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.

摘要

松果体是三个等效的鸟类生物钟结构之一，也是密集神经类固醇合成（7α-羟基孕烯醇酮和别孕烯醇酮）的部位。松果体神经类固醇生物合成涉及六种酶：细胞色素 P450 侧链裂解 – Cyp11a1编码、细胞色素 P450 _7α – Cyp7b1、3β-羟基类固醇脱氢酶 – Hsd3b2、5α-还原酶 – Srd5a1、3α-羟基类固醇脱氢酶–Sdr15rd3β - reduce. 神经甾体生物合成的调节尚未完全了解；尽管已知 E4BP4 转录因子诱导生物合成胆固醇基因的激活，这些基因是 SREBP（元件结合蛋白转录因子）的目标。SREBP 在松果体中的主要活动是对Period2经典时钟基因的抑制和抑制，这表明我们的假设是编码参与神经类固醇生成的酶的基因受生物钟控制并且是时钟控制基因 (CCG)。因此，通过研究Cyp11a1、Cyp7b1、Hsd3b2、Akr1d1、Srd5a1和Srd5a3的日常变化，在体内和体外测试了松果体基因, 在培养的松果体细胞中。对取自 16 日龄鸡的体内松果体或使用从 16 日龄动物收集的松果体细胞的体外培养物进行了实验。体内实验中的鸟类和松果体细胞都保持在受控光照条件下 (LD 12:12) 或持续黑暗 (DD)。随后，准备材料用于 RT-qPCR 分析。结果显示，六个测试基因中的三个：Cyp11a1、Cyp7b1和Srd5a3在体内和体外表现出显着的 24 小时变异. 这项研究的结果证实，这些基因可能受时钟控制，并满足许多被鉴定为 CCG 的要求。