The broad goal of this project of the U-54 center is to extend our clinical studies of gonadotrope regulation to the cellular and molecular level so that we may better understand the physiology and pathophysiology of gonadotropin biosynthesis and secretion.
In Specific Aim 1, GnRH regulation of gonadotropin secretion and subunit gene expression will be explored in perifused pituitary cells. The perfusion system allows the administration of precisely controlled patterns of pulsatile GnRH. Specific issues to be addressed are the role of desensitization in the physiological regulation of gonadotropin secretion; the effects of GnRH pulse contour on gonadotropin secretion; the effects of GnRH pulse contour on gonadotropin secretion; second messenger pathway involved in gonadotrope desensitization; the regulation of gonadotropin subunit mRNA levels by different patterns of pulsatile GnRH; and the contributions of transcription and post-transcription factors will be characterized using the alpha gene as a model system. The gonadotrope-derived alphaT3-1 cells will be used as an alternative to primary pituitary cells. DNA response elements will be defined using deletion and site-directed mutants, as well as transfer to heterologous promoters. Active regions will be confirmed by transfection of primary pituitary cells. Transcription factors will be characterized by gel-shift, DNase footprinting, methylation interference and southwestern blot in anticipation of future efforts to clone these factors.
In Specific Aim 3, the mechanisms by which inhibin and activin regulate FSH biosynthesis and secretion will be explored. Specific issues to be addressed include the production of activin by pituitary gonadotropes; the relative contributions of inhibin, activin, and GnRH to the regulation of FSH biosynthesis and secretion; and the contributions of transcription and post-transcriptional processing to the levels of FSHbeta mRNA after treatment with inhibin and activin. Attempts will also be made to establish FSHbeta-expressing clonal cell lines as an alternative to the use of primary pituitary cells.
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