Vol. 6, Suppl. 1 195 The effect of aromatase inhibitor, fadrozole, on sgnrha-stimulated LH secretion in goldfish (Carassius auratus) and common carp (Cyprinus carpio) Tomasz Mikolajczyk 1, Jaroslaw Chyb, Miroslawa Sokolowska- Mikolajczyk, Pawel Szczerbik, Magdalena Socha, Piotr Epler Department of Ichthyobiology and Fisheries, University of Agriculture, Kraków, Poland SUMMARY The aromatase inhibitor, fadrozole, was applied to common carp and goldfish in order to examine its ability to increase the spontaneous and sgnrha-stimulated LH secretion. First, trials in goldfish in 2003 showed fadrozole s moderate ability to potentiate sgnrha-stimulated LH secretion. However, this ability was much weaker than that obtained with dopamine antagonist, pimozide. There was no ovulation in fadrozole-treated fish. Several experiments on goldfish and carp during the two consecutive years with the different treatment regimes and doses of fadrozole did not confirm the optimistic results obtained in 2003. This shows that fadrozole is unable to replace the antidopaminergic drugs being used in fisheries practice. Reproductive Biology 2006 6 Suppl. 1:195 199. Key words: aromatase inhibitor, LH, estradiol, goldfish, carp 1 Corresponding author: Department of Ichthyobiology and Fisheries, University of Agriculture, Spiczakowa 6, 30-199 Kraków-Mydlniki, Poland; e-mail: tmikolaj@ar.krakow.pl Copyright 2006 by the Society for Biology of Reproduction
196 Fadrozole and LH secretion Starting from the late 1980s, the most effective and widespread technique of inducing ovulation in cultured fish was the use of synthetic GnRH analogues. However, in cyprinids, due to strong dopamine inhibitory tonus upon LH secretion [4], GnRH analogue needed to be combined with dopamine antagonist [14]. Unfortunately, the presence of antidopaminergic drugs in the formulations used for induction of ovulation is a major obstacle for their common use in the European Union. Antidopaminergic drugs are well-known neuroleptic compounds exhibiting a wide range of physiological effects, which rise serious problems for carp farmers and commercial hatcheries. It has been recently discovered that the high level of dopamine, affecting LH secretion, is determined by the high levels of circulating estradiol [12]. In the last stages of ovarian maturity, just before ovulation, a rapid drop in estradiol levels is followed by the drop in dopamine production. Thanks to this phenomenon, GnRH action may be strenghthened and results in the ovulatory LH surge and ovulation [6, 13]. The aim of our investigation was the attempt to potentiate the stimulatory action of GnRH analogues on LH secretion in cyprinids by the use of an aromatase inhibitor fadrozole. Cytochrome P450 aromatase (CYP19) is a key enzyme in steroidogenesis in vertebrates, enabling the conversion of C19 androgens into C18 estrogens. The activity of brain aromatase in fish was shown to be about 1000 times higher than in mammals and birds [7]. Estrogens can be produced in the brain of fish of both sexes [2]. Since there is no aromatase in the testes of males [5], the main source of estrogens in blood circulation in males is the brain. There are several data indicating the use of aromatase inhibitors in modulation of the reproductive cycle in fish. Afonso et al. [1, 2] showed that maturation and spawning in Coho salmon accelerated with the use of fadrozole. In Black porgy aromatase inhibitors increased LH secretion [8], but it also blocked the natural sex change [9]. On the contrary, in tilapia it was possible to achieve sex reversal after treatment with aromatase inhibitors [3]. The activity of brain aromatase changes significantly during the reproductive cycle, being associated with the changes in the brain catecholamines. It was shown that applying estradiol to fish evokes increased
Mikolajczyk et al. 197 activity of pituitary neurones producing dopamine [10, 12]. Moreover, there is immunohistochemical evidence that the dopamine containing neurons reaching the pituitary gland possess estrogenic receptors [10]. In the last stages of ovarian maturation, just before ovulation, the rapid estradiol level drop is followed by the diminution of dopamine production. Thanks to this phenomenon there is potentiation of GnRH action and the appearance of LH surge and ovulation as observed in the rainbow trout [13]. The results of our experiments performed in 2003 on goldfish [11] supported the above findings. A single injection of fadrozole at a dose of 50 µg/kg, performed 72 h prior to salmon GnRH analogue (sgnrha) injection, had a stimulatory effect on spontaneous and sgnrha-stimulated LH secretion in comparison to the control group. The highest LH levels were detected in the group treated with sgnrha and pimozide (dopamine antagonist, a positive control) and only in the latter group females (80%) ovulated. In a subsequent experiment, one dose of fadrozole (50 µg/kg) was used at different treatment times; four consecutive injections of fadrozole (from 72 h to 0 h prior to GnRH treatment) had a more pronounced effect on sgnrha action than a single injection. LH levels in this group reached half of the level found in the (sgnrha+pimozide)-treated group. The highest LH level as well as ovulation of 80% of females were observed in the group treated with sgnrha+pimozide. Four injections of fadrozole at a much higher dose (1 mg/kg) also significantly increased sgnrha-stimulated LH secretion at 12 and 24 h after sgnrha injection in comparison with the group treated with only sgnrha (Mikolajczyk et al., unpublished). Four separate experiments performed on goldfish in 2004 did not confirm the results obtained in 2003. Surprisingly, in all four experiments there was no effect of the same doses of fadrozole on the spontaneous and sgnrhastimulated LH secretion. In 2005 there were four separate experiments performed on sexually mature female carps. Initially, fadrozole treatment, regardless the dose applied (4 50, 100 or 200 µg/kg) had no effect on the spontaneous LH secretion. However, 24 h following the sgnrha injection, the LH levels in groups treated with 100 or 200 µg/kg of fadrozole were significantly lower when compared with control group (sgnrha alone). In another trial,
198 Fadrozole and LH secretion despite the prolonged (six days) exposure to fadrozole (50 µg/kg) there were no changes in the spontaneous as well as in sgnrha-stimulated LH secretion. In the last two experiments performed in common carp, instead of sgnrha a commercially available product, GONAZON TM was used. Fadrozole (4 1 mg/kg) had no effect on the spontaneous as well as Gonazonstimulated LH release. In the last experiment which was performed under commercial hatchery conditions the same fadrozole treatment evoked significant inhibition of Gonazon-stimulated LH secretion in comparison to control (Mikolajczyk et al., unpublished). All these observations draw a confusing picture of the role and the effects of aromatase inhibitors in the processes involved in the regulation of LH secretion in cyprinids. It seems that the effects of fadrozole are highly dependent on the actual hormonal status of fish, state of gonad development and the origin of fish. At the moment, it is impossible to explain why, in some cases, fadrozole stimulated LH secretion while in others its action was opposite. In our opinion the only explanation for this phenomenon is the insufficient time of fish s exposure to fadrozole. In most cases this time was limited to three days before sgnrha or Gonazon injection, while other authors applied a much longer treatment (several weeks or months). Such a protocol would be unacceptable to carp farmers. In conclusion, it has to be stated that because of striking differences in the results obtained so far, the treatment with the aromatase inhibitor, fadrozole, cannot be recommended as a potential tool for spawning induction in cyprinids. REFERENCES 1. Afonso LO, Iwama GK, Smith J, Donaldson EM 1999 Effects of the aromatase inhibitor fadrozole on plasma sex steroid secretion and oocyte maturation in female coho salmon (Oncorhynchus kisutch) during vitellogenesis. Fish Physiology and Biochemistry 20 231-241. 2. Afonso LO, Iwama GK, Smith J, Donaldson EM 2000 Effects of the aromatase inhibitor fadrozole on reproductive steroids and spermiation in male coho salmon (Oncorhynchus kisutch) during sexual maturation. Aquaculture 188 175-187. 3. Afonso LO, Wasserman CJ, Terezihna de Oliveira R 2001 Sex reversal in Nile tilapia (Oreochromis niloticus) using a nonsteroidal aromatase inhibitor. Journal of Experimental Zoology 290 177-181.
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