The long-term objectives of this project are to elucidate the mechanisms by which gonadal steroids affect the GnRH neurons to induce GnRH synthesis and secretion at the time of the LH surge. It is believed that estrogen and progesterone do not act directly on the GnRH neurons to induce the LH surge. These steroids, however, indirectly affect GnRH production and/or release. Experiments are planned that will lead to information about the effects of gonadal steroids on transcription, translation and processing of proGnRH to GnRH.
The specific aims are as follows: The first series of experiments will assess the activation of proGnRH gene expression during proestrus. We will measure heterogenous nuclear RNA (primary transcript) as well as mature proGnRH mRNA in the rat preoptic area at specific times during proestrus. Quantitative polymerase chain reaction (PCR) will be used to amplify and quantify the respective RNAs using intron and exon specific probes. Secondly, we will study the role of estrogen and progesterone in controlling proGnRH gene expression. This will be accomplished by measuring and quantifying heterogenous nuclear RNA (primary transcript) and cytoplasmic mRNA at specific times before and during the estrogen or estrogen/progesterone induced LH surge, using intron and exon specific probes and quantitative PCR. We will, in the same animal model, also perform in situ hybridization to measure the effects of the ovarian steroids on the cellular content and distribution of proGnRH primary transcript and mRNA. Lastly, we will explore the roles of estrogen and progesterone in regulating proGnRH mRNA translation and posttranslational events. We will quantify proGnRH mRNA, proGnRH and GnRH in the POA and the BH at specific times before and during the estrogen/progesterone induced LH surge. These experiments will be performed using HPLC fractionation and specific RIAs to measure proGnRH and GnRH, and solution hybridization and in situ hybridization to measure proGnRH mRNA. In these experiments, we expect to elucidate some of the basic regulatory mechanisms of GnRH neurons. Therefore, important information will be obtained to understand the control of the mammalian reproductive cycle.
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