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

Published: January 06, 2023

doi:

Yanjun Yang, Chunxue Zhang, Xiaoqiang Sheng

¹Department of Obstetrics and Gynecology, the Third Affiliated Hospital,Soochow University, ²Department of Obstetrics and Gynecology, Nanjing First Hospital,Nanjing Medical University, ³Center for Reproductive Medicine and Obstetrics and Gynecology, Nanjing Drum Tower Hospital,Nanjing University Medical School

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 …

Disclosures

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

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