Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes
Summary
Here, we describe the isolation of mitochondria from mouse adipose-derived mesenchymal stem cells, and then transfer the mitochondria into aged mouse oocytes to improve the quality of the oocytes.
Abstract
Due to the decline in the quantity and quality of oocytes related to age, the fertility of women over 35 years of age has declined sharply. The molecular mechanisms that maintain oocyte quality remain unclear, thus it is difficult to increase the birth rate of women over 35 years old at present. Oocytes contain more mitochondria than any type of cell in the body, and any mitochondrial dysfunction can lead to reduced oocyte quality. In the 1990s, oocyte cytoplasmic transfer resulted in great success in human reproduction but was accompanied by ethical controversies. Autologous mitochondrial transplantation is expected to be a useful technique to increase the quality of oocytes that have decreased due to age. In the present study, we used adipose-derived stem cells from aged mice as a mitochondria donor to increase the quality of oocytes of aged mice. Further development of autologous mitochondrial transfer technology will provide a new and effective treatment for infertility in aged women.
Introduction
One of the important factors that affects female fertility is oocyte aging; decline in oocyte quality is the main cause of infertility in aged women. However, the main cause of oocyte aging and the molecular mechanism that regulates oocyte quality are still unclear. Previous studies have indicated that both the number and quality of mitochondria are involved in the quality control of oocytes and embryonic development1,2,3. The decrease in the quantity and quality of mitochondria is closely related to aging3.
Many attempts have been made to improve the function of mitochondria in aged oocytes, including the nutritional supplement of mitochondria and mitochondria transfer. Well-known, effective, nutritional supplements of mitochondria include Coenzyme Q10 (CoQ10), Alpha-lipoic acid (α-LA), and resveratrol (RSV)4. Studies have shown that CoQ10 supplementation can not only improve the age-related decline in the quantity and quality of oocytes, but also promote the normal development and ovulation of oocytes5. α-LA slows the oocyte quality decline related to aging and the metabolic phenotype of patients with polycystic ovary syndrome (PCOS)6,7. Resveratrol can reduce the number of oocytes with abnormal spindles and improper chromosome alignment increased in aging mice, while affecting the embryonic development in a dose-dependent manner8. However, as the clinical effect of nutritional supplements of mitochondria has not reached expected levels, other effective treatments need to be explored.
The first attempt of mitochondria transfer was carried out in 1997. The transfer of young donor oocyte cytoplasm into aged recipient oocytes improved the oocyte quality of the aged patients, who gave birth to healthy infants successfully9, which was the rationale behind the use of this technique. However, allogeneic oocyte cytoplasmic transfer cannot be applied to clinical practice due to two main reasons-the problem of genetic heterogeneity and regulatory issues caused by donor mitochondria transplantation. A previous study showed that autologous cell mitochondrial transplantation could improve the quality of oocytes, embryo development, and fertility of aged mice10, which had no ethical problems or genetic heterogeneity issues and solved some problems caused by the transfer of donor oocyte cytoplasm into recipient oocytes10,11.
Meanwhile, autologous cell mitochondrial transfer was superior to the effect of previous nutritional supplements of mitochondria on improving the quality of oocytes11. Therefore, autologous cell mitochondrial transplantation is the appropriate choice for the clinical application of this technology12. Adipose-derived stem cells (ADSCs) can be obtained by minimally invasive technology, are easy to isolate and culture, and can be an ideal "seed" cell for regenerative medicine. Mitochondria are rich in ADSCs, and the function of mitochondria does not decline with age, which suggests that ADSCs are an excellent source of mitochondria13,14. In this protocol, we introduce a method to transfer the mitochondria of mouse adipose-derived mesenchymal stem cells into aged mouse oocytes to improve the oocyte quality. This is a useful model for human ADSC autologous mitochondrial transfer technology.
Protocol
Representative Results
Discussion
Oocytes contain more mitochondria than any type of cell in the body, with ~1-5 × 105 mtDNA copy numbers. Mitochondria are essential for oocyte maturation, fertilization, and embryonic development, thus, any mitochondrial dysfunction can lead to decreased oocyte quality. Decreased mitochondrial quantity and quality are closely related to physiological aging. In this protocol, a simple method for isolating mitochondria from the ADSCs of aged mice and transfer to aged mouse oocytes was introduced to attempt …
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
The authors wish to acknowledge support from the National Nature Science Foundation of China (82001629 to X.Q.S.), the Basic Research Project of Changzhou science and Technology Bureau under grant number CJ20200110 (to Y.J.Y.), the Youth Program of Natural Science Foundation of Jiangsu Province (BK20200116 to X.Q.S.), and Jiangsu Province Postdoctoral Research Funding (2021K277B to X,Q.S.).
Materials
| 0.05% trypsin/EDTA | Gibco | 25300054 | Cell Culture |
| 4% paraformaldehyde | beyotime | P0099 | immunofluorescence |
| 40 μm cell strainer | Corning | 352340 | ADSC isolation |
| adipogenic induction | Cyagen | HUXXC-90031 | Multidirectional differentiation |
| Alizarin red staining solution | Sigma | A5533 | Multidirectional differentiation |
| Antibody against CD29 | BD Biosciences | 558741 | flow analysis |
| Antibody against CD34 | BD Biosciences | 560942 | flow analysis |
| Antibody against CD90 | BD Biosciences | 553016 | flow analysis |
| Antibody against HLA-DR | BD Biosciences | 555560 | flow analysis |
| β-actin | Abcam | ab-8226 | Mitochondrial function test |
| BSA | Sigma | V900933 | immunofluorescence |
| CCCP | Solarbio | C6700 | mitochondria JC-1 flow analysis |
| ChamQ Universal SYBR qPCR Master Mix | Vazyme | Q711 | qPCR |
| collagenase type I | Sigma | SCR103 | ADSC isolation |
| DAPI | Invitrogen | D1306 | immunofluorescence |
| DMEM-F12 | Gibco | 11320033 | Cell Culture |
| DMSO | Sigma | 276855 | mitochondria JC-1 flow analysis |
| EGTA | Sigma | 324626 | Mitochondria isolation |
| FBS | Gibco | 10100147 | Cell Culture |
| Flow cytometry | BD Biosciences | FACSCanto™ II | Characteristics of ADSCs |
| fluorescence microscope | leica | DM2500 | immunofluorescence |
| gelatin | Sigma | 48722 | Multidirectional differentiation |
| glass homogenization tube | Sangon | F519062 | Mitochondria isolation |
| hCG | Aibei | M2520 | Ovarian superstimulation |
| hyaluronidase | Sigma | H1115000 | Ovarian superstimulation |
| Inverted microscope | Olympus | IMT-2 | Microinjection |
| Isolated Mitochondria Staining Kit | Sigma | CS0760 | mitochondria JC-1 flow analysis |
| JC-1 | Sigma | T4069 | Mitochondrial function test |
| KCl | Sigma | P5405 | Mitochondria transfer |
| KH2PO4 | Sigma | P5655 | Mitochondria transfer |
| LaminB | Abcam | ab-16048 | Mitochondrial function test |
| M16 Medium | Sigma | M7292 | embryo cell culture |
| M2 Medium | Sigma | M7167 | embryo cell culture |
| mannitol | Sigma | M9546 | Mitochondria transfer |
| Microinjector | Olympus+ eppendorf | IX73 | Mitochondria transfer |
| MitoTracker red | Invitrogen | M22425 | Mitochondria staining |
| MOPS | Sigma | M1442 | Mitochondria isolation |
| neurofilament mediator polypeptide (NFM) | Santa Cruz Biotechnology | sc-16143 | Multidirectional differentiation |
| neurogenic induction | Gibco | A1647801 | Multidirectional differentiation |
| Neuron-specific enolase (NSE) | Santa Cruz Biotechnology | sc-292097 | Multidirectional differentiation |
| Oil Red O | Sangon | E607319 | Adipogenic differentiation |
| oil red O solution | Sigma | O1516 | Multidirectional differentiation |
| osteogenic induction | Cyagen | HUXXC-90021 | Multidirectional differentiation |
| PBS (phosphate buffered saline) | Hyclone | SH30256.LS | Cell Culture |
| penicillin and streptomycin | Hyclone | SV30010 | Cell Culture |
| PMSG | Aibei | M2620 | Ovarian superstimulation |
| protease Inhibitor cocktail | Sigma | P8340 | Mitochondria isolation |
| sucrose | Sigma | V900116 | Mitochondria isolation |
| Tris | Sigma | 648314 | Mitochondria isolation |
| Tris-HCl | Sigma | 108319 | Mitochondria transfer |
| Triton X-100 | beyotime | P0096 | immunofluorescence |
| VDAC | Abcam | ab-14734 | Mitochondrial function test |
Referenzen
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