Keywords: Aromatase inhibitor; Testosterone; Atlantic salmon; Gonadotropins; Luteinizing Hormone; Follicle-Stimulating Hormone

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1 Preliminary results on the effects of different aromatase inhibitors and different doses of testosterone on gonadotropins in one year old male Atlantic salmon (Salmo salar) Efthimia Antonopoulou a,b and Bertil Borg a a Department of Zoology, University of Stockholm, S Stockholm, Sweden b NAGREF-Fisheries Research Institute, Nea Peramos, Kavala, Greece; efiantonopolou@hotmail.com Aromatization of certain androgens, such as testosterone (T), to estrogens is particularly active in teleosts, having unique potential of influencing the physiological balance between the sex steroid hormones, and therefore involved in the control of reproduction in fishes. Both positive and negative aromatase-dependent and aromatase-independent feedback mechanisms on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) occur in salmonids. The aim of the present study was to investigate the involvement of aromatization in feedback mechanisms at the early stages of sexual development of Atlantic salmon, Salmo salar. One-year-old males were implanted with Silastic capsules filled with three different aromatase inhibitors, 1,4,6- androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4OH), and 4-benzonitrile monohydrochloride (CGS), or T in different doses, and the effects on the plasma and pituitary LH and FSH levels were studied. T treated fish always had high pituitary LH levels. In addition, treatment with ATD increased pituitary LH, though to a lesser extent than T, in immature salmon parr, whereas ATD and CGS treatment suppressed pituitary LH in maturing fish. However, ATD combined with T diminished pituitary LH levels, compared to T or ATD alone in immature fish, indicating an aromatase-dependent positive feedback of T on LH. 4OH, which was less effective as an aromatase inhibitor, increased LH content. ATD treatment resulted in increased pituitary FSH, although FSH levels did not differ significantly from those of mature controls. Positive effects of ATD on plasma FSH were found, indicating the presence of an aromatase-dependent negative feedback. T exerted both positive and negative effects on pituitary FSH and testis growth, depending on dose and season. The combined treatment of T with ATD did not affect the positive effect of T alone on pituitary and plasma FSH, indicating the presence of a nonaromatase dependent positive feedback on FSH. Keywords: Aromatase inhibitor; Testosterone; Atlantic salmon; Gonadotropins; Luteinizing Hormone; Follicle-Stimulating Hormone

2 Προκαταρτικά αποτελέσµατα στις επιδράσεις αναστολέων αρωµατάσης και τεστοστερόνης στις γοναδοτροπίνες σε νεαρά αρσενικά άτοµα σολωµού (Salmo salar) Ευθυµία Αντωνοπούλου a,b και Bertil Borg a a Department of Zoology, University of Stockholm, S Stockholm, Sweden b ΕΘΙΑΓΕ-Ινστιτούτο Αλιευτικής Έρευνας, Νέα Πέραµος, Καβάλα; efiantonopolou@hotmail.com Στα ψάρια, όπως και στα θηλαστικά, οι γονάδες παράγουν και εκκρίνουν κάτω από την επίδραση των γοναδοτροπινών, στεροειδή. Τα στεροειδή αυτά ρυθµίζουν, µε τη σειρά τους, την παραγωγή και έκκριση των δύο γοναδοτρόπων ορµονών της υπόφυσης, της ωοθυλακιοτρόπου (FSH) και της ωχρινοτρόπου (LH), δρώντας είτε άµεσα στην υπόφυση ή έµµεσα µέσω του υποθαλάµου. Σε πολλά ψάρια έχουν βρεθεί τόσο θετικοί όσο και αρνητικοί µηχανισµοί ανάδρασης της παραγωγής και έκκρισης των FSH και LH. Το αντικείµενο της παρούσας έρευνας ήταν η αναζήτηση του πιθανού ρόλου της τεστοστερόνης καθώς και η εµπλοκή της αρωµατοποίησης στον έλεγχο της φυλετικής ωρίµανσης στα ψάρια. Για το σκοπό αυτό χορηγήθηκαν εµφυτεύµατα µε διάφορους αναστολείς αρωµατάσης σε νεαρά αρσενικά άτοµα σολωµού του Ατλαντικού κατά την πρώτη περίοδο της φυλετικής τους ωρίµανσης, καθώς και εµφυτεύµατα µε τεστοστερόνη σε δύο δόσεις. Η χορήγηση τεστοστερόνης είχε θετική δράση στην LH σε νεαρά άτοµα. Το ίδιο ισχύει και για τον αναστολέα της αρωµατάσης ATD, εφόσον χoρηγηθεί σε µη ώριµα άτοµα. Η χορήγηση αναστολέων της αρωµατάσης αύξησε το ποσοστό ωρίµανσης στα νεαρά άτοµα, ενώ οι τιµές της FSH παραµένουν στα ίδια επίπεδα µε αυτά των ώριµων µαρτύρων, ωστόσο αυξηµένες σε σχέση µε όλους τους µάρτυρες. Η χορήγηση χαµηλής δόσης τεστοστερόνης είχε θετική δράση στην FSH, ενώ η χορήγηση υψηλής δόσης σε νεαρά άτοµα είχε αρνητική δράση Ο συνδυασµός τεστοστερόνης µε τον αναστολέα της αρωµατάσης ATD δεν επηρέασε τη θετική επίδραση της τεστοστερόνης στην FSH. Λέξεις κλειδιά: Αναστολείς Αρωµατάσης, Τεστοστερόνη, Σολωµός, Γοναδοτροπίνες, Ωοθυλακιοτρόπος, Ωχρινοτρόπος

3 INTRODUCTION The Atlantic salmon spawns in rivers. After a few years young fish migrate to the sea, and return to the same river a number of years later to spawn as large fish. Some of the males, however, undergo sexual maturation already as small parr males in the river. This precocious sexual maturation is of an all-or-nothing character; either the fish mature fully or not at all. The phenomenon of early sexual maturation occurs not only in salmonids, but also in other teleosts. From an economic point of view, early sexual maturation is highly undesirable to the aquaculture industry. Thus, a better understanding of the causal factors controlling this phenomenon would be a great benefit not only for a scientific point of view, but also to the aquaculture. The gonadotropic (GTH) hormones, follicle-stimulating hormone (FSH or GTH I) and luteinizing hormone (LH or GTH II) are important factors regulating the two main gonadal functions, hormone production (steroidogenesis) and germ cell production (spermatogenesis / oogenesis), in fish. Sex steroids, in turn, exert feedback effects on the brain-pituitary-gonad axis, and control gonadotropic cells both directly on the pituitary and/or indirectly via the brain. A lot of information is available concerning LH production and/or secretion in fishes, whereas feedback regulation of FSH has received less attention. However, castration experiments in salmonids have demonstrated both negative (Larsen and Swanson, 1997) and positive (Borg et al., 1998) feedback effects on both LH and FSH. Administration of the androgen, testosterone (T) exerts always postive effects on LH in Atlantic salmon males, whereas the effects of T on FSH appear to be more complex than thοse on LH and more dependent on the season and physiological conditions (Borg et al., 1998; Antonopoulou et al., 1999). Previous experiments by Berglund et al. (1995) with juvenile Atlantic salmon (Salmo salar) males showed that different doses of T had stimulatory and inhibitory effects on gonadal development (gonad weight and spermatogenesis), with the later being more pronounced in fish treated with high dose of T. The effects, however, of different doses of T on gonadotropic hormones had not been studied earlier. Certain androgens, such as T, exert feedback effects after have been converted to estrogens. This conversion is called aromatization and is particularly active in the brain of teleost fishes. Antonopoulou et al. (1999) find both positive and negative aromatase dependent feedback effects of T on LH and FSH in two year mature male salmon parr. Furthermore, treatment with aromatase inhibitors increases the proportion of young Atlantic salmon males undergoing sexual maturation (Antonopoulou et al., 1995). The aim of the present study was to investigate the role of aromatization in the regulation of the very early stages of sexual maturation in Atlantic salmon male parr. To that end, intact one year old males which had not been mature previously were treated with different aromatase inhibitors and T in two different doses, at different seasons and their effects on gonadal development and on pituitary and plasma LH and FSH levels were measured. MATERIALS AND METHODS Several experiments were conducted, using one-year old Atlantic salmon male parr that had not yet undergone maturation reared at a swedish salmon hatchery. Fish were randomly divided into the respective experimental groups and were implanted in late spring in the abdominal cavity through a c. 2 mm long incision with Silastic capsules (Dow Corning, Midland, MI, USA) sealed with silicone glue. Capsules were filled with: (a) 1,4,6-androstatriene-3,17- dione (ATD, Steraloids Inc., Wilton, NH, USA); or (b) 4-hydroxy-4-androstene-3,17-dione (4OH, Sigma Chemical Co., St. Louis, MO, USA), or (c) fadrozole (CGS, Ciba, Geigy Corp. USA) or (d) T (Sigma Chemical Co. St. Louis, USA) in two different doses (3 mm or 10 mm); or (e) ATD in combination with T or (f) with empty capsules. During the experiments, the fish were

4 maintained in 2 m 3 tanks supplied with through-flowing river water and kept under simulated natural photoperiod and at ambient temperature. Fish were sampled in summer, at the time of gonadal development and in autumn, at the spawning season. All fish were measured, weighed and dissected. Blood samples were taken by means of severing the caudal peduncle and collecting the blood in heparinized tubes. The tubes were kept on ice until centrifugation, after which the plasma was drawn off, frozen in plastic tubes on dry ice, and stored at -70 o C until assay. Pituitaries were excised, frozen and stored as above. Testes were excised and weighed in order to estimate the gonadosomatic index [GSI=100 X (gonad weight/total weight)]. The Silastic capsules were retrieved, and the fish in which the appropriate capsule could not be found were excluded from the analysis. FSH and LH were measured using heterologous RIA for coho salmon gonadotropins. RESULTS AND DISCUSSION Significant difference was found in GSIs between the different groups. All treated groups showed gonadal development, with GSI values significantly higher than the immature controls. Testosterone treatment resulted in higher GSIs compared to control. However, T3 treated fish had significantly higher GSI levels than the T10 treated ones. Moreover, T3+ATD group showed significantly (p < 0.001) higher GSI values when compared to the T10+ATD fish. T treated fish always had high pituitary LH levels. In addition, treatment with ATD increased pituitary LH, though to a lesser extent than T, in immature salmon parr, whereas ATD and CGS treatment suppressed pituitary LH in maturing fish. However, ATD combined with T diminished pituitary LH levels, compared to T or ATD alone in immature fish, indicating an aromatase-dependent positive feedback of T on LH. 4OH, which was less effective as an aromatase inhibitor, increased LH content. ATD treatment resulted in increased pituitary FSH, although FSH levels did not differ from those of mature controls. Treatments with different aromatase inhibitors increased significantly the proportion of mature fish compared to the controls. However, the site at which the aromatase inhibitors influenced maturation rates is not known. The brain and the pituitary are possible candidates, but not the only ones. T exerted both positive and negative effects on pituitary FSH and testes growth, depending on dose and season. The combined treatment of T with ATD did not affect the positive effect of T alone on pituitary and plasma FSH, indicating the presence of a non-aromatase dependent positive feedback on FSH. References Antonopoulou, E., Mayer, I., Berglund, I. & Borg, B. (1995) Effects of aromatase inhibitors on sexual maturation in Atlantic salmon, Salmo salar, male parr. Fish Physiol. Biochem., 14, Antonopoulou, E., Swanson, P., Mayer, I. & Borg, B. (1999) Feedback control of gonadotropins in Atlantic salmon, Salmo salar, mature parr II. Aromatase inhibitor and androgen effects. Gen. Comp. Endocrinol., 114, Berglund, I., Antonopoulou, E., Mayer, I. & Borg, B. (1995) Stimulatory and inhibitory effects of testosterone on testes in Atlantic salmon male parr. J. Fish Biol., 47, Borg, B., Antonopoulou, E., Mayer, I., Andersson, E., Berglund, I. & Swanson, P. (1998) Effects of gonadectomy and androgen-treatments on pituitary and plasma levels of gonadotropins in

5 Atlantic salmon, Salmo salar, mature male parr - Physiological positive feedback control of both gonadotropins. Biol. Reprod., 58, Larsen, D.A. & Swanson, P. (1997) Effects of gonadectomy on plasma gonadotropins I and II in coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol., 108,