目的:在本研究中,我们研究了双酚A(BPA)及其主要类似物的作用,双酚F(BPF),和双酚-S(BPS),使用体外SSC培养和体内移植模型对精原干细胞(SSC)群体进行研究。
方法:用不同浓度的双酚处理从6天至8天龄的C57BL/6-eGFP+雄性小鼠睾丸中富集的SSC,持续7天,以检查双酚衍生的细胞毒性和SSC特征的变化。我们利用流式细胞仪,免疫细胞化学,实时定量逆转录-PCR,和蛋白质印迹分析。通过体内移植和随后的睾丸分析检查供体SSC来源的精子发生评估,进一步研究了SSC的功能改变。
结果:BPF对SSC的抑制作用与BPA相似,证明SSC存活率显著下降,抑制增殖,和诱导细胞凋亡。另一方面,虽然BPS相对弱于BPA和BPF,它仍然显示出显著的SSC细胞毒性。重要的是,暴露于BPA的SSC,BPF,和BPS显示出供体SSC来源的生殖细胞集落/培养细胞总数的显着减少,表明,比如BPA,BPF,和BPS可以诱导受体动物中功能性SSC的相当减少。然而,精子发生的进展,如组织化学和PCNA和SSC特异性标志物的表达所证明,统称为BPA,BPF,BPS可能不会对精子发生产生不利影响。
结论:我们的研究结果表明,主要的BPA替代品,BPF和BPS,对SSCs有显著的细胞毒性作用,类似于BPA。这些效应可能导致功能性自我更新干细胞群的减少和对男性生育力的潜在影响。
OBJECTIVE: In this study, we investigated the effect of bisphenol-A (BPA) and its major analogs, bisphenol-F (BPF), and bisphenol-S (BPS), on spermatogonial stem cells (SSCs) populations using in vitro SSC culture and in vivo transplantation models.
METHODS: SSCs enriched from 6- to 8-day-old C57BL/6-eGFP+ male mice testes were treated with varying concentrations of bisphenols for 7 days to examine bisphenol-derived cytotoxicity and changes in SSC characteristics. We utilized flow cytometry, immunocytochemistry, real-time quantitative reverse transcription-PCR, and western blot analysis. The functional alteration of SSCs was further investigated by examining donor SSC-derived spermatogenesis evaluation through in vivo transplantation and subsequent testis analysis.
RESULTS: BPF exhibited a similar inhibitory effect on SSCs as BPA, demonstrating a significant decrease in SSC survival, inhibition of proliferation, and induction of apoptosis. On the other hand, while BPS was comparatively weaker than BPA and BPF, it still showed significant SSC cytotoxicity. Importantly, SSCs exposed to BPA, BPF, and BPS exhibited a significant reduction in donor SSC-derived germ cell colonies per total number of cultured cells, indicating that, like BPA, BPF, and BPS can induce a comparable reduction in functional SSCs in the recipient animals. However, the progress of spermatogenesis, as evidenced by histochemistry and the expressions of PCNA and SSC specific markers, collectively indicates that BPA, BPF, and BPS may not adversely affect the spermatogenesis.
CONCLUSIONS: Our findings indicate that the major BPA substitutes, BPF and BPS, have significant cytotoxic effects on SSCs, similar to BPA. These effects may lead to a reduction in the functional self-renewal stem cell population and potential impacts on male fertility.