Summary

Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes

Published: January 06, 2023
doi:

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

All the animal experiments described were approved by the Animal Research Ethics Committee of the Third Affiliated Hospital, Soochow University. All operations follow appropriate animal care and use agency and national guidelines. See the Table of Materials for details of all materials, instruments, and reagents used in this protocol. 1. Isolation and characterization of aged mouse adipose-derived mesenchymal stem cells (ADSCs) Sacrifice the aged m…

Representative Results

In this protocol, we isolated and characterized ADSCs from mouse fat (Figure 1). To obtain isolated mitochondria, the cell membrane must be disrupted using a glass homogenizer. (Figure 2A). It is important to obtain a uniform mitochondrial fraction without large clumps so that the microinjection tube is not blocked. First, 200 µL, and then 10 µL, pipette tips must be used to resuspend the homogenates gently; finally, a 29 G needle must be used to slowl…

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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Yang, Y., Zhang, C., Sheng, X. Mitochondrial Transfer from Mouse Adipose-Derived Mesenchymal Stem Cells into Aged Mouse Oocytes. J. Vis. Exp. (191), e64217, doi:10.3791/64217 (2023).

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