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ABSTRACT

In tilapia, there are two forms of prolactin (PRL) whose effects on sodium and chloride movements differ and depend on the living environment of the fish. To see whether different receptors or the same receptor mediates these different effects, we have characterized the specific binding of both forms of tilapia (ti)PRL in two osmoregulatory organs, the gill and kidney. Two recombinant tiPRLs were used for this analysis. The recombinant hormones had the same properties as the native hormones in a tilapia gill radioreceptor assay. Specific binding to gill and kidney membranes was increased by optimizing the quality of the tissue preparations (physiological state of fish, membrane preparation) and the incubation conditions (pH, salt concentrations, temperature, time). Under these optimized conditions, we detected only one class of high affinity PRL receptor in gill and kidney. Its binding affinity was higher for tiPRL_I than for tiPRL_II in both gill and kidney (for tiPRL_I the respective affinity values were 2·9 and 2·3 × 10¹⁰ per m, for tiPRL_II they were 1·9 and 0·5 × 10¹⁰ per m). In competition studies, tiPRL_I was more potent, followed by tiPRL_II and ovine (o)PRL. tiGH and oGH did not significantly displace either tiPRL. The receptor we have characterized thus recognizes quite specifically both tiPRLs.

ABSTRACT

Two forms of prolactin (tiPRL_I and tiPRL_II), with only 69% sequence identity, have been previously described in the cichlid fish tilapia (Oreochromis species). In the present study we have attempted to investigate the biological activity of these two prolactin forms during adaptation to a hyperosmotic environment. For this purpose, we have developed two highly sensitive (sensitivity: 0·05 ng/ml) and specific (cross-reactivity <0·04%) radioimmunoassays for tiPRL_I and tiPRL_II, using recombinant hormones.

When fish were directly transferred from fresh to brackish water, the measured levels of plasma tiPRL_I and tiPRL_II dropped abruptly until 12 h after transfer. Thereafter, plasma tiPRL_II remained stable (around 0·5 ng/ml) until the end of the experiment, whereas plasma tiPRL_I continued to decrease to undetectable levels. These different patterns of change are reflected in the calculated ratio of plasma tiPRL_II to tiPRL_I, which increased from 2–3 in fresh water-adapted fish to over 10 in fish which had spent 3 days or more in brackish water. The pituitary contents of tiPRL_I and tiPRL_II varied in a qualitatively similar fashion after transfer to brackish water. The tiPRL_I content dropped continuously after 12 h, reaching one-twelfth of its initial level after 2 weeks. The pituitary tiPRL_II content, on the other hand, did not decrease significantly until day 7, and after a 2-week exposure to brackish water it had only decreased by 50%.

When injected into tilapia adapted to brackish water, both ovine prolactin and recombinant tiPRL_I induced a clear dose-dependent ion-retaining effect. In contrast, the effect induced by tiPRL_II treatment was markedly smaller and not dose-dependent.

Northern blot analysis of tiPRL mRNAs using either a tiPRL_I or a tiPRL_II cDNA probe indicated the presence of two mRNAs differing in size: a 1·7 kb mRNA coding for tiPRL_I and a 1·3 kb mRNA coding for tiPRL_II. After transfer to brackish water, levels of the two mRNAs decreased similarly.

The present study indicates that, in O. niloticus, the two forms of prolactin have different osmoregulatory roles during adaptation to brackish water. Accordingly, their syntheses are differentially regulated after transfer to a hyperosmotic environment, presumably at a post-transcriptional level.