Project: Research project

Project Details


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.
Effective start/end date9/1/883/31/99


  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $192,474.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health


  • Medicine(all)


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