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Rejuvenation of aged oocyte through exposure to young follicular microenvironment

Nature Aging volume 4, pages 1194–1210 (2024)Cite this article

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Abstract

Reproductive aging is a major cause of fertility decline, attributed to decreased oocyte quantity and developmental potential. A possible cause is aging of the surrounding follicular somatic cells that support oocyte growth and development by providing nutrients and regulatory factors. Here, by creating chimeric follicles, whereby an oocyte from one follicle was transplanted into and cultured within another follicle whose native oocyte was removed, we show that young oocytes cultured in aged follicles exhibited impeded meiotic maturation and developmental potential, whereas aged oocytes cultured within young follicles were significantly improved in rates of maturation, blastocyst formation and live birth after in vitro fertilization and embryo implantation. This rejuvenation of aged oocytes was associated with enhanced interaction with somatic cells, transcriptomic and metabolomic remodeling, improved mitochondrial function and higher fidelity of meiotic chromosome segregation. These findings provide the basis for a future follicular somatic cell-based therapy to treat female infertility.

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Fig. 1: Follicular somatic cells accumulate age-related abnormalities.
Fig. 2: Aged follicular environment compromises young oocyte quality and developmental competence.
Fig. 3: Young r-follicles restore the developmental potential of aged oocytes.
Fig. 4: Regeneration of TZP in RCF.
Fig. 5: Transcriptomic and metabolomic remodeling in aged oocytes cultured in young r-follicles.
Fig. 6: Restored mitochondrial fitness in aged oocytes cultured in young r-follicles.
Fig. 7: Decreased chromosomal abnormalities and cohesin loss in aged oocytes cultured in young follicular environment.
Fig. 8: Young follicular somatic cells improve chromosome segregation fidelity of aged oocytes.

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Data availability

All raw RNA-seq data, as well as processed datasets, can be found in the Gene Expression Omnibus database under accession number GSE270016. Metabolomics data are available in Supplementary Table 5. The rest of the data generated or analyzed during this study are all included in the published article and its Supplementary Information files. Source data are provided with this paper. All other data are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank S. Xiao (Rutgers University) for the helpful discussion on mouse follicle in vitro culture method. We thank M.l Lampson (University of Pennsylvania) for providing Rec8 antibody. We thank T. S. Kitajima (RIKEN Center for Developmental Biology) for providing pGEMHE–2mEGFP–CENP-C plasmid. Graphics from Figs. 2a,f, 3a, 4a,e and 8g and Extended Data Figs. 3b, 6a and 7a were created with BioRender. This work was supported by a grant from the National University of Singapore Bia-Echo Asia Centre for Reproductive Longevity and Equality and by the National Research Foundation, Singapore, under its mid-sized grant (NRF-MSG-2023-0001) to R.L. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Mechanobiology Institute, National University of Singapore, Singapore, Singapore

    HaiYang Wang, Xingyu Shen, Yaelim Lee, XinJie Song, Chang Shu, Jin Zhu & Rong Li

  2. NUS Bia Echo Asia Centre for Reproductive Longevity and Equality, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Zhongwei Huang

  3. Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Zhongwei Huang

  4. Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Zhongwei Huang

  5. Cardiovascular Research Institute, National University Health System, Singapore, Singapore

    Lik Hang Wu, Leroy Sivappiragasam Pakkiri, Poh Leong Lim & Chester Lee Drum

  6. Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore

    Lik Hang Wu, Leroy Sivappiragasam Pakkiri, Poh Leong Lim & Chester Lee Drum

  7. Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore, Singapore

    Lik Hang Wu

  8. Center for Cell Dynamics and Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Xi Zhang & Rong Li

  9. Department of Biological Sciences, National University of Singapore, Singapore, Singapore

    Rong Li

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Contributions

H.W. and R.L. conceived the study. H.W. and R.L. designed the experiments and methods for data analysis. H.W. performed experiments and analyzed the data with assistance from Z.H., X.J.S., X.S. and C.S., with the following exceptions: L.H.W., P.L.L. and L.S.P. performed the MS experiments and data analysis; C.S. measured the distance between sister kinetochores; X.Z. generated MTS–mCherry–GFP1–10 mice strain; J.Z. supervised the RNA-seq experiments and analyzed the data with Y.L.; and C.L.D. and L.S.P. supervised the MS analysis. H.W. and R.L. wrote the paper and prepared the figures with input from all authors. R.L. supervised the study.

Corresponding authors

Correspondence to HaiYang Wang or Rong Li.

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Competing interests

We disclose that we have filed a patent for this study. The applicants and inventors for this patent are R.L. and H.W. The patent application, titled ‘Somatic Cell-Based Therapy to Treat Female Infertility’, was filed under number PCT/SG2023/050339 and has been published with the publication number WO 2023/224556 A1. The other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Follicles accumulate age-related abnormalities.

a,b, Representative images of Ki-67 staining in ovarian sections (a). F-actin was stained with phalloidin. Scale bar, 50 μm. Quantitative analysis of the percentage of Ki-67-positive cells per follicle is shown in (b). n = 31 (young), 25 (aged) follicles. c, Quantification of γH2AX foci in GCs from follicles in ovarian sections. n = 37 (young), 38 (aged) follicles. d-f, CM-H2DCFDA staining in isolated oocyte-GC complexes (d). Scale bar, 30 μm. Scatter plots (e) show the correlation between ROS levels in GCs and oocytes (simple linear regression and two-tailed analysis). Gray areas around fit lines indicate 95% confidence intervals, with Pearson’s correlation coefficient (r). Comparison of ROS intensity in young and aged oocytes or GCs is shown in (f). n = 76 (young), 43 (aged). 2-month-old (young) and 14-month-old (aged) mice were used in (b, c, e, f). Box plots in (b, c, f) show mean (black square), median (center line), quartiles (box limits), and 1.5× interquartile range (whiskers). Box plots inside the violins in (f) show mean (black circle), quartiles (box limits), and 1.5× interquartile range (whiskers). Two-tailed unpaired t-tests for (b, c, f). P value: ****P < 0.0001, ***P < 0.001. Exact P values are in the Source Data. Data are from at least three independent experiments.

Source data

Extended Data Fig. 2 Comparison of in vivo and in vitro grown oocytes.

a, Diameter of oocytes grown in vivo or in vitro. n = 109 (in vivo), 90 (in vitro). b, Quantification of oocyte maturation rate. Sample sizes: n = 126 (in vivo) and n = 98 (in vitro) oocytes, with 4 biological replicates in each group. Data are shown as mean ± SD. c, Analysis of embryo development potential. n = 83 (in vivo) and 75 (in vitro). d-f, Transcriptome analysis of oocytes grown in vitro and in vivo. Volcano plot (d) of DEGs (p.adjust < 0.05 and log2 fold change > 0.5 or < −0.5) between in vitro and in vivo oocytes. Two-sided Wald-test adjusted with Benjamini-Hochberg method. Correlation heatmap (e) with hierarchical clustering to show the sample-to-sample distances. PCA analysis (f) of the normalized gene expression data. Ellipses fit a multivariate t-distribution at confidence level of 0.8. n = 8 in vivo and 8 in vitro. g, Dot plots illustrating follicle size changes over time during 3D ex vivo culture. Color bar and circle size represent follicle size. 2-month-old (young, n = 18) and 14-month-old (aged, n = 18) mice were used. h,i, Representative images (h) of 3D ex vivo cultured young and aged follicles. Follicles were considered atretic if there was disruption of contact between the oocyte (red asterisk) and GCs, leading to the release of oocytes from the follicles (bottom left), or if the follicles contained apoptotic or dead oocytes (bottom right). Antrum is indicated by the white arrowhead. Scale bar, 100 μm. Atresia rate was quantified (i) in young (2-3 months) and aged (14-15 months) follicles after 3D ex vivo culture. The median is represented by the center line, with individual dots representing biological replicates for each group. Sample sizes: n = 166 (young), 199 (aged) follicles, with 5 biological replicates in each group. 2-3 month-old mice were used in (a-f). Box plots inside the violins in (a) show mean (black circle), quartiles (box limits), and 1.5× interquartile range (whiskers). Two-tailed unpaired t-tests for (a, b, i). Two-tailed Fisher’s exact test for (c). P value: **P < 0.01, ns, not significant (P > 0.05). Exact P values are in the Source Data. All data are from at least three independent experiments.

Source data

Extended Data Fig. 3 Growth and maturation of oocytes from RCFs in 3D ex vivo culture.

a, Procedure for generating reconstituted chimeric follicles. Red arrow points to the oocyte used for transplantation. Red asterisk indicates the oocyte within the r-follicle that will be replaced. Refer to Supplementary Video 1 and Methods for further details. b, To distinguish between the donor oocyte and the r-follicle, we employed oocytes from mTmG transgenic mice exhibiting membrane-localized tdTomato (pseudo-colored yellow). In contrast, the r-follicles were sourced from non-fluorescent wild-type mice. The mTmG oocytes served as donors as referenced in Fig. 2g and Extended Data Fig. 3c–e. c, RCF size increased during 3D ex vivo culture. Oocytes from transgenic mTmG mice and follicular somatic cells from wild-type mice, as shown in (b). Scale bars, 50 μm. d, Cumulus-oocyte complexes (COCs) isolated from antral RCFs were induced for oocyte maturation with hCG for 16 hours in vitro. Note that cumulus cells surrounding the oocytes (from mTmG mice) expanded, and oocytes resumed meiosis, extruded the PB1 as shown in (e). Scale bars, 200 μm. e, Representative image of mature eggs derived from RCFs as shown in (c and d). The cumulus cells were removed after maturation to visualize mature eggs with the first polar body (PB1, arrows). Scale bars, 40 μm. All images are representative of at least three independent experiments.

Extended Data Fig. 4 Aged follicular somatic cells elevate ROS levels and reduce mitochondrial membrane potential in young oocytes.

a. Representative confocal images of cellular ROS stained with CM-H2DCFDA in oocytes from YY and YA RCFs. Scale bar, 100 μm. b. Quantification of CM-H2DCFDA fluorescence intensity in oocytes from YY and YA RCFs, as well as Y. n = 104 (Y), 122 (YY), 97 (YA). 2-month-old (young) and 14-month-old (aged) wide-type ICR mice were used. c. Fluorescence images of oocyte stained with MitoTracker Green (MTG, cyan) and mitochondrial membrane potential-sensitive dye TMRM (red). Scale bar, 100 μm. d. Quantification of the fluorescence intensity ratio of TMRM to MTG in oocytes from YY and YA RCFs, as well as Y. n = 110 (Y), 94 (YY), 72 (YA). 2-month-old (young) and 14-month-old (aged) wide-type ICR mice were used. Box plots in (b, d) show mean (black square), median (center line), quartiles (box limits), and 1.5× interquartile range (whiskers). One-way ANOVA, Tukey’s multiple comparisons test for (b, d). P value: ****P < 0.0001, ns, not significant (P > 0.05). The exact P values are presented in the Source Data. All data are from at least three independent experiments.

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Extended Data Fig. 5 Impact of young follicular somatic cells on aged oocyte quality.

a, Quantification of oocyte death rates. Data are presented as mean ± SD. Sample sizes: n = 77 (YY), 65 (AA), 142 (AY) oocytes. Individual dots represent biological replicates for each group. Young: 2-3 months old, aged: 14-15 months old. b-e, Representative live-cell images (b) showing spindle and chromosomes in MII oocytes. Scale bar, 10 µm. Quantification of the percentage of chromosomal misalignment (c) and spindle abnormalities (d). Panel (e) presents a separate quantitative analysis of various classes of spindle abnormalities. Young: 2-3 months old, aged: 14-17 months old. f,g, Representative image of DAPI-stained blastocysts (f). Scale bars, 20 μm. Cell numbers per blastocyst were quantified in (g). n = 57 (YY), 43 (AY), 26 (AA). h-m, Comparison of various parameters between AA and AY RCFs and A: (h) cellular ROS levels, (i) oocyte maturation rates, (j) chromosomal misalignment, (k) spindle abnormalities, (l) blastocyst formation rate, and (m) blastocyst size. For (h), n = 72 (A), 72 (AA), 72 (AY); for (m), n = 25 (A), 28 (AA), 31 (AY). In (c, d, i, j, k, l), the oocyte numbers are specified in brackets. 2-month-old (young) and 14-month-old (aged) mice were used in (g-m). Box plots in (g, m) show mean (black square), median (center line), quartiles (box limits), and 1.5× interquartile range (whiskers). Box plots inside the violins in (h) show mean (black circle), quartiles (box limits), and 1.5× interquartile range (whiskers). One-way ANOVA with Tukey’s multiple comparisons test was used for (a, g, h, m). Two-tailed Fisher’s exact test for (c, d, i-l). P value: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant (P > 0.05). Exact P values are in the Source Data. All data are from at least three independent experiments.

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Extended Data Fig. 6 TZP regeneration and oocytes transcriptomic remodeling in RCFs.

a, Schematic demonstrating TZPs from GCs that pass through the zona pellucida, forming either adherens junctions or gap junctions on the oocyte surface. b, TZP regenerated within 3 hours of RCF culturing. RCF containing follicular somatic cells from mTmG mouse and wild-type oocytes were cultured within Alginate-rBM beads for 3 h. Somatic cells were then removed to visualize TZP regeneration. Scale bars, 20 μm. c, Histogram displays the number of up-regulated or down-regulated DEGs between oocytes from YY and AA, AY and AA, or YY and AY RCFs. d,e, Representative GO terms associated with the genes that were downregulated (d) and upregulated (e) in aged oocytes from AA RCFs when compared to young oocytes from YY RCFs. One-sided hypergeometric test with FDR adjustment for multiple comparisons.

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Extended Data Fig. 7 Investigating possible GC-to-oocyte mitochondrial transport in RCFs.

a. Experimental design to study mitochondrial transport within RCFs. RCFs were created using somatic cells from transgenic MTS-mCherry-GFP1-11 mice, which express mitochondria-targeted mCherry, and unlabelled oocytes from wild-type mice. b. Confocal microscopy images of mCherry-labelled mitochondria in oocytes. Top panel: Positive control, an RCF formed by transplanting an MTS-mCherry-GFP1-11 oocyte into an MTS-mCherry-GFP1-10 r-follicle. Middle panel: RCF generated by transplanting a wild-type oocyte into an MTS-mCherry-GFP1-10 r-follicle. Bottom panel: Negative control, an RCF generated by transplanting a wild-type oocyte into a wild-type r-follicle. Rightmost panel of each row: overexposed images corresponding to the second column (mCherry). Somatic cells were partially removed before imaging to better observe oocyte fluorescence. Scale bar, 20 µm. All images are representative of at least three independent experiments.

Extended Data Fig. 8 Comparative analysis of oocytes from YY, YA, AY, AA RCFs.

This analysis examines various parameters of oocyte quality and developmental potential across four different RCF groups (YY, YA, AY, AA): (a) oocyte maturation rates (n = 12 YY, 4 YA, 10 AY, 9 AA), (b) chromosome misalignment (n = 9 YY, 4 YA, 5 AY, 5 AA), (c) spindle abnormalities (n = 9 YY, 4 YA, 5 AY, 5 AA), (d) blastocyst formation rates (n = 9 YY, 5 YA, 7 AY, 7 AA), (e) cellular ROS accumulation (n = 150 YY, 97 YA, 38 AY, 26 AA), (f) mitochondrial membrane potential (n = 153 YY, 72 YA, 57 AY, 53 AA). All metrics were normalized to those of the YY group in the same experiments using the non-normalized data as in Figs. 2h, j, k, l, 3b, d, 6f, j, Extended Data Fig. 4b, d, and 5c, d. The data were analyzed by one-way ANOVA, Tukey’s multiple comparisons test. P value: ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant (P > 0.05). The exact P values are presented in the Source Data. All results are presented as mean ± SD. All data are from at least three independent experiments.

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Supplementary information

Reporting Summary

Supplementary Video 1

Chimeric follicle generation process. This video demonstrates a step-by-step example of creating a RCF, highlighting the process of transplanting an oocyte into an r-follicle.

Supplementary Video 2

An example of sister kinetochore pair distance measurement. This video demonstrates the measurement of sister kinetochore pair distances in oocytes expressing 2mEGFP–CENP-C (green) and H2B–mCherry (red) to label kinetochores and chromosomes, respectively. See Methods for a detailed description of the measurement protocol.

Supplementary Tables 1–5

Supplementary Table 1. Differential gene expression analysis in vitro oocytes versus in vivo oocytes. Two-sided Wald test adjusted with the Benjamini–Hochberg method. Supplementary Table 2. Differential gene expression analysis in oocytes from AA RCFs versus YY RCFs. Two-sided Wald test adjusted with the Benjamini–Hochberg method. Supplementary Table 3. Differential gene expression analysis in oocytes from AA RCFs versus AY RCFs. Two-sided Wald test adjusted with the Benjamini–Hochberg method. Supplementary Table 4. Differential gene expression analysis in oocytes from AY RCFs versus AA RCFs. Two-sided Wald test adjusted with the Benjamini–Hochberg method. Supplementary Table 5. Metabolomic profiling of oocytes from YY, AA, and AY RCFs. Two-sided Wald test.

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Wang, H., Huang, Z., Shen, X. et al. Rejuvenation of aged oocyte through exposure to young follicular microenvironment. Nat Aging 4, 1194–1210 (2024). https://doi.org/10.1038/s43587-024-00697-x

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