M. V. Alvarado

Expression of kisspeptins and their Receptors, gnrh-1/gnrhr-II-1a and gonadotropin genes in the brain of adult male and female European sea bass during different gonadal stages

Kisspeptins play a critical role in the control of hypothalamic-gonadotropic function and puberty onset in mammals. Studies in fish have all supported the hypothesis that they might play similar roles in the reproduction of this animal group, however, their physiological relevance in the occurrence of key reproductive events still remains to be determined. This study examines the relative mRNA expression profiles of the duplicate kisspeptin system (kiss1, kiss2, gpr54-1b, and gpr54-2b) in the hypothalamus and pituitary of adult male and female sea bass (Dicentrarchus labrax L.) during different gonadal stages using qRT-PCR. We also report the changes in the expression levels of gnrh-1, gnrhr-II-1a, fshβ, and lhβ and the relationships observed between both kisspeptin and GnRH systems. Our data show clear sex differences in the dynamics of kisspeptin and kisspeptin receptor gene expression in the hypothalamus of sea bass during gonadal development. Overall, all four kisspeptin system genes increased either before or during the advanced stages of oogenesis and declined during atresia, exhibiting profiles that are identical to those observed for gnrhr-II-1a, fshβ, lhβ, and the gonadosomatic index (GSI). While the situation was not as clear in males, the high kiss2 expression levels observed in the hypothalamus during mid recrudescence suggest that it might be playing a role in the neuroendocrine signaling that regulates germ cell proliferation at the testicular level. In this sense, the proposed role attributed to kisspeptins as key factors in the onset of reproduction in fish receives an additional support from the data obtained in the present work. Nevertheless, further research is required to clarify their precise role in sea bass.

Melatonin-induced changes in kiss/gnrh gene expression patterns in the brain of male sea bass during spermatogenesis

Evidence exists that melatonin may drive the seasonal changes in kisspeptin-expressing cells and GnRH/gonadotropin secretion in mammals, thus modulating their reproductive activity. This study established the influence of long-term melatonin administration (as an implant) on growth performance and reproduction of adult male sea bass. Melatonin reduced the fish weight and condition factor, thus affecting the performance of fish. Melatonin also affected gonadogenesis, as shown by a decrease in the gonadosomatic index after 150 days of treatment and the lower percentage of running males during the spermatogenesis and full spermiation stages of this species. Exogenous melatonin also resulted in lower plasma androgen levels during the reproductive period, and showed a significant decrease in serum Lh and Fsh concentration after 30 and 60 days of treatment, respectively. Thus, melatonin elicited seasonal changes in key reproductive hormones that affected testicular maturity. The hypothalamic expression of kiss1 was significantly higher in melatonin-treated fish than in controls after 30 days of treatment, while a significant increase in kiss2 expression was detected on day 90 of treatment. By contrast, melatonin showed a significant decrease in kisspeptin expression in the dorsal brain on day 150 of treatment and also affected the expression of gnrh-1 and gnrh-3 and gnrhr-II-1a and 2b and the fshβ gene in the pituitary. These results suggest that in this species, melatonin evokes changes in the mRNA levels of kisspeptin and gnrh system genes that appear to mirror disturbances in spermatogenesis.

Updating control of puberty in male European sea bass: A holistic approach

Puberty is the process by which an immature animal acquires the ability to reproduce for the first time; its onset occurs soon after sexual differentiation and is characterized by the beginning of gametogenesis in both sexes. Here we present new insights on when and how the onset of puberty occurs in male European sea bass, its dependence on reaching a critical size, and how it can be controlled by photoperiod, revealing the existence of a photolabile period with important applications in aquaculture. Regarding size, apparently only European sea bass above a certain size threshold attain the ability to carry out gametogenesis during their first year of life, while their smaller counterparts fail to do so. This could imply that fish need to achieve an optimal threshold of hormone production, particularly from the kisspeptin/Gnrh/Gth systems, in order to initiate and conclude puberty. However, a long-term restricted feeding regime during the second year of life did not prevent the onset of puberty, thus suggesting that the fish are able to maintain the reproductive function, even at the expense of other functions. Finally, the study of daily hormonal rhythms under different photoperiod regimes revealed the equivalence between their core values and those of seasonal rhythms, in such a way that the daily rhythms could be considered as the functional units of the seasonal rhythms.

Responsiveness of pituitary to galanin throughout the reproductive cycle of male European sea bass ( Dicentrarchus labrax )

The neuropeptide galanin (Gal) is a putative factor regulating puberty onset and reproduction through its actions on the pituitary. The present study investigated the pituitary responsiveness to galanin and the patterns of galanin receptors (Galrs) expression throughout the reproductive cycle of two years old male European sea bass (Dicentrarchus labrax), an important aquaculture species. Quantitative analysis of pituitary and hypothalamus transcript expression of four galr subtypes revealed differential regulation according to the testicular developmental stage, with an overall decrease in expression from the immature stage to the mid-recrudescence stage. Incubation of pituitary cells with mammalian 1-29Gal peptide induced significant changes in cAMP concentration, with sensitivities that varied according to the testicular development stages. Furthermore 1-29Gal was able to stimulate both follicle stimulating hormone (Fsh) and luteinizing hormone (Lh) release from pituitary cell suspensions. The magnitude of the effects and effective concentrations varied according to reproductive stage, with generalized induction of Fsh and Lh release in animals sampled in January (full spermiation). The differential expression of galrs in pituitary and hypothalamus across the reproductive season, together with the differential effects of Gal on gonadotropins release in vitro strongly suggests the involvement of the galaninergic system in the regulation the hypothalamus-pituitary-gonad axis of male sea bass. This is to our knowledge the first clear evidence for the involvement of galanin in the regulation of reproduction in non-mammalian vertebrates.