USE OF PRIMARY CULTURES OF SALMON HEPATOCYTES FOR THE STUDY OF HORMONAL-REGULATION OF INSULIN-LIKE GROWTH FACTOR-I EXPRESSION IN-VITRO

Hepatocytes isolated from sockeye salmon (Oncorhynchus nerka) were see ded on an uncoated plastic dish with a positively charged surface, and cultured in hormone-free defined medium. Initially single cells began to aggregate and became flat after being attached to the dish. After approximately 6 days in culture, hepatocytes formed a monolayer sheet with cells in side-by-side contact, and remained in this form for at l east 2 weeks. Hepatocytes in multicellular spheroids were also observe d. The viability of the cells remained high (85-90%) during culture as judged by trypan blue exclusion. The cells actively incorporated [H-3 ]leucine into cellular and secreted proteins. Salmon and bovine insuli ns stimulated the leucine incorporation in a dose-dependent manner, su ggesting that the cells remained responsive to hormones during culture . The basal IGF-I mRNA level in cultured hepatocytes was much lower th an that of in vivo status. Salmon GH increased the level of IGF-I mRNA , whereas salmon prolactin had no effect. These findings suggest that the primary culture system of salmon hepatocytes can be used for the i n vitro study of hormonal regulation of IGF-I gene expression in salmo n.