WT1 suppresses follicle-stimulating hormone-induced progesterone secretion by regulating ERK1/2 pathway in chicken preovulatory granulosa cells
WT1 suppresses follicle-stimulating hormone-induced progesterone secretion by regulating ERK1/2 pathway in chicken preovulatory granulosa cells
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WT1通过调节鸡排卵前颗粒细胞中的ERK1/2通路抑制促卵泡激素诱导的黄体酮分泌
DOI：
10.1016/j.gene.2021.146097
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发表时间：
2021-12-16
期刊：
影响因子：
3.5
通讯作者：
Gong, Yanzhang
中科院分区：
文献类型：
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作者：
Tang, Shuixin;Li, Xuelian;Gong, Yanzhang
多种Wilms肿瘤基因1(WT1)剪接变异体在哺乳动物中表达，这些变异体调节肿瘤的发生，并调节包括性腺在内的多个组织和器官的发育。然而，WT1剪接变异体(+KTS或-KTS)只在两种非哺乳动物脊椎动物中表达，令人惊讶的是，它们在鸡卵巢中的功能仍然难以捉摸。鸡颗粒细胞(GCs)分泌的孕酮(P4)参与多种生理过程，在维持繁殖性能方面起着重要作用。本研究旨在探讨WT1(+KTS)和WT1(-KTS)对排卵前GCs分泌P4的影响。首先，我们用实时定量聚合酶链式反应(RT-qPCR)检测了不同发育阶段的卵泡GCs中WT1mRNA的表达，发现排卵前GCs中WT1mRNA的表达明显高于层级前GCs。用WT1(+KTS)或WT1(-KTS)过表达载体处理排卵前卵泡的原代细胞，然后在不加或不加促卵泡激素(FSH)的情况下进行培养。用RT-qPCR法检测FSH受体(FSHR)和类固醇生成基因的mRNA水平，用放射免疫法测定细胞上清液中P4的水平。WT1(+KTS)和WT1(-KTS)均可显著降低FSHR、STAR和Cyp11a1mRNA水平，从而显著降低P4的分泌。Western blotting显示WT1(+KTS)或WT1(-KTS)过表达可抑制ERK1/2和BRAF的磷酸化水平，而总蛋白和mRNA水平无明显变化。此外，WT1(+KTS)或WT1(-KTS)过表达后，CREB蛋白和磷酸化水平受到抑制。综上所述，WT1(+kts)和WT1(-kts)抑制CREB蛋白活性，并显著降低FSHR、STAR和Cyp11a1的mRNA水平，从而通过调节ERK1/2信号通路抑制FSH诱导的排卵前GCs分泌P4。
Multiple Wilms tumor gene 1 (WT1) splicing variants are expressed in mammals, and these variants regulate tumorigenesis and mediate the development of multiple tissues and organs, including gonads. However, WT1 splicing variants (+KTS or -KTS) are expressed in only two nonmammalian vertebrates, and unexpectedly, their functions in chicken ovaries remain elusive. Progesterone (P4) secreted by chicken granulosa cells (GCs) participates in various physiological processes and plays an important role in maintaining reproductive performance. The purpose of this study was to investigate the effect of WT1(+KTS) and WT1(-KTS) on chicken P4 secretion in preovulatory GCs. First, we detected WT1 mRNA expression in GCs from follicles of different developmental stages by Quantitative real-time PCR (RT-qPCR) and found that WT1 mRNA expression was considerably increased in preovulatory GCs compared with prehierarchical GCs. Primary cells collected from preovulatory follicles were treated with WT1(+KTS) or WT1(-KTS) overexpression vectors and subsequently cultured in the absence or presence of follicle-stimulating hormone (FSH). The mRNA levels of FSH-receptor (FSHR) and steroidogenesis genes were determined by RT-qPCR, and the P4 levels in the cell supernatants were measured by radioimmunoassay (RIA). Both WT1(+KTS) and WT1(-KTS) significantly decreased P4 secretion due to a reduction in FSHR, STAR and CYP11A1 mRNA levels. Western blotting revealed that ERK1/2 and BRAF phosphorylation levels were suppressed after overexpression of WT1(+KTS) or WT1(-KTS), whereas total protein and mRNA levels were not significantly changed. In addition, CREB protein and phosphorylation levels were inhibited after overexpression of WT1(+KTS) or WT1(-KTS). In conclusion, WT1(+KTS) and WT1 (-KTS) inhibited CREB protein activity and significantly reduced FSHR, STAR and CYP11A1 mRNA levels, which subsequently suppressed FSH-induced P4 secretion in preovulatory GCs by modulating ERK1/2 signaling.