The P450 side-chain cleavage enzyme Cyp11a2 facilitates steroidogenesis in zebrafish
The P450 side-chain cleavage enzyme Cyp11a2 facilitates steroidogenesis in zebrafish
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DOI：
10.1530/joe-19-0384
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发表时间：
2020-02-01
影响因子：
4
通讯作者：
Krone, Nils P.
中科院分区：
文献类型：
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作者：
Li, Nan;Oakes, James A.;Krone, Nils P.
由CYP 11 A1基因编码的细胞色素P450侧链裂解酶催化类固醇激素生物合成的第一步和限速步骤。先前在斑马鱼中的吗啉敲低研究表明cyp 11 a2是人类CYP 11 A1的功能等同物，并且对于斑马鱼幼体的肾间类固醇合成是必需的。Cyp 11 a2在成年斑马鱼中的作用，特别是在性腺类固醇生成中，仍然是难以捉摸的。为了探索Cyp 11 a2在成年人中的作用，我们通过使用CRISPR/Cas9基因组工程方法在Cyp 11 a2中创建缺失来开发斑马鱼突变系。纯合子cyp 11 a2突变斑马鱼幼虫表现出下丘脑-垂体-肾间轴的上调。此外，这些Cyp 11 a2缺陷的斑马鱼表现出严重的糖皮质激素和雄激素缺乏。Cyp 11 a2纯合子只能发育成第二性征女性化的雄性。成年cyp 11 a2（-/-）突变鱼表现出缺乏自然繁殖行为。组织学特征显示睾丸结构紊乱，成熟精子数量显著减少。cyp 11 a2纯合子斑马鱼睾丸中几个促雄性基因的表达下调进一步支持了这些发现，包括sox 9a，dmrt 1和amh。此外，在cyp 11 a2纯合突变体的睾丸中，精原细胞标记nanos 2和piwil 1表达上调，而精母细胞标记sycp 3和精子细胞标记odf 3b表达下调。我们的表达分析与我们的组织学研究一致，表明精原细胞是cyp 11 a2纯合突变体睾丸中的主要细胞类型。因此，我们的工作表明Cyp 11 a2在斑马鱼幼体和成体肾间和性腺类固醇合成中的关键作用。
Cytochrome P450 side-chain cleavage enzyme, encoded by the CYP11A1 gene, catalyzes the first and rate-limiting step of steroid hormone biosynthesis. Previous morpholino-knockdown studies in zebrafish suggested cyp11a2 is a functional equivalent of human CYP11A1 and is essential for interrenal steroidogenesis in zebrafish larvae. The role of Cyp11a2 in adult zebrafish, particularly in gonadal steroidogenesis, remains elusive. To explore the role of Cyp11a2 in adults, we developed zebrafish mutant lines by creating deletions in cyp11a2 using the CRISPR/Cas9 genomic engineering approach. Homozygous cyp11a2 mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal axis. Furthermore, these Cyp11a2-deficient zebrafish demonstrated profound glucocorticoid and androgen deficiencies. Cyp11a2 homozygotes only developed into males with feminized secondary sex characteristics. Adult cyp11a2(-/-) mutant fish showed a lack of natural breeding behaviors. Histological characterization revealed disorganized testicular structure and significantly decreased numbers of mature spermatozoa. These findings are further supported by the downregulation of the expression of several pro-male genes in the testes of cyp11a2 homozygous zebrafish, including sox9a, dmrt1 and amh. Moreover, the spermatogonia markers nanos2 and piwil1 were upregulated, while the spermatocytes marker sycp3 and spermatids marker odf3b were downregulated in the testes of cyp11a2 homozygous mutants. Our expression analysis is consistent with our histological studies, suggesting that spermatogonia are the predominant cell types in the testes of cyp11a2 homozygous mutants. Our work thus demonstrates the crucial role of Cyp11a2 in interrenal and gonadal steroidogenesis in zebrafish larvae and adults.