Kiss2, a paralogous gene for kiss1, has recently been identified in several vertebrates. However, their relative potencies for the regulation of reproductive functions appear to differ among species. Here we used medaka as a model animal to examine the kiss1 and kiss2 expression dynamics by in situ hybridization under different conditions: breeding or nonbreeding and ovariectomized or sham operated. Medaka kiss1-expressing neurons and kiss2-expressing neurons were mainly localized in two hypothalamic nuclei, nucleus ventralis tuberis (NVT) and nucleus recessus lateralis (NRL), respectively. NRL kiss2 expression did not change according to differences in breeding condition, whereas NVT kiss1 expression was strongly correlated with breeding condition. In addition, ovariectomy did not change kiss2 expression but significantly decreased the kiss1 expression. Moreover, double-label in situ hybridization revealed that NVT Kiss1 neurons coexpress estrogen receptor-alpha, whereas NRL Kiss2 neurons do not. From these results, we conclude that the NVT Kiss1 neurons are positively regulated by ovarian estrogen via their coexpressed estrogen receptor-alpha and are directly involved in the central regulation of reproduction in medaka. In contrast, we argue that the NRL Kiss2 neurons in medaka may serve nonreproductive functions. These functional differences between Kiss1 and Kiss2 neurons are discussed from a phylogenetic viewpoint.

To dissect the molecular and cellular basis of sexual differentiation of the teleost brain, which maintains marked sexual plasticity throughout life, we examined sex differences in neural expression of all subtypes of nuclear oestrogen and androgen receptors (ER and AR) in medaka. All receptors were differentially expressed between the sexes in specific nuclei in the forebrain. The most pronounced sex differences were found in several nuclei in the ventral telencephalic and preoptic areas, where ER and AR expression were prominent in females but almost completely absent in males, indicating that these nuclei represent female-specific target sites for both oestrogen and androgen in the brain. Subsequent analyses revealed that the female-specific expression of ER and AR is not under the direct control of sex-linked genes but is instead regulated positively by oestrogen and negatively by androgen in a transient and reversible manner. Taken together, the present study demonstrates that sex-specific target sites for both oestrogen and androgen occur in the brain as a result of the activational effects of gonadal steroids. The consequent sex-specific but reversible steroid sensitivity of the adult brain probably contributes substantially to the process of sexual differentiation and the persistent sexual plasticity of the teleost brain.

Estrogen and androgen play crucial roles in coordinating reproductive functions through estrogen receptors (ERs) and androgen receptors (ARs), respectively. These receptors are considered important for regulation of the hypothalamo-pituitary-gonadal (HPG) axis. Despite their biological importance, the distribution of sex steroid receptors has not been fully analyzed anatomically in the teleost brain. The teleosts have many characteristic features, which allow unique approaches toward an understanding of the regulatory mechanisms of reproductive functions. Medaka serves as a good model system for studying the mechanisms by which steroid receptor-mediated systems are regulated, because (1) their breeding conditions can be easily manipulated; (2) we can take advantage of the genome database; and 3) molecular genetic tools, such as transgenic techniques, are applicable. We analyzed the distribution of ERα, ERβ1, ERβ2, ARα, and ARβ mRNA by in situ hybridization in the brain of female medaka. We found that all subtypes of ERs and ARs were expressed in the following nuclei: the dorsal part of the ventral telencephalic area (Vd), supracommissural part of the ventral telencephalic area (Vs), postcommissural part of the ventral telencephalic area (Vp), preoptic area (POA), and nucleus ventralis tuberis (NVT). These regions are known to be involved in the regulation of sexual behavior (Vd, Vs, Vp, POA) or the HPG axis (NVT). These ER- and/or AR-expressing neurons may regulate sexual behavior or the HPG axis according to their axonal projections. Future analysis should be targeted to the neurons described in the present study to extend our understanding of the central regulatory mechanisms of reproduction.

In vertebrates, sex steroids play crucial roles in multiple systems related to reproduction. In females, estrogens and their receptor estrogen receptor (ER or Esr) play indispensable roles in the negative sex steroid feedback regulation of pituitary gonadotropin secretion, which prevents excessive development of ovarian follicles. However, the mechanism of this feedback regulation of a gonadotropin, follicle stimulating hormone (FSH), which is essential for folliculogenesis throughout vertebrates, is poorly understood. In the present study, we generated knockouts of all subtypes of nuclear estrogen receptors in a model teleost medaka, which is suitable for the study of endocrine control and behavioral assays, and analyzed fertility, behavior and functionality of estrogen feedback in each knockout line. Among the estrogen receptors, we revealed that an estrogen receptor Esr2a plays an essential role in this feedback regulation. In addition to this, we also found that esr2a −/− females showed oviduct atresia, which causes complete infertility. Interestingly, esr2a −/− females showed apparently normal sexual behavior but without oviposition in response to male courtship. This phenotype indicates that physical readiness and motivation of sexual behavior is independently controlled.