Murine blastocysts generated by in vitro fertilization show increased Warburg metabolism and altered lactate production
- Center for Reproductive Sciences, Department of Obstetrics and Gynecology, University of California, San Francisco, United States
- Division of Cardiovascular Medicine, Department of Medicine, Stanford University, United States
Figures
Figure 1 with 1 supplement
The levels of glutathione (GSH) and reactive oxygen species (ROS).
(A) Analysis of glutathione (GSH) and reactive oxygen species (ROS) in blastocysts. (B) GSH levels are lower in in vitro fertilization-generated blastocysts, while ROS levels are higher both (C) …
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Figure 1—source data 1
The ROS and GSH level in embryos (Figure 1A–C).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig1-data1-v2.xlsx
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Figure 1—source data 2
The extracellular ROS level in embryo culture medium (Figure 1D).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig1-data2-v2.xlsx
Figure 1—figure supplement 1
Morphology of blastocyst generated after natural mating (in vivo or flushed blastocyst-group, (A) or by in vitro fertilization (IVF) using physiologic oxygen (B) IVF5%O2 group, or IVF20%O2 (C) IVF 20%O2 group).
In addition, (D) embryos generated by IVF and cultured in physiologic oxygen (5%O2), showed higher 2 cell rate (71.0 ± 3.1%, p<0.0001) and (E) blastocyst rate (82.8 ± 10.9%, p<0.0001) compared to …
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Figure 1—figure supplement 1—source data 1
The cleavage and blastocyst rate in embryos (Figure 1D-E).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig1-figsupp1-data1-v2.xlsx
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Figure 1—figure supplement 1—source data 2
The total cell number of blastocysts (Figure 1F).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig1-figsupp1-data2-v2.xlsx
Figure 2
Immunocytochemical analysis of oxidative-related markers in blastocysts.
(A and B) In vitro fertilization (IVF) generated blastocysts show increase in 8-OHdG staining (DNA damage marker, green), (C and D) PGF2-alpha (lipid damage marker, green) and (E and F) …
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Figure 2—source data 1
The level of oxidative damage (DNA, lipids, and proteins) in embryos (Figure 2A–F).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig2-data1-v2.xlsx
Figure 3
Metabolic analysis of mitochondrial and glycolytic function using Agilent Seahorse XF HS Mini.
(A) Mitochondrial respiration was analyzed at baseline or after injection of oligomycin (Adenosine triphosphate [ATP] synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, …
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Figure 3—source data 1
The mitochondrial activity in embryos (Figure 3A).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig3-data1-v2.xlsx
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Figure 3—source data 2
The mitochondrial activity in embryos (Figure 3B–D).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig3-data2-v2.xlsx
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Figure 3—source data 3
The glycolytic function in embryos (Figure 3E–H).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig3-data3-v2.xlsx
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Figure 3—source data 4
The ratio of oxygen consumption rate and extracellular acidification rate in embryos (Figure 3I–J).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig3-data4-v2.xlsx
Figure 4 with 1 supplement
IVF-generated embryos show several metabolic alterations.
(A) Confocal microscopy images of blastocysts using phase contrast or confocal microscopy. Two fluorescence emission wavelengths were used, 640 nm (pH sensitive, green) and 600 nm (pH insensitive, …
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Figure 4—source data 1
Intracellular pH level in embryos (Figure 4A–B).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig4-data1-v2.xlsx
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Figure 4—source data 2
The NAD level in embryos (Figure 4D).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig4-data2-v2.xlsx
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Figure 4—source data 3
The intracellular pyruvate level in embryos (Figure 4E).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig4-data3-v2.xlsx
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Figure 4—source data 4
The intracellular lactate level in embryos (Figure 4F).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig4-data4-v2.xlsx
Figure 4—figure supplement 1
Lactate (A) and pyruvate (B) levels are lower in media where in vitro fertilization (IVF)-generated embryos are cultured compared to media maintained in incubator without embryos.
Seven biological replications were performed for each experiment. Error bar indicates standard deviation. * if p<0.05; ** if p<0.01, *** if p<0.001 and **** if p<0.0001.
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Figure 4—figure supplement 1—source data 1
The extracellular lactate and pyruvate level in embryo culture medium (Figure 4—figure supplement 1A-B).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig4-figsupp1-data1-v2.xlsx
Figure 5
Analysis of glycolysis, pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle proteins dysregulated in in vitro fertilization (IVF)-generated blastocysts vs control.
Overall, 3965 proteins were identified and 1029 were differentially expressed between the groups. IVF5%O2-generated blastocysts had 334 proteins upregulated relative to the control (flushed …
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Figure 5—source data 1
The proteomic analysis data (Figure 5).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig5-data1-v2.xlsx
Figure 6
IVF-generated embryos show downregulation of enzymes involved in lactate metabolism.
(A) Expression levels of lactate dehydrogenase (LDH)-A and LDH-B (red color: LDHA, green color: LDHB) and (B and C) their quantification. (D and E) MCT1 enzyme is downregulated in in vitro …
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Figure 6—source data 1
The fluorescence intensity of LDHB, LDHA, and MCT1 in embryos (Figure 6A–E).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig6-data1-v2.xlsx
Figure 7 with 1 supplement
Western blot analysis of protein levels of lactate dehydrogenase (LDH)-B and MCT1 enzymes in selected insulin sensitive tissues of 40-week-old mice conceived in vivo (FB n=8, 50% females) or by IVF5%O2 (n=8, 50% females).
In vitro fertilization (IVF)-conceived mice show reduced levels of LDHB and monocarboxylate transporter 1 (MCT1) in gastrocnemius muscle (A–C) gonadal adipose tissue (D–F) and liver (G–I). Selected …
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Figure 7—source data 1
Eight biological replicates for FB and IVF were compared.
The value of each biological replicate was the average of 4 technical replicates. (i) Muscle, Western blot images (ppt), source data (xls). (ii) Fat, Western blot images (ppt), source data (xls). (iii) Liver, Western blot images (ppt), source data (xls).
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig7-data1-v2.zip
Figure 7—figure supplement 1
Unsupervised metabolomic analysis by LC/MS was performed in liver, gonadal adipose tissue, and serum of in vivo conceived mice or mice conceived by in vitro fertilization (IVF) (5%O2).
Data are from Feuer et al., 2014b and at least three independent replicates were performed. Error bar indicates standard deviation.Relative lactic acid levels trend lower in IVF conceived mice.
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Figure 7—figure supplement 1—source data 1
The lactate level in liver, fat, and serum in adult mice.
- https://cdn.elifesciences.org/articles/79153/elife-79153-fig7-figsupp1-data1-v2.xlsx
Figure 8
Summary of changes observed in IVF generated blastocysts and offspring.
In vitro fertilization (IVF) generated embryos show increase in ROS and oxidative damage and significant metabolic derangement including decreased NAD levels, increased intracellular pH and …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-8-Hydroxy-2- deoxyguanosine (Mouse monoclonal) | Abcam | Cat#: ab48508 | IF (1:200) |
| Antibody | Anti-8 iso Prostaglandin F2 alpha (Rabbit polyclonal) | Abcam | Cat#: ab2280 | IF (1:200) |
| Antibody | Anti-DNP antibody (Rabbit polyclonal) | Sigma | Cat#: D9656 | IF (1:200) |
| Antibody | Anti-MCT1 antibody (Mouse monoclonal) | Santa Cruz | Cat#: sc365501 | IF (1:200) |
| Antibody | Anti-LDHA antibody (Rabbit monoclonal) | Cell Signaling | Cat#: 3582 | IF (1:200) |
| Antibody | Anti-LDHB antibody (Mouse monoclonal) | Santa Cruz | Cat#: sc100775 | IF (1:200) |
| Antibody | Goat anti-Rabbit (goat polyclonal, Alexa Fluor conjugate) | Abcam | Cat#: ab150077 | IF (1:200) |
| Antibody | Donkey anti-Rabbit (donkey polyclonal, Alexa Fluor conjugate) | Abcam | Cat#: ab150075 | IF (1:200) |
| Antibody | Goat anti-Mouse (goat polyclonal, Alexa Fluor conjugate) | Abcam | Cat#: ab150113 | IF (1:200) |
| Biological sample (mouse) | Blastocyst | This paper | Female | |
| Biological sample (mouse) | Liver | This paper | Male/Female | |
| Biological sample (mouse) | Muscle | This paper | Male/Female | |
| Biological sample (mouse) | Fat | This paper | Male/Female | |
| Commercial assay or kit | Seahorse XF Cell Mito Stress Test kit | Agilent | 103010–100 | |
| Commercial assay or kit | Seahorse XF Glycolysis Stress Test kit | Agilent | 103020–100 | |
| Commercial assay or kit | Pyruvate assay kit | Cayman | Cat#: 700470 | |
| Commercial assay or kit | Lactate assay kit | Cayman | Cat#: 700510 | |
| Commercial assay or kit | NAD Quantitation kit | Sigma | Cat#: MAK037 | |
| Chemical compound, drug | SNARF-1 | Thermo fisher | Cat#: C1272 | |
| Software, algorithm | GraphPad Prism | GraphPad | ||
| Strain, strain background (mouse) | CF-1 | ENVIGO | Hsd:NSA | Oocyte donor |
| Strain, strain background (mouse) | B6D2F1 | The Jackson Laboratory | 100006 | Sperm donor |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/79153/elife-79153-mdarchecklist1-v2.pdf
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Source data 1
The source data for Figures 1—7 and Figure 1—figure supplement 1, Figure 4—figure supplement 1, Figure 7—figure supplement 1.
- https://cdn.elifesciences.org/articles/79153/elife-79153-data1-v2.zip
