Animal Science Department

 

Date of this Version

8-2005

Comments

A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Animal Science, Under the Supervision of Professor Brett R. White
Lincoln, Nebraska: August, 2005
Copyright 2005 Emily A. McDonald

Abstract

The interaction between GnRH and its receptor on gonadotropes within the anterior pituitary gland represents a central point for regulation of reproduction. Additionally, the close proximity of the porcine GnRHR gene to a quantitative trait locus for ovulation rate makes it a positional candidate for genes influencing ovulation rate. We isolated the GnRHR gene promoter from three lines of swine (Control, Index and Meishan) with different ovulation rates. Transient transfection of gonadotrope-derived αT3-1 cells with vectors containing approximately 5000 bp of GnRHR gene promoter fused to the cDNA encoding luciferase resulted in a 2- to 4-fold difference in luciferase activity (Index and Meishan, respectively) over the Control promoter. Reduction of the full-length promoter to 1900 bp, mutation of the steroidogenic factor-1 binding element within the GnRHR gene promoter for all three breeds, and promoter “swap” experiments using the -1900/-1400 bp region of 5’ flanking sequence failed to eliminate the differences in promoter activity among lines. Divergence in promoter activity among lines was ablated following reduction of the promoter from 1400 to 1000 bp, indicating the presence of a breed-specific element within this region. Sequence analysis of this region revealed five single bp substitutions among lines. Electrophoretic mobility shift assays (EMSA) using nuclear extracts from αT3-1 cells demonstrated unique binding for the oligonucleotide corresponding to -1245/-1225 bp of the Meishan promoter compared to the Control/Index promoter, with competition for binding by NF-κB and Sp1 consensus oligonucleotides. Furthermore, EMSA using an antibody directed against the p65 subunit of NF-κB resulted in a supershift. However, an Sp1-specific antibody failed to supershift the DNA:protein complex. Transient transfection of αT3-1 cells with vectors containing the full-length Meishan promoter harboring mutations spanning this region reduced luciferase activity compared to native sequence. Our results demonstrate that increased transcriptional activity of the Meishan GnRHR gene promoter is conferred in part via the novel utilization of NF-κB within gonadotropes.

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