An Exploratory Study Of Transplanted MSCs Exerting Resistance To Ovarian Dysfunction Caused By Natural Aging Via Potential Mechanism

Part 1 Mouse-age determination for BMSCs^mT/mG transplantation and establishment of optimal transplantation pathway[Purpose] To systematically evaluate the period of decline in ovarian function and to determinate the appropriate mouse-age for BMSCs^mT/mGtransplantation;By comparative analysis of the multiple transplantation methods,the effect of BMSCs^mT/mGon ovarian dysfunction caused by natural aging was evaluated to establish the optimal pathway for BMSCs^mT/mGtransplantation.[Methods] To determine the appropriate age for BMSCs^mT/mGtransplantation,we distributed these mice into seven groups: 16 weeks old(16w),20 weeks old(20w),24 weeks old(24w),28 weeks old(28w),32 weeks old(32w),36 weeks old(36w)and 40 weeks old(40w)(n=30,per group).We observed the pregnancy and average litter size of the mice and collected the serum at the same menstrual cycle of each group for the FSH,LH,E₂ and AMH level detection by CLIA assay.Highly purified BMSCs^mT/mGwere subsequently selected to administer an intraperitoneal injection(Intra-P-group),ovarian local injection(Oc-group)or tail-intravenous injection(Ti-group)for transplantation,and moderate normal saline(NS)was used as the vehicle.Serum hormone levels and follicular pool reserve were preliminary assessed.Furthermore,estrous cycles,ovarian function and fertility potential were further compared and analyzed,and the optimal model of BMSCs^mT/mGtransplantation(+BMSCs)was established with equivalent normal saline transplantation(+NS)as the control group.[Results] We observed the pregnancy and average litter size of the mice and collected the serum at the same menstrual cycle of each group for the FSH,LH,E₂ and AMH level detection,respectively.The results showed that both the pregnancy rate(21.43%)and the average little size(about 5 cubs per pregnant mouse)were markedly declined at 28w (P<0.05).Moreover,the hormone levels in each group were detected;the CLIA assay demonstrated the serum AMH level(3.558±0.188ng/ml)and E₂(42.002±2.355pg/ml)level gradually decreased with the increased age and significantly decreased at 28 w and32w,respectively(P<0.01).The levels of FSH(30.786±1.953ng/ml)and LH(5.097±0.415ng/ml)at different observed weeks gradually increased with age,and both levels were significantly increased at 32w(P<0.01).The transplanted BMSCs^mT/mG exerted a more sensitive effect on the upward trends about AMH and E₂ levels and downward trends about FSH levels in the Oc-and Ti-groups compared with those in the Intra-P-group,with significant rising about AMH levels in Oc-group compared with that in Intra-P-group(P<0.01).Furthermore,follicles with HE-staining in each stage were shown in the Oc-,Tiand Intra-P-groups.When compared to the Intra-P-group,the numbers of primordial follicles(21.00±6.38 vs.5.60±1.50,P<0.01),primary follicles(22.67±6.02 vs.6.20±1.16,P<0.01),secondary follicles(17.00 ± 5.72 vs.11.80 ± 1.94,P<0.05)were significantly increased after transplantation for 4 weeks in the Oc-group with slightly increased levels in Ti-group just for primordial follicles(11.60±3.61 vs.5.60±1.50;P<0.05).Ultimately,the Oc-and Ti-groups were selected for further assessment.The immunofluorescence analysis tracked the ovarian location of transplanted BMSCs^mT/mG,and the results indicated the homing to ovaries after transplantation for 4 and 8 weeks.The red staining(anti-td Tomato)cells were defined as BMSCs^mT/mG,which were scattered and randomly distributed in the ovarian tissue without developing into follicles,even after 8 weeks of transplantation(40w mice)by the tail intravenous injection and ovarian local injection with decreasing amount of transplanted BMSCs^mT/mGwithout obvious proliferation,and the transplanted BMSCs^mT/mGwere not detected in the control group.By the two month evaluation of the mouse estrous cycle after BMSCs^mT/mGtransplantation by tail-intravenous and ovarian local injection,it was evident that BMSCs^mT/mGin the Oc+BMSCs or Ti+BMSCs group can increase the number of estrous cycles compared with the Oc+NS group(P<0.001)or Ti+NS group(P<0.01)with more pronounced increase in Oc+BMSCs group(9.25±0.96 vs.7.9± 0.83,P<0.001)compared with that in Ti+BMSCs group.Moreover,the percent of mice with an estrous cycle was also increased after 4 weeks of BMSCs^mT/mG transplantation,and in particular,the Oc+BMSCs group exhibited a conspicuous effect.Compared with the Ti+BMSCs group,the method of ovarian local injection was more effective at 37w-38w(100% vs.80.0%)and 39w-40w(88.2% vs.60.0%).In addition,the average duration of estrus was significantly prolonged in the BMSCs^mT/mG-transplantation groups,particularly for the durative days in the Oc+BMSCs group compared with that in the Oc+NS group at 37w-38w(2.00±0.58 vs.1.00±0.88,P<0.05)and 39w-40w(2.24±1.16 vs.0.93 ± 0.83,P<0.05);moreover,the results in the Oc+BMSCs group also showed a remarkable difference at 37w-38w(2.0±0.58 vs.0.78±1.09,P<0.05)and 39w-40w(2.24±1.16 vs.0.38±0.74,P<0.01)compared to those in the Ti+BMSCs group,and proestrus or diestrus decreased in their duration.In addition,the results showed that these ovaries had a ruddy and normal appearance with some follicle protuberances on the surface.In the Ti+BMSCs group,the ovarian tissues were enlarged in size after 8 weeks of BMSCs^mT/mG transplantation(40w)compared to that in the Ti+NS group without a significant change in size at 36 w,and the weight index of the ovaries(ovarian weight/mouse weight)also showed the same increasing tendency.By comparison,BMSCs^mT/mGexhibited a conspicuous effect in the Oc+BMSCs group with a clear enlargement in size after 4 weeks of BMSCs^mT/mGtransplantation(36w)and a significant difference in the weight index,particularly at 40w(P<0.01).The CLIA test demonstrated that the levels of FSH(declined as much as 15.5%)and LH(declined as much as 22.7%,P<0.05)in the Ti+BMSCs group were slightly rescued by BMSCs^mT/mG,and both the AMH and E₂ levels maintained an upward trend in the Ti+BMSCs group compared to those in the Ti+NS group.However,in comparison,the levels of FSH significantly decreased up to 17.6%(P<0.05),and LH remarkably decreased up to 27.3%(P<0.01).Moreover,the AMH and E₂ levels showed significant increases up to 24.8%(P<0.05)and 29.0%(P<0.05).All measured outcomes indicated that BMSCs^mT/mGtransplantation into mice could improve the reproductive capability,including the pregnancy rate and the average litter size per pregnancy.Compared with the Ti+NS and Oc+NS groups,respectively,both the rate of pregnancy and the average litter size showed escalating trends in the Ti+BMSCs and Oc+BMSCs groups,and the effect in the Oc+BMSCs group was more conspicuous.[Conclusions] After comprehensive evaluation,the female C57BL/6J mice showed a significant decline in ovarian function at 32 weeks,and the mice of 32 weeks were ultimately determined to be the appropriate mouse-age for BMSCs^mT/mGtransplantation.The transplanted BMSCs^mT/mGthrough ovarian local injection had the most efficient resistance to ovarian natural aging,so ovarian local injection was selected as the optimal approach for BMSCs^mT/mGtransplantation.Part 2 In vivo effect of BMSCs^mT/mG transplantation on ovarian dysfunction caused by natural aging in mice and preliminary assessment of transplanted BMSCs^mT/mG for the long-term safety[Purpose] To observe and analyze the resistance of BMSCs^mT/mGto ovarian natural aging,and demonstrate that BMSCs^mT/mGmay improve the quality of oocytes by enhancing mitochondrial function and increase the storage of the follicle pool by inhibiting oocyte and granulosa cell apoptosis,and to evaluate the long-term safety of transplanted stem cells in vivo.[Methods] Ovarian local injection was performed on C57BL/6J mice at 32 weeks using highly purified BMSCs^mT/mGabout 500,000 ¹ million cells with moderate normal saline as vehicle,and equivalent normal saline(+NS)was transplanted as the control group.The treated or untreated groups were as follows: ovarian local injection with BMSCs^mT/mG(Oc+BMSCs)and ovarian local injection with normal saline(Oc+NS).Ovarian tissue was collected from each group at 4 weeks(36 weeks old,36w),6 weeks(38 weeks old,38w)and 8 weeks(40 weeks old,40w)after transplantation,and the follicle numbers in each stage were statistically analyzed.In vitro fertilization(IVF)and blastocysts culture in vitro were conducted respectively at two time points of 4 weeks and 8 weeks after ovarian local transplantation,and to observe the fertilization rate,cleavage rate and blastocyst formation rate of MII oocytes.Meanwhile,anti-tubulin monoclonal antibody and DAPI staining solution were used for MII oocytes to observe the spindle morphology and chromosome arrangement under confocal microscopy,and immunofluorescence co-localization analysis about chromosome centromeres and nucleic acids was conducted with specific antibody CREST and DAPI to analyze aneuploidy formation of MII oocytes.In addition,Mito-Tracker Green and mitochondrial membrane potential detection kit(JC-1)was respectively used to observe the mitochondrial morphology and distribution of MII oocytes and mitochondrial membrane potential level(? ? m)of ovarian cells,with mitochondrial dynamics-related proteins(MFN1 / 2,OPA1,DRP1)and mt DNA(mitochondrial specific expression gene ND-1)levels detected by western blot and RT-q PCR at different time points after BMSCs^mT/mGtransplantation,as well as the mitochondrial ultramicrostructure of the ovaries in the two groups was detected by transmission electron microscopy(TEM).Furthermore,TUNEL assay,western blotting and TEM were used to determine the cell apoptosis and the expression of apoptosis-related factors(such as Bcl-2,Bax,Caspase3 and Caspase9)at different time points in the two groups.After BMSCs^mT/mG transplantation,the mice were observed in the same batch for 6 months to 1 year,the natural mortality rate and the rate of abnormalities under anatomy were calculated,and the abnormal tissues were further obtained for histopathological detection and analysis.[Results] After BMSCs^mT/mGwere administered via ovarian local injection for 4,6and 8 weeks,the follicles with HE-staining in each stage in the Oc+BMSCs group and the control group(Oc+NS)were analyzed.The numbers of primordial follicles(21.00±7.81 vs.6.67±3.79),primary follicles(22.67±7.37 vs.7.00±1.00)and secondary follicles(17.00 ± 2.00 vs.11.00 ± 2.65)were significantly increased after transplantation for 4weeks in the Oc+BMSCs group compared with those in the Oc+NS group(P<0.05);however,there was no significant difference after transplantation for 8 weeks.Nevertheless,the number of antral follicles markedly increased with the duration of BMSCs^mT/mGtherapy at 36 w,38w,even at 40 w after BMSCs^mT/mGtransplantation for 8weeks(15.67±4.16 vs.6.33±4.04;14.33±4.04 vs.5.67±2.52;7.57±1.59 vs.3.75±0.83;P<0.05).In addition,we found that the antresia follicle count substantially decreased with the duration of BMSCs^mT/mG treatment compared to the control group(26.00 ±5.76 vs.42.75±4.02,P<0.01).The results indicated that BMSCs^mT/mGtransplantation affected the fertilization and cleavage of oocytes,and the early embryo formation rate in the Oc+BMSCs group(75%,69%,64%,and 55%)was substantially increased compared with that in the Oc+NS group(61%,54%,48%,and 34%)at 36 w after BMSCs^mT/mG transplantation.Aberrant changes in the spindle apparatus and chromosome alignment and separation,such as spindles with abnormal polarization and disordered microtubules,misalignment chromosome and the homologous chromosome without separation despite the first polar body eduction,was observed in the Oc+NS group.Distinctly,compared to the Oc+NS group,the transplanted BMSCs^mT/mGcould improve the abnormal situation with declined disordered spindle assembly(51.06% vs.70.83%,P<0.05)and the decreased percentage of aneuploid arrested at the MII stage with 18 or 21 sister chromatids(20.37%vs.39.13%,P<0.05).Our results showed that there were three patterns of mitochondrial distribution,including polarity(normal mitochondrial redistribution of MII oocytes),homogeneous distribution and heterogeneous clusters.Surprisingly,in the Oc+BMSCs group,the normal polarity distribution of mitochondria was apparently increased compared to the Oc+NS group(57.14% vs.35.42%,P<0.05),while the pattern of heterogeneous clusters(35.71% vs.52.08%)and homogeneous distribution(7.14% vs.12.50%)were decreased.The ovarian mitochondrial membrane potential(??m)was measured using the JC-1 Assay Kit in the Oc+NS and Oc+BMSCs groups,and the fluorescence intensity was apparently enhanced in the Oc+BMSCs group compared with in the Oc+NS group after BMSCm T/m Gtransplantation for 4 and 8 weeks.Furthermore,we measured the expression levels of mitochondrial dynamics-related proteins(such as MFN1/2,OPA1,and DRP1)by western blotting and found that in the Oc+BMSCs group,the MFN1/2 and OPA1 expression levels were differentially increased and the DRP1 protein levels were significantly decreased compared to the Oc+NS group(P<0.01).Moreover,the mt DNA copy levels after BMSCs^mT/mGtransplantation were found increased approximately more than 2-fold from the ovaries in the Oc+NS group.In addition,TEM showed a conspicuous difference in the mitochondrial morphologies and number.In the Oc+NS group,the mitochondria presented a short rod-like structure and spheroids with fewer numbers,serious vacuolation and a disordered mitochondrial cristae arrangement.Nevertheless,after BMSCs^mT/mGtransplantation in the Oc+BMSCs group,we could observe ameliorative mitochondrial cristae alignment and vacuolation,as well as a small number of long rod-like structures.Four and eight weeks after BMSCs^mT/mGtransplantation,the average Int Den of TUNEL-positive ovarian cells in the Oc+BMSCs group was significantly weaker than that in the Oc+NS group(P<0.000).In addition,the results of the ultramicrostructure from granulosa cells around oocytes in the Oc+NS group showed many apoptotic cells with nuclear shrinkage and severe cavitation;however,the apoptotic cells were clearly decreased in the Oc+BMSCs group as indicated by TEM observation.Western blotting further indicated that the Bcl-2 expression levels were increased in the Oc+BMSCs group compared to the Oc+NS group,while the expression levels of Bax,Caspase3 and Caspase9 were decreased in the Oc+BMSCs group(P<0.05).To further evaluate the potential for abnormal differentiation of BMSCs^mT/mGafter transplantation for long-term retention in vivo,long-term observation and histopathological data showed that no neoplasms and obvious prosoplasia were found after transplantation for six months to one year,just with fibrous hyperplasia,decreased macrophages,increased cell proliferation and inflammatory response.In addition,when compared with the control group,the rate of natural death decreased observably,but without significant difference about the rate of abnormality.[Conclusions] BMSCs^mT/mGin mice were not at risk of tumorigenesis for at least 6months after transplantation.The transplanted BMSCs^mT/mGaround ovaries can exert resistance to the ovarian natural aging,which may reversed the poor developmental potential and arrested meiosis of oocytes extracted form mice with advanced age by changing the mitochondrial structure,regulating mitochondrial function of cumulus granulosa cells,and attenuating cell apoptosis,reducing follicular atresia at all stages to resist follicular pool depletion.Part 3 In vitro verification and deep exploration of the mechanism related to the prominent role of BMSCs^mT/mG in ovarian dysfunction caused by natural aging[Purpose] To explicate the underlying mechanisms that BMSCs^mT/mGmay act on the target cells through the potential FOXO3 a signaling pathway,and further to explore the underlying mechanism.[Methods] Immunocytochemical analysis was used to further verify the intracellular localization of FOXO3 a in oocytes and cumulus granulosa cells.The ovarian tissues were obtained in different transplantation groups and at different time points,which were grouped as follows: Young group(Y-age-group,6 to 8 weeks old),Middle-age group (M-age-group,about 36 weeks old),Older-age group(O-age-group,about 44 weeks old).FOXO3 a expression,including m RNA and protein levels,were detected and verified in each group using RT-q PCR and Western blot technology.Efficient si RNA was designed and screened to silence the FOXO3 a expression of target cells(CELL-Y),which were distributed as follows: CELL-Y+blank-control,CELL-Y+BMSCs^mT/mG,CELL-Y+FOXO3a-si RNA,CELL-Y+FOXO3a-si RNA+BMSCs^mT/mG.After silencing with FOXO3a-si RNA and co-culture with BMSCs^mT/mG,RT-q PCR and western blot were used to verify the silencing efficiency of FOXO3 a and its expression levels in target cells of each group.The supernatants extracted from culture medium in each group were collected to detect the changes of hormone levels by CLIA technique.The cell viability in each group was measured by CCK-8 assay to indirectly reflect the cell proliferation ability.Annexin V-EGFP cell apoptosis assay kit was used to analyze the cell apoptosis under FACS(Fluorescent-activated cell sorting).In addition,Mito-Tracker Green and Hochest33342 staining solution were used for target cells to observe the mitochondrial morphology and distribution under confocal microscopy.At four weeks after BMSCs^mT/mG transplantation,MII oocytes and cumulus granulosa cells(3 cases / per group)were collected for RNA-seq sequencing using illumina Hiseq sequencing platform.DEGseq analysis,combined with Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment of DE-m RNAs,was applied to screened out the genes with significant up-regulation and down-regulation,which was related to the ovarian natural aging.[Results] It was also found that FOXO3 a was mainly located around nuclear membrane of oocytes with a few scattered within the nucleus,and even uniformly distributed within the whole nucleus of cumulus granulosa cells.Furthermore,immunocytochemical analysis exhibited weak fluorescence intensity about the FOXO3 a expression in the GV oocytes and strong fluorescence intensity in cumulus granulosa cells extracted from MII oocytes both in Oc+NS group and Oc+BMSCs group,however,the transplanted BMSCs^mT/mGcould significantly increase FOXO3 a expression in cumulus granulosa cells extracted from small follicles(P<0.05).Both of western blot and RT-q PCR results showed that FOXO3 a expression levels decreased significantly with increasing age(P<0.05),and FOXO3 a expression remarkably increased after BMSCs^mT/mG transplantation(P<0.01).In addition,the five FOXO3a-si RNAs were designed and synthesized,among which si-m-foxo3a₀04 showed the highest silencing efficiency after transfecting target cells multiple times with 100 n M FOXO3a-si RNA,with higher transfection efficiency in granulosa cells after the FOXO3a-si RNA sequences were modified with cholesterol(5cy3/3chol/2OMe)compared with FOXO3a-si RNA(Cy3)(P<0.001),and the levels of FOXO3 a expression could be silenced to 20% 30% with decreased secretion function.After co-culture with BMSCs^mT/mGfor 48 hours,both FOXO3 a m RNA and protein levels increased significantly(P<0.05),as well as the conspicuously increased secretion levels of estrogen(E₂,P<0.05)in culture supernatants,which was significantly lower than the FOXO3 a non-silencing group(P<0.01),however,there was no significant difference in progesterone(PG)levels.In addition,CCK-8 showed that cell proliferation decreased significantly after FOXO3 a was silenced(P<0.001),and cell viability and proliferation ability increased obviously after co-culture with BMSCs^mT/mGfor 48 hours(P<0.001).Moreover,the BMSCs^mT/mGcould inhibit cell apoptosis caused by FOXO3 a silencing according to FACS detection,but still significantly higher than the FOXO3 a non-silencing group(P<0.01).After FOXO3 a was silenced,mitochondrial division was significantly increased under confocal microscopy(P<0.001),and the number of mitochondria distributed around the nucleus was decreased with poor fluorescence intensity(P<0.05).After co-culture for 48 hours with BMSCs^mT/mG,it was found that the number of mitochondria increased in the peri-nuclear distribution with significantly enhanced fluorescence intensity(P<0.05)and significantly reduced mitochondrial division(P<0.001),and the proportion of fusion mitochondria was lower than that in the non-silent group(P<0.05).Furthermore,for the cumulus granulosa cells,a total of 25408 m RNAs were identified in the post-transplanted ovaries,of which 99 m RNAs were differentially expressed(DE)based on DEGseq analysis(|log2FC|>=1,Pvalue<=0.05 and FDR<=0.05),consisting of 10 up-regulated and 89 down-regulated m RNAs.After the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment of DE-m RNAs were performed and analyzed,two up-regulated genes(Retnla,Cd5l)and six down-regulated genes(Irgm1,7420426K07 Rik,Nlrp4f,Gm13084,Nlrp5?Lrrc17)were screened out,suggesting that these genes were related to ovarian natural aging and could be used for further study of the deeper mechanism.[Conclusions] FOXO3 a plays crucial role in the process of follicular development and maturation.BMSCs^mT/mGmay act on cumulus granulosa via regulating FOXO3 a signaling pathway after ovarian transplantation,and improve the quality of oocytes by enhancing mitochondrial function and inhibiting oocyte and granulosa cell apoptosis,inhibiting follicular atresia at all stages to resist ovarian natural aging.Part 4 In situ repair abilities of human UC-MSCs on ovarian dysfunction caused by natural aging in non-human primates with advanced age[Purpose] To investigate the effect of human umbilical cord mesenchymal stem cells on ovarian dysfunction caused by natural aging in non-human primates with advanced age by ovarian injection.[Methods] Six rhesus monkeys(14¹6 years old)were randomly divided into two groups: the Control Group and Hu MSCs-treated Group,three million human UC-MSCs(Hu MSCs)were transplanted by ovarian local injection,the moderate normal saline was used as the vehicle.The serum of rhesus monkey during the menstrual period were collected before and after Hu MSCs transplantation for 2 to 4 menstrual cycles,and chemiluminescence immunoassay(CLIA)assay was used to detect serum hormone levels.Human testicular tissue(including Xp11.1-Xq11.1 and Yp11.3 sequences)was used as positive control,and the FISH Probe "Vysis SRY Probe LSI SRY Spectrum Orange/Vysis CEP X Spectrum Green" was used for the location detection of transplanted Hu MSCs in ovarian tissue with the ovarian tissue of rhesus monkeys used as negative control in the Control Group(excluding Xp11.1-Xq11.1 and Yp11.3 sequences).After Hu MSCs transplantation for two or four months,the laparotomy was performed respectively to observe the ovarian morphology and obtain ovarian tissues,followed by paraffin embedding,sectioning and HE staining,the ovarian follicle count was conducted,as well as the ovarian ultramicrostructure in the two groups at different time points was detected by transmission electron microscopy(TEM).[Results] After the highly purified Hu MSCs were injected into ovaries for two months,the probe “Vysis SRY Probe LSI SRY Spectrum Orange/Vysis CEP X Spectrum Green” was used to mark the human umbilical cord-derived mesenchymal stem cells by a FISH technique.The results showed that male testicular tissue(Containing XY chromosome)was obtained as a positive control group with green/orange double signal,and control group derived-ovary was randomly selected as a negative control without positive signal.And the presence of a positive signal(green signal)in the ovaries was showed after Hu MSCs treatment for 2 months by FISH detection,suggesting that transplanted Hu MSCs can locate into the ovarian tissues at least for 2 months.The results showed that FSH levels increased significantly in Control Group at three months after transplantation compared with that before transplantation(Pre-treatment,P<0.01),with rising much hither than 5 times.In Hu MSCs-treated Group,FSH level increased by 78%compared with that before transplantation after transplantation for three months,and was significantly lower than that in Control Group(P<0.01).In addition,at three months after transplantation,E₂ level in Control Group decreased by 20.36% compared with that before transplantation,however,E₂ level only decreased by 3.72% in Hu MSCs-treated Group compared with that before transplantation.With the advanced age,the total follicles and primordial follicles in ovarian tissue of old rhesus monkeys were decreased significantly compared with that in young group(Young-Control)(P<0.05,P<0.01).Two months after Hu MSCs transplantation,the total number of follicles was significantly higher than that in Control Group(P<0.05).At 4 months after transplantation,the total number of follicles in the ovarian cortex was significantly lower than that in the younger group(P<0.01),but still higher than that in Control Group.In addition,the number of follicles at all levels(primordial follicles,primary follicles and antral follicles)in old rhesus monkeys was significantly lower than that in young group(P<0.05).Two months after Hu MSCs transplantation,the number of follicles at all levels was significantly higher than that in Control Group(P<0.05),especially the number of antral follicles was still significantly higher than that of the control group at 4 months after Hu MSCs transplantation(P<0.05).In addition,the ovarian ultramicrostructure in the control group and Hu MSCs-treated group was also detected by transmission electron microscopy(TEM).The mitochondrial morphologies showed a conspicuous improvement with obvious mitochondrial cristae alignment and a small number of long rod-like structures displayed in the Hu MSCs-treated group,compared with the short rod-like structure and spheroids,serious vacuolation and disordered mitochondrial cristae arrangement in the Control Group ang in oocytes before transplantation.[Conclusions] Human umbilical cord mesenchymal stem cells can repair ovarian dysfunction caused by natural aging in rhesus monkeys,i.e.,Hu MSCs transplantation in situ can exert resistance to ovarian natural aging in non-human primates.