The decapeptide, gonadotropin releasing hormone (GnRH), is the central regulator of reproductive function. It binds to receptors on the gonadotrope cells of the pituitary and stimulates release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Eleven different structural forms of GnRH have now been identified in various animal species. Chimaeric analogues of some of the variant forms of GnRH were synthesized in order to study the functional significance of the most common amino acid substitutions, which occur in positions 5, 7 and 8. Peptide binding affinities for sheep and rat GnRH receptors and potencies in stimulating LH and FSH release from cultured sheep pituitary cells and LH release from cultured chicken pituitary cells were measured. Histidine in position 5 decreased LH releasing potency in chicken cells, but slightly increased receptor binding affinity in rat and sheep membranes. Tryptophan in position 7 had minimal effect on GnRH activity in mammals, but increased LH release in chicken cells. Although differences in the structural requirements of mammalian and chicken GnRH receptors were anticipated, it was also found that rat GnRH receptors exhibited higher affinity for analogues with Tryptophan in position 7, than did sheep GnRH receptors. Substitutions in position 8 revealed the most marked differences in the structural requirements of mammalian and chicken GnRH receptors. Arginine was required for high GnRH activity in mammalian systems, but analogues with neutral substitutions in position 8 were more potent in chicken pituitary cells. The tolerance of position 8 substitutions, combined with the relatively small effects, in chicken cells, of incorporating a D-amino acid in position 6, indicate that the chicken GnRH receptor is less stringent than mammalian receptors in its recognition of peptide conformation. To examine how changes in ligand structure cause changes in receptor binding affinity and receptor activation, it was necessary to know the structures of the GnRH receptors. A protocol was developed for the purification of GnRH binding proteins from detergent-solubilized pituitary membranes, by affinity chromatography. This procedure yielded a protein which migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, but was different from the recently cloned GnRH receptor. To test the proposal that the arginine residue in mammalian GnRH interacts with an acidic receptor residue, eight conserved acidic residues of the cloned mouse GnRH receptor were mutated to asparagine or glutamine. Mutant receptors were transiently expressed in COS-1 cells and tested for decreased preference for Arg⁸-containing ligands by ligand binding and inositol phosphate production. One mutant receptor, in which the glutamate residue in position 301 was mutated, exhibited decreased affinity for mammalian GnRH. The mutant receptor also exhibited decreased affinity for [Lys⁸]-GnRH, but unchanged affinity for [Gln⁸]-GnRH compared with the wildtype receptor, and increased affinity for the acidic analogue, [Glu⁸]-GnRH. This loss of affinity was specific for the residue in position 8, because the mutant receptor retained hiszh affinity for analogues with favourable substitutions in positions 5, 6 and 7. Thus, the Glu³⁰¹ residue of the GnRH receptor plays a role in receptor recognition of Arg⁸ in the ligand, consistent with an electrostatic interaction between these two residues. The Glu³⁰¹ and Arg⁸ residues were not required for the high affinity interactions of conformationally constrained peptides. This indicates that an interaction which involves these two residues may induce changes in the conformation of GnRH after it has bound to the receptor.

The existence of an ultrashort feedback mechanism regulating GnRH secretion has been supported from in vivo and in vitro studies. However, the complex synaptic connections of GnRH neurons with other neural elements made it difficult to determine whether the regulation was mediated by direct actions on the GnRH neurons or through actions on other interneurons. The recent development of the GnRH-secreting neuronal cell line, GT1, provided a model system for the study of neural regulation of a pure population of GnRH neurons. The present studies utilized GT1 -7 cells to investigate whether GnRH (at the level of the nerve terminal) influences the control of its own release. Preliminary studies determined the presence of GnRH mRNA in GT1-7 cells and established a cell culture system for the analysis of secretagogue-induced GnRH release. In this system GnRH release was shown to be spontaneous and was enhanced by the addition of K⁺, L-GLU, forskolin and PMA. Furthermore, K⁺- and forskolin-induced GnRH release was dependent on extracellular Ca²⁺. For the analysis of an ultrashort feedback mechanism, GT1-7 cells were cultured in 6-well plates to near confluence and then incubated in serum-free medium in the presence (1 nM- 1 μM) or absence of GnRH antagonist, Ant 27. Basal, K⁺-and forskolin-induced secretion of GnRH was monitored with antiserum 1076 which does not cross-react with Ant 27 at> 1 μM. Ant 27 treatment increased basal, K⁺- and forskolin-stimulated GnRH release in a dose-dependent manner. Total content was unaffected by 18 h treatment of GT1-7 cells with Ant 27. This suggests that the effects of Ant 27 are at the level of release and not biosynthesis. The presence of GnRH binding sites in the cells was demonstrated with ¹²⁵I-GnRH analog. These findings support the concept that GnRH, acting via autoreceptors, negatively controls its own release.

by Lau Hoi Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 83-96). / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iv / Abbreviations --- p.v / Table of contents --- p.vi / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Endocrine control of normal and abnormal growth of prostate --- p.1 / Chapter 1.1.1 --- Androgen regulation of prostate gland --- p.1 / Chapter 1.1.2 --- Estrogen regulation of prostate gland --- p.4 / Chapter 1.2 --- Gonadotropin-releasing hormone plays a central role in reproduction --- p.6 / Chapter 1.2.1 --- GnRH gene --- p.7 / Chapter 1.2.2 --- GnRH receptor --- p.9 / Chapter 1.3 --- Therapeutic strategies using GnRH analogs to treat prostate cancer --- p.12 / Chapter 1.4 --- Expression of GnRH or its receptor in reproductive tissues --- p.12 / Chapter 1.4.1 --- Expression of GnRH in reproductive --- p.13 / Chapter 1.4.2 --- Expression of GnRH and its receptor in pituitary and reproductive tissues --- p.13 / Chapter 1.5 --- Animal models for the study of prostate cancer --- p.15 / Chapter 1.5.1 --- Nobel rat inducible model --- p.15 / Chapter 1.5.2 --- Androgen dependent rat Dunning prostatic tumor --- p.16 / Chapter 1.5.3 --- Androgen-independent prostatic carcinoma line of Noble rat --- p.18 / Chapter 1.6 --- Aim of study --- p.18 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Origin and supply of Nobel rat --- p.20 / Chapter 2.2 --- Induction of dysplasia in Nobel rat prostate gland by long-term treatment with steroids --- p.20 / Chapter 2.2.1 --- Chemicals --- p.20 / Chapter 2.2.2 --- Preparation of steroid hormone-filled Silastic tubings --- p.20 / Chapter 2.2.3 --- Surgical implantation of Silastic® tubings --- p.21 / Chapter 2.2.4 --- Protocols of hormonal treatments --- p.21 / Chapter 2.3 --- Androgen- dependent Dunning rat prostatic adenocarcinoma --- p.22 / Chapter 2.4 --- Androgen- independent prostatic carcinoma line (ALT) of Noble rat --- p.22 / Chapter 2.5 --- Detection of mRNA expression of gonadotropin- releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.23 / Chapter 2.5.1 --- Preparation of tissue for total RNA extraction --- p.23 / Chapter 2.5.2 --- Total RNA extraction --- p.24 / Chapter 2.5.3 --- Reverse-transcription Polymerase Chain Reaction (RT-PCR) --- p.25 / Chapter 2.5.4 --- Purification of DNA fragments from agarose gels --- p.27 / Chapter 2.5.5 --- Subcloning of DNA into vector --- p.27 / Chapter 2.5.6 --- Nucleotide sequencing --- p.30 / Chapter 2.5.7 --- Southern blot analysis --- p.32 / Chapter 2.5.7.1 --- Southern blotting --- p.32 / Chapter 2.5.7.2 --- Preparation of α-32P-dCTP labelled GnRH probe --- p.32 / Chapter 2.5.7.3 --- Hybridization --- p.33 / Chapter 2.6 --- Detection of mRNA expression of gonadotropin-releasing hormone receptor (GnRH-R) in normal and neoplastic rat prostates --- p.34 / Chapter 2.6.1 --- Cloning of GnRH-R cDNA and synthesis of its probe --- p.34 / Chapter 2.6.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates by Southern blot --- p.36 / Chapter 2.6.3 --- Detection of GnRH receptor mRNA expression in Dunning tumor --- p.37 / Chapter 2.6.4 --- Detection of the GnRH receptor mRNA expression in AIT tumor by RT-PCR --- p.37 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Detection of mRNA expression of gonadotropin-releasing hormone (GnRH) in normal and neoplastic rat prostates --- p.38 / Chapter 3.1.1 --- Reverse -transcription Polymerase Chain Reaction (RT-PCR) --- p.38 / Chapter 3.1.2 --- Purification of DNA fragments amplified by PCR from the agarose gel --- p.38 / Chapter 3.1.3 --- Subcloning of DNA into vector --- p.39 / Chapter 3.1.4 --- Nucleotide sequencing --- p.39 / Chapter 3.1.5 --- Southern-blot analysis --- p.39 / Chapter 3.2 --- Detection of gonadotropin-releasing hormone receptor mRNA expression in normal and neoplastic rat prostates --- p.40 / Chapter 3.2.1 --- Cloning of gonadotropin-releasing hormone receptor (GnRH) cDNA and synthesis of probe from the normal Noble rat pituitary gland --- p.40 / Chapter 3.2.2 --- Detection of GnRH receptor mRNA expression in normal and dysplastic Nobel rat prostates --- p.42 / Chapter 3.2.3 --- Detection of GnRH receptor mRNA expression in rat Dunning tumor by PCR --- p.43 / Chapter 3.2.4 --- Detection of GnRH receptor mRNA expression in AIT tumor --- p.43 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Detection of mRNA expression of gonadotropin-releasing releasing hormone(GnRH) in normal and neoplastic rat prostates --- p.69 / Chapter 4.1.1 --- Expression of GnRH mRNA in normal Nobel rat prostate gland --- p.69 / Chapter 4.1.2 --- Expression of GnRH mRNA in dysplastic Nobel rat prostate --- p.71 / Chapter 4.1.3 --- Expression of GnRH mRNA in androgen-dependent rat Dunning prostatic tumor --- p.72 / Chapter 4.1.4 --- Expression of GnRH mRNA in AIT tumor --- p.74 / Chapter 4.2 --- Detection of GnRH receptor in normal and dysplastic rat prostates --- p.75 / Chapter 4.2.1 --- Negative expression of GnRH receptor in normal and dysplastic Nobel in rat prostates --- p.75 / Chapter 4.2.2 --- Positive expression of GnRH receptor mRNA in rat Dunning tumor --- p.77 / Chapter 4.2.3 --- Negative expression of GnRH receptor mRNA in ALT tumor --- p.78 / Chapter Chapter 5 --- Summary and Conclusions --- p.80 / References --- p.83