Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel.
IVF Unit, Chaim Sheba Medical Centre, Tel-Hashomer, 52621, Ramat-Gan, Israel.
J Ovarian Res. 2020 Feb 26;13(1):22. doi: 10.1186/s13048-020-00623-w.
Fragile X premutation (Amplification of CGG number 55-200) is associated with increased risk for fragile X-Associated Premature Ovarian Insufficiency (FXPOI) in females and fragile X-associated tremor/ataxia syndrome (FXTAS) predominantly in males. Recently, it has been shown that CGG repeats trigger repeat associated non-AUG initiated translation (RAN) of a cryptic polyglycine-containing protein, FMRpolyG. This protein accumulates in ubiquitin-positive inclusions in neuronal brain cells of FXTAS patients and may lead to protein-mediated neurodegeneration. FMRpolyG inclusions were also found in ovary stromal cells of a FXPOI patient. The role of FMRpolyG expression has not been thoroughly examined in folliculogenesis related cells. The main goal of this study is to evaluate whether FMRpolyG accumulates in mural granulosa cells of FMR1 premutation carriers. Following FMRpolyG detection, we aim to examine premutation transfected COV434 as a suitable model used to identify RAN translation functions in FXPOI pathogenesis.
FMRpolyG and ubiquitin immunostained mural granulosa cells from six FMR1 premutation carriers demonstrated FMRpolyG aggregates. However, co-localization of FMRpolyG and ubiquitin appeared to vary within the FMR1 premutation carriers' group as three exhibited partial ubiquitin and FMRpolyG double staining and three premutation carriers demonstrated FMRpolyG single staining. None of the granulosa cells from the five control women expressed FMRpolyG. Additionally, human ovarian granulosa tumor, COV434, were transfected with two plasmids; both expressing 99CGG repeats but only one enables FMRpolyG expression. Like in granulosa cells from FMR1 premutation carriers, FMRpolyG aggregates were found only in COV434 transfected with expended CGG repeats and the ability to express FMRpolyG.
Corresponding with previous studies in FXTAS, we demonstrated accumulation of FMRpolyG in mural granulosa cells of FMR1 premutation carriers. We also suggest that following further investigation, the premutation transfected COV434 might be an appropriate model for RAN translation studies. Detecting FMRpolyG accumulation in folliculogenesis related cells supports previous observations and imply a possible common protein-mediated toxic mechanism for both FXPOI and FXTAS.
脆性 X 前突变（CGG 数 55-200 扩增）与女性脆性 X 相关卵巢早衰（FXPOI）和男性脆性 X 相关震颤共济失调综合征（FXTAS）的风险增加有关。最近，已经表明 CGG 重复触发隐匿性多聚甘氨酸蛋白 FMRpolyG 的重复相关非 AUG 起始翻译（RAN）。这种蛋白质在 FXTAS 患者的神经元脑细胞中积累，与泛素阳性包涵体有关，可能导致蛋白介导的神经退行性变。在 FXPOI 患者的卵巢基质细胞中也发现了 FMRpolyG 包涵体。FMRpolyG 表达在卵泡发生相关细胞中的作用尚未得到彻底研究。本研究的主要目的是评估 FMR1 前突变携带者的颗粒细胞中是否存在 FMRpolyG 积累。在检测到 FMRpolyG 后，我们旨在检查转染 COV434 的前突变，作为鉴定 FXPOI 发病机制中 RAN 翻译功能的合适模型。
六个 FMR1 前突变携带者的 FMRpolyG 和泛素免疫染色的颗粒细胞显示 FMRpolyG 聚集。然而，在前突变携带者组中，FMRpolyG 和泛素的共定位似乎不同，因为三个表现出部分泛素和 FMRpolyG 双重染色，而三个前突变携带者表现出 FMRpolyG 单染色。五个对照女性的颗粒细胞均未表达 FMRpolyG。此外，用两个质粒转染人卵巢颗粒细胞瘤 COV434，均表达 99CGG 重复序列，但只有一个能使 FMRpolyG 表达。与 FMR1 前突变携带者的颗粒细胞一样，只有在转染扩展 CGG 重复序列且能够表达 FMRpolyG 的 COV434 中转染的细胞中才发现 FMRpolyG 聚集。
与 FXTAS 的先前研究一致，我们证明了 FMR1 前突变携带者的颗粒细胞中 FMRpolyG 的积累。我们还建议，在进一步研究后，转染前突变的 COV434 可能是 RAN 翻译研究的合适模型。在卵泡发生相关细胞中检测到 FMRpolyG 积累支持了先前的观察结果，并暗示 FXPOI 和 FXTAS 可能存在共同的蛋白介导的毒性机制。
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