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The European eel is known for its complex life cycle and long migration fromcontinental habitats in Europe and North Africa to their spawning area in the Sargasso Sea. Still, significant gaps in knowledge remain on their reproductive development, spawning and early life history. As for all vertebrates, gonadal development in fish is controlled through endocrine regulation on the brain-pituitary-gonad axis triggered by external and internal cues. In eels, a dual neuro-endocrine control on the brain-pituitary axis prevents sexual maturation in continental habitats. In captivity, this inhibition remains and consequently, farmed eels do not reproduce naturally. From a commercial perspective, this impedes closed cycle production and development of a sustainable aquaculture of this species that presently is critically endangered. Nonetheless, gonadal development can be induced through hormonal treatmentsin captivity. For female eels, this is achieved using treatments with pituitary extracts from carp (CPE) or salmon. The extracts contain the gonadotropins follicle-stimulating hormone (FSH) and luteinizing hormone (LH) that stimulate the activity of two key organs, the ovary and liver, which are responsible for ovarian development and vitellogenesis. Furthermore, treatment with a maturation inducing steroid is needed to complete oocyte maturation and ovulation. Current hormonal treatments are suboptimal and the resultant reproduction success and egg quality are exceedingly variable. In this context, the focus of this PhD thesis was on studying characteristics of hormonally induced ovarian development and vitellogenesis in European eel with emphasis on the endocrine regulation in and between the ovary and liver. The thesis includes three interrelated experimental studies.The objective of the first study was to expand insights into the endocrineregulation of induced ovarian development and vitellogenesis with focus on the ovary and liver, as well as subsequent reproductive success. Two commonly used CPE treatment protocols, using different doses (i.e. constant and a stepwise increasing), were compared to study potential dose dependent effects on the regulation of the developmental process. Results showed that both CPE treatments led to similar ovarian development and vitellogenesis as assessed biometrically and histologically, but rate of progression differed. A qPCR analysis showed that the expression of ovarian gonadotropin and steroid receptor genes, essential for gonad development, were upregulated during vitellogenesis using either CPE treatment and showed similar expression profiles regardless of CPE treatment dose. Correspondingly, the reproductive success was not different between treatment groups. Three novel discoveries were made in this study: i) vitellogenin genes were expressed not only in hepatic tissue, but also in ovarian tissue, which indicated that the ovary is a secondary site of vitellogenin synthesis, besides the liver, ii) the receptor gene of FSH was expressed and upregulated in the liver, suggesting an FSH regulatory role here, and iii) heat shock protein 90 (Hsp90) was expressed and upregulated in the liver, which suggested a role in binding steroid receptors (a known Hsp90 function) during hepatic vitellogenesis. Altogether, these results increased our understanding on the complexity of vitellogenesis.The objective of the second study was to elucidate transcriptomic responses toinduced vitellogenesis in the ovary of CPE-treated eels. Changes in the ovariantranscriptome (the complete set on RNA in a sample) were elucidated using RNA-Seq, a high-throughput technique. RNA was extracted from ovarian tissue from eight female eels sampled prior to CPE treatment (week 0) and after nine weeks of CPE treatment given at a constant dose. During this period, the status of females changed from a previtellogenic to late vitellogenic developmental stage. RNA-Seq was performed to identify differentially expressed genes in ovarian tissue between females at these two stages. The analysis showed that key genes involved in processes such as vitellogenesis, steroidogenesis, vitamin uptake, ovulation and tissue growth were upregulated, while genes linked to early oocyte development were downregulated. Thus, biological processes and pathways regulated during induced ovarian development paralleled expression patterns observed in other teleosts. In accordance with study 1, genes for vitellogenins were highly upregulated, thus confirming that the ovary besides the liver likely is a site of local vitellogenin synthesis in eel.The objective of the last study was to unravel the role of the liver during inducedvitellogenesis in the same female eels as in the second study. The liver is essential for vitellogenesis, as it produces vitellogenins through sex steroid action. Moreover, the liver has an important role in reallocating resources for energy metabolism as well as for reproductive development. This is an effect of eels ceasing to feed in nature during silvering, when eels enter their migratory phase. These fasting conditions are simulated during induced vitellogenesis. In this study, RNA-Seq was performed using RNA extracted from livers to compare transcriptomes of female eels sampled in week 0 and week 9. Results showed that the liver grew in size during induced vitellogenesis, following a growth pattern reported in other female teleosts during reproductive development, however different from a typical pattern for fasting. At the molecular level, upregulated genes were mainly involved in processes related to energy management biosynthesis, transport and reproduction, whereas downregulated were linked to developmental processes, organ maintenance and the immune system. Within the reproductive biological processes, pathways related to steroid production were upregulated. The analysis detected steroid production genes that have not previously been investigated in eel, such as lectin, mannose binding 1 (lman1) and nuclear protein 1 transcriptional regulator (nupr1). These findings may be important as benchmark for further investigations on hepatic vitellogenesis.In conclusion, the three studies expanded our knowledge on the endocrine regulation during induced ovarian development and vitellogenesis through experimental work and interdisciplinary methodologies with focus on the ovary and liver. Firstly, insights were substantiated as hormonal treatments led to regulation of known genes and biological pathway during reproductive development. Furthermore, novel information about genes involved in reproductive processes in the ovary and liver expanded our understanding of the complex regulation in and between these two organs. Particularly, the new findings do not only present possibilities for further studies on the reproductive physiology in eel, but they also open up for broader discussions on the regulation of vitellogenesis in teleosts, as well as the applicability of high-throughput technologies in aquaculture research. |
