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. 2016 Feb 16;11(2):e0148577.
doi: 10.1371/journal.pone.0148577. eCollection 2016.

Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes

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Free PMC article

Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes

Hyuck Jun Mok et al. PLoS One. .
Free PMC article

Abstract

The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H2O2), a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H2O2-treated oocytes, we identified 35 decreased and 26 increased lipids in H2O2-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylserine (LPS) significantly decreased both in H2O2-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG) was only noted in H2O2-treated oocytes, indicating that the acute effect of H2O2-caused oxidative stress is distinct from aging-associated lipidome alteration. In H2O2-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Fluorescence staining using BODIPY 500/510 and CellMask in young, aged, and H2O2-treated oocytes.
Five oocytes from each group were stained with BODIPY 500/510 (10 mg/mL, green fluorescence) and CellMask Deep Red (2.5 mg/mL, red fluorescence) in PBS. Oocytes from 4-week-old mice were treated with 50 μM H2O2 for 20 min. White scale bar, 10 μm.
Fig 2
Fig 2. Workflow for lipidomics analysis in three different oocyte samples.
(A) A schematic diagram showing the workflow. Three groups of samples were prepared: 60 MII oocytes from 4-week-old ICR mice (young oocytes), 60 H2O2-treated oocytes from 4-week-old mice (H2O2-treated), and 60 oocytes from 40-week-old mice or older (aged oocytes). Each sample was subjected to lipid extraction. One half of each extract was methylated with TMSD for anionic lipid detection, and the other half used for non-TMSD detection for neutral- and positive-lipid analysis. Samples were then pooled and analyzed by MS to profile oocyte lipids. The identified peaks [signal-to-noise ratio (S/N) > 3] were selected as target MRMs for quantitative analysis of oocyte lipids in three experimental conditions. To mine differentially regulated lipids (DRLs), data were normalized with internal standards and validated using Student’s t-test. (B) This panel shows the process of DRL mining in oocytes. First, 200 oocytes from 4-week-old mice (100 with H2O2 treatment and 100 untreated) were pooled and analyzed using MS to identify detectable target lipids (S/N > 3) in MII oocytes. From this run, we selected 307 lipid peaks as target transition. In the main experiment, three groups (A) were each subjected to lipid extraction and MS analysis in triplicate with three these target MRM transitions. Among 307 lipids, 249 (S/N > 10) were quantifiable in young oocytes, 260 in aged oocytes, and 228 in H2O2-treated oocytes. After data validation, we selected 61 and 32 DRLs in H2O2-treated and aged oocytes, respectively, compared to young oocytes (control). These DRLs are shown in Tables 1 and 2.
Fig 3
Fig 3. Principal component analysis (PCA) plot for MS analysis of young, aged, and H2O2-treated oocyte lipids.
Young oocytes, blue dots; aged oocytes, green dots; H2O2-treated oocytes, red dots.
Fig 4
Fig 4. Hierarchical clustering of datasets from triplicate MS analysis showing differentially regulated lipids in young, H2O2-treated, and aged MII oocytes.
Fig 5
Fig 5. The volcano plots of detected lipids of each set of compared groups.
Grey dots indicate lipids showing no differences in two sets of compared groups. (A) Red dots indicate DRLs in aged oocytes. (B) Red dots indicate DRLs in H2O2-treated oocytes. Red dots indicate detected lipid species with fold changes greater than 1.5 (p < 0.05). Blue triangles in both plots indicate common DRLs between two compared groups. FC, fold change.
Fig 6
Fig 6. Lipid classes that are significantly changed between two sets of compared groups.
(A) Upregulated lipid classes and (B) downregulated lipid classes in comparison with young oocytes (control). Some of the downregulated lipid classes are similar in aged and H2O2-treated oocyte groups, but no significantly increased lipid class was identified in aged oocytes. Fold changes greater than 1.5 are shown (p < 0.05). ISTD, internal standard.
Fig 7
Fig 7. Western blotting of LPCAT1 in H2O2-treated oocytes.
MII oocytes from 4-week-old mice were oxidized with 50 μM H2O2 for 20 min. Control oocytes were incubated in PBS. Oocytes (100 oocytes each) were directly collected in 12 μL of 1× sample buffer and run in a 10% SDS-PAGE gel. After transfer, western blotting was performed with anti-LPCAT1 and anti-α-tubulin primary antibodies. Ovarian extract was used as a positive control. The asterisk indicates LPCAT1 product.

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Grant support

This study was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C0055) (https://www.htdream.kr/).
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