Date of Award

Winter 2005

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Stacia A Sower

Abstract

The vertebrate hypothalamic-pituitary-gonadal (HPG) axis is regulated by gonadotropin-releasing hormone (GnRH), a decapeptide that is produced and released from the hypothalamus. At the anterior pituitary, GnRH action is mediated through high affinity binding with the GnRH receptor, a rhodopsin-like seven transmembrane G-protein coupled receptor (GPCR). Interest in the evolution of reproductive physiology has led scientists to study the lamprey, a member of the oldest extant class of vertebrates, the agnathans. The studies presented herein contribute to the field of reproductive neuroendocrinology through developing our understanding of ancestral, or ancestral-like characteristics and mechanisms of the HPG axis. This dissertation is divided into two major components: (1) functional characterization and kinetic studies of the lamprey GnRH receptor (chapters II and III), and (2) an analysis of the lamprey GnRH-III cDNA (chapter IV).

A type II lamprey GnRH receptor was recently identified via cDNA cloning, BLAST analysis and in situ hybridization, however the classification by these homology and expression studies was insufficient. Demonstration of function, through binding capacity or efficacy is a vital and required component of receptor characterization. To this end, a heterologous expression system was developed using COS7 cells transiently transfected with the lamprey GnRH receptor. The lamprey GnRH receptor was shown to be functional as well as lamprey GnRH-III selective based on a series of efficacy and kinetic studies. Ligand dependant internalization was characterized, which was dependant on a motif within the first forty amino acids of the C-terminal tail. Further function and kinetics studies were performed using C-terminal tail truncation mutants.

The objective of the second component of this dissertation was to clone and characterize the cDNA encoding lamprey GnRH-III from eight species of lamprey, which were analyzed by phylogenetics methodology to address the molecular evolution of the GnRH family and the lamprey lineage. The lamprey GnRH-III sequences formed three groups, supporting the current view of the lamprey lineage at the family level. Phylogenetic analysis of these sequences together with 64 previously described GnRH sequences suggested that the lamprey GnRHs are unique, as they group together separately from the three previously described paralogous lineages of the GnRH family.

Download

Share

COinS