The overall objective of this research program is to elucidate the molecular mechanisms involved in both the nucleus and the cytoplasm which are responsible for the alterations in mRNA levels of three important reproductive hormones, proGnRH, beta- LH and beta-FSH. An interdisciplinary approach will be taken using the tools of molecular genetics and histology in a well characterized rodent reproductive model system, that of castration and estrogen/androgen replacement in female and male. There are two aspects to the proposal which can be divided upon anatomical lines. The first is the study of gonadotropin releasing hormone (GnRH) gene expression in the preoptic area of the rat. Studies will be performed using in situ hybridization techniques, solution hybridization/nuclease protection techniques and transcription run on assays to determine how the proGnRH gene is regulated at the molecular level in this model system. In addition, the proGnRH gene promoter will be fused to a chloramphenocol acetyl-transferase reporter gene and used in subsequent gene transfection studies into various types of cultured cells to determine which second messenger systems and/or steroids are responsible for regulation of proGnRH gene expression. In situ hybridization studies in the proGnRH expressing region of the preoptic area of the rat will be performed using exon and intron specific probes to determine levels of cytoplasmic mRNA and nuclear hnRNA gene expression, respectively. Secondly, studies will also be performed in the pituitary to analyze the molecular mechanisms of beta-FSH gene expression using the castration with steroid replacement model in male and female rats as well as in primary cultures of anterior pituitary cells. Studies will be performed in primary cultures using GnRH/GAP/inhibin/sex steroids to determine which of these factors are responsible for the molecular mechanisms of regulation of beta-FSH gene expression. Analyses will be performed by transcription run on assays as well as solution hybridization/nuclease protection assays using intron/exon junctional probes developed for the beta-FSH gene in rat. In situ hybridization studies will also be performed on beta-FSH and beta-LH using mRNA and intron specific probes in the castration plus estrogen/androgen replacement paradigm in male and female using pituitary gland sections. Finally, studies on beta-LH gene expression will be extended to analyze the molecular mechanism by which the non-transcriptional effects of GnRH elicits increases in beta-LH gene expression. If regulation of beta-FSH gene expression is also found to be non-transcriptional, similar studies will be performed for beta-FSH. These studies will focus upon electron microscopic in situ hybridization studies analyzing the subnuclear level of expression of beta-LH hnRNA as well as studies on regulation of polyadenylation of beta-LH hnRNA. These studies should provide a more detailed understanding the basic molecular mechanisms involved in the regulation of these extremely important reproductive peptide/protein hormones.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK039029-02
Application #
3238670
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1988-09-01
Project End
1991-08-31
Budget Start
1989-09-01
Budget End
1990-08-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10029
Wu, T J; Mani, Shaila K; Glucksman, Marc J et al. (2005) Stimulation of luteinizing hormone-releasing hormone (LHRH) gene expression in GT1-7 cells by its metabolite, LHRH-(1-5). Endocrinology 146:280-6
Jakubowski, M; Lenoir, V; Jimenez-Linan, M et al. (2002) Long-term effects of the mammary carcinogen 7,12-dimethylbenz(a) anthracene on hypothalamic gonadotropin-releasing hormone and its pituitary receptor gene expression, during the promotion stage, in female Sprague-Dawley rats. Breast Cancer Res Treat 73:23-9
Wu, T J; Gibson, M J; Roberts, J L (2000) Effect of N-methyl-D,L-aspartate (NMA) on gonadotropin-releasing hormone (GnRH) gene expression in male mice. Brain Res 862:238-41
Sun, Y; Gore, A C; Roberts, J L (1998) The role of calcium in the transcriptional and posttranscriptional regulation of the gonadotropin-releasing hormone gene in GT1-7 cells. Endocrinology 139:2685-91
Gore, A C; Yeo, T T; Ho, A et al. (1997) Post-transcriptional regulation of the gonadotropin-releasing hormone gene in GT1-7 cells. J Neuroendocrinol 9:271-7
Yeo, T T; Gore, A C; Jakubowski, M et al. (1996) Characterization of gonadotropin-releasing hormone gene transcripts in a mouse hypothalamic neuronal GT1 cell line. Brain Res Mol Brain Res 42:255-62
Gore, A C; Wu, T J; Rosenberg, J J et al. (1996) Gonadotropin-releasing hormone and NMDA receptor gene expression and colocalization change during puberty in female rats. J Neurosci 16:5281-9
Gore, A C; Ho, A; Roberts, J L (1995) Translational efficiency of gonadotropin-releasing hormone messenger ribonucleic acid is negatively regulated by phorbol ester in GT1-7 cells. Endocrinology 136:1620-5
Yu, K L; Yeo, T T; Dong, K W et al. (1994) Second messenger regulation of mouse gonadotropin-releasing hormone gene expression in immortalized mouse hypothalamic GT1-3 cells. Mol Cell Endocrinol 102:85-92
Gore, A C; Roberts, J L (1994) Regulation of gonadotropin-releasing hormone gene expression by the excitatory amino acids kainic acid and N-methyl-D,L-aspartate in the male rat. Endocrinology 134:2026-31

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