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Neuroendocrine Regulation of Reproduction in Fish

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Neuroendocrine Regulation of Reproduction in Fish Neuroendocrine Regulation of Reproduction in Fish A Thesis Submitted to the College of Graduate and Postdoctoral Studies In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Veterinary Biomedical Sciences Western College of Veterinary Medicine University of Saskatchewan Saskatoon By Jithine Jayakumar Rajeswari © Copyright Jithine Jayakumar Rajeswari, August 2020. All rights reserved. Permission to use and disclaimer statement In presenting this thesis/dissertation in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for scrutiny. I further agree that permission for copying this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis/dissertation work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis/dissertation or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use, which may be made of any material in my thesis/dissertation. The company/corporation/brand name and website were exclusively created to meet the thesis and/or exhibition requirements for the degree of Postgraduate studies at the University of Saskatchewan. Reference in this thesis/dissertation to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation, or favoring by the University of Saskatchewan. The views and opinions of the author expressed herein do not state or reflect those of the University of Saskatchewan and shall not be used for advertising or product endorsement purposes. Requests for permission to copy or to make other uses of materials in this thesis/dissertation in whole or part should be addressed to: Head of the Department of Veterinary Biomedical Sciences Western College of Veterinary Medicine University of Saskatchewan, S7N 5B4, Canada. OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan S7N 5C9 Canada i Abstract Fish is an essential source of food, and it is important to improve fish yield in aquaculture to enhance food production and sustainability. Several lines of research are in progress to increase reproductive capacity in cultured fishes. Hormones play a critical role in fish reproductive success and endocrine approaches are being successfully tested in stimulating fish reproduction. In this context, my thesis research aimed to further characterize three relatively new orphan ligands and their role in the regulation of reproductive hormones and oocyte maturation in fish. Phoenixin-20 (PNX-20) is a newly identified peptide with endocrine-like functions in mammals. Nesfatin-1 is an 82 amino acid multifunctional peptide with hormone-like actions in vertebrates. Nesfatin-1-like peptide (NLP) is 77 amino acids long, resembles nesfatin- 1, and share similar biological actions. Based on the available information from mammals and fish, I hypothesized that PNX is stimulatory, while both nesfatin-1 and NLP are inhibitory to reproductive hormones and oocyte maturation in teleosts. I tested this hypothesis using goldfish and zebrafish, two well-characterized models in neuroendocrinology. qPCR-based gene expression results indicated a widespread distribution of PNX in central and peripheral tissues of zebrafish. Immunohistochemical localization of PNX and its putative receptor (SREB3) in the gonads suggested a role of PNX in zebrafish reproduction. In vivo administration of PNX-20 upregulated reproductive regulatory hormonal transcripts in the hypothalamus and gonads of zebrafish. In vitro incubation of PNX-20 upregulated all vitellogenin transcripts in zebrafish liver cells. In addition, incubation of ovarian follicles with PNX-20 promoted oocyte maturation in zebrafish. Together, my research outcomes supported my hypothesis that PNX-20 promotes reproductive functions in zebrafish, by positively influencing hormones, vitellogenesis and oocytes. For both nesfatin-1 and NLP studies, I used male and female goldfish as a research model. Single intraperitoneal (IP) injection of synthetic gfnesfatin-1 and gfNLP (50 ng/g body weight) suppressed several reproductive regulatory hormonal mRNAs in both sexes. These include the gonadotropin-releasing hormones (hypothalamus), kisspeptin system (multiple tissues), gonadotropin β subunits (pituitary), gonadotropin receptors and genes involved in sex steroidogenic synthetic pathway (gonads). In addition, both peptides upregulated the expression of several reproductive suppressors in goldfish, including the gonadotropin inhibitory hormone and anti-Müllerian hormone. Both nesfatin-1 and NLP decreased serum testosterone and ii estradiol levels in male and female goldfish at 60 minutes post-injection. Incubation of ovarian follicles with NLP suppressed oocyte maturation in zebrafish. Overall, my results suggested that both nesfatin-1 and NLP negatively influence aspects of reproductive biology in fish by suppressing the expression of critical hormonal mRNAs, gonadal steroids, and oocyte maturation. The results supported my hypotheses about nesfatin-1 and NLP. While limitations exist, my research identified PNX-20 as a positive regulator, and both nesfatin-1 and NLP as negative regulators of reproductive biology of fish. This is the major contribution of my thesis research. Future research to extend these findings to male fish and commercially cultured fish, and to understand the mechanism of action of these hormones is essential. iii Acknowledgments I would like to extend my sincere thanks and gratitude to my supervisor Dr. Suraj Unniappan for his unconditional support, encouragement and help that he provided throughout my research program. I would also like to extend my sincere thanks to my graduate chairs Drs. Ali Honaramooz, Daniel McPhee, Gregg Adams and my committee members Drs. David Janz, Dinesh Dadarwal and Som Niyogi for their constant support and valuable inputs and advice during my Ph.D. research studies. I would also like to thank Dr. Chun Peng for her acceptance to serve as the external examiner for my thesis. I wish to express my gratitude and thanks to all my fellow lab members. I also extend my sincere thanks to Cindy Pollard (VBMS Graduate Program Coordinator), Cheryl Hack (Administrative Assistant), animal care unit technicians, endocrine lab members (Susan Cook, Kim Hue Tran and Danielle Carriere) and animal order desk of the University of Saskatchewan for their help and support for the successful completion of my program of study. The generous funding for this research was supported by the Discovery Grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada and the University of Saskatchewan Centennial Enhancement Chair in Comparative Endocrinology to Dr. Suraj Unniappan (SU). Infrastructure support was provided by the John Evans Leaders Fund from the Canada Foundation for Innovation, and an Establishment grant from the Saskatchewan Health Research Foundation (SHRF) to SU. The facilities and research infrastructure provided by the Department of Veterinary Biomedical Sciences and the University of Saskatchewan are deeply appreciated. iv Permission to reproduce A substantial part (data chapters) of this thesis has been published or under the submission process to consider for publication (introduction chapter) in peer-reviewed journals. All published parts of the thesis are distributed under the terms of the Creative Commons CC BY license, which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. The copyright rules of and Nature Publishing Group (Scientific Reports; Chapter 2), Bioscientifica on behalf of the Society for Reproduction and Fertility (Reproduction: Chapter 3), Oxford Journals (Biology of Reproduction; Chapter 4) allow reproduction of published work in the author’s thesis. v Dedication I dedicate my Ph.D. thesis to everyone who has helped, guided and assisted me throughout my graduate studies. First, I would like to dedicate this thesis to my family members, who have always encouraged me to pursue my ambitions to date. This thesis is dedicated to my father, mother, and brothers who are eagerly waiting for my return home for the past three and a half years. Next, I would like to dedicate this thesis to my supervisor Dr. Suraj Unniappan for his continued guidance, encouragement and support throughout my graduate studies. You always serve as an inspiration to me and corrected me for the endless mistakes that I made with my research and writing. Your trust in me during the research program helped me in the completion of my program of study. I am deeply appreciative and thankful for your advice as a mentor, friend, science family member and a well-wisher over the past years at the University of Saskatchewan. You will always be a role model in my life, you and your family will be remembered in my whole life. I am privileged to complete my Ph.D. degree from your lab and also grateful to have worked under various operating grants that you have
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