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Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

The fish pineal organ, through its 24 h rhythmic release of melatonin, acts as a transducer of the photoperiod, influencing different physiological functions (e.g. reproduction, growth). We have investigated the binding of 2-[125I]iodomelatonin to whole brain membrane preparations from pikes (Esox lucius L., teleost) maintained for 24-48 h under different photoperiodic conditions. Specific binding was stable, reversible, saturable and sensitive to the presence of a GTP analogue. Scatchard analysis revealed one class of binding sites. Displacement experiments suggested the presence of two components with affinities in the femtomolar and nanomolar range of concentrations, respectively. The Bmax exhibited monophasic nycthemeral variations, with higher values at the light-to-dark transition (34.0 +/- 4.5 fmol/mg protein) and low values during the second half of night (10.0 +/- 1.0 fmol/mg protein). Under the same conditions, the KD exhibited biphasic variations: values were low during daytime and at the middle of the dark phase (approximately 100 pM); they were high at the beginning (approximately 225 pM) and at the end (approximately 330 pM) of the night. These variations were maintained under constant light (LL) and constant darkness (DD). Thus, the variations in the number and affinity of the melatonin binding sites were controlled by circadian oscillators, synchronized by the photoperiod. The nature of these oscillators is not known. Therefore, in fish, we suggest that the photodependent effects of melatonin result from the circadian variations of both its production by the pineal and its binding sites in the brain.