Vickers, Elaine Denise2024-08-152024-08-1520022002https://hdl.handle.net/1828/19997Gonadotropin-releasing hormone (GnRH) is a small peptide hormone that is essential for reproduction in vertebrates and some invertebrates. GnRH is synthesized in the brain and acts on the pituitary gland stimulating the synthesis and release of gonadotropins into the blood. These gonadotropins circulate to the gonads where they stimulate gametogenesis and the synthesis and release of reproductive steroid hormones such as estrogen and testosterone. Seventeen GnRH forms have been isolated and named after the species in which they were first identified. The structure of this ten-amino-acid hormone is highly conserved among vertebrates, except for the four amino acids located in positions five to eight. To date, species contain either two or three GnRH forms. Lake whitefish was chosen as the study animal because it is a representative of the earliest subfamily of salmonids ( Coregoninae). All salmonids studied so far express only two GnRH forms: salmon (s)GnRH and chicken (c)GnRH-II. However, our previous protein work done on lake whitefish identified three GnRH forms : sGnRH, cGn.RH-II, and the third form called whitefish (wf)Gn.RH. There are three objectives in this study: ( 1) to obtain the cDNA sequences of each form of GnRH in lake whitefish, (2) to determine the releasing potential of wfGnRH on pituitary cells, and (3) to detem1ine the location of each GnRH form in the whitefish brain using in situ hybridization. An examination of the structure (both protein and DNA), specific brain function, and location of each GnRH form within a species provides clues to the evolution of the hormone. The cDNA sequence of each form of GnRH in lake whitefish was isolated using 5' and 3' rapid amplification of cDNA ends (RACE) and reverse transcription polymerase chain reaction (RT- PCR). Each sequence consists of a 5' untranslated region (UTR), signal peptide, GnRH-encoding region, cut site, GnRH-associated peptide (GAP), and 3 'UTR. The lake whitefish cGnRH-II precursor has 88% nucleotide and 79% amino acid identity with rainbow trout cGnRH-II. The lake whitefish sGnRH precursor shows 93% nucleotide and 79% amino acid identity when compared to Atlantic salmon sGnRH. As seen in other vertebrate GnRH sequences, the regions that are most conserved encode the GnRH peptide and the cut site. The novel wfGnRH cDNA reveals little nucleotide sequence identity to any known GnRH sequences but shows 60% amino acid identity with Haplochromis burtoni seabream (sb)GnRH. This suggests the ancestral forms ofwfGnRH and sbGnRH were closely related. Whitefish GnRH is biologically active, as it stimulated cultured pituitary cells to increase the expression of the a-subunit of gonadotropin mRNA. This suggests that wfGnRH has a role in gonadotropin-release from the pituitary in lake whitefish. Also, cGnRH-II had some biological activity, but is unlikely to cause gonadotropin-release in vivo due to its location in the midbrain. Localization of brain GnRH by in situ hybridization revealed an overlap of regions expressing sGnRH or wfGnRH in the anterior brain from olfactory bulb to preoptic area (POA). The only form located in the hypothalamus was wfGnRH and in the midbrain was cGnRH-II. It is concluded that wfGnRH plays the main role in gonadotropin release in lake whitefish and that sGnRH may have a minor role. Also, both sGnRH and cGnRH-II probably have neuromodulatory roles as well.121 pagesAvailable to the World Wide WebThesis