchus mykiss spermiation: implication with 17alpha, 20beta-dihydroxyprogesterone on the milt fluidity?

BackgroundIn rainbow trout (Oncorhynchus mykiss), the endocrine control of spermiation is not fully understood. Besides 11ketotestosterone (11KT) and 17alpha, 20beta-dihydroxyprogesterone (MIS), the potential physiological ligand of the mineralocorticoid receptor (MR) 11-deoxycorticosterone (DOC), is a credible candidate in O. mykiss spermiation regulation as spermiation is accompanied with changes in aqueous and ionic flows.MethodsIn this study, we investigated potential roles of DOC during spermiation 1) by describing changes in blood plasma DOC level, MR mRNA abundance during the reproductive cycle and MR localization in the reproductive tract 2) by investigating and comparing the effects of DOC (10 mg/kg) and MIS (5 mg/kg) supplementations on sperm parameters 3) by measuring the in vitro effect of DOC on testis MIS production.ResultsThe plasma concentration of DOC increased rapidly at the end of the reproductive cycle to reach levels that were 10–50 fold higher in mature males than in immature fish. MR mRNA relative abundance was lower in maturing testes when compared to immature testes, but increased rapidly during the spermiation period, immediately after the plasma rise in DOC. At this stage, immunohistochemistry localized MR protein to cells situated at the periphery of the seminiferous tubules and in the efferent ducts. Neither DOC nor MIS had significant effects on the mean sperm volume, although MIS treatment significantly increased the percentage of males producing milt. However, a significant reduction in the spermatocrit was observed when DOC and MIS were administrated together. Finally, we detected an inhibitory effect of DOC on testis MIS production in vitro.ConclusionThese results are in agreement with potential roles of DOC and MR during spermiation and support the hypothesis that DOC and MIS mechanisms of action are linked during this reproductive stage, maybe controlling milt fluidity. They also confirm that in O. mykiss MIS is involved in spermiation induction.

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch, Perca fluviatilis.

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated. Plasma cortisol and glucose levels increased markedly 1h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1-72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses. Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.

Implication of the mineralocorticoid axis in rainbow trout osmoregulation during salinity acclimation

Cortisol and glucocorticoid receptors (GRs) play an important role in fish osmoregulation, whereas the involvement of the mineralocorticoid receptor (MR) and its putative ligand 11-deoxycorticosterone (DOC) is poorly investigated. In this study, we assessed the implication of DOC and MR in rainbow trout (Oncorhynchus mykiss) osmoregulation during hypo- and hypersaline acclimation in parallel with the cortisol-GR system. A RIA for DOC was developed to measure plasma DOC levels, and a MR-specific antibody was developed to localize MR protein in the gill, intestine, and kidney. This is the first study to report DOC plasma levels during salinity change and MR localization in fish osmoregulatory tissue. Corticosteroid receptor mRNA abundance was investigated in osmoregulatory tissue during salinity acclimation, and the effect of cortisol and DOC on ionic transporters gene expression was assayed using an in vitro gill incubation method. Differential tissue-, salinity-, and time-dependent changes in MR mRNA levels during both hyper- and hyposaline acclimations and the ubiquitous localization of MR in osmoregulatory tissue suggest a role for the MR in osmoregulation. Presumably, DOC does not act as ligand for MR in osmoregulation because there were no changes in plasma DOC levels during either freshwater-seawater (FW-SW) or SW-FW acclimation or any effect of DOC on gill ionic transporter mRNA levels in the gill. Taken together, these results suggest a role for MR, but not for DOC, in osmoregulation and confirm the importance of cortisol as a major endocrine regulator of trout osmoregulation.

Evidence that Elevated Water Temperature Affects the Reproductive Physiology of the European Bullhead Cottus Gobio

Climate change is predicted to increase the average water temperature and alter the ecology and physiology of several organisms including fish species. To examine the effects of increased water temperature on freshwater fish reproduction, adult European bullhead Cottus gobio of both genders were maintained under three temperature regimes (T1: 6–10, T2: 10–14 and T3: 14–18°C) and assessed for gonad development (gonadosomatic index—GSI and gonad histology), sex steroids (testosterone—T, 17β-estradiol—E2 and 11-ketotestosterone—11-KT) and vitellogenin (alkali-labile phosphoprotein phosphorus—ALP) dynamics in December, January, February and March. The results indicate that a 8°C rise in water temperature (T3) deeply disrupted the gonadal maturation in both genders. This observation was associated with the absence of GSI peak from January to March, and low levels of plasma sex steroids compared with T1-exposed fish. Nevertheless, exposure to an increasing temperature of 4°C (T2) appeared to accelerate oogenesis with an early peak value in GSI and level of plasma T recorded in January relative to T1-exposed females. In males, the low GSI, reduced level of plasma 11-KT and the absence of GSI increase from January to March support the deleterious effects of increasing water temperature on spermatogenesis. The findings of the present study suggest that exposure to elevated temperatures within the context of climate warming might affect the reproductive success of C. gobio. Specifically, a 4°C rise in water temperature affects gametogenesis by advancing the spawning, and a complete reproductive failure is observed at an elevated temperature of 8°C.

Physiological and proteomic evidences that domestication process differentially modulates the immune status of juvenile Eurasian perch (Perca fluviatilis) under chronic confinement stress.

The current study aimed to evaluate the influence of domestication process on the stress response and subsequent immune modulation in Eurasian perch juveniles (Perca fluviatilis) submitted to chronic confinement. Briefly, F1 and F4 generations were confined into small-size tanks and sampled 7 and 55 days after stocking. Cortisol and glucose levels as well as lysozyme activity and immunoglobulin level were evaluated in the serum. Spleen Somatic Index and spleen ROS production were also measured. A proteomic analysis was performed on serum sampled on day 7. Finally, both generations were genetically characterized using a microsatellite approach. Globally, results revealed that chronic confinement did not elicit a typical stress response but resulted in a prolonged immune stimulation. Proteomic results suggested that domestication process influenced the immune status of perch submitted to chronic confinement as the F1 confined fish displayed lower abundance of C3 complement component, transferrin and Apolipoprotein E. Microsatellite data showed a strong genetic drift as well as reduced genetic diversity, allelic number and heterozygosity along with domestication process. The present work is the first to report that fish under domestication can develop an immune response, assessed by a combined approach, following recurrent challenges imposed by captive environment despite a reduced genetic variation.

Does domestication process affect stress response in juvenile Eurasian perch Perca fluviatilis

The objective was to evaluate the impact of domestication process on the physiological stress response of cultured Eurasian perch confronted to a chronic stress situation. Briefly, F1 and F4 juveniles were submitted to chronic confinement and investigated on days 5, 15 and 30. Capture and 15min-anesthesia were imposed on fish to assess the effect of preceding confinement on acute stress response. On day 30, the fish were finally challenged with Aeromonas hydrophila and sampled after 5 and 10 days for immune parameter measurements. Cortisol and glucose levels were not affected by confinement but increased significantly after acute stressor exposure. Moreover, cortisol rise following capture and anesthesia was higher in F1 confined-fish, suggesting that they have previously been affected by chronic confinement. A higher HSP70 level was also observed on day 30 in F1 confined-juveniles. During bacterial challenge, regardless of confinement level, F4 juveniles displayed higher lysozyme activity and agglutination response than F1 which may indicate a higher immune capacity in domesticated fish. In conclusion, chronic confinement stressor induced few physiological responses but may increase the responsiveness to other aquacultural stressors. Domestication process also seems to improve chronic stress resistance, growth as well as the immune status of the fish.

Physiological and proteomic responses to single and repeated hypoxia in juvenile Eurasian perch under domestication – Clues to physiological acclimation and humoral immune modulations

We evaluated the physiological and humoral immune responses of Eurasian perch submitted to 4-h hypoxia in either single or repeated way. Two generations (F1 and F5) were tested to study the potential changes in these responses with domestication. In both generations, single and repeated hypoxia resulted in hyperglycemia and spleen somatic index reduction. Glucose elevation and lysozyme activity decreased following repeated hypoxia. Complement hemolytic activity was unchanged regardless of hypoxic stress or domestication level. A 2D-DIGE proteomic analysis showed that some C3 components were positively modulated by single hypoxia while C3 up- and down-regulations and over-expression of transferrin were observed following repeated hypoxia. Domestication was associated with a low divergence in stress and immune responses to hypoxia but was accompanied by various changes in the abundance of serum proteins related to innate/specific immunity and acute phase response. Thus, it appeared that the humoral immune system was modulated following single and repeated hypoxia (independently of generational level) or during domestication and that Eurasian perch may display physiological acclimation to frequent hypoxic disturbances.