The Improvement Of The Developmental Potential Of Aging Oocytes By Mitochondrial Transfer From Induced Pluripotent Stem Cells

With the change of lifestyle and concept,the postponement of the childbearing has become a common phenomenon.However,the increasing age at childbearing is often accompanied by a rapid decline in fertility.The decline in oocyte quality is one of the main reasons among the physiological factors that result in a decline in female fertility.Mitochondria are the most critical organelles for the quality of oocytes,because the number,distribution and function of mitochondria in oocytes are crucial for subsequent normal embryonic development.The mitochondria of aging female animals have a series of abnormalities due to their long period of oxidative stress,which leads to the damage of the developmental potential of aging oocytes.There are currently three mitochondrial supplement strategies to improve the mitochondrial abnormalities in oocytes,including ooplasmic transfer,mitochondrial replacement,and mitochondrial transfer.Mitochondrial transfer is currently the main technical to improve the developmental potential of aging oocytes.Mitochondria derived from somatic cells are frequently used for mitochondrial transfer.Although it is easy to obtain,the effect is not ideal.Upon reprogramming into induced pluripotent stem cells(iPSCs),somatic cells exhibit mitochondrial remodeling to an immature state,which may be more similar in the oocytes.In order to solve the problem of poor developmental potential of aging oocytes,we look for the mitochondria to supplement into aging oocytes which were closer to the oocyte mitochondria by comparing the differences of mitochondria between the aging and young oocytes and different cell types.Firstly,we verified the abnormality of mitochondria in aging oocytes.We compared oocytes derived from young mouse and aging mouse through the observation by transmission electron microscopy and detection of ATP production,membrane potential,and mtDNA copy number.We observed that the mitochondria of aging mice suffered from severe structural damage,decreased ATP production,decreased mtDNA copy number,and abnormally activated glycolysis.Further,we compared mitochondrial features of pluripotent cells,differentiated somatic cells,and oocytes.We observed the mitochondrial morphology using ultra-high resolution confocal laser microscopy,and the mitochondrial ultrastructure using transmission electron microscopy,then detected mitochondrial membrane potential by JC-1 staining.In addition,we analyzed the expression of mitochondrial-related genes in pluripotent cells,differentiated somatic cells and oocytes through RNA-seq.The results showed that the expression of mitochondrial related genes have significant differences not only between various cell types,but also between iPSCs in different pluripotent states.Compared with somatic cells,the mitochondrial morphology,ultrastructure,membrane potential and gene expression patterns of iPSCs are more similar to those of oocyte.The up-regulation of amino acid metabolism,down-regulation of fatty acid metabolism,and related mitochondrial transport in iPSCs are closely related to pluripotency and the quality of iPSCs.Finally,we chose the mitochondria of iPSCs,which are very similar to the mitochondria of oocytes,to transfer into the zygotes of aging mouse by microinjection.Meanwhile,buffer using to suspend mitochondria and mitochondria extract from MEF were also transferred as controls.The results showed that the implantation rates of embryos developed from IVF zygotes injected with iPSCs mitochondria was significantly higher than that of MEF mitochondria transfer and buffer transfer.And the E 7.5 and E 12.5 fetuses developed from zygotes supplemented with iPS mitochondria developed normal.In addition,we detected ATP production and mtDNA copy numbers of embryos supplemented with iPSCs mitochondria,the results showed that the abnormally activated glycolysis in aging oocytes supplemented with iPSCs mitochondria was restored,and the mtDNA copy number of aging embryos increased to the level of young embryos.In summary,there are mitochondrial damage in aging oocytes,and the morphology,structure and function of iPSCs mitochondria are closer to the oocytes mitochondria.The developmental potential of aging oocytes can be effectively improved by mitochondrial transfer from iPSCs.It's the first time to improve the developmental potential of aging oocytes by mitochondrial transfer from iPSCs.Our study provided an important reference for improving the developmental potential of oocytes from reproductive aging women or animals using autologous iPSCs.