背景:Cyp19a1a是将雄激素转化为雌激素的途径中的关键酶,并受TGF-β信号传导的调节。Smad4和FoxH1是TGF-β信号传导的下游效应子,可能在黄曲霉的卵巢发育中起重要作用。
方法:我们使用qRT-PCR和免疫荧光研究了Smad4和FoxH1的表达模式,然后通过qRT-PCR检测卵巢与FSH体外孵育后smad4和foxh1的变化,并通过双荧光素酶报告基因分析分析Smad4和FoxH1对cyp19a1a转录的调控。
结果:我们发现Smad4编码449个氨基酸的推定蛋白质,并包含该蛋白质家族典型的三个保守结构域。Smad4和foxh1在卵巢发育和FSH孵育后表现出相似的表达模式,皮尔逊系数分别为0.873和0.63-0.81。此外,Smad4,FoxH1和Cyp19a1a共定位在卵巢的颗粒细胞和卵泡膜细胞中。Smad4通过cyp19a1a启动子中的SBE1(-1372/-1364)和SBE2(-415/-407)抑制cyp19a1a活性,而突变SBE1或SBE2恢复了cyp19a1a启动子的活性。Smad4和FoxH1的共过表达显着降低了cyp19a1a启动子的活性。
结论:这项研究为转录因子Smad4和FoxH1在卵巢发育中的潜在功能以及cyp19a1a在大黄分枝杆菌中的转录调控机制提供了新的见解,这将揭示Smad4/FoxH1介导的TGF-β信号在生殖和cyp19a1a的调节。芳香化酶,由cyp19a1a编码,参与卵巢发育,对卵子质量起着重要作用,还有性别比例,硬骨鱼,M.阿不思。对cyp19a1a转录调控的研究有助于理解其在卵巢发育中的作用。在以前的研究中,显示FoxH1抑制cyp19a1a转录。在本研究中,Smad4被确认为cyp19a1a转录抑制因子,Smad4也可能与FoxH1协调以抑制cyp19a1a转录。目前,我们为硬骨鱼卵巢中转录因子Smad4和FoxH1对cyp19a1a的转录调控提供了新的视角。在未来,将进一步研究Smad4和FoxH1的调控网络,并将基因编辑技术用于筛选cyp191a1a基因的特异性调控因子,从而改变雌性周期并调节产卵的性别比。
BACKGROUND: Cyp19a1a is a key enzyme in the pathway that converts androgens into estrogen and is regulated by TGF-β signaling. Smad4 and FoxH1 are downstream effectors of TGF-β signaling and may play important roles in ovarian development in M. albus.
METHODS: We investigated the expression pattern of the Smad4 and FoxH1 using qRT‒PCR and immunofluorescence, then tested the changes of smad4 and foxh1 by qRT‒PCR after ovary incubation with FSH in vitro, and analysed the regulation of cyp19a1a transcription by Smad4 and FoxH1 by dual-luciferase reporter assays.
RESULTS: We found that Smad4 encoded a putative protein of 449 amino acids and harbored the three conserved domains typical of this protein family. Smad4 and foxh1 exhibited similar expression patterns during ovarian development and after FSH incubation, with Pearson\'s coefficients of 0.873 and 0.63-0.81, respectively. Furthermore, Smad4, FoxH1 and Cyp19a1a colocalized in the granulosa cells and theca cells of ovaries during the mid-to-late vitellogenic stage. Smad4 repressed cyp19a1a activity via SBE1 (- 1372/-1364) and SBE2 (- 415/-407) in the cyp19a1a promoter, whereas mutating SBE1 or SBE2 restored cyp19a1a promoter activity. Co-overexpression of Smad4 and FoxH1 significantly reduced cyp19a1a promoter activity.
CONCLUSIONS: This study provides new insights into the potential functions of transcription factors Smad4 and FoxH1 in ovarian development and the transcriptional regulation mechanism of cyp19a1a in M. albus, which will reveal Smad4/FoxH1-mediated TGF-β signaling in reproduction and the regulation of the cyp19a1a. Aromatase, encoded by cyp19a1a, is involved in ovarian development and plays an important role in the quality of eggs, as well the sex ratio, of the teleost fish, M. albus. The research on the transcriptional regulation of cyp19a1a has contributed to the understanding of its role in ovarian development. In previous study, it was shown that FoxH1 inhibits cyp19a1a transcription. In the present study, Smad4 was confirmed as a cyp19a1a transcriptional repressor and Smad4 may also coordinate with FoxH1 to repress cyp19a1a transcription. At present, we provide a new perspective for the transcriptional regulation of cyp19a1a by transcription factors Smad4 and FoxH1 in teleost fish ovary. In the future, the regulatory networks of Smad4 and FoxH1 will be further studied and the gene editing technology will be applied to screen specific regulatory factors of cyp191a1a gene, so as to alter the female cycle and modulate the sex ratio of the eggs production.