Prolactin and cortisol mediate the maintenance of hyperosmoregulatory ionocytes in gills of Mozambique tilapia: Exploring with an improved gill incubation system

Endocrine control of osmoregulation is essential for teleosts to adapt to various aquatic environments. Prolactin (PRL) is known as a fundamental endocrine factor for hyperosmoregulation in teleost fishes, acting on ionocytes in the gills to maintain ion concentrations of body fluid within narrow physiological ranges in freshwater conditions. Cortisol is also known as an osmoregulation-related steroid in teleosts; however, its precise function is still controversial. Here, we investigated more detailed effects of PRL and roles of cortisol on ionocytes of Mozambique tilapia (Oreochromis mossambicus) in freshwater, using an improved gill filament incubation system. This incubation system resulted in enhanced cell viability, as evaluated using the dead cell marker propidium iodide. PRL was shown to maintain the density of freshwater-type ionocytes in isolated gill filaments; this effect of PRL is not achieved by the activation of cell proliferation, but by the maintenance of existing ionocytes. Cortisol alone did not show any distinct effect on ionocyte density in isolated gill filaments. We also assessed effects of PRL and cortisol on relative mRNA levels of NCC2, NHE3, NKAa1a, and NKAa1b. PRL maintained relative NCC2 and NKAa1a mRNA abundance, and cortisol showed a stimulatory effect on relative NCC2 and NKAa1a mRNA levels in combination with PRL, though cortisol alone exerted no effect on these genes. An increase in NKAa1b mRNA abundance was detected in cortisol-treated groups. PRL treatment also maintained normal NCC2 localization at the apical membrane of the ionocytes. These results indicate that PRL maintains freshwater-type ionocytes, and that cortisol stimulates the function of ionocytes maintained by PRL.