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Granulosa cell mevalonate pathway abnormalities contribute to oocyte meiotic defects and aneuploidy

Abstract

With aging, abnormalities during oocyte meiosis become more prevalent. However, the mechanisms of aging-related oocyte aneuploidy are not fully understood. Here we performed Hi-C and SMART-seq of oocytes from young and old mice and reveal decreases in chromosome condensation and disrupted meiosis-associated gene expression in metaphase I oocytes from aged mice. Further transcriptomic analysis showed that meiotic maturation in young oocytes was correlated with robust increases in mevalonate (MVA) pathway gene expression in oocyte-surrounding granulosa cells (GCs), which was largely downregulated in aged GCs. Inhibition of MVA metabolism in GCs by statins resulted in marked meiotic defects and aneuploidy in young cumulus–oocyte complexes. Correspondingly, supplementation with the MVA isoprenoid geranylgeraniol ameliorated oocyte meiotic defects and aneuploidy in aged mice. Mechanically, we showed that geranylgeraniol activated LHR/EGF signaling in aged GCs and enhanced the meiosis-associated gene expression in oocytes. Collectively, we demonstrate that the MVA pathway in GCs is a critical regulator of meiotic maturation and euploidy in oocytes, and age-associated MVA pathway abnormalities contribute to oocyte meiotic defects and aneuploidy.

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Fig. 1: Oocytes from aged ovaries show chromatin structure abnormalities at MI.
Fig. 2: Meiosis-associated gene expression decreases in aged oocytes at MI.
Fig. 3: MVA pathway is abnormally downregulated in aged GCs at MI.
Fig. 4: Abnormal MVA pathway in GCs causes aging-related meiotic defects in oocytes.
Fig. 5: Supplementation with the MVA isoprenoid GGOH in vitro reduces meiotic defects in aged oocytes.
Fig. 6: MVA pathway in GCs regulates meiosis-associated genes in oocytes via EGF signaling at MI.
Fig. 7: Supplementation with the MVA isoprenoid GGOH in vivo reduces meiotic defects and aneuploidy in aged oocytes.

Data availability

All main and extended data figures have associated original data. Original data have been deposited to Mendeley Data: https://data.mendeley.com/datasets/d27fbh375b/draft?a=9dbcb577-58aa-45f7-8698-dd6ccc036424. All raw Hi-C sequencing data and processed files for young and aged oocytes used in this study have been deposited in the National Center for Biotechnology Informationʼs Gene Expression Omnibus under accession number GSE175830. The mouse oocyte and GC RNA-seq data can be found under accession numbers GSE175835 and GSE175834. Human GC RNA-seq data can be found under accession number GSE175832. In total, 406 meiosis-associated genes and 2,986 metabolism-associated genes from the Gene Ontology database have been deposited into source data of Fig. 2 and Extended Data Fig. 6. Source data files are provided for all main and extended data figures, and other data are available from the corresponding authors upon reasonable request.

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Acknowledgements

This work was supported by a grant from the National Key Research and Development Program of China (2018YFC1004701) to L.D.; a grant from the National Natural Science Foundation of China (82201830) to C. Liu; grants from the Jiangsu Province Social Development Project (BE2018602) and the self-determination research program of the State Key Laboratory of Reproductive Medicine (SKLRM-2022D2) to H.S.; grants from the National Key Research and Development Program of China (2018YFC1004703) and the National Natural Science Foundation of China (32170544 and 31970585) to Q.B.; and grants from the National Key Research and Development Program of China (2018YFC1004703) and the National Natural Science Foundation of China (31530046) to C. Li. The authors would like to thank Jabrehoo for VeriSeq analysis of oocytes.

Author information

Authors and Affiliations

Authors

Contributions

L.D., H.S., Q.B. and C. Liu. conceived this project. L.D. and C. Liu. isolated oocytes and GCs and performed RNA-seq library construction and bioinformatics analysis of the database. Q.B., W.Z. and C. Liu. isolated the oocytes and performed the Hi-C experiments and analysis. C. Liu., C.C., H.W., W.L., S.W., J.F., Y.Z., J.Z., X.Z., T.F. and L.D. performed the oocyte-related and COC-related experiments. S.S. and Z.W. performed chromosome spread. L.D., Q.B., H.S., C. Liu., W.Z., G.Y., Y.H., S.L., X.T., Y.L. and C. Li. wrote and revised the paper.

Corresponding authors

Correspondence to Chaojun Li, Qian Bian, Haixiang Sun or Lijun Ding.

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Competing interests

The authors declare no competing interests.

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Nature Aging thanks Karen Schindler and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Diminished follicle reserve and meiotic defects in aged ovaries.

a, Images of ovaries stained with PSR. The experiments were repeated three times independently with similar results. Scale bar, 500 µm. b, HE staining of ovaries from 6-week-old and 10-month-old mice. The experiments were repeated three times independently with similar results. Scale bar, 50 µm, 500 µm. c, Left: Images of MIIOs from 6-week-old and 10-month-old mice. The arrowheads denote fragmented oocytes. Right: Images of oocytes with PBE from 6-week-old and 10-month-old mice after IVM. The arrowheads denote oocytes that failed to extrude a polar body. The experiments were repeated at least three times independently with similar results. Scale bar, 100 µm. d, Spindle and chromosome morphology in oocytes from 6-week-old (n = 97) and 10-month-old (n = 104) mice. Scale bar, 25 µm. e, Proportions of different spindle and chromosome abnormalities in oocytes from 6-week-old (n = 97) and 10-month-old (n = 104) mice. 6W, 6 weeks; 10M, 10 months; PSR, picrosirius red; PBE, polar body extrusion.

Extended Data Fig. 2 Hi-C sample preparation and reproducibility assessment of Hi-C replicates.

a, Heatmap showing the correlations for pairwise combinations of Hi-C datasets generated in this study. The Pearson correlation coefficients were calculated using balanced chromatin interactions binned at 10 kb and the HiCRep package in R. Overall, the two biological replicates for each stage were highly correlated with each other, indicating a high degree of reproducibility. YGO, YFGO, YMIO, and YMIIO: oocytes from young mice. OGO, OFGO, OMIO, and OMIIO: oocytes from aged mice. b, Pearson correlation coefficients calculated with HiCRep at 10 kb resolution for normal saline-treated and GGOH-treated MI (meiosis I) oocytes Hi-C datasets. Overall, the two biological replicates for each stage were highly correlated with each other, indicating a high degree of reproducibility.

Extended Data Fig. 3 P(s) curves of young and aged oocytes at MI for individual autosomes.

P(s) curves indicate the relationships between chromatin contact probability and genomic distances for chromatin interactions on individual autosomes (chr1-19 and chromosome X) in young and aged oocytes. The dotted line corresponding to P(s) ~ s−0.6 is shown as a reference. p values were calculated from two-sided paired Wilcoxon signed rank test.

Extended Data Fig. 4 Derivatives of P(s) plots for young and aged oocytes.

Derivative of P(s) plots for young and aged oocytes Hi-C datasets show significant differences of the second diagonal size, indicated by the arrows.

Extended Data Fig. 5 Analysis of DEGs between young and aged oocytes.

a, Violin plots showing the average numbers of detected genes (FPKM > 1) in young (6-week-old) and aged (10-month-old) oocytes at different stages. The data are shown with at least three independent experiments each group. b, PCA plot of young and aged oocytes of different stages based on gene expression patterns separated by PC1 and PC2. The size and color of the dots represent the stage and age of the oocytes, respectively. c, Heatmaps showing the distributions of DEGs in young and aged oocytes. Red, upregulated genes; blue, downregulated genes; gray, unchanged genes. The pink bars on the left of the heatmaps point to DEGs shared by at least two oocyte stages, and the others are oocyte subtype-specific DEGs. d, Gene Ontology analysis of the upregulated DEGs in the four aged oocyte subtypes. The red font denotes biological processes associated with inflammation. Statistical significance was determined by two-sided Fisher’s precision probability test.

Extended Data Fig. 6 Analysis of metabolism-associated genes in young GCs.

a, Violin plots showing the average number of detected genes (FPKM > 1) in young and aged GCs at different stages. YGC, YFGC, YMIGC, and YMIIGC: GCs from young mice. OGC, OFGC, OMIGC, and OMIIGC: GCs from aged mice. The data are shown with at least three independent experiments each group. b, PCA plot of young and aged GCs of different stages based on gene expression patterns separated by PC1 and PC2. The size and color of the dots represent the stage and age of the GCs, respectively. c, Proportions of lipid-associated genes among the highly expressed metabolic genes at each stage. d, KEGG map of MIGC stage-specific highly expressed genes. Many genes highlighted with red rectangles are involved in steroid biosynthesis. 1.14.1417: SQLE; 5.4.99.7: LSS; 1.3.1.72: DHCR24; 1.14.14154: CYP51; 1.3.1.70: TM7SF2; 1.14.189: MSMO1; 1.1.1.170: NSDHL; 5.3.3.5: EBP; 1.14.19.20: SC5D; 1.3.1.21: DHCR7. e, Heatmap showing the expression levels of MVA pathway-associated genes in the four GC subtypes. The value for each gene is row-scaled Z score. GC, granulosa cell.

Source data

Extended Data Fig. 7 Analysis of DEGs between young and aged GCs.

a, Heatmaps showing the distribution of DEGs in young and aged GCs. Red, upregulated genes; blue, downregulated genes; gray, unchanged genes. The pink bars on the left of the heatmaps point to DEGs shared by at least two GC stages, and the others are GC subtype-specific DEGs. b, Left: Percentage of lipid-associated genes among the metabolic downregulated DEGs in aged MIGCs. Right: Gene Ontology analysis of the lipid-associated DEGs. Significance is indicated as the -log10 P value. Statistical significance was determined by two-sided Fisher’s precision probability test. c, Immunofluorescence analysis of HMGCS in young and aged ovaries. The experiments were repeated three times independently with similar results. Scale bar, 50 µm. d, Immunofluorescence analysis of GGPPS in young and aged ovaries. The experiments were repeated three times independently with similar results. Scale bar, 50 µm. e, mRNA levels of HMGCS and GGPPS in young (n = 12) and aged (n = 12) MIGCs. f, Gene Ontology analysis of the upregulated DEGs in the four mouse aged GC subtypes. The red font denotes biological processes associated with inflammation. Statistical significance was determined by two-sided Fisher’s precision probability test. g, Heatmap showing the expression levels of DEGs associated with the MVA pathway in human young and aged luteinized GCs as determined by RNA-seq. The value for each gene is row-scaled Z score. h, Gene Ontology biological processes of the upregulated DEGs in human aged luteinized GCs. The red font denotes biological processes associated with inflammation. Significance is indicated as the -log10 P value. Statistical significance was determined by two-sided Fisher’s precision probability test. GC, granulosa cell. For bar graphs in e, data are presented as mean ± SEM. Statistical significance was determined by two-sided unpaired t-test; exact P values are labeled above the graphs.

Source data

Extended Data Fig. 8 Mevalonate pathway is important for oocyte meiosis process.

a, mRNA levels of MVK and GGPPS in mouse GCs from the CTL (n = 15) and ATO (n = 15) groups. CTL group: young COCs cultured in IVM medium with 40 µM DMSO. ATO group: young COCs cultured in IVM medium with 40 µM atorvastatin. b, Rate of GVBD in the CTL (DO: n = 76, COC: n = 75) and ATO (DO: n = 78, COC: n = 126) groups. The data are shown with at least three independent experiments. c, Images of PBE oocytes from the CTL and ATO groups. The black arrowheads denote oocytes that failed to extrude a polar body. The experiments were repeated at least three times independently with similar results. Scale bar, 100 µm. d, COCs were treated with inhibitors of specific steps in the MVA pathway: 6-fluoromevalonate (40 µM), FTI-227 (20 µM), GGTI-298 (20 µM), and Zaragozic Acid A (20 µM). The rate of GVBD was recorded. The data are shown with at least three independent experiments. e, Images of PBE from oocytes in the CTL and GGTI-298 groups. CTL group: young COCs cultured in M199 IVM medium with 20 µM DMSO. GGTI-298 group: young COCs cultured in IVM medium supplemented with 20 µM GGTI-298. Black arrowheads denote oocytes that failed to extrude a polar body. The experiments were repeated at least three times independently with similar results. Scale bar, 100 µm. f, The levels of isoprenoid modification in GCs from young and aged mice (dot blotting). g, The rate of GVBD was recorded in different groups. The data are shown with at least three independent experiments. h, Images of PBE from oocytes in the CTL and GGOH groups. The experiments were repeated at least three times independently with similar results. Scale bar, 100 µm. i, PBE rate of DOs in the CTL and GGOH groups. The data are shown with at least three independent experiments. j, mRNA levels of MVK and GGPPS in GCs from the CTL (n = 18) and GGOH (n = 18) groups. For bar graphs in a,b,d,g,i,j, data are presented as mean ± SEM. Statistical significance was determined by two-sided unpaired t-test; exact P values are labeled above the graphs of a,b,i,j.

Source data

Extended Data Fig. 9 EGF signaling is regulated by the MVA pathway in GCs.

a, Expression levels of meiosis signal-associated genes in the four GC subtypes in 6-week-old mice. The value for each gene is row-scaled Z score. b, Number of potential ligand–receptor pairs in GCs and oocytes predicted by CellPhoneDB 2 in young and aged mice. c, Number of ligand–receptor pairs in GCs and oocytes at each stage in young and aged mice. d, mRNA levels of LHR, AREG, and EREG in GCs from young (n ≥ 17) and aged (n ≥ 16) donors. e, Western blot analysis of MVK, LHR and AREG expression in KGN cells treated with atorvastatin or GGOH. The data are shown with three independent experiments. For bar graphs in d,e, data are presented as mean ± SEM. Statistical significance was determined by two-sided unpaired t-test; exact P values are labeled above the graphs.

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Extended Data Fig. 10 GGOH supplementation improves aged oocyte quality.

a, Body weights of normal saline-treated (n = 6) and GGOH-treated (n = 6) mice. CTL group: aged mice treated with normal saline. GGOH group: aged mice treated with 4 mg kg−1 d−1 GGOH. b, Images of ovaries stained with PSR. The experiments were repeated three times independently with similar results. Scale bar, 500 µm. c, Immunofluorescence analysis of GGPPS in the ovaries of normal saline-treated and GGOH-treated mice. The experiments were repeated three times independently with similar results. Scale bar, 50 µm. d, Rates of GVBD in the CTL (DO: n = 28, COC: n = 34) and GGOH (DO: n = 47, COC: n = 41) groups. The data are shown with at least three independent experiments. e, Images of oocytes with PBE from normal saline-treated and GGOH-treated mice. The black arrowheads denote oocytes that extrude a polar body. The experiments were repeated at least three times independently with similar results. Scale bar, 100 µm. f, Rate of PBE in the normal saline-treated (DO: n = 28, COC: n = 34) and GGOH-treated (DO: n = 47, COC: n = 41) groups. The data are shown with at least three independent experiments. g, Number of normal MII oocytes per mouse. The data are shown with five independent experiments. h, Micrographs of IVF outcomes from normal saline-treated and GGOH-treated mice. The black arrowheads denote oocytes that failed to develop into blastulas. The experiments were repeated three times independently with similar results. Scale bar, 100 µm. i, The rates of oocytes developing into blastulas in the normal saline-treated and GGOH-treated groups. The data are shown with three independent experiments. j, The implanted embryos in the CTL and GGOH group at E14.5. The experiments were repeated six times independently with similar results. k, The number of implanted embryos at E14.5 in two groups. The data are shown with six independent experiments. CTL, control; GGOH, geranylgeraniol. For bar graphs in a,d,f,g,i,k, data are presented as mean ± SEM. Statistical significance was determined by two-sided unpaired t-test; exact P values are labeled above the graphs.

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Liu, C., Zuo, W., Yan, G. et al. Granulosa cell mevalonate pathway abnormalities contribute to oocyte meiotic defects and aneuploidy. Nat Aging 3, 670–687 (2023). https://doi.org/10.1038/s43587-023-00419-9

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