THE GENERATION OF AN ARRAY OF CLONAL, IMMORTALIZED CELL MODELS FROM THE RAT HYPOTHALAMUS: ANALYSIS OF MELATONIN EFFECTS ON KISSPEPTIN AND GONADOTROPIN-INHIBITORY HORMONE NEURONS

Significant information on reproductive function has been generated based on the rat model, including many seminal discoveries. Yet little is known about the molecular and cellular events involved in control of reproductive function, mainly due to the pervasive lack of cell models from rat. We have therefore generated a wide array of cell lines using primary cell culture from the rat hypothalamus. Immortalization of the primary cells was achieved through retroviral transfer of T-antigen, followed by selection with geneticin. The mixed cell populations were subcloned and each clonal cell line was analyzed for expression of specific cellular markers. Each line has a distinct phenotypic profile, with expression of key neuroendocrine markers. We have functionally analyzed two clonal cell lines, rHypoE-7 and rHypoE-8, for hormones implicated in the control of gonadotropin-releasing hormone neuronal function through melatonin, specifically kisspeptin (KISS) and RF-amide-related peptide-3 (RFRP-3, the mammalian ortholog of the avian gonadotropin-inhibiting hormone, GnIH). We detected functional melatonin receptor activity, as each cell line exhibited inhibition of forskolin-stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation. Upon treatment with 10 nM melatonin, we found that KISS gene expression was decreased in the rHypoE-8 cell line, while RFRP-3 was increased in the rHypoE-7 cell line. These results are in accordance with the differential regulatory functions of these two peptides, particularly on GnRH neuronal control. These cell lines will serve as novel tools for the analysis of the cellular and molecular mechanisms involved in hypothalamic control of a number of physiological processes described in the rat animal model. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.