VDR promotes testosterone synthesis in mouse Leydig cells via regulation of cholesterol side chain cleavage cytochrome P450 (Cyp11a1) expression,Genes & Genomics

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VDR promotes testosterone synthesis in mouse Leydig cells via regulation of cholesterol side chain cleavage cytochrome P450 (Cyp11a1) expression
Genes & Genomics ( IF 1.6 ) Pub Date : 2023-09-25 , DOI: 10.1007/s13258-023-01444-z
Yuanyuan Hu ₁ , Ling Wang _{1,

2} , Ge Yang ₁ , Shanshan Wang ₁ , Miaomiao Guo ₁ , Hongzhao Lu _{1,

3} , Tao Zhang _{1,

3,

4}

Affiliation

Background

The vitamin D receptor (VDR) mediates the pleiotropic biological actions that include osteoporosis, immune responses and androgen synthesis.VDR is widely expressed in testis cells such as Leydig cells, Sertoli cells, and sperm. The levels of steroids are critical for sexual development. In the early stage of steroidogenesis, cholesterol is converted to pregnenolone (precursor of most steroid hormones) by cholesterol side-chain lyase (CYP11A1), which eventually synthesizes the male hormone testosterone.

Objective

This study aims to reveal how VDR regulates CYP11A1 expression and affects testosterone synthesis in murine Leydig cells.

Methods

The levels of VDR, CYP11A1 were determined by quantitative real-time polymerase chain reaction (RT-qPCR) or western blot. Targeted relationship between VDR and Cyp11a1 was evaluated by dual-luciferase reporter assay. The levels of testosterone concentrations in cell culture media serum by enzyme-linked immunosorbent assay (ELISA).

Results

Phylogenetic and motif analysis showed that the Cyp11a1 family had sequence loss, which may have special biological functions during evolution. The results of promoter prediction showed that vitamin D response element (VDRE) existed in the upstream promoter region of murine Cyp11a1. Dual-luciferase assay confirmed that VDR could bind candidate VDREs in upstream region of Cyp11a1, and enhance gene expression. Tissue distribution and localizatio analysis showed that Cyp11a1 was mainly expressed in testis, and dominantly existed in murine Leydig cells. Furthermore, over-expression VDR and CYP11A1 significantly increased testosterone synthesis in mice Leydig cells.

Conclusions

Active vitamin D3 (VD₃) and Vdr interference treatment showed that VD₃/VDR had a positive regulatory effect on Cyp11a1 expression and testosterone secretion. VDR promotes testosterone synthesis in male mice by up-regulating Cyp11a1 expression, which played an important role for male reproduction.

中文翻译：

VDR 通过调节胆固醇侧链裂解细胞色素 P450 (Cyp11a1) 表达促进小鼠睾丸间质细胞的睾酮合成

背景

维生素 D 受体 (VDR) 介导多效性生物学作用，包括骨质疏松、免疫反应和雄激素合成。VDR 广泛表达于睾丸细胞，如间质细胞、支持细胞和精子。类固醇的水平对于性发育至关重要。在类固醇生成的早期阶段，胆固醇通过胆固醇侧链裂解酶（CYP11A1）转化为孕烯醇酮（大多数类固醇激素的前体），最终合成雄性激素睾酮。

客观的

本研究旨在揭示 VDR 如何调节CYP11A1表达并影响小鼠 Leydig 细胞中的睾酮合成。

方法

通过定量实时聚合酶链反应（RT-qPCR）或蛋白质印迹测定VDR、 CYP11A1的水平。通过双荧光素酶报告基因测定评估VDR 和Cyp11a1之间的靶向关系。通过酶联免疫吸附测定 (ELISA) 检测细胞培养基血清中睾酮浓度的水平。

结果

系统发育和基序分析表明Cyp11a1家族存在序列丢失，可能在进化过程中具有特殊的生物学功能。启动子预测结果表明，小鼠Cyp11a1的上游启动子区存在维生素D反应元件(VDRE) 。双荧光素酶测定证实VDR可以结合Cyp11a1上游区域的候选VDRE ，并增强基因表达。组织分布和定位分析表明，Cyp11a1主要在睾丸中表达，并主要存在于小鼠Leydig细胞中。此外，过表达 VDR 和CYP11A1显着增加小鼠 Leydig 细胞中睾酮的合成。