In all vertebrate species, reproduction is a hormonally controlled process, important for growth and maturation of gonads and germ cells. Production of functional germ cells is of outmost importance to secure the survival of a species. Fish comprises 50% of the known vertebrates and are found in aquatic habitats all over the world. Even though fish have evolved a wide variety of morphological and physiological characteristics, due to large differences in the living environment, the growth an maturation of germ cells follows the same pattern in all species. In this thesis the focus has been directed on oocyte growth and development in Arctic char (Salvelinus alpinus), and if stress might inflict disturbances on the reproductive systems. All sexually mature female egg laying vertebrates produces yolky eggs surrounded by an eggshell. Production of yolk and egg shell is under estrogenic control and it is known that production of egg components can be induced in male and juvenile fish by estrogenic substances. Many manmade chemicals have been found to interfere with hormonally controlled processes. Therefore production of the egg yolk precursor, vitellogenin (VTG), and the egg shell components, vitelline envelope proteins (VEP), have been used as biomarkers for estrogenic effect. Exposure to endocrine disrupting substances (EDS) does not only give rise to hormonal effects on the organism, but in addition it also gives rise to an increase in stress hormone, cortisol (F), levels. It is evident that a wide variety of substances may affect Arctic char oocyte growth and maturation. VTG and VEP production is found to be under dose dependent estrogenic control, but the production was directly affected by F. Under natural condition it has been found that F increases towards ovulation. Even though both VTG and VTG is under estrogenic control, these studies showed that stress lead to a decrease of VTG while the VEP production increased. These effects was only observed on protein levels indicating that a post transcriptional down regulation of VTG production is mediated by F in Arctic char. In order for an egg to become fertilizatible, it must undergo a maturation phase. This maturation phase is primarily induced by gonadotropins, which in turn induce the production of species specific maturation inducing substances (MIS). To investigate oocyte development in Arctic char a characterization of its MIS receptor was made. The MIS receptor is localized on the oocyte surface and displays a single class of high affinity and low capacity binding sites. The binding moieties displays association and dissociation kinetics typical of steroid membrane receptors. Even though high specificity for Arctic char MIS was observed, it was found that some EDS bind to the Arctic char oocyte membrane receptor. This suggest that certain EDS might affect oocyte maturation and thereby might alter the reproductive success. Furthermore, it was found that F did not bind to the MIS receptor in Arctic char. It is therefore suggested that oocytes are more sensitive to stress during the growth phase than during maturation

<p></p><p>In salmonids, the decision to migrate or remain resident is influenced by the status, and hence condition, of individuals. Status has been suggested to arise from the temperament of fish. In this study the links between standard metabolic rate and the levels of aggressiveness and shy/boldness were examined for 0+, hatchery-raised brown trout (<em>Salmo trutta</em>). I hypothesized, from the results of earlier studies (Cutts <em>et al</em>., 1998; Yamamoto <em>et al</em>., 1998), that high metabolic rates (MR) would be positively correlated to levels of aggression and boldness. The study was conducted in 200 L aquaria in which aggressiveness was measured by allowing each fish to interact with a mirror image of itself, and shy/boldness was tested by measuring the amount of time a fish used before exploring a new area. Standard metabolic rate was measured in a flow-through respirometer. In contrast to my expectations, there was no correlation between the different behavioural measures and the metabolic rate of fish. Also, no correlation between boldness and aggressiveness of fish was found. In additional testing aggressiveness correlated positively with the condition of fish (in coherence with Harwood <em>et al</em>., 2003) but, contrary to earlier studies (Överli <em>et al.,</em> 2004; Schjolden & Winberg, 2007), not with the speed of acclimatization. The difference in results between this test and earlier studies, concerning the degree of correlation between MR and aggressiveness, suggests that the strength of this link differs between species of salmonids. Also, it may suggest changeability in the MR – behaviour link in different environments. Last, the status and condition of individuals cannot be unambiguously explained by temperament alone, but arise from a wider array of physiological and environmental factors.</p><p> </p>

The neuropeptides arginine-vasotocin (AVT) and corticotropin-releasing hormone (CRH) are involved in the hypothalamic-pituitary-interrenal (HPI) axis. During stress, the HPI axis is activated and cortisol is released into the blood. In addition to their role in the HPI axis, AVT and CRH also have behavioural effects. The roles of AVT and CRH in stress responses and agonistic behaviour were studied in this thesis, using two different models. In the first model, two strains of rainbow trout (Onchorhynchus mykiss) divergent in stress-induced release of cortisol were investigated. This was done by observing behaviour and stress responses under different conditions. These strains were found to have divergent stress coping strategies based on the observed behaviour and levels of plasma cortisol. This divergence in behaviour could be associated with the CRH system, since the mRNA levels of CRH differed between the strains during stress. However, no differences between strains were observed in AVT or its receptor expressions. In the second model, non-selected rainbow trout were paired and the effect of intracerebroventricular (icv) injections of an active substance (AVT, CRH or the CRH related peptide Urotensin-I (UI)) on fights for dominance was investigated. One fish of the pair received the active substance icv and the other received saline icv. Fish receiving AVT became subordinate in accordance with the suggestion that AVT attenuates aggression in territorial vertebrates. Fish receiving CRH became subordinate whereas UI showed no effect on fights for dominance. Further, both CRH and UI induced an anxiety-related behaviour similar to non-ambulatory motor activity in rats. In addition, CRH appeared to affect the dopaminergic and serotonergic systems. In this thesis, it is suggested that CRH is involved in the behavioural modulation of the stress coping strategies in teleost fish. Further, AVT and CRH seem to act inhibitory on aggressive behaviour.

The first objective of this thesis was to investigate effects of endocrine disruptors on the developing brain and gonads of bird embryos. The substances studied were the insecticide methoxychlor, and nine UV-filters (3-benzylidene camphor (3BC), 4 methyl benzylidene camphor (4MBC), benzophenone (BP) 1,2 and 3, 4 hydroxy benzophenone (4 HB), 4 dihydroxy benzophenone (4DHB), benzyl salicylate (BS), and ethyl-4-aminobenzoate Et-PABA)), commonly used in cosmetic products. Some of these substances have no estrogenic effect in vitro, but have been shown to be estrogenic in vivo. The PCB-mixture Clophen A50 is a well-known inducer of biotransformation enzymes and was co-administered with methoxychlor and the UV-filters 3BC and 4MBC. Exposure to 3BC or 4MBC caused ovotestis formation and malformations of the Müllerian ducts in Japanese quail embryos. Co-exposure to one of these compounds and Clophen A50 enhanced the effects, indicating that Clophen A50 potentiates the effects of the UV-filters. Embryonic co-exposure to Clophen A50 and methoxychlor caused a disturbed male sexual behaviour. The metabolites of methoxychlor are estrogen receptor (ER)α-selective, which indicates that the effects on behaviour following embryonic treatment were mediated by ERα. Another objective in this thesis was to localize estrogen receptors (ERs) in the brain of adult and embryonic Japanese quail. The ER localization provides a basis for mechanistic studies on effects of endocrine disruptors, by the identification of estrogen-responsive areas in the brain. We found that ERβ, not previously implicated in sex-differentiation of the brain, was the only ER-subtype present in a sexually dimorphic brain area during differentiation. In conclusion, the estrogenic effects of 3BC, 4MBC and methoxychlor were increased by co-exposure to PCB. These results raise concern since many wildlife species, as well as humans, carry large body burdens of persistent organic pollutants like PCBs, which potentially can interact and enhance the effects of other endocrine disruptors.

In salmonids, the decision to migrate or remain resident is influenced by the status, and hence condition, of individuals. Status has been suggested to arise from the temperament of fish. In this study the links between standard metabolic rate and the levels of aggressiveness and shy/boldness were examined for 0+, hatchery-raised brown trout (Salmo trutta). I hypothesized, from the results of earlier studies (Cutts et al., 1998; Yamamoto et al., 1998), that high metabolic rates (MR) would be positively correlated to levels of aggression and boldness. The study was conducted in 200 L aquaria in which aggressiveness was measured by allowing each fish to interact with a mirror image of itself, and shy/boldness was tested by measuring the amount of time a fish used before exploring a new area. Standard metabolic rate was measured in a flow-through respirometer. In contrast to my expectations, there was no correlation between the different behavioural measures and the metabolic rate of fish. Also, no correlation between boldness and aggressiveness of fish was found. In additional testing aggressiveness correlated positively with the condition of fish (in coherence with Harwood et al., 2003) but, contrary to earlier studies (Överli et al., 2004; Schjolden &amp; Winberg, 2007), not with the speed of acclimatization. The difference in results between this test and earlier studies, concerning the degree of correlation between MR and aggressiveness, suggests that the strength of this link differs between species of salmonids. Also, it may suggest changeability in the MR – behaviour link in different environments. Last, the status and condition of individuals cannot be unambiguously explained by temperament alone, but arise from a wider array of physiological and environmental factors.

Two distinct stress coping strategies, proactive and reactive, have been stated in various animal studies, each associated with a set of behavioural and physiological characteristics. In a given challenging situation, proactive animals show more aggression, a higher general activity and a predominant sympathetic reaction. In contrast, the reactive copers respond more with immobility and avoidance, and a predominant parasympathetic/hypothalamic activation. This divergence in coping has also been indicated in salmonid fish. Interestingly, many of the differences reported between sea-ranched and wild fish resembles characteristics that differentiate proactive and reactive copers. In the present thesis it is shown that individuals with divergent stress coping styles are identifiable in several brown trout (Salmo trutta) populations. Further, the results show that the distribution of individuals displaying these distinct stress coping strategies differs between populations. This strongly indicates that these traits are heritable and that the variation in selection regime in the native rivers influences these traits. In addition, the results show that populations with hatchery origin are biased towards having higher frequencies of trout displaying a proactive style than populations having wild origin. Also, even though the frequency of early sexual maturation, known as a viable alternative life history in salmonids, differs between populations of brown trout, no link between stress coping strategy and early sexual maturation were found. However, this thesis show that maternal contribution, in the form of egg size, is of major importance whether the progeny will sexually mature early and that it also might be of importance for stress coping strategy. Further, correlations of traits commonly associated with stress coping strategies and behavioural syndromes across context and over time is investigated. The results show that individuals with a strong sympathetic reactivity are more prone to change their behaviour than others.