Center for Reproductive Medicine, Shandong Provincial Hospital Affiliated to Shandong University, Shandong 250001, P.R. China.
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, P.R. China.
Mol Med Rep. 2017 Nov;16(5):6489-6498. doi: 10.3892/mmr.2017.7463. Epub 2017 Sep 12.
The present study aimed to identify the molecular mechanisms underlying the effects of the fructose‑1,6‑bisphosphatase 1 (FBP1) signaling pathway within normal follicle development and in hyperandrogenism‑induced abnormal follicle growth. To achieve this, murine primary follicles, granulosa cells (GCs) and theca‑interstitial cells (TICs) were isolated, cultured in vitro and treated with a high concentration of androgens. A concentration of 1x10‑5 mol/l testosterone was considerable to induce hyperandrogenism by MTT assay. All cells were divided into four groups, as follows: Control group, testosterone group, androgen receptor antagonist‑flutamide group and flutamide + testosterone group. Flutamide was used in the present study as it blocks the effects of the androgen receptor. The mRNA expression levels of FBP1 were detected using reverse transcription‑quantitative polymerase chain reaction. The expression levels and localization of FBP1 were analyzed by western blot analysis and immunofluorescence staining. The experimental results demonstrated that androgen presence stimulated follicle development, whereas excessive testosterone inhibited development. FBP1 was identified as being mainly expressed in follicles; FBP1 protein was significantly expressed in GCs of the 14‑day‑cultured follicle, as well as in the cytoplasm and nuclei of GCs and TICs in vitro. Testosterone increased FBP1 expression during a specific range of testosterone concentrations. Testosterone increased the expression of FBP1 within GCs. Furthermore, FBP1 and phosphoenolpyruvate carboxykinase 1 (PCK1) mRNA expression was increased in GCs treated with testosterone, whereas forkhead box protein O1 (FOXO1) and peroxisome proliferator‑activated receptor γ coactivator‑1α mRNA expression was significantly decreased in the testosterone group. In TICs, testosterone and flutamide inhibited the mRNA expression levels of FOXO1 and glucose‑6‑phosphatase enzyme, and promoted the expression of PCK1. These results suggested that the FBP1 signaling pathway may serve an important role in normal follicle growth and hyperandrogenism‑induced abnormal development, which may be associated with abnormal glucose metabolism induced by high concentrations of testosterone.
本研究旨在鉴定果糖-1,6-二磷酸酶 1 (FBP1) 信号通路在正常卵泡发育和高雄激素诱导的异常卵泡生长中的分子机制。为此，分离培养了小鼠初级卵泡、颗粒细胞（GC）和间质细胞（TIC），并用高浓度雄激素处理。MTT 检测显示，1x10-5mol/L 睾酮浓度可诱导高雄激素血症。所有细胞均分为四组：对照组、睾酮组、雄激素受体拮抗剂氟他胺组和氟他胺+睾酮组。本研究中使用氟他胺阻断雄激素受体的作用。采用逆转录定量聚合酶链反应检测 FBP1 的 mRNA 表达水平。通过 Western blot 分析和免疫荧光染色分析 FBP1 的表达水平和定位。实验结果表明，雄激素存在刺激卵泡发育，而过量的睾酮抑制发育。鉴定出 FBP1 主要在卵泡中表达；FBP1 蛋白在 14 天培养的卵泡的 GC 中以及 GC 和 TIC 的细胞质和核中均有明显表达。在特定的睾酮浓度范围内，睾酮增加了 FBP1 的表达。睾酮增加了 GC 中 FBP1 的表达。此外，GC 中经睾酮处理后 FBP1 和磷酸烯醇丙酮酸羧激酶 1（PCK1）mRNA 表达增加，而 FOXO1 和过氧化物酶体增殖物激活受体 γ 共激活因子 1α mRNA 表达在睾酮组中显著降低。在 TIC 中，睾酮和氟他胺抑制 FOXO1 和葡萄糖-6-磷酸酶的 mRNA 表达水平，并促进 PCK1 的表达。这些结果表明，FBP1 信号通路可能在正常卵泡生长和高雄激素诱导的异常发育中发挥重要作用，这可能与高浓度睾酮诱导的异常葡萄糖代谢有关。
