Lecante Laetitia L, Gaye Bintou, Delbes Geraldine
Institut National de la Recherche Scientifique, Centre Armand-Frappier Santé Biotechnologie, Laval, QC, Canada.
Front Toxicol. 2022 Jun 2;4:893050. doi: 10.3389/ftox.2022.893050. eCollection 2022.
Although the decline in male fertility is believed to partially result from environmental exposures to xenoestrogens during critical developmental windows, the underlying mechanisms are still poorly understood. Experimental exposures in rodents have demonstrated the negative impact of xenoestrogens on reproductive development, long-term adult reproductive function and offspring health. In addition, transcriptomic studies have demonstrated immediate effects on gene expression in fetal reproductive tissues, However, the immediate molecular effects on the developing germ cells have been poorly investigated. Here, we took advantage of a transgenic rat expressing the green fluorescent protein specifically in germ cells allowing purification of perinatal GFP-positive germ cells. Timed-pregnant rats were exposed to ethinylestradiol (EE2, 2 μg/kg/d), genistein (GE, 10 mg/kg/d) or vehicle by gavage, from gestational days (GD) 13-19; testes were sampled at GD20 or post-natal (PND) 5 for histological analysis and sorting of GFP-positive cells. While EE2-exposed females gained less weight during treatment compared to controls, neither treatment affected the number of pups per litter, sex ratio, anogenital distance, or body and gonadal weights of the offspring. Although GE significantly decreased circulating testosterone at GD20, no change was observed in either testicular histology or germ cell and sertoli cell densities. Gene expression was assessed in GFP-positive cells using Affymetrix Rat Gene 2.0 ST microarrays. Analysis of differentially expressed genes (DEGs) ( < 0.05; fold change 1.5) identified expression changes of 149 and 128 transcripts by EE2 and GE respectively at GD20, and 287 and 207 transcripts at PND5, revealing an increased effect after the end of treatment. Only about 1% of DEGs were common to both stages for each treatment. Functional analysis of coding DEG revealed an overrepresentation of olfactory transduction in all groups. In parallel, many non-coding RNAs were affected by both treatments, the most represented being small nucleolar and small nuclear RNAs. Our data suggest that despite no immediate toxic effects, fetal exposure to xenoestrogens can induce subtle immediate changes in germ cell gene expression. Moreover, the increased number of DEGs between GD20 and PND5 suggests an effect of early exposures with latent impact on later germ cell differentiation.
尽管人们认为男性生育能力下降部分是由于在关键发育窗口期环境接触到外源性雌激素所致，但其潜在机制仍知之甚少。对啮齿动物的实验性暴露已证明外源性雌激素对生殖发育、成年后的长期生殖功能和后代健康有负面影响。此外，转录组学研究已证明其对胎儿生殖组织中的基因表达有即时影响。然而，对外源性雌激素对发育中的生殖细胞的即时分子影响的研究却很少。在此，我们利用了一种在生殖细胞中特异性表达绿色荧光蛋白的转基因大鼠，从而能够纯化围产期绿色荧光蛋白阳性生殖细胞。将处于特定孕期的大鼠从妊娠第13天至第19天通过灌胃给予乙炔雌二醇（EE2，2μg/kg/d）、染料木黄酮（GE，10mg/kg/d）或溶剂；在妊娠第20天或出生后第5天采集睾丸进行组织学分析和绿色荧光蛋白阳性细胞分选。与对照组相比，暴露于EE2的雌性大鼠在治疗期间体重增加较少，但两种处理均未影响每窝幼崽数量、性别比例、肛门与生殖器距离或后代的体重及性腺重量。尽管GE在妊娠第20天显著降低了循环睾酮水平，但睾丸组织学、生殖细胞和支持细胞密度均未观察到变化。使用Affymetrix大鼠基因2.0 ST微阵列评估绿色荧光蛋白阳性细胞中的基因表达。对差异表达基因（DEGs）（<0.05；倍数变化>1.5）的分析确定，EE2和GE在妊娠第20天时分别有149个和128个转录本表达发生变化，在出生后第5天时分别有287个和207个转录本表达发生变化，表明在治疗结束后影响增加。每种处理在两个阶段中只有约1%的差异表达基因是相同的。对编码差异表达基因的功能分析表明，嗅觉转导在所有组中均过度表达。同时，许多非编码RNA受到两种处理的影响，其中最具代表性的是小核仁RNA和小核RNA。我们的数据表明，尽管胎儿暴露于外源性雌激素没有即时毒性作用，但仍可诱导生殖细胞基因表达发生细微的即时变化。此外，妊娠第20天和出生后第5天之间差异表达基因数量的增加表明早期暴露对后期生殖细胞分化有潜在影响。
