24 hour patterning in gene expression of pineal neurosteroid biosynthesis in young chickens (Gallus gallus domesticus L.)

The pineal gland, one of the three equivalent avian biological clock structures, is also the site of intensive neurosteroid synthesis (7α-hydroxypregnenolone and allopregnanolone). Pineal neurosteroid biosynthesis involves six enzymes: cytochrome P450 side-chain cleavage – Cyp11a1 encoded, cytochrome P4507α – Cyp7b1, 3β-hydroxysteroid dehydrogenase – Hsd3b2, 5α-reductase – Srd5a1, 3α-hydroxysteroid dehydrogenase – Akr1d1, and 5β-reductase – Srd5a3. Regulation of neurosteroid biosynthesis is not fully understood; although it is known that the E4BP4 transcription factor induces activation of biosynthetic cholesterol genes, which are the targets for SREBP (element-binding protein transcription factor). SREBP principal activity in the pineal gland is suppression and inhibition of the Period2 canonical clock gene, suggesting our hypothesis that genes encoding enzymes involved in neurosteroidogenesis are under circadian clock control and are the Clock Control Genes (CCGs). Therefore, through investigation of daily changes in Cyp11a1, Cyp7b1, Hsd3b2, Akr1d1, Srd5a1, and Srd5a3, pineal genes were tested in vivo and in vitro, in cultured pinealocytes. Experiments were carried out on pineal glands taken from 16-day-old chickens in vivo or using in vitro cultures of pinealocytes collected from 16-day-old animals. Both the birds in the in vivo experiments and the pinealocytes were kept under controlled light conditions (LD 12:12) or in constant darkness (DD). Subsequently, materials were prepared for RT-qPCR analysis. Results revealed that three of the six tested genes: Cyp11a1, Cyp7b1, and Srd5a3 demonstrated significant 24-hour variation in in vivo and in vitro. Findings of this study confirm that these genes could be under clock control and satisfy many of the requirements to be identified as CCGs.