Kisspeptins and their receptors in the brain-pituitary-gonadal axis of Odonthestes bonariensis: Their relationship with gametogenesis along the reproductive cycle.

In vertebrates, the reproduction is controlled by the brain-pituitary-gonadal (BPG) axis and kisspeptin has emerged as a key player of this axis. In this study, we analyzed changes in the expression levels of kiss1, kiss2, and their receptors, kissr2 and kissr3 during gametogenesis in the BPG axis of feral Odontesthes bonariensis. In females, levels of brain kiss1 showed an increase at final maturation (Fm), while kiss2 levels were shown to be high at primary growth (Pg) stage, with no differences in the expression of their receptors. In the pituitary, kiss1 and kiss2 peaked at the cortical alveoli (Ca) stage, and kissr3 at initial vitellogenesis. In parallel, there was an increase of kiss1, kissr2 and kissr3 in the ovary during the Ca stage and both receptors again at Fm stage. In males, the four genes were highly expressed in the brain at the arrested (A) stage. In the pituitary, kiss2 peaked at spermatogonial (SG) and spermatocytary (SC) stages; while kissr3 reached a peak at the spermiogenic stage (SP). In testes, kiss1 and kiss2 significantly increased during the SG and SC stages; meanwhile, kissr2 increased at SG and SC, whereas kissr3 levels were significantly high at SC and SP stages. Taken together these results showed that the kisspeptin system in pejerrey is expressed in the three levels of the BPG axis with different expression profiles during the gonadal cycle. These findings pointed that kisspeptins have different roles in gametogenesis in this species.

Kisspeptin system in pejerrey fish (Odontesthes bonariensis). Characterization and gene expression pattern during early developmental stages.

In vertebrates, kisspeptins and their receptors are known to be related to puberty onset and gonadal maturation, however, there are few studies concerning their role in early development. Here, we characterize the kisspeptin system in the pejerrey, Odontesthes bonariensis, a fish with strong temperature-dependent sex determination. We reconstructed the phylogenetic history of the two ligands (kiss1 and kiss 2) and two receptors (kissr2 and kissr3) in pejerrey in the context of recent classifications of bony fishes, determined their tissue distribution and documented the early expression pattern of these ligands and receptors. Phylogenetic analysis of these gene families clearly resolved the percomorph clade and grouped pejerrey with Beloniformes. Paralogous sets of genes putatively arising from the teleost-specific genome duplication event (3R) were not detected. Kisspeptins and their receptors showed a wide tissue distribution in adult pejerrey, including tissues not related to reproduction. In larvae reared at 24°C, the four kisspeptin elements were expressed in the head from week 1 to week 8 of life, with no differences in transcript levels. Larvae kept at a female-producing temperature (17°C) did not show statistically significant differences in the transcript levels of all analyzed genes during the sex determination/differentiation period; however, in those larvae raised at male producing temperature (29°C), kiss2 levels were increased at week 4 after hatching. These results showed that all members of the kisspeptin system are expressed at this early period, and the increase of kiss2 transcripts at week 4 could be interpreted as it would be related to the differentiation of the brain-pituitary axis in male development.

Steroid production in toads.

In Bufo arenarum, androgen biosynthesis occurs through a complete 5-ene pathway, including 5-androstane-3beta,17beta-diol as the immediate precursor of testosterone. Besides, steroidogenesis changes during the breeding period, turning from androgens to C(21)-steroids such as 5alpha-pregnan-3alpha,20alpha-diol, 3alpha-hydroxy-5alpha-pregnan-20-one and 5alpha-pregnan-3,20-dione. In B. arenarum, steroid hormones are not involved in hCG-induced spermiation, suggesting that the steroidogenic shift to C(21)-steroids during the breeding be not related to spermiation. The activity of 17-hydroxylase-C(17-20) lyase (CypP450(c17)) decreases during the reproductive season, suggesting that this enzyme would represent a key enzyme in the regulation of seasonal changes. However, the increase in the affinity for pregnenolone of 3beta-hydroxysteroid dehydrogenase (3alphaHSD)/isomerase could also be involved. Moreover, the reduction in CypP450(c17) leading to a reduction in C(19)-steroids, among them dehydroepiandrosterone (DHE), would contribute to the conversion of pregnenolone into progesterone, avoiding the non-competitive inhibition exerted by DHE on this transformation. Additionally, CypP450(c17) possesses a higher affinity for pregnenolone than for progesterone, explaining the predominance of the 5-ene pathway for testosterone biosynthesis. Animals in reproductive condition showed a significant reduction in circulating androgens, enhancing the physiological relevance of all the in vitro results. The in vitro effects of mGnRH and hrFSH on testicular steroidogenesis revealed that both hormones inhibited CypP450(c17) activity. In summary, these results demonstrate that, in B. arenarum, the change in testicular steroidogenesis during the reproductive period could be partially due to an FSH and GnRH-induced decrease in CypP450(c17) activity.

Effects of mGnRH on testicular steroidogenesis in the toad Bufo arenarum.

GnRH controls vertebrate reproduction in several ways. This hormone not only affects the secretion of gonadotropins from the pituitary gland but also has a direct influence on several gonadal functions such as steroidogenesis, spermatogenesis, and spermiation. In the present paper we have studied the in vitro effects of GnRH on the testicular steroidogenesis of Bufo arenarum to ascertain the role of this peptide in the control of the steroidogenic pathway previously described in this species. It was found that GnRH is able to reduce basal as well as hCG-stimulated testosterone release, having an inhibitory effect on P450(c17) activity. Thus, GnRH could be involved in the mechanism that regulates the metabolic change in the testicular steroidogenesis. Additionally, testicular GnRH binding site has been characterised, showing a K(d) of 34 nM and a maximum binding of 4.7 pmol/mg protein.