- Title
- Development of a novel injectable chemical agent for the sterilisation of cats and dogs
- Creator
- Fraser, Barbara Anne
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2022
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- We have a worldwide problem with free-roaming cats and dogs that imposes social, economic, and environmental costs as well as impacting human health. A major source of free-roaming animals is the dumping of unwanted animals, from uncontrolled breeding, into the environment. The surgical sterilisation of dogs and cats has been used to prevent unwanted breeding for decades. However, surgical sterilisation is often inconvenient, comes with some risk, and is relatively costly. Indeed, the expense of surgical sterilisation remains as a major impediment to many pet-owners as well as organisations that host humane programs for neutering and rehoming of free-roaming animals. Immunocontraceptive and chemosterilant strategies have been investigated as alternatives to surgical interventions and a small number of these products have entered the market. However, a limitation of these agents is that they require periodic administration, or multiple doses, to provide contraception or temporary infertility. A sterilisation agent that could be administered in a single injection would not only eliminate those risks imposed by surgery but also be a much more cost effective solution to a worldwide problem. The targeting of a cell ablation agent to the non-renewable cells of the testes using a cell selective peptide may present one solution to this problem. This approach would permit targeted delivery of the chemosterilant to the immediate vicinity of the target cell, thereby ensuring both the efficacy and safety of the approach. This research project was formulated to develop such peptides and to explore the utility of this novel approach to sterilisation. Initially, to identify appropriate targets expressed by the cells within the adult testes that, once ablated, would result in permanent infertility, we reviewed the current literature (Chapter 1). Additionally, we sought to discover a peptide that selectively binds to gonocytes. These cells are the embryonic germ cell precursors and, if they could be targeted and ablated in uteri, it would mean that the offspring would be infertile at birth. As these cells do not express any unique receptors, we used a process of ‘phage panning and identified several bacteriophage clones that exhibited selective binding to these cells. The corresponding synthetic peptides, however, did not retain binding capability. This led us to modify the ‘phage itself with the cell ablation agent, menadione (Chapter 2). Furthermore, in a proof-of-concept study, the menadione-modified ‘phage was able to significantly decrease the vitality of gonocytes in vitro. In the adult testes, there are two glycoprotein hormone receptors, follicle-stimulating hormone receptor and luteinising hormone receptor, which are uniquely expressed. Based on the crystal structure of the glycoprotein hormone-receptor complex we designed peptides, (Chapters 3 and 4), to target adult Sertoli and Leydig cells. These peptides exhibited binding capability in vitro and localised to the testes in vivo. When cytotoxic agents were covalently attached to these peptides we observed an increase in oxidative damage and extensive germ cell apoptosis. Given the selective binding capabilities of these peptides, we wondered if it was possible to develop peptides that target other types of receptors such as the anti-Mullerian hormone receptor, belonging to the TGFβ superfamily of G-coupled protein receptors. Whilst, the anti-Mullerian hormone receptor is expressed in the testes, it is also expressed, albeit at a much lower level, by GnRH-secreting neurones in the hypothalamus. The synthetic AMH peptide (Chapter 5) selectively targeted Leydig cells in vitro and also localised to the testes in vivo. However, when applied in vivo, an AMH peptide-menadione construct produced no discernible effect on spermatogenesis or the oxidative status of the testes. Although, notably, the treated males were infertile. The data presented within this thesis confirms that we are able to intelligently design peptides that selectively bind to testis-specific cell receptors. Additionally, we have confirmed that bacteriophage, discovered by ‘phage panning, can be modified to selectively deliver cell ablation reagents to any cell of interest. Although we have not yet been able to permanently delete the target cells within the testes in vivo, we propose that peptide instability and low solubility may have limited the amount of cytotoxic agent that reached the target cells. Encapsulation of these agents within nanoparticles that have been modified with our cell-specific targeting peptides may present an ideal solution. The utility of targeting nanoparticles to the male reproductive system is discussed in a review article in Chapter 6. Additionally, further justification for the use of nanoparticles to enhance the efficacy of our peptide-cell ablation reagents is detailed in the final discussion chapter, Chapter 7. Taken together, the novel targeting reagents that have been discovered and developed within this project, provide the basis for the future development of an injectable sterilisation agent.
- Subject
- sterilisation; chemical; injectable agent; dogs and cats
- Identifier
- http://hdl.handle.net/1959.13/1513700
- Identifier
- uon:56760
- Rights
- Copyright 2022 Barbara Anne Fraser
- Language
- eng
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