目的:研究环境内分泌干扰物双酚A(BPA)对生殖细胞囊肿破裂的影响,并探讨调节该活性的可能机制。
方法:在妊娠第11天通过管饲法将BPA(2μg/kg/d或20μg/kg/d)或生育酚剥离的玉米油(载体对照)给予妊娠小鼠,并在出生后第4天(PND)和PND22处死后代(产前处理的小鼠)并切除卵巢。卵巢形态记录在第一孝子(F1)代雌性后代中,并在PND4上对卵泡进行形态学分析和分类。发现差异表达的基因和相关的靶途径,我们用RNA-seq,京都基因和基因组百科全书(KEGG)途径分析,和基因本体论(GO)分析。通过Q-PCR评估了毛喉素诱导的KGN细胞中关键类固醇激素合成相关基因的mRNA表达。免疫印迹(WB)和qRTPCR检测脑源性神经营养因子(BDNF)蛋白和基因表达水平。
结果:BPA,一种典型的内分泌干扰化学物质(EDC),降低了关键的类固醇激素合成相关基因P450scc和芳香化酶的表达,而Star的表达显著增加,Cyp17a1或HSD3β在毛喉素诱导的KGN细胞中的表达无显著差异。此外,我们证实,在子宫内暴露于环境相关浓度的BPA(2μg/kg/d和20μg/kg/d)可以显着破坏生殖细胞囊肿的分解,导致原始卵泡的产生少于对照组。介导抑制作用的因素包括PI3K-Akt信号通路和BDNF的显著下调。
结论:这些研究结果表明,在子宫内低剂量暴露于BPA,低于推荐的“安全”剂量,可能通过抑制类固醇激素合成相关基因的表达,部分通过调节BDNF介导的PI3K/Akt通路来影响原始卵泡的形成。
OBJECTIVE: To determine the influence of the environmental endocrine disruptor bisphenol A (BPA) on germ cell cyst breakdown and explore the possible mechanisms regulating this activity.
METHODS: BPA (2 μg/kg/d or 20 μg/kg/d) or tocopherol-stripped corn oil (vehicle control) was administered to pregnant mice by gavage at gestational day 11, and the offspring (prenatally treated mice) were sacrificed and ovariectomized at postnatal day (PND) 4 and PND22. Ovarian morphology was documented in the first filial (F1) generation female offspring, and the follicles were analyzed and classified morphologically on PND 4. To discover differentially expressed genes and associated target pathways, we used RNA-seq, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Ontology (GO) analysis. The mRNA expression of key steroid hormone synthesis-related genes was evaluated by Q-PCR in forskolin-induced KGN cells. Western blotting (WB) and qRTPCR were used to determine the protein and gene expression levels of brain-derived neurotrophic factor (BDNF).
RESULTS: BPA, a typical endocrine disrupting chemical (EDC), decreased the expression of the key steroid hormone synthesis-related genes P450scc and aromatase, while the expression of Star increased significantly and caused no significant difference in the expression of Cyp17a1 or HSD3β in forskolin-induced KGN cells. Moreover, we confirmed that in utero exposure to environmentally relevant concentrations of BPA (2 μg/kg/d and 20 μg/kg/d) could significantly disrupt germ cell cyst breakdown, leading to the generation of fewer primordial follicles than in the control group. The factors mediating the inhibitory effects included the PI3K-Akt signaling pathway and a significant downregulation of BDNF.
CONCLUSIONS: These findings indicate that in utero exposure to BPA at low doses, which are lower than recommended as \'safe\' dosages, may influence the formation of primordial follicles by inhibiting the expression of steroid hormone synthesis-related genes and partly by regulating the BDNF-mediated PI3K/Akt pathway.