Maternal Gliadin Intake Reduces Oocyte Quality with Chromosomal Aberrations and Increases Embryonic Lethality through Oxidative Stress in a Caenorhabditis elegans Model
<p>Gliadin peptide (GP) intake increases mitochondrial ROS production (mtROS) in adult <span class="html-italic">C. elegans</span>. (<b>A</b>) Experimental scheme of GP treatment. The synchronized wild-type N2 worms at L4 stage were incubated on NGM plates containing 0 (DMSO) or 3 µM of GP at 20 °C, and then worms were examined at day 1 or day 3 post L4 stage. (<b>B</b>–<b>F</b>) Comparison of the mtROS levels in worms (<b>B</b>,<b>C</b>), in the dissected gonad (<b>D</b>,<b>E</b>), or in the oocytes (<b>F</b>) treated with 0 (CT) and 3 µM of GP at the L4 stage for 1 day (<b>B</b>,<b>D</b>) or 3 days (<b>C</b>,<b>E</b>,<b>F</b>) using CellROX<sup>®</sup>Green staining. The dashed line indicates the outline of the whole body (<b>B</b>,<b>C</b>) or distal gonad (<b>D</b>,<b>E</b>). Scale bars are 50 µm (<b>B</b>–<b>E</b>) or 20 μm (<b>F</b>). Error bars represent SD. n.s.: not significant, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.005, *** <span class="html-italic">p</span> < 0.001 (Student’s <span class="html-italic">t</span>-test).</p> "> Figure 2
<p>Chromosomal aberrations in the oocytes, embryonic lethality, and oxidative stress sensitivity in offspring are increased by GP intake. (<b>A</b>) Chromosomal morphology was observed using DNA staining. The type of aberration was classified into three categories depending on the condensation of chromosomes: aligned and condensed (a), under-condensed (b), and misaligned and condensed (c). The graph indicates the chromosomal morphology of -1 oocytes produced from control (CT) or GP-treated adult worms. Scale bars are 10 μm. ** <span class="html-italic">p</span> < 0.05 (chi-square test). (<b>B</b>) The graph indicates the percentages of unhatched embryos for 24 h in day-3 adult-stage worms treated with 0 (CT) or 3 µM of GP. Error bars represent SD. * <span class="html-italic">p</span> < 0.05 (Student’s <span class="html-italic">t</span>-test). (<b>C</b>) Experimental scheme for survival assay against 100 mM paraquat (PQ) in the offspring produced from DMSO-treated (CT) or GP-treated worms (GP). (<b>D</b>) The graph indicates survival rate in the F1 progenies produced by worms treated with 0 (CT) or 3 µM GP and 100 mM PQ for 6 h. Error bars represent SD. * <span class="html-italic">p</span> < 0.05 (two-way ANOVA with Tukey’s post hoc test).</p> "> Figure 3
<p>N-acetyl-L-cysteine (NAC) treatment suppresses effects of GP intake in adult <span class="html-italic">C. elegans</span>. (<b>A</b>) Experimental scheme of NAC treatment. The synchronized wild-type N2 worms at L4 stage were incubated on NGM plates containing 0 (DMSO) or 3 µM of GP at 20 °C, and treated with NAC for 24 h, and then worms were examined at day 3 post L4 stage. (<b>B</b>) Comparison of mtROS levels in oocytes produced from control (CT) and GP-treated adult worms without (CT, GP) or with NAC (CT + NAC, GP + NAC) treatment using CellROX<sup>®</sup>Green staining. The bar graph indicates the relative pixel intensity of fluorescence of CellROX<sup>®</sup>Green. Scale bars are 20 μm. Error bars represent SD. * <span class="html-italic">p</span> < 0.05 (Student’s <span class="html-italic">t</span>-test). (<b>C</b>) The graph indicates chromosomal morphology of -1 oocytes produced from control (CT) or GP-treated adult worms without (CT, GP) or with NAC (CT + NAC, GP + NAC) treatment using DNA staining. The types of aberrations were classified into three categories depending on the chromosomal morphology: aligned and condensed (a), under-condensed (b), and misaligned and condensed (c). Scale bars are 10 µm, * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.005 (chi-square test). (<b>D</b>) The graph indicates the percentages of unhatched embryos for 24 h in day-3 adult worms treated with 0 (CT) or 3 µM of GP with or without NAC. Error bars represent SD. n.s.: not significant. * <span class="html-italic">p</span> < 0.05 (Student’s <span class="html-italic">t</span>-test). (<b>E</b>) The graph indicates survival rate in the F1 progenies produced by 0 (CT) or 3 µM of GP-treated worms without (CT, GP) or with NAC (CT + NAC, GP + NAC) after 100 mM paraquat treatment for 6 h. Error bars represent SD. n.s.: not significant. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.005 (two-way ANOVA with Tukey’s post hoc test).</p> "> Figure 4
<p>GP intake regulates the expression of antioxidant genes and DAF-16 activity in adult <span class="html-italic">C. elegans</span>. (<b>A</b>) The mRNA levels of antioxidant genes in GP-treated adult <span class="html-italic">C. elegans</span> were detected by three independent quantitative reverse transcription-polymerase chain reaction analyses using <span class="html-italic">cdc-42</span> mRNA in each sample as an internal control for normalization. Error bars represent SD. * <span class="html-italic">p</span> < 0.05 (Student’s <span class="html-italic">t</span>-test). (<b>B</b>) Nuclear localization of DAF-16::GFP was observed in worms treated without (CT) or with GP. The graph indicates the percentage of nuclear localization of DAF-16::GFP in neurons, hypodermis, and intestines. HEAT indicates heat shock at 37 °C for 15 min, which is used for the positive control of nuclear localization of DAF-16::GFP. Scale bars are 50 µm.</p> "> Figure 5
<p>GP intake promotes intestinal disruption in adult <span class="html-italic">C. elegans</span>. (<b>A</b>) The presence of blue food dye in the body cavity indicates leakage of the intestinal barrier after 0 (CT) or 3 µM of GP treatment. Error bars represent SD. n.s.: not significant. ** <span class="html-italic">p</span> < 0.05 (chi-square test). (<b>B</b>) Transgenic worms expressing <span class="html-italic">actin 5 (ACT-5)::GFP</span> were treated with 0 (CT) or 3 µM of GP at the L4 stage for 3 days. The intestinal actin localization was classified into four stages depending on mislocalization in the apical side of the intestine. The percent distributions of the respective stage in worms are presented in the graph. Scale bars are 50 µm, ** <span class="html-italic">p</span> < 0.05 (chi-square test).</p> "> Figure 6
<p>Working model: maternal gliadin peptide (GP) intake in day-3 adult <span class="html-italic">C. elegans</span> increases the level of mitochondrial ROS (mtROS) and decreases oocyte quality with chromosomal aberrations, consequently increasing embryonic lethality and decreasing oxidative stress resistance in offspring.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Caenorhabditis elegans Strains and Gliadin Peptide (GP) Treatment
2.2. Mitochondrial Reactive Oxygen Species (mtROS) Analysis and N-Acetyl-L-Cysteine (NAC) Treatment
2.3. DNA Staining in Oocytes, Embryonic Lethality, and Survival Assay
2.4. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
2.5. Live Image Observation of Fluorescence-Tagged Transgenic Animals
2.6. Intestinal Barrier Function Assay
2.7. Statistical Analysis
3. Results
3.1. Mitochondrial Reactive Oxygen Species (mtROS) Production Was Increased in Gliadin Peptide (GP)-Treated Adult C. elegans
3.2. Oocyte Quality, Embryonic Lethality, and Oxidative Stress Sensitivity in GP-Treated Adult C. elegans and Offspring
3.3. N-Acetyl-L-Cysteine (NAC) Treatment Suppresses Adverse Effects of GP Intake
3.4. Expression of Oxidative Stress-Responding Genes Increased with GP Intake
3.5. GP Intake Causes Disruption of Intestinal Barrier in Adult C. elegans
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
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Lee, J.H.; Lee, M.; Min, H.; Youn, E.; Shim, Y.-H. Maternal Gliadin Intake Reduces Oocyte Quality with Chromosomal Aberrations and Increases Embryonic Lethality through Oxidative Stress in a Caenorhabditis elegans Model. Nutrients 2022, 14, 5403. https://doi.org/10.3390/nu14245403
Lee JH, Lee M, Min H, Youn E, Shim Y-H. Maternal Gliadin Intake Reduces Oocyte Quality with Chromosomal Aberrations and Increases Embryonic Lethality through Oxidative Stress in a Caenorhabditis elegans Model. Nutrients. 2022; 14(24):5403. https://doi.org/10.3390/nu14245403
Chicago/Turabian StyleLee, Jae Hyuck, Mijin Lee, Hyemin Min, Esther Youn, and Yhong-Hee Shim. 2022. "Maternal Gliadin Intake Reduces Oocyte Quality with Chromosomal Aberrations and Increases Embryonic Lethality through Oxidative Stress in a Caenorhabditis elegans Model" Nutrients 14, no. 24: 5403. https://doi.org/10.3390/nu14245403