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

ABSTRACT

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 P450_7α – 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.

KEYWORDS:

• Pineal gland
• circadian rhythm
• Clock Controlled Genes
• neurosteroids
• allopregnanolone
• 7α-hydroxypregnenolone
• chicken

Acknowledgements

We would like to thank Dr. Krystyna Zuzewicz from Central Institute for Labour Protection – National Research Institute for her invaluable help in performing the cosinor analysis. We thank Dr. Zuzewicz for her help and great patience in explaining the complexities of mathematics. The authors would like to thank Agata Bogdanska, Agnieszka Wojtaszko, and Marta Lopacinska from the Department of Animal Physiology, Faculty of Biology, University of Warsaw, Poland for their assistance and suggestions.

Author contribution

MC performed in vitro experiments, evaluated results and prepared the manuscript; NB performed in vivo experiments and commented on the manuscript; PMM planned the study, evaluated results and critically revised the manuscript; IA planned, funded, designed, and coordinated the study, evaluated the results, and critically revised the manuscript.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

Additional information

Funding

This work was supported by National Science Centre of Poland Grant [UMO-2016/21/B/NZ3/00364].