Department of Gynecology and Obstetrics, Ruijin Hospital Affiliated with the Medical School of Shanghai Jiao Tong University, Shanghai, 200025, China.
Department of Histology, Embryology, Genetics and Developmental Biology, Shanghai Key Laboratory for Reproductive Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China.
Reprod Biol Endocrinol. 2021 Sep 9;19(1):139. doi: 10.1186/s12958-021-00823-z.
Granulosa cells (GCs) in cumulus oophorus highly express follicle stimulating hormone receptor (FSHR), which is the most important mediator of both estradiol synthesis and oocyte maturation. Obese women have elevated free fatty acids (FFAs) levels in their follicular fluids and decreased FSHR expression in GCs, which is related to an altered protein kinase B/glycogen synthase kinase 3β (Akt/GSK3β) signaling pathway. Such FFA increases accompany 3-fold rises in pseudokinase 3 (TRIB3) expression and reduce the Akt phosphorylation status in both the human liver and in insulinoma cell lines. Therefore, in a high FFA environment, we determined if TRIB3 mediates regulation of FSHR via the Akt/GSK3β signaling pathway in human GCs.
GCs from women undergoing in vitro fertilization were collected and designated as high and low FFAs cohorts based on their follicular fluid FFA content. GCs with low FFA levels and a human granulosa-like tumor (KGN) cell line were exposed to palmitic acid (PA), which is a dominate FFA follicular fluid constituent. The effects were assessed of this substitution on the Akt/GSK3β signaling pathway activity as well as the expressions of TRIB3 and FSHR at both the gene and protein levels by qPCR, Western blot and immunofluorescence staining analyses. Meanwhile, the individual effects of TRIB3 knockdown in KGN cells and p-AKT inhibitors were compared to determine the mechanisms of FFA-induced FSHR downregulation.
The average FSH dose consuming per oocyte (FSH dose/oocyte) was elevated and Top embryo quality ratio was decreased in women with high levels of FFAs in their follicular fluid. In these women, the GC TRIB3 and ATF4 protein expression levels were upregulated which was accompanied by FSHR downregulation. Such upregulation was confirmed based on corresponding increases in their gene expression levels. On the other hand, the levels of p-Akt decreased while p-GSK3β increased in the GCs. Moreover, TRIB3 knockdown reversed declines in FSHR expression and estradiol (E2) production in KGN cells treated with PA, which also resulted in increased p-Akt levels and declines in the p-GSK3β level. In contrast, treatment of TRIB3-knockdown cells with an inhibitor of p-Akt (Ser473) resulted in rises in the levels of both p-GSK3β as well as FSHR expression whereas E2 synthesis fell.
During exposure to a high FFA content, TRIB3 can reduce FSHR expression through stimulation of the Akt/GSK3β pathway in human GCs. This response may contribute to inducing oocyte maturation.
卵丘颗粒细胞（GCs）高度表达卵泡刺激素受体（FSHR），FSHR 是雌二醇合成和卵母细胞成熟的最重要介质。肥胖女性的卵泡液中游离脂肪酸（FFAs）水平升高，GCs 中的 FSHR 表达降低，这与蛋白激酶 B/糖原合成酶激酶 3β（Akt/GSK3β）信号通路的改变有关。这种 FFA 的增加伴随着拟激酶 3（TRIB3）表达增加 3 倍，以及人肝和胰岛素瘤细胞系中 Akt 磷酸化状态的降低。因此，在高 FFA 环境中，我们确定了 TRIB3 是否通过 Akt/GSK3β 信号通路在人 GCs 中调节 FSHR。
根据卵泡液 FFA 含量，从接受体外受精的女性中收集 GCs，并将其分为高和低 FFA 队列。用棕榈酸（PA）处理低 FFA 水平的 GCs 和人颗粒细胞样肿瘤（KGN）细胞系，PA 是卵泡液中主要的 FFA 成分。通过 qPCR、Western blot 和免疫荧光染色分析评估这种替代物对 Akt/GSK3β 信号通路活性以及 TRIB3 和 FSHR 基因和蛋白水平表达的影响。同时，比较了 KGN 细胞中 TRIB3 敲低和 p-AKT 抑制剂的个体作用，以确定 FFA 诱导的 FSHR 下调的机制。
卵泡液中 FFAs 水平较高的女性，每个卵母细胞消耗的 FSH 剂量（FSH 剂量/卵母细胞）升高，优质胚胎比例降低。在这些女性中，GC 的 TRIB3 和 ATF4 蛋白表达水平上调，同时 FSHR 下调。这种上调是通过相应的基因表达水平增加来证实的。另一方面，GC 中的 p-Akt 水平降低，而 p-GSK3β 水平升高。此外，在 PA 处理的 KGN 细胞中，TRIB3 敲低逆转了 FSHR 表达和雌二醇（E2）产生的下降，这也导致了 p-Akt 水平的升高和 p-GSK3β 水平的下降。相比之下，用 p-Akt（Ser473）抑制剂处理 TRIB3 敲低的细胞会导致 p-GSK3β 和 FSHR 表达水平升高，而 E2 合成下降。
在高 FFA 含量暴露下，TRIB3 可以通过刺激人 GCs 中的 Akt/GSK3β 通路来降低 FSHR 表达。这种反应可能有助于诱导卵母细胞成熟。
