Disruption of the Salmon Reproductive Endocrine Axis Through Prolonged Nutritional Stress: Changes in Circulating Hormone Levels and Identification of Differentially Expressed Ovarian Genes.

Abstract

Environmental stressors (e.g., reduced food availability, hypoxia, endocrine disrupting chemicals) are known to disrupt endocrine function of fish, potentially delaying the age of puberty and affecting gamete quality. Assessing the reproductive health of fish in early stages of gametogenesis using histological and hormonal indices provides useful information, but has limited prognostic value for future reproductive potential. Our long-term aim is to develop tools for monitoring environmental impacts on ovarian growth in fish using large-scale ovarian gene expression analyses. However, little is known about the regulation of gonadal gene expression during previtellogenic oocyte growth in either healthy growing fish or fish exposed to environmental conditions that have been shown to disrupt the reproductive endocrine system and inhibit gonadal growth. The goal of this study was to identify genes that are specifically up- or down-regulated in the previtellogenic ovary of coho salmon when ovarian growth is impaired by prolonged fasting. Two year-old fish were individually tagged and fasted over a 17-week period, which resulted in less than 20% loss of body weight. Prolonged fasting caused regression of the ovaries and disrupted the reproductive endocrine axis, as fasted salmon showed significantly lower gonad weights, oocyte diameters, and plasma estradiol-17β (E2) levels relative to fed animals by week 14. Further, a higher proportion of atretic oocytes was observed in ovaries of fasting fish at week 14 and numbers increased by week 17. Reciprocal suppression subtractive hybridization (SSH) cDNA libraries using ovaries from fed and fasted animals at week 14 were generated. A total of 480 clones were sequenced per library and their putative identity determined by comparing sequences to the NCBI and fish EST databases. The library representing genes up-regulated in the fed ovary included steroidogenesis related genes, such as steroidogenic acute regulatory protein (star), 3β-hydroxysteroid dehydrogenase (hsd3b), P450-aromatase (cyp19a1a), and TGF-β superfamily members, such as anti-Mullerian hormone (amh) and inhibin (inha). The library representing genes up-regulated in the fasted ovary included genes that have been associated with apoptosis, such as programmed cell death protein 4 (pdcd4), lipopolysaccharide-induced TNF factor (litaf), cullin 3 (cul3), and kruppel-like factor 6 (klf6). The genes described above were examined with quantitative PCR and results confirmed that the SSH libraries were enriched in transcripts differentially expressed between ovaries of fed and fasted salmon. This study has revealed ovarian genes that are important to normal ovarian development and those that are affected by nutritional stress and may relate to apoptosis/atresia within the ovary. Ultimately, this and subsequent studies should provide new tools that will enable molecular based assessment of fish reproductive health. This research was funded by Washington Sea Grant project number R/B-49. (poster)