Thesis (M. Phil.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 193-235).

Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 70-85).

Thesis (M. Phil.)--University of Hong Kong, 1997. / Includes bibliographical references (leaf 119-120).

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 56-69).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1981. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 125-131).

Thesis (M.S.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vi, 84 p. : ill. Vita. Includes abstract. Includes bibliographical references (p. 76-83).

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 44-48).

Two forms of gonadotropin-releasing hormone (GnRH) were extracted from brain-pituitary tissues of two species of Thai catfish, Clarias inacrocephalus and C. batrachus. The peptides were detected using high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA), The amino acid sequences of both forms were determined using Edman degradation. One form of GnRH in the brain-pituitary tissues of the Thai catfish was novel, whereas the second form of GnRH was identical to chicken GnRH-XI. The presence of the N-terminal pGlu residue in both peptides was established by digestion with pyroglutamyl aminopeptidase. In addition, catfish GnRH-I was studied by mass spectrometry. The localization of these two peptides was determined to be in the discrete brain areas and in the pituitary of female and male catfish, C. macrocaphalus, using heterologous and homologous radioimmunoassays. Initially a heterologous RIA was used with mammalian GnRH as iodinated tracer and standard, and an antiserum made against salmon GnRH. Catfish GnRH-I (novel form) was found in most areas of the female and male brain with the highest content and concentration in the female pituitary and in the male hypothalamus,, Catfish GnRH-II (chicken GnRH-II) was found with the highest content in the female Cerebellum and highest concentration in the pituitary, catfish GnRH-II (chicken GnRH-II) was found with the highest content and concentration for males in the same area, hypothalamus. Additionally, a homologous RIA was used with catfish GnRH-II (chicken GnRH-II) as iodinated tracer and standard, and an antiserum prepared against chicken GnRH-II. Catfish GnRH-II was detected with the highest content and concentration in the cerebellum of both sexes. These values are higher than the results obtained in the heterologous assay. The location of catfish GnRH-I suggests that it plays a role in regulating the release of gonadotropin from the pituitary since the high content and concentration of this immunoreactive GnRH are detected in the hypothalamus and pituitary gland. In contrast, catfish GnRH-II may act as a neurotransmitter in the catfish brain, in particular in tha cerebellum where a high content and concentration of immunoreactive GnRH are detected. Physiological in vivo studies indicate that catfish GnRH-II is more effective than catfish GnRH-I and other forms of GnRH such as mammalian and dogfish GnRH for induction of ovulation in catfish, C. macrocephalus. Eight GnRH analogs had varying potencies for the induction of ovulation, but the most effective forms were two forms of catfish GnRH-II (chicken GnRH-II) modified in positions six and ten. In vitro studies found that catfish GnRH-I not only causes the release of gonadotropin but also the release of growth hormone in a dose-dependent manner. The primary structures of the two catfish GnRH peptides are important for understanding the evolution of this family peptide. The novel catfish GnRH shows that only positions 5, 7 and 8 vary in the GnRH molecule in jawed vertebrates, whereas catfish GnRH-II provides direct evidence that the structure of this GnRH is conserved in teleosts. / Graduate

Growth hormone-releasing hormone (GHRH) is a neuropeptide which stimulates the synthesis and release of growth hormone (GH) from the pituitary gland. The primary structure of this peptide has been identified in 7 mammalian species while the gene has been isolated from only rat and human. GHRH is a member of the glucagon superfamily which includes vasoactive intestinal peptide (VIP), glucagon, secretin, peptide histidine methionine (PHM), gastric inhibitory peptide (GIP) and a recently identified peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The evolutionary relationships of this superfamily are not well understood because the gene structure of these molecules has only been identified in mammals. This thesis presents immunological evidence of a GHRH-like molecule, and identifies a GHRH/PACAP precursor and gene that encode two peptides, a GHRH-like molecule structurally related to PACAP-related peptide (PRP) and PACAP, from sockeye salmon brain. An antiserum directed against a topologically assembled epitope of human GHRH 1-44 (NH2) was produced and used to develop a radioimmunoassay for detection of immunoreactive GHRH in brain extracts of salmon, guinea pig, mouse and alligator. An immunoreactive GHRH from salmon brain extracts with a retention time on reverse phase high-performance liquid chromatography (HPLC) distinct from human GHRH was present. In alligator, the same antiserum also detected a GHRH-like molecule. During attempts to purify alligator GHRH, alligator brain neuropeptide Y (NPY) was identified. Alligator NPY is the first non-mammalian vertebrate to have 100% sequence identity to human NPY. The sequence identity between alligator and human NPY suggests that this sequence is the same as the ancestral amniote NPY. Molecular biological techniques were used for the structural identification of the salmon GHRH-like molecule and another peptide. The salmon GHRH/PACAP precursor contains 173 amino acids and has dibasic and monobasic processing sites for cleavage of a 45 amino acid GHRH-like peptide with a free acid C-terminus and a 38 amino acid PACAP with an amidated C-terminus. The salmon GHRH-like peptide has 40% amino acid sequence identity with the human GHRH and 56% identity with human PACAP-related peptide (PRP). Salmon PACAP-38 is highly conserved (89%) with only 4 amino acid substitutions compared with the human, ovine and rat PACAP-38 peptides. Nucleotide sequencing and use of the polymerase chain reaction show the exon/intron organization of the salmon GHRH/PACAP gene to be similar to the human PACAP gene. Unlike the mammalian PACAP genes, the salmon gene produces two precursor forms by post-transcriptional processing. One form is similar to the mammalian PACAP precursors, while the second form is shorter due to the excision of exon 4. This deletion results in the loss of the first 32 amino acids of the GHRH-like peptide from the precursor. The high sequence identity and structural organization between the GHRH(PRP)/PACAP and PHM(PHI)/VIP genes suggest a duplication event occurred in an ancestral gene after the divergence from the other glucagon superfamily members. GHRH in mammals may have arisen by gene duplication after the divergence of the tetrapods from the other vertebrate lines. Thus, GH in fish may be controlled by the two molecules, GHRH-like peptide and PACAP, located on a single GHRH/PACAP gene. / Graduate

The growth and development of an organism requires the coordinated actions of many factors. During development individual cells undergo proliferation, migration and differentiation to form the adult organism. Two structurally related members of the glucagon superfamily, growth hormone releasing hormone (GRF) and pituitary adenylate cyclase-activating polypeptide (PACAP), are thought to modulate vertebrate development. In mammals, GRF modulates the development of pituitary somatotrophs and the release of fetal growth hormone. In contrast, PACAP appears to have a more general role during development. PACAP may be involved in the patterning of the embryonic axis and in the development of the neural tube. The objectives of my study were to isolate GRF, PACAP and their receptors from the zebrafish, characterize their expression in the developing embryo and adult embryo and examine the role of PACAP during brain development. To study the role of GRF and PACAP, I isolated a genomic clone encoding the GRF and PACAP peptides from the zebrafish genomic library and characterized its gene copy number and adult tissue expression pattern. The GRF-PACAP gene isolated from the zebrafish was comprised of five exons with the GRF peptide encoded on the fourth exon and the PACAP peptide encoded on the fifth exon. This gene structure is similar to that found in other non-mammalian vertebrates and supports the hypothesis that the gene duplication leading to the encoding of the GRF and PACAP peptides on separate genes occurred later in evolution. In addition, the zebrafish genome was found to contain only one copy of the GRF-PACAP gene. The GRF-PACAP gene was widely expressed in the adult zebrafish in tissues developmentally derived from all three germ layers, suggesting that the gene may be widely expressed in the embryo as well. To examine the functional significance of the co-expression of GRF and PACAP in zebrafish, I isolated the GRF and PACAP receptors and characterized their expression pattern. I isolated three distinct cDNAs from zebrafish encoding the GRF receptor, the PACAP specific PAC1 receptor and the shared vasoactive intestinal peptide/PACAP receptor VPAC1. In addition, four isoforms of the PAC1 receptor were isolated from zebrafish including a novel isoform found in the gill. All three receptors were widely expressed in adult zebrafish and receptors for both GRF and PACAP were found in most tissues. This indicates that GRF and PACAP may modulate each other’s function. To determine the developmental role of GRF and PACAP, I characterized the expression pattern of the GRF-PACAP gene and the GRF, PAC1 and VPAC1 receptors in the zebrafish embryo. The GRF and PAC1 receptors are the earliest to be expressed in development starting at the cleavage stage. Later, the GRF-PACAP gene and the VPAC1 receptor are first expressed at the late blastula/early gastrula stage in the zebrafish and are expressed throughout the developmental period. Strong expression of the GRF, PACAP and their receptors during mid gastrulation indicates that these peptides may be involved in modulating the formation of the embryonic axis. During the segmentation period the GRF-PACAP gene is widely expressed in the zebrafish embryo and the PAC1 receptor short and hop isoforms are differentially expressed. Therefore, PACAP may regulate cell cycle exit or cell proliferation through activation of different PAC1 receptor isoforms during the segmentation stage. In the subsequent pharyngula period, the GRF-PACAP transcript is localized mainly to the hatching gland. However, expression is seen also in tissues that undergo differentiation during this stage. Therefore, the timing of the expression of the GRF-PACAP gene indicates that it may be involved in early patteming events and promoting cell cycle exit prior to differentiation. To investigate the role of GRF and PACAP in the developing brain, I localized the expression of GRF, PACAP and the PAC1 receptor in neuroblasts derived from an embryonic day 3.5 chick. PACAP was found to stimulate the cAMP pathway in these cells, indicating that PACAP may modulate brain development. This work indicates that GRF and PACAP play an important role in vertebrate development. / Graduate