Investigation Of Melatonin Receptor Subtype Protein And Gene Expression In Human Embryos

The pineal honnone melatonin, N-acetyl-5-methoxytryptaniine, which is small (mol wt, 132.3) and lipopbilic substance, is synthesized and secreted from pineal gland during the hours of darkness. Wbist the activity of melatonin as a pigment-concentrating hormone remains an important physiological activity, at least in lower vertebrates, discovery of the chemical structure of melatonin led to the realization of many other of its biological activity. These include its action in regulating circadian rhythms, sleep, mood, sexual maturation, reproduction, tumor-suppression, and immune response. Melatonin elicits its biological effects principally via Pharmacologically specific, high-affinity; guanine-protein coupled receptors (GPCR). The high-affinity mclatomn receptor subtypes (Isoform) have been cloned so far and classified as mti (previously termed Mel Ia), MT2 (previously termed Mel lb), and Mel lc. The mt1 and MT2 subtype have been found in mammalian tissues including humans. Radioligands binding assays and in vitro autoradiography (ARG) using 2~I1~Ij iodomelatonin have identified high-affinity mclatonin receptors in the many tissues, e.g., the suprachiasmatic nuclei (SCN), the pars tuberalis (PT), and caudal artery, of various mammals (hamster and rat Ct a]). Co-expression of mt1 and MT2 subtypes in SCN, PT and caudal artery have been further identified by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. The MT2 receptor subtype is believed to mediate phase advance of circadian rhythms generated by a master clock located in SCN, to mediate inhibiting dopamine release in the retina and to mediate vasodilator responses in the rat caudal artery. By contrast, activation of the mtm receptor subtype by melanin appears to mediate vasoconstriction in rat caudal artery. Co-expression of the mt1 and MT2 receptor subtypes in human fetal kidney was determined using RT-PCR. In situ hybridization confirmed the expression of mt1 mRNA. According to basical clinical experiment, it is well documented that melatonin may regulate many biological processes, such as circadian rhythms, sleep, mood, sexual maturation, reproduction, anti-tumor, and immune response and so on. However, precise cellular localization of melatonin receptor subtypes is still an open matter, especially in human. We cannot localize mtt or MT2 receptor subtype to special tissue or target cells although our laboratory have identified many sites for melatonin in various human fetal organs and tissues using 2-(?51j iodomelatomn as a lig~nd. In order to fi.uther investigate the possible implication of melatonin-mediated physiological effects and the signaling transudation mechanisms by melatonin and its 4 receptor subtype, we combined immunohistochemistry, in situ hybridization and RT-PCR techniques to identified and localize the nit1 or MT2 receptor subtypes and its mRNA expression in the human embryonic tissues. Material wad methods Following termination of pregnancy (4? months), we collected hypothalamus, pituitary, retina, pancreas, adrenals, kidneys, liver, spleen, thymus, heart, and lung. For immunohistochcmiszry and in situ hybridization, the tissues were dissected, immersed in 4% paraformaldehyde in addition of 0.1% DEPC, embedded with paraffin wax, and stored in 4 ~ until evaluating. The tissues for RT-PCR were in liquid nitrogen and stored at -70 C until use. The strcptavidin-biotin method was applied to irnmununob...