Quantification and localization of StAR expression in Atlantic cod ovaries

Abstract

Steroidogenesis is essential for the production of sex steroids, and therefore also the sexual maturation process. The rate-limiting step in steroidogenesis is the movement of cholesterol from the outer to the inner mitochondrial membrane, a process that is controlled by the steroidogenic acute regulatory (StAR) protein. To date, limited information exists about StAR expression and regulation. Today's cod farming industry is facing different challenges, among them are problems associated with final gonad maturation and spawning, especially in females. Numerous factors may affect this, and suboptimal nutrition is suggested as one of them. One ingredient in cod broodstock feed that an optimal amount is not yet established for is Arachidonic acid (ARA). In addition to affect different aspects of spawning results, ARA is suggested to be involved in the regulation of steroid production. In this thesis work StAR transcripts (mRNA) in cod ovaries were quantified over a whole reproductive cycle to investigate if the transcript levels changed over time, and to check if they showed a connection with the amount of ARA in the diet; this was performed by applying quantitative real-time PCR (qPCR). Furthermore, StAR mRNA was localized in ovaries by applying In Situ hybridization. The samples used for quantification of StAR originated from cod from a previous experiment at the Institute of Marine Research (IMR), Austevoll, where they were fed different amounts of dietary ARA; 0.5, 1, 2 and 4% ARA of total fatty acids. StAR expression levels showed the same general pattern in all groups, with a peak during the spawning season (February and March 2006), but also elevated and variable values after their first spawning season (June 2005). There were no significant differences in StAR expression between groups except in July (p = 0.03485) and January 2005 (p = 0.00349), but which group was different could only be revealed for January; here fish fed 0.5% ARA had significantly higher StAR expression than fish fed 4% ARA. Fish used for localization of StAR were reared at IMR, Austevoll, and randomly sampled during the thesis study. No StAR expression was detected in follicles of early primary growth (early previtellogenic). However, in both late primary growth (late previtellogenic)-, and late vitellogenic follicles StAR was detected in the ooplasm. Furthermore, StAR was detected in granulosa- and theca cells of late vitellogenic follicles. These findings suggest that StAR expression levels change over a reproductive cycle in cod, with a peak in the spawning months February and March. Furthermore, ARA may affect StAR expression levels in early spawning. Additionally, StAR expression changes with degree of follicle development.