Mito-TEMPO Improves the Meiosis Resumption and Mitochondrial Function of Vitrified Sheep Oocytes via the Recovery of Respiratory Chain Activity
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Experimental Design
2.2. Collection of COCs
2.3. Vitrification and COC Warming
2.4. In Vitro Maturation
2.5. Oocyte Survival and the First Polar Body Extrusion (PBE)
2.6. Immunofluorescence (IF) Staining
2.7. Intracellular ROS and GSH Level Assay
2.8. Quantification of Mitochondrial Function Staining
2.9. Quantification of Mitochondrial Membrane Potentials (∆Ψm)
2.10. ATP Content Determination
2.11. Quantification of Cytoplasmic Ca2+ ([Ca2+]c), Mitochondrial Ca2+ ([Ca2+]m), and Endoplasmic Reticulum Ca2+ ([Ca2+]ER)
2.12. Building a Single-Cell RNA Library and Sequencing the Transcriptome
2.13. RNA Isolation and Real-Time Quantitative PCR (RT-qPCR)
2.14. Statistical Analysis
3. Results
3.1. Mito-TEMPO Improved the Oocyte Viability and Meiotic Maturation after Vitrification
3.2. Mito-TEMPO Inhibits Oxidative Stress after Vitrification
3.3. Mito-TEMPO Restores Mitochondrial Function in Vitrified Oocytes
3.4. Mito-TEMPO Restores Mitochondrial and Cytoplasmic Ca2+ Levels in Vitrified Oocytes
3.5. Investigating the Target Effectors of Mito-TEMPO in Vitrified Oocytes Using Single-Cell Transcriptome Analysis
3.6. Mito-TEMPO at a Concentration of 1 μM Had No Potential Toxic Effect on Oocyte Mitochondrial Function
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | NCBI Reference Sequences | Primer Sequence (5′ to 3′) | Annealing Temperature (°C) |
---|---|---|---|
GADPH | NM_001190390 | F: GGTTGTCTCCTGCGACTTCA R: CAGGGCCTTGAGGATGGAAA | 55 |
ATP8 | XR_003587932.1 | F: GCCACAACTAGACACATCAACG R: AGGGGTAATGAAAGAGGCAAA | 55 |
FAU | XM_004019665.4 | F: TAGCTTCGTTGGAGGGCATC R: TTAGGGCCCTTCTTCTTGCC | 55 |
FST | XM_012096672.3 | F: TATGGGACTTCAAGGTTGGC R: GGTGTCTTCCGAAATGGAGT | 50 |
NACA | XM_012174719.3 | F: CAACAATGCCCTCTGGCAAC R: TTCTGAGCGAGCAACTGGAG | 50 |
LAMTOR2 | XM_004009674.3 | F: AGACCGTTGGCTTCGGAATG R: TGATGCTGCTACTTGGGTGAG | 55 |
ND4L | XR_003587932.1 | F: TCACAGTATCCCTCACAGGACT R: CTCGCAAGCTGCGAAAACTA | 55 |
SPP1 | NM_001009224.1 | F: TCCGCCCTTCCAGTTAAACC R: TCAGGGGTTTCAGCATCGTC | 55 |
EMD | XM_027963104.1 | F: CGCCAGTACAACATCCCACA R: ACGGACGCCGAATCTAAGTC | 55 |
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Zhao, X.; Dilixiati, A.; Zhang, L.; Aihemaiti, A.; Song, Y.; Zhao, G.; Fu, X.; Wang, X.; Wusiman, A. Mito-TEMPO Improves the Meiosis Resumption and Mitochondrial Function of Vitrified Sheep Oocytes via the Recovery of Respiratory Chain Activity. Animals 2024, 14, 152. https://doi.org/10.3390/ani14010152
Zhao X, Dilixiati A, Zhang L, Aihemaiti A, Song Y, Zhao G, Fu X, Wang X, Wusiman A. Mito-TEMPO Improves the Meiosis Resumption and Mitochondrial Function of Vitrified Sheep Oocytes via the Recovery of Respiratory Chain Activity. Animals. 2024; 14(1):152. https://doi.org/10.3390/ani14010152
Chicago/Turabian StyleZhao, Xi, Airixiati Dilixiati, Luyao Zhang, Aikebaier Aihemaiti, Yukun Song, Guodong Zhao, Xiangwei Fu, Xuguang Wang, and Abulizi Wusiman. 2024. "Mito-TEMPO Improves the Meiosis Resumption and Mitochondrial Function of Vitrified Sheep Oocytes via the Recovery of Respiratory Chain Activity" Animals 14, no. 1: 152. https://doi.org/10.3390/ani14010152
APA StyleZhao, X., Dilixiati, A., Zhang, L., Aihemaiti, A., Song, Y., Zhao, G., Fu, X., Wang, X., & Wusiman, A. (2024). Mito-TEMPO Improves the Meiosis Resumption and Mitochondrial Function of Vitrified Sheep Oocytes via the Recovery of Respiratory Chain Activity. Animals, 14(1), 152. https://doi.org/10.3390/ani14010152