Male and female animals display innate sex-specific mating behaviors. In teleost fish, altering the adult sex steroid milieu can effectively reverse sex-typical mating behaviors, suggesting remarkable sexual lability of their brains as adults. In the teleost medaka, neuropeptide B (NPB) is expressed female-specifically in the brain nuclei implicated in mating behavior. Here, we demonstrate that NPB is a direct mediator of estrogen action on female mating behavior, acting in a female-specific but reversible manner. Analysis of regulatory mechanisms revealed that the female-specific expression of NPB is dependent on direct transcriptional activation by estrogen via an estrogen-responsive element and is reversed in response to changes in the adult sex steroid milieu. Behavioral studies of NPB knockouts revealed that female-specific NBP mediates female receptivity to male courtship. The female-specific NPB signaling identified herein is presumably a critical element of the neural circuitry underlying sexual dimorphism and lability of mating behaviors in teleosts.

In the brain of medaka (Oryzias latipes), a teleost fish, we recently found that the supracommissural/posterior nuclei of the ventral telencephalic area (Vs/Vp) and the magnocellular/gigantocellular portions of the magnocellular preoptic nucleus (PMm/PMg) express estrogen receptor (ER) and androgen receptor (AR) specifically in females. This finding led us to postulate that sex steroid hormones might induce gene expression unique to females in these nuclei. In the present study, we searched for genes differentially expressed between the sexes in the medaka brain and identified the gene encoding neuropeptide B (npb) as being female-specifically expressed in Vs/Vp and PMm/PMg. As expected, the neurons expressing npb female-specifically constituted a significant proportion of the ER/AR-expressing neurons in these nuclei. Subsequent analyses provided evidence that the female-specific expression of npb in Vs/Vp and PMm/PMg results from the reversible and transient action of estrogens secreted from the ovary and that this estrogenic action is most likely mediated by the direct transcriptional activation of npb through an estrogen-responsive element in its proximal promoter region. Vs/Vp and PMm/PMg are generally recognized in teleost fish as the sites where neurons expressing 2 other neuropeptides, isotocin and vasotocin, are present, but the female-specific npb/ER/AR-expressing neurons were distinct from, although adjacent to, isotocin and vasotocin neurons. Taken together, these data demonstrate that npb is female-specifically expressed in novel, as-yet undefined populations of Vs/Vp and PMm/PMg neurons, resulting from the direct stimulatory action of ovarian estrogens via female-specific ER in these neurons.

The CRH family of neuropeptides, including CRH and urocortins, plays pivotal roles in the regulation of physiological and behavioral stress responses in vertebrates. In this study, we identified a previously undescribed member of the CRH family of peptides in a teleost fish species (medaka; Oryzias latipes) and named this peptide teleocortin (Tcn). Medaka Tcn is a 41-amino acid polypeptide derived from the C terminus of a larger precursor protein that is encoded by a 2-exon gene, thus sharing common structural features with known CRH family peptides. tcn was found exclusively in teleost fish. Phylogenetic analysis suggested that tcn probably has an ancient origin but was lost from the tetrapod lineage shortly after the divergence of the teleost and tetrapod lineages. In the medaka brain, tcn was expressed in nuclei of the telencephalon, preoptic area, hypothalamus, tegmentum, and isthmic region. Because none of these nuclei have been implicated in the control of ACTH secretion from the pituitary, Tcn may exert its effects centrally in the brain rather than via stimulation of the pituitary-adrenal/interrenal axis. Most, if not all, tcn-expressing neurons also expressed crh, suggesting that Tcn and Crh share common physiological functions. Moreover, Tcn activated Crh receptors 1 and 2 with equivalent or slightly higher potency than Crh, further suggesting that these peptides share common functions. Taken together, these data identified Tcn as a novel, teleost-specific member of the CRH family of peptides that may act centrally with Crh to regulate physiological and behavioral stress responses.

Recent studies in mice demonstrate that a subset of neurons in the medial preoptic area (MPOA) that express galanin play crucial roles in regulating parental behavior in both sexes. However, little information is available on the function of galanin in social behaviors in other species. Here, we report that, in medaka, a subset of MPOA galanin neurons occurred nearly exclusively in males, resulting from testicular androgen stimulation. Galanin-deficient medaka showed a greatly reduced incidence of male–male aggressive chases. Furthermore, while treatment of female medaka with androgen induced male-typical aggressive acts, galanin deficiency in these females attenuated the effect of androgen on chases. Given their male-biased and androgen-dependent nature, the subset of MPOA galanin neurons most likely mediate androgen-dependent male–male chases. Histological studies further suggested that variability in the projection targets of the MPOA galanin neurons may account for the species-dependent functional differences in these evolutionarily conserved neural substrates.

The neuroplastic mechanisms in the fish brain that underlie sex reversal remain unknown. Gonadotropin-releasing hormone 3 (GnRH3) neurons control male reproductive behaviours in Mozambique tilapia and show sexual dimorphism, with males having a greater number of GnRH3 neurons. Treatment with androgens such as 11-ketotestosterone (KT), but not 17β-estradiol, increases the number of GnRH3 neurons in mature females to a level similar to that observed in mature males. Compared with oestrogen, the effect of androgen on neurogenesis remains less clear. The present study examined the effects of 11-KT, a non-aromatizable androgen, on cellular proliferation, neurogenesis, generation of GnRH3 neurons and expression of cell cycle-related genes in mature females. The number of proliferating cell nuclear antigen-positive cells was increased by 11-KT. Simultaneous injection of bromodeoxyuridine and 11-KT significantly increased the number of newly-generated (newly-proliferated) neurons, but did not affect radial glial cells, and also resulted in newly-generated GnRH3 neurons. Transcriptome analysis showed that 11-KT modulates the expression of genes related to the cell cycle process. These findings suggest that tilapia could serve as a good animal model to elucidate the effects of androgen on adult neurogenesis and the mechanisms for sex reversal in the fish brain.