Department of Production Animal Health, Faculty of Veterinary Medicine, 3330 Hospital Drive NW, University of Calgary, Calgary, AB T2N 4N1, Canada.
Department of Production Animal Health, Faculty of Veterinary Medicine, 3330 Hospital Drive NW, University of Calgary, Calgary, AB T2N 4N1, Canada; and Corresponding author. Email:
Reprod Fertil Dev. 2020 Jun;32(10):929-940. doi: 10.1071/RD19460.
The critical role of insulin-like growth factor (IGF) 1 in promoting Sertoli cell proliferation invivo and invitro has been established, but its downstream signalling mechanisms remain unknown. In addition to mitogenic effects, a role for IGF1 in mediating cholesterol biosynthesis within testes has been implied. The aims of this study were to investigate the roles of: (1) phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) signalling in IGF1-mediated Sertoli cell proliferation; and (2) IGF1 in mediating cholesterol biosynthesis in Sertoli cells. Primary cultures of Sertoli cells were prepared from 1-week-old porcine testes. On Day 3 of culture, Sertoli cells were treated with 300ng mL-1 IGF1, alone or in combination with inhibitors of IGF1 receptor (2μM picropodophyllotoxin), Akt (1μM wortmannin) or mTOR (200nM rapamycin). Cells were cultured for 30min and phosphorylation levels of Akt, mTOR and p70 ribosomal protein S6 kinase (p70S6K) were determined by immunoblotting. Cell proliferation and quantitative polymerase chain reaction assays were conducted using cells cultured for 24h. IGF1 increased phosphorylation of Akt, mTOR and p70S6K and cell proliferation, and these effects were inhibited by inhibitors of IGF1R, Akt and mTOR. Furthermore, IGF1 upregulated the expression of cholesterol biosynthetic genes (3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS1) and cytochrome P450, family 5, subfamily A, polypeptide 1 (CYP5A1)), but not sterol regulatory element-binding transcription factor 1 (SREBF1). Increased phosphorylation of p70S6K, a major downstream target of mTOR, and upregulated expression of genes involved in cholesterol biosynthesis are indicative of the key role played by IGF1 in regulating the synthesis of cholesterol, the precursor for steroid hormones.
胰岛素样生长因子 1（IGF-1）在促进体内和体外支持细胞增殖中的关键作用已经确立，但它的下游信号机制尚不清楚。除了有丝分裂作用外，IGF-1 在介导睾丸内胆固醇生物合成中的作用也已暗示。本研究的目的是探讨以下两个方面的作用：（1）磷脂酰肌醇 3-激酶/蛋白激酶 B/哺乳动物雷帕霉素靶蛋白（PI3K/Akt/mTOR）信号通路在 IGF-1 介导的支持细胞增殖中的作用；（2）IGF-1 在介导支持细胞胆固醇生物合成中的作用。从 1 周龄猪睾丸中制备原代支持细胞培养物。在培养的第 3 天，用 300ng/ml IGF-1 单独或与 IGF-1 受体抑制剂（2μM 苦鬼白毒素）、Akt 抑制剂（1μM 渥曼青霉素）或 mTOR 抑制剂（200nM 雷帕霉素）联合处理支持细胞。培养 30min 后，通过免疫印迹法测定 Akt、mTOR 和 p70 核糖体蛋白 S6 激酶（p70S6K）的磷酸化水平。用培养 24h 的细胞进行细胞增殖和定量聚合酶链反应检测。IGF-1 增加了 Akt、mTOR 和 p70S6K 的磷酸化和细胞增殖，这些作用被 IGF-1R、Akt 和 mTOR 的抑制剂所抑制。此外，IGF-1 上调了胆固醇生物合成基因（3-羟-3-甲基戊二酰基辅酶 A 还原酶（HMGCR）、3-羟-3-甲基戊二酰基辅酶 A 合酶（HMGCS1）和细胞色素 P450，家族 5，亚家族 A，多肽 1（CYP5A1））的表达，但不调节固醇调节元件结合转录因子 1（SREBF1）。mTOR 的主要下游靶标 p70S6K 的磷酸化增加和胆固醇生物合成相关基因的表达上调表明 IGF-1 在调节胆固醇合成中起关键作用，胆固醇是类固醇激素的前体。
