Specific and sensitive radioimmunoassays (RIAs) were newly developed for two types of gonadotropin-releasing hormone (GnRH), namely, seabream (sb) GnRH and chicken (c) GnRH-II. We employed these two RIAs together with a previously reported RIA for salmon (s) GnRH to study the presence and regional distribution of these three GnRHs in the brains and pituitaries of four perciform fishes (red seabream, Pagrus major; black seabream, Acanthopagrus schlegeli; striped knifejaw, Oplegnathus fasciatus; and Nile tilapia, Oreochromis niloticus), as well as clarify seasonal changes in levels of these GnRHs in the brain and pituitary of red seabream. All three GnRHs were found in brains of all fishes examined, with regional distributions in the brains of the three GnRHs being rather similar. sbGnRH was abundant in telencephalon and hypothalamus. cGnRH-II was concentrated from the middle to posterior part of the brain and distributed throughout the brain. sGnRH was concentrated in the olfactory bulb and distributed all over the brain, as was cGnRH-II. The dominant form of GnRH in the pituitary was sbGnRH, with levels 500- to 2400-fold higher than those of sGnRH, while cGnRH-II was undetectable in all four species. In the brain and pituitary of female red seabream, levels of both brain and pituitary sbGnRH increased from October (immature phase) and reached a peak in April (spawning phase), reflecting the increase in gonadosomatic index and vitellogenesis. However, levels of sbGnRH remained high only in the pituitary of completely regressed fish in June. Levels of both sGnRH and cGnRH-II in the brain were higher in the regressed phase and remained lower during the spawning phase. From these and previous results, it appears that sbGnRH is physiologically the most important form of GnRH in reproduction in red seabream and, probably, in other perciforms also.

Stratum intermedium is a transient and subtle epithelial structure closely associated with inner dental epithelium in tooth germs. Little is known about its development and roles. To facilitate analysis, we used bovine tooth germs, predicting that they may contain a more conspicuous stratum intermedium. Indeed, early bell stage bovine tooth germs already displayed an obvious stratum intermedium with a typical multilayered organization and flanking the enamel knot. Strikingly, with further development, the cuspally located stratum intermedium underwent thinning and involution, whereas a multilayered stratum intermedium formed at successive sites along the cusp-to-cervix axis of odontogenesis. In situ hybridization and immunohistochemistry showed that stratum intermedium produces the signaling molecule Sonic hedgehog (Shh). Maximal Shh expression was invariably seen in its thickest multilayered portions. Shh was also produced by inner dental epithelium; expression was not constant but varied with development and cytodifferentiation of ameloblasts along the cusp-to-cervix axis. Interestingly, maximal Shh expression in inner dental epithelium did not coincide with that in stratum intermedium. Both stratum intermedium and inner dental epithelium expressed the Shh receptor Patched2 (Ptch2), an indication of autocrine signaling loops. Shh protein, but not RNA, was present in underlying dental mesenchyme, probably resulting from gradual diffusion from epithelial layers and reflecting paracrine loops of action. To analyze the regulation of Shh expression, epithelial and mesenchymal layers were separated and maintained in organ culture. Shh expression decreased over time, but was maintained in unoperated specimens. Our data show for the first time that stratum intermedium is a highly regulated and Shh-expressing structure. Given its dynamic and apparently interactive properties, stratum intermedium may help orchestrate progression of odontogenesis from cusp to cervix.

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Lingual epithelial cells, including those of the taste buds, are regularly replaced by proliferative stem cells. We found that integrin beta(1), a keratinocyte stem cell marker, was expressed at the basal layer and taste buds of adult mouse tongue epithelium. We purified and cultured integrin beta(1)-positive cells (termed KT-1 cells), whose growth was stimulated by epidermal growth factor (EGF) and basic fibroblast growth factor (FGF-2). FGF-2 stimulation induced translocation of the FGF type I receptor (FGFR1) into nuclei, suggesting that the growth-stimulating effect of FGF-2 was mediated through FGFR1. EGF and FGF-2 also regulated cell surface expression of the neural cell adhesion molecule (N-CAM) in KT-1 cells. Anti-N-CAM antibody immunoreactivity was restricted to the gustatory epithelium and the nerves in the tongue epithelium, giving rise to the possibility that KT-1 may contain gustatory epithelial cells. KT-1 cells may thus be useful for analyzing the factors that regulate the growth and differentiation of lingual and gustatory epithelial cells in vitro.

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