Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of Foxl2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. Foxl2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, Foxl2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. Foxl2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous Foxl2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17beta-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia Foxl2 (tFoxl2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17beta-estradiol and 11-ketotestosterone. Altogether, these results suggest that Foxl2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway.

The neuropeptide gonadotropin-releasing hormone (GnRH) has a central role in the neural control of vertebrate reproduction. This review describes an overview of what is currently known about GnRH in vertebrates in the context of its structural and functional evolution. A large body of evidence has demonstrated the existence of three paralogous genes for GnRH (GnRH1, GnRH2 and GnRH3) in the vertebrate lineage. They are most probably the products of whole-genome duplications that occurred early in vertebrate evolution. Although GnRH3 has been identified only in teleosts, comparative genomic analyses indicated that GnRH3 has not arisen from a teleost-specific genome duplication, but has been derived from an earlier genome duplication in an ancestral vertebrate, followed by its loss in the tetrapod lineage. A loss of other paralogous genes has also occurred independently in different vertebrate lineages, leading to species-specific differences in the organization of the GnRH system. In addition to the GnRH3 gene, the GnRH2 gene has been deleted or silenced in certain mammalian species, while some teleosts seem to have lost the GnRH1 or GnRH3 gene. The duplicated GnRH genes have undergone subfunctionalization during the evolution of vertebrates; GnRH1 has become the major stimulator of gonadotropins and probably other pituitary hormones as well, whereas GnRH2 and GnRH3 would have functioned as neuromodulators, affecting reproductive behaviour. Conversely, in cases where a paralogous gene for GnRH has been lost, one of the remaining paralogues appears to have adopted its role.

Neurons that synthesize and release GnRH are essential for the central regulation of reproduction. Evidence suggests that forebrain GnRH neurons originate in the olfactory placode and migrate to their final destinations, although this is still a matter of controversy. X-linked Kallmann syndrome (X-KS), characterized by failed gonadal function secondary to deficient gonadotropin secretion, is caused by a mutation in KAL1, which is suggested to regulate the migration of forebrain GnRH neurons. Because rodents lack Kal1 in their genome and have GnRH neurons scattered throughout their forebrain, the development of forebrain GnRH neurons and the pathogenesis of X-KS have been difficult to study. In the present study, we generated transgenic medaka that expressed green fluorescent protein under the control of the gnrh1 and gnrh3 promoters for analyzing forebrain GnRH neuronal development. Our data revealed the presence of the following four gnrh1 neuronal populations: an olfactory region-derived ventral preoptic population, a dorsal preoptic population that migrates from the dorsal telencephalon, a medial ventral telencephalic population that migrates from the anterior telencephalon, and a nonmigratory ventral hypothalamic population. We found that all forebrain gnrh3 neurons, extending from the terminal nerve ganglion to the anterior mesencephalon, arise from the olfactory region and that trigeminal ganglion neurons express gnrh3. Maternal gnrh3 expression was also observed in oocytes and early embryos. We subsequently identified a KAL1 ortholog and its paralogous form in the medaka. Consistent with the X-KS phenotype, antisense knockdown of the medaka KAL1 ortholog resulted in the disruption of forebrain GnRH neuronal migration. Thus, these transgenic medaka provide a useful model system for studying GnRH neuronal development and disorders of GnRH deficiency.

Social familiarity affects mating preference among various vertebrates. Here, we show that visual contact of a potential mating partner before mating (visual familiarization) enhances female preference for the familiarized male, but not for an unfamiliarized male, in medaka fish. Terminal-nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons, an extrahypothalamic neuromodulatory system, function as a gate for activating mating preferences based on familiarity. Basal levels of TN-GnRH3 neuronal activity suppress female receptivity for any male (default mode). Visual familiarization facilitates TN-GnRH3 neuron activity (preference mode), which correlates with female preference for the familiarized male. GnRH3 peptides, which are synthesized specifically in TN-GnRH3 neurons, are required for the mode-switching via self-facilitation. Our study demonstrates the central neural mechanisms underlying the regulation of medaka female mating preference based on visual social familiarity.

The kiss1 gene product kisspeptin is now considered to be an essential regulator of the hypothalamic-pituitary-gonadal (HPG) axis in most vertebrate species. Recent findings in fishes are beginning to set a new stage for the kisspeptin study; the existence of paralogous kisspeptin genes as well as kisspeptin receptor (formerly called GPR54) genes has quite recently been reported in several fish and amphibian species. The fishes may provide excellent animal models for the study of general principles underlying the kisspeptin and kisspeptin receptor systems of vertebrates from the evolutionary viewpoint. Unlike placental and marsupial mammalian species mainly studied so far, many teleost species have two paralogous genes of kisspeptin, kiss1 and kiss2. Medaka, Oryzias latipes, in which kiss1 and kiss2 are expressed in distinctive hypothalamic neuron populations, is a good model system for the study of central regulation of reproduction. Here, the kiss1 system but not the kiss2 system shows expression dynamics strongly indicative of its direct involvement in the HPG axis regulation via its actions on GnRH1 neurons. On the other hand, the kiss1 gene is missing, and only kiss2 is expressed in some fish species. Also, there are some recent reports that Kiss2 peptide may be a potent regulator of reproduction in some fish species. The ancestral vertebrate probably already had two paralogous kiss genes, and their main function was the HPG axis regulation. In the species that retained both paralogues during evolution, either Kiss1 or Kiss2 predominantly retains its ability for the HPG axis regulation, while the other may assume new non-reproductive functions (neofunctionalization). Alternatively, both the paralogues may assume complementary functions in the HPG axis regulation (subfunctionalization). After the divergence of teleost and tetrapod lineages, either one of the two paralogues, or even both in birds, have been lost (degradation) or became a pseudogene (non-functionalization), but the remaining paralogue retained its original function of HPG axis regulation. The identification of multiple forms of kisspeptin receptors and the rather promiscuous ligand-receptor relationships has led to the further proposal that such promiscuousness may be the basis for the functional robustness of kisspeptin and kisspeptin receptor systems in the HPG axis regulation, when one or both paralogous genes are lost or functionally partitioned during evolution.