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Differential regulation of gonadotropins

Periodic Reporting for period 1 - GTHREG (Differential regulation of gonadotropins)

Reporting period: 2015-09-01 to 2017-08-31

As wild fisheries catches are declining, they can no longer sustain the world needs and aquaculture is becoming a more prominent supplier of fish for human consumption. In the last three decades global fish production from aquaculture has expanded by almost 12 fold at an average annual rate of almost 9%, making aquaculture one of the fastest growing sectors worldwide. Aquaculture is set to remain one of the fastest-growing animal food-producing sectors and, in the next decade, total production from both capture and aquaculture will exceed that of beef, pork or poultry. Despite these global trends, European aquaculture accounts for only 18% of total European fish production. One of the major bottlenecks in captive rearing of fish is the control of their reproductive biology and in many foodfish species this concerns remains the main challenge hindering their production. As most aquacultured species are teleosts, comprehensive knowledge and understanding of the factors regulating the teleost reproductive axis is paramount for enabling the future growth of fish production through aquaculture.
Our research focuses on the key regulators of reproduction in vertebrates, namely the hypothalamic peptide gonadotropin-releasing hormone (GnRH) and its pituitary targets, the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH). The zebrafish genetic toolbox and the anatomy of the fish hypothlamo-pituitary (HP) axis provide a unique, accessible and relevant model to dissect out the mechanisms underlying gonadotropin release. Using the zebrafish toolbox we aim to better understand how the GnRH system develops in fish and how it affects the secretion of the two gonadotropins in the adult pituitary. These insights are expected to increase our ability to control and manipulate reproduction in commercially important species.
Our work focused on two main aspects: 1. The migration of the GnRH neurons during early development. 2. The regulation of gonadotropin secretion from the adult fish pituitary.
GnRH migration: The migration of GnRH neurons from the olfactory placode into the brain is a hallmark of GnRH neurons in all studied vertebrates. We use in vivo calcium imaging and electrophysiology in conscious fish to analyze activity patterns of migrating GnRH neurons. Our findings reveal highly synchronized activity of GnRH cells both within and between the two brain hemispheres that is driven by internal electrical activity. We used single-cell genetic labeling, electrophysiology and genetically-encoded lectin tracing to show that this synchronization is mediated within the GnRH circuit rather than by synchronized inputs. By genetically-targeted silencing of individual GnRH cells we show that the electrical and calcium activity of GnRH neurons is critical for their migration. Finally, by knocking down GnRH expression we are able to un-synchronize the activity of the cells, leading to perturbed and non-controlled migration. Together, our data suggests that the synchronized activity of GnRH cells plays an important role in controlling their migration. Furthermore, the GnRH circuit develops in a largely autonomous and isolated manner, thus contributing to its developmental robustness that is paramount for guaranteeing its function as a main regulator of reproduction.
Regulation of gonadotropin secretion: Using transgenic zebrafish lines that express genetically-encoded calcium indicators in their gonadotropes we monitored the activity of gonadotropes and the mechanisms controlling those hormones. By measuring calcium activity in whole brain and pituitary tissue, we found that calcium events in the gonadotropes always showed a rapid rise and were correlated in time across cells, suggesting that the cells share information and synchronize the timing of secretion. By employing this advanced live-imaging techniques in fish we are able to visualize the activity of those cell and to better understand the mechanisms controlling LH and FSH release.
During the course of the current project we have shown, for the first time in a living, conscious vertebrate, synchronized activity of migrating GnRH neurons. These studies, impossible to perform in mammals, reveal a completely new form of communication between migrating GnRH cells and suggest a new pathway for the regulation of GnRH circuit development. This synchronization is extremely robust and is present both within the GnRH cells in a single hemisphere as well as between the cells of the two hemispheres. Moreover, instead of being driven by external inputs, the observed synchronization is mediated within the GnRH circuit. Perturbing the synchronization (generally, by expressing botulinum toxin in GnRH cells, or by knocking-down GnRH expression) causes significant migration defects. This notion of an independent, isolated, self-regulated circuit contributes to the evolutionary robustness of the circuit that controls the critical process of reproduction.
By monitoring calcium dynamics in zebrafish gonadotropes we have succeded, for the first time in any organism, to simultaneously record spontaneous and stimulated activity patterns of FSH and LH cells in situ. We describe different spontaneous activity patterns and coupling dynamics between the two types of gonadotropes that underlie their differential mode of their secretion.
Due to the promising initial results, we have decided to continue the project beyond the current funding period. The study now concentrates on identifying the molecular signals that are used by the GnRH cells to communicate and identifying the reproductive consequences of impaired GnRH migration in fish. In gonadotropes we are concentrating on understanding how different GnRH patterns elicit LH or FSH-specific responses.
Our results pave the way for the development of new tools to control reproduction in fish through manipulating GnRH neuronal migration and deepen our understanding of the secretion patterns of LH and FSH in fish. These insights will be used in the future to design better strategies for controlling and manipulating reproduction in commercially important species. In addition, we revealed a new mechanism for the regulation of GnRH cell migration that sheds light on the evolution of the reproductive axis in vertebrates.
GnRH projections (green) in a clarified brain of an adult female zebrafish.