Grant: $72,500 - National Institutes of Health - May. 1, 2009
100% voted satisfied - 0% voted not satisfied - 2 vote(s) cast
Award Description: Significance of Pituitary Leptin to Gonadotropes - Pituitary leptin changes in a cyclic manner that indicates it may facilitate or permit the secretion of gonadotropins. There are parallel reductions in pituitary leptin and LH during food deprivation and a parallel recovery mediated by glucose. This circumstantial evidence suggests that pituitary leptin may serve as a metabolic gateway to permit gonadotrope function, if the nutritional status is optimal. This proposed pilot study is designed to develop cellular and animal models that test the hypothesis that pituitary leptin from somatotropes is vital for the normal development and responses of gonadotropes to gonadotropin releasing hormone (GnRH). The studies will test the hypothesis with in vitro approaches in Aim 1 in which siRNA for leptin mRNA will be used to knock out pituitary leptin. The pituitary gonadotropes exposed to siRNA will be exposed to gonadotropin releasing hormone or neuropeptide Y, in vitro to determine the effect of the absence of pituitary leptin on basal or stimulated secretory responses. In Aim 2, the hypothesis will be tested by in vivo studies, in which leptin expression is deleted in somatotropes by Cre-loxP technology. To ablate the leptin gene in somatotropes, mutant mice bearing the transgene rGHp-Cre will be crossed with mice that bear loxP flanking exon 3 of the leptin gene. The mutants will include heterozygotes (bearing one deleted allele of leptin exon 3) and homozygotes, bearing two deleted alleles of the leptin exon 3. The deletion mutant mice will be tested for changes in serum leptin, Luteinizing hormone, Follicle Stimulating hormone, Growth hormone, prolactin, Adrenocorticotropin, Thyroid stimulating hormone, insulin, testosterone, estrogen, and leptin. They will also be evaluated for changes in growth rates, weight, the timing of puberty, and fertility. Gonadotrope responses to GnRH will be tested to determine if ablation of pituitary leptin in vitro or in vivo (in somatotropes) affects gonadotrope functions. Gonadotrope development during neonatal states will also be tested in the mutant mice that have leptin excised in somatotropes. This pilot study provides a focused approach to answer the question of the significance of pituitary leptin to gonadotrope functions, which avoids metabolic problems seen with global leptin knockouts that affect gonadotropes and confound interpretation of leptin functions. The pilot study will lay the foundation for the development of future studies that test the effect of pituitary leptin on gonadotrope responses to other regulators (steroids, neuropeptide Y and activin). The ultimate aim of the study will be to determine how leptin supports reproductive competence and how information about malnutrition may be linked with infertility.
Project Description: Aim 1 studies- We have designed the siRNA probes for mouse leptin mRNA and have improved the yield of our cell dispersion protocol for mice pituitaries. We will be setting up these experiments in October. In the meantime, we have developed a number of assays that will be needed to evaluate the results for Aim 1 studies. With a new Luminex Assay system and Multiplex pituitary hormone assay kits, we are now able to quantify serum or culture GH, LH, FSH, ACTH, BDNF, leptin, and TSH. We have also developed all immunolabeling techniques needed to detect changes in the pituitary cell populations, including GH, LH, FSH, Prolactin, and leptin. These were developed by student workers coming in during the summer, funded by this R03 proposal. We have expanded the colonies of mice to allow us to collect sets of cells for the siRNA studies to be done this Fall.Aim 2 studies-The production of the founders bearing floxed leptin exon 3 is ongoing and has reached Phase III. The transgene was designed during Phase I and it was transfected into embryonic stem cells (ES) during Phase II. This resulted in clones bearing the transgene and after Southern Analysis confirmed the presence of the transgene in the cells, the cells are being implanted into blastocysts prior to implanting them into foster mothers. We expect the first chimeric mice to be produced late this year. The planned experiments in the second quarter are as follows: 1) Develop in situ hybridization applications for mouse tissues or pituitary cells; 2) Run the siRNA experiments (Aim 1) to test pituitary leptin’s effects on gonadotrope responses; 3) Receive the heterozygote founders bearing one floxed allele of leptin. Breed to homozygosity and remove the neo cassette (with flipase mice) (Aim 2); 4)Develop qRT-PCR assays for leptin, LH, FSH and GH mRNAs.
Infrastructure Description: n/a
Jobs Summary: Student research technicians for College and/or Medical students ;Two were fulltime for 3.5 months and one was parttime for 1 month. (Total jobs reported: 2)
Project Status: Less Than 50% Completed
This award's data was last updated on May. 1, 2009. Help expand these official descriptions using the wiki below.