[Regulatory role and mechanism of HSD3B7 in bovine follicular development].

内容摘要

This study aimed to explore the regulatory effect and mechanism of 3β-hydroxy-Δ5-C27-steroid oxidoreductase (HSD3B7) on bovine follicular development. Bovine primary granulosa cells (GCs) with HSD3B7 knockdown and overexpression were constructed, and the proliferation efficiency of GCs was examined by the cell counting kit-8 (CCK-8) method. Annexin V-EGFP/PI double staining was performed to detect the apoptosis level of GCs. The concentration of estradiol (E2) in the culture medium and that of 2-methoxyestrone in GCs were determined by enzyme-linked immunosorbent assay (ELISA). The relative expression levels of the CCND2, Bim, Caspase-3, CYP19A1 and STAR were determined by qRT-PCR. The protein levels of related genes were determined by Western blotting. The results showed that HSD3B7 knockdown decreased the proliferation efficiency and CCND2 expression level of primary bovine follicular GCs, promoted the apoptosis, and increased Bim and Caspase-3 expression in GCs. Additionally, it decreased E2 secretion and CYP19A1 and STAR expression, and increased the concentration of 2-methoxyestrone, the main metabolite of E2. The opposite results were obtained after HSD3B7 overexpression. These results suggested that HSD3B7 positively regulated the development of bovine follicles by promoting the proliferation and E2 secretion and inhibiting the apoptosis of GCs. The results provide a new theoretical basis for further understanding the molecular regulatory network of bovine follicle development, and also lay a foundation for related research and practical application to improve bovine reproductive efficiency. 本研究旨在探究3β-羟基-Δ5-C27-类固醇氧化还原酶(3β-hydroxy-Δ5-C27-steroid oxidoreductase, HSD3B7)对牛卵泡发育的调节作用及其机制。构建敲降和过表达HSD3B7基因的牛原代颗粒细胞(granulosa cells, GCs)，用细胞计数试剂-8 (cell counting kit-8, CCK-8)法检测GCs增殖效率，用Annexin V-EGFP/PI双染法检测GCs凋亡水平，用酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)技术检测培养液中雌二醇(estradiol, E2)和胞内2-甲氧基雌酮的浓度，用实时荧光定量PCR (real-time quantitative-polymerase chain reaction, qRT-PCR)技术检测CCND2、Bim、Caspase-3、CYP19A1、STAR的相对表达水平，用Western blotting技术检测相关基因蛋白水平的表达情况。结果显示，HSD3B7敲降后降低了牛卵泡原代GCs增殖效率和CCND2蛋白的表达水平;促进了GCs凋亡和Bim蛋白、Caspase-3蛋白的表达;减少E2的分泌量和CYP19A1蛋白、STAR蛋白的表达量;增加E2主要代谢物2-甲氧基雌酮的浓度。HSD3B7过表达后取得相反结果。本研究结果表明，HSD3B7通过促进牛GCs增殖和E2分泌、抑制GCs凋亡，正向调控牛卵泡发育进程。研究结果为深入解析牛卵泡发育的分子调控网络提供了新的理论依据，也为提高牛繁殖效率的相关研究和实践应用奠定了基础。.