All genes encoding enzymes participating in melatonin biosynthesis in the chicken pineal gland are transcribed rhythmically.

Our recent research on the pineal gland of young chickens confirmed that three genes encoding enzymes involved in pineal melatonin biosynthesis, tryptophan hydroxylase 1 (Tph1), arylalkylamine-N-acetyltransferase (Aanat) and acetylserotonin O-methyltransferase (Asmt), are transcribed rhythmically under light:dark (L:D) 12:12 conditions in vivo. Additionally, in the pineal gland of maturing chickens, the dopa decarboxylase (Ddc) gene is transcribed rhythmically at a specific stage of the developmental process. Therefore, the aim of the present study was to verify whether all of these genes are transcribed rhythmically in vivo under constant darkness (D:D) and in pinealocyte cultures under both L:D and D:D. Experiments were performed on chickens maintained under L:D 12:12 conditions. Chickens at 15 days of age were divided into two groups; chickens from the first group remained under the same conditions, whereas those from the second group were kept in darkness. Subsequently, 16-day-old animals were sacrificed every 2 hours over a 24-h period. For the in vitro experiments, 16-day-old chickens were sacrificed at ZT 6, and their pineal glands were isolated. Pineal cultures were maintained for up to two days in L:D conditions. Then, the pinealocyte cultures were divided into two groups: the first remained under L:D conditions, whereas the second was transferred to D:D conditions. Pinealocytes were subsequently collected every 2 hours over a 24-h period. Transcription was evaluated using the RT-qPCR method, and the rhythm percentage was calculated through Cosinor analysis. The mRNA levels of all genes examined were rhythmic under all conditions. Moreover, in silico analysis of the promoters of all of the genes examined revealed the presence of enhancer box sequences in all of the promoters as well as DBP/E4BP4 binding elements in the promoters of Tph1 and Asmt. This suggests that these genes may all be regulated transcriptionally by the molecular clock mechanism and may be considered clock as controlled genes.

Season-dependent postembryonic maturation of the diurnal rhythm of melatonin biosynthesis in the chicken pineal gland.

Previously, we have demonstrated that the timing of the nocturnal peak of activity of the pineal arylalkylamine-N-acetyltransferase - a key enzyme in the melatonin biosynthesis pathway - in 3-wk-old chickens kept from the day of hatch under controlled laboratory conditions (L:D 12:12) varies depending on the season of hatch (summer vs. winter). The present study was undertaken to answer the following questions: (1) are season-related differences seen in the level of transcription of genes encoding enzymes of the melatonin biosynthesis pathway? (2) Does the pineal content of the main precursor (serotonin) and the final product (melatonin) exhibit age- and season-related changes? (3) At which step in postembryonic development are these season-related variations in pineal gland function most pronounced? Male Hy-line chickens hatched in the summer or winter, from eggs laid by hens held on L:D 16:8, were kept from the day of hatch under L:D 12:12 conditions. At the age of 2, 9, or 16 d, chickens were sacrificed every 2 h over a 24-h period and their pineal glands, isolated under dim red light, were processed for the measurement of (i) the level of Aanat and Asmt (acetylserotonin O-methyltransferase) mRNAs encoding the two last enzymes involved in melatonin biosynthesis, (ii) the activity of these enzymes, and (iii) the pineal content of serotonin and melatonin. Circadian rhythmicity of all the measured parameters was evaluated by the cosinor method. The levels of Aanat mRNA, AANAT enzymatic activity, and the pineal melatonin content changed during postembryonic development in a manner that was dependent on the season of hatch. Furthermore, the diurnal profile of Asmt mRNA was elevated during the light phase. In "winter" birds, the pattern and amplitude of the diurnal rhythm of accumulation of this transcript did not change with age, while in "summer" birds it increased in an age-related way. In contrast, the enzymatic activity of hydroxyindole-O-methyltransferase (HIOMT; encoded by the Asmt gene) did not change rhythmically, although it increased with age in a season-related way. In "winter" chickens, the pineal serotonin content was low, regardless of age, and did not change rhythmically, whereas in "summer" individuals the serotonin rhythm was already well established by day 2, with the amplitude increasing with age. These results confirm the existence of a "seasonal memory" operating within the chicken pineal gland, although the mechanism(s) underlying this phenomenon have yet to be characterized.