Molecular cloning of bullfrog corticotropin-releasing factor (CRF): effect of homologous CRF on the release of TSH from pituitary cells in vitro.

Corticotropin-releasing factor (CRF) plays multiple roles in vertebrate species. In non-mammalian vertebrates, CRF has been considered to be the major thyrotropin (TSH)-releasing factor. This notion, however, was derived from experimental data on CRF of mammalian origin. Moreover, in the case of amphibians it has never been directly proved that CRF stimulates the release of TSH from the pituitary. The presently described experiment was conducted to provide direct evidence that homologous CRF enhances the release of TSH from the bullfrog (Rana catesbeiana) pituitary. First, cloning of cDNA encoding bullfrog CRF (fCRF) was accomplished. The cDNA encoding fCRF precursor was isolated from a cDNA library of the bullfrog hypothalamus. The amino acid sequence of fCRF predicted from the amplified cDNA sequence showed 83 and 95% identities with the sequences of ovine and human CRFs, respectively. An antiserum against the fCRF synthesized on the basis of the amino acid sequence was raised and used for immunohistochemical staining of the hypothalamus-hypophyseal region of the bullfrog brain. It stained some of the cell bodies situated mainly in the preoptic area, the nucleus infundibularis dorsalis and nucleus hypothalamicus ventralis and the axons that terminate in the median eminence and neural lobe. The synthetic fCRF was tested for its TSH-releasing activity toward anterior pituitary cells of adult bullfrogs in an in vitro system. As a result, the fCRF caused the release of TSH from the dispersed pituitary cells into the culture medium concentration-dependently, as measured by a specific radioimmunoassay for bullfrog TSH. The potency of the fCRF was almost equivalent to that of ovine CRF. Human urocortin III (hUCN III), a CRF receptor type 2 (CRF-R2) specific agonist enhanced the release of TSH from the pituitary cells in culture, suggesting the involvement of CRF-R2 in the CRF-induced TSH release in the bullfrogs. Culture of pituitary cells in the presence of the hypothalamic extract (HE) and alpha-helical CRF(9-41), a CRF-R antagonist, revealed that the antagonist suppressed the TSH-releasing activity of the HE by approximately 50%, suggesting that endogenous CRF contributes as a TSH-releasing factor.