García Elina V, Mansilla Mariano J, LeBlanc Jean Guy, Barrera Antonio D
Instituto Superior de Investigaciones Biológicas (INSIBIO), CONICET-UNT, Chacabuco 461, T4000ILI, San Miguel de Tucumán, Tucumán, Argentina; Instituto de Biología "Dr. Francisco D. Barbieri", Facultad de Bioquímica, Química y Farmacia, UNT, Chacabuco 461, T4000ILI, San Miguel de Tucumán, Tucumán, Argentina.
Instituto de Biología "Dr. Francisco D. Barbieri", Facultad de Bioquímica, Química y Farmacia, UNT, Chacabuco 461, T4000ILI, San Miguel de Tucumán, Tucumán, Argentina.
Theriogenology. 2020 Sep 15;154:161-170. doi: 10.1016/j.theriogenology.2020.05.022. Epub 2020 May 21.
Folate plays a specific role as methyl donor for nucleotide synthesis and genomic methylation patterns, which in turn are important epigenetic determinants in gene expression. Previous studies have revealed the presence of folate in bovine oviductal fluid as well as the existence of a fine-tuned regulation of the gene expression of folate receptors and transporters in bovine oviduct epithelial cells (BOECs). However, the functional implications of folate in the oviduct remain unknown. The present study aimed to assess the effect of folic acid (FA) on expression levels of selected genes that potentially respond to the folate status in in vitro BOECs. To obtain an insight into the optimization of a culture system for assays, gene expression of folate receptors and transporters was compared between BOECs grown in monolayers and in suspension. The results showed that BOECs from isthmus and ampulla in suspension culture better preserved the region-dependent gene expression profile than in monolayers. Subsequently, BOECs from both anatomical regions were separately cultured in suspension for 24 h assaying different FA concentrations: I) TCM-199 (control); II) TCM-199 + 1 μM FA (similar to the oviduct concentration); III) TCM-199 + 10 μM FA and IV) TCM-199 + 100 μM FA. Expression analysis of genes related to important cellular processes including folate transport, DNA methylation, cell-cell interaction, antioxidant activity and signaling pathways was performed in BOECs using RT-qPCR. Our data demonstrated that addition of 1 μM FA did not affect mRNA levels of most genes analyzed. In contrast, BOECs cultured with 10 μM FA exhibited increased mRNA expression levels of genes involved in folate intake, DNA methylation and antioxidant protection. It is worth noting that at 100 μM FA, transcriptional response in BOECs mainly resulted in decreased mRNA levels of the majority of the genes assayed. Interestingly, cytotoxicity analysis showed a similar LDH activity in the culture media of the experimental groups, indicating that cell integrity was not affected by the FA concentrations assayed. In conclusion, our findings suggest that folate can affect BOECs, promoting changes in gene activity in a framework of functional readjustments in response to environmental conditions.
叶酸作为核苷酸合成和基因组甲基化模式的甲基供体发挥着特定作用，而这些反过来又是基因表达中重要的表观遗传决定因素。先前的研究揭示了牛输卵管液中存在叶酸，以及牛输卵管上皮细胞（BOECs）中叶酸受体和转运蛋白基因表达存在精细调节。然而，叶酸在输卵管中的功能意义仍不清楚。本研究旨在评估叶酸（FA）对体外培养的BOECs中可能对叶酸状态作出反应的选定基因表达水平的影响。为了深入了解用于检测的培养系统的优化，比较了单层培养和悬浮培养的BOECs中叶酸受体和转运蛋白的基因表达。结果表明，与单层培养相比，悬浮培养的峡部和壶腹部BOECs能更好地保留区域依赖性基因表达谱。随后，将来自两个解剖区域的BOECs分别在悬浮液中培养24小时，检测不同浓度的FA：I）TCM - 199（对照）；II）TCM - 199 + 1μM FA（类似于输卵管浓度）；III）TCM - 199 + 10μM FA和IV）TCM - 199 + 100μM FA。使用RT - qPCR在BOECs中对与重要细胞过程相关的基因进行表达分析，这些过程包括叶酸转运、DNA甲基化、细胞间相互作用、抗氧化活性和信号通路。我们的数据表明，添加1μM FA对大多数分析基因的mRNA水平没有影响。相反，用10μM FA培养的BOECs表现出参与叶酸摄取、DNA甲基化和抗氧化保护的基因的mRNA表达水平增加。值得注意的是，在100μM FA时，BOECs中的转录反应主要导致大多数检测基因的mRNA水平下降。有趣的是，细胞毒性分析显示实验组培养基中的乳酸脱氢酶（LDH）活性相似，表明细胞完整性不受所检测的FA浓度影响。总之，我们的研究结果表明，叶酸可以影响BOECs，在响应环境条件的功能调整框架内促进基因活性的变化。
