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Polyamine metabolite spermidine rejuvenates oocyte quality by enhancing mitophagy during female reproductive aging

Nature Aging volume 3, pages 1372–1386 (2023)Cite this article

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Abstract

Advanced age is a primary risk factor for female infertility due to reduced ovarian reserve and declining oocyte quality. However, as an important contributing factor, the role of metabolic regulation during reproductive aging is poorly understood. Here, we applied untargeted metabolomics to identify spermidine as a critical metabolite in ovaries to protect oocytes against aging. In particular, we found that the spermidine level was reduced in ovaries of aged mice and that supplementation with spermidine promoted follicle development, oocyte maturation, early embryonic development and female fertility of aged mice. By microtranscriptomic analysis, we further discovered that spermidine-induced recovery of oocyte quality was mediated by enhancement of mitophagy activity and mitochondrial function in aged mice, and this mechanism of action was conserved in porcine oocytes under oxidative stress. Altogether, our findings suggest that spermidine supplementation could represent a therapeutic strategy to ameliorate oocyte quality and reproductive outcome in cis-gender women and other persons trying to conceive at an advanced age. Future work is needed to test whether this approach can be safely and effectively translated to humans.

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Fig. 1: Metabolome profiling of ovaries in aged mice.
Fig. 2: Effects of spermidine supplementation on the ovarian spermidine level, follicle development and female fertility in aged mice.
Fig. 3: Effects of spermidine supplementation on in vivo maturation of oocytes from aged mice.
Fig. 4: Effects of spermidine supplementation on the fertilization capacity and embryonic development potential of oocytes from aged mice.
Fig. 5: Effects of spermidine supplementation on in vitro maturation of oocytes from aged mice.
Fig. 6: Effects of spermidine supplementation on the transcriptome level of oocytes from aged mice.
Fig. 7: Effects of spermidine supplementation on autophagy in oocytes from aged mice.
Fig. 8: Effects of spermidine supplementation on mitophagy activity of oocytes from aged mice.

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Data availability

Transcriptomic raw data have been deposited in the Gene Expression Omnibus database under accession number GSE239551.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (2021YFC2700100 to B.X.), the Fundamental Research Funds for the Central Universities (KYT2023002 to B.X.) and the National Natural Science Foundation of China (32070836 to B.X.).

Author information

Authors and Affiliations

  1. College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, China

    Yu Zhang, Jie Bai, Zhaokang Cui, Yu Li, Yilong Miao & Bo Xiong

  2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China

    Qian Gao

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Contributions

B.X. designed the research; Y.Z., J.B., Z.C., Y.L., Q.G. and Y.M. performed experiments; Y.Z. and B.X. analyzed data; Y.Z. and B.X. wrote the manuscript.

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Correspondence to Bo Xiong.

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Extended data

Extended Data Fig. 1 Supplementary data related to Fig. 2.

a, Effects of different doses of spermidine on the in vivo maturation of aged oocytes. The percentage of PB1 extrusion was quantified in young (n = 132), aged (n = 71), and SPD+aged (25 mg/kg: n = 30, 50 mg/kg: n = 36, 100 mg/kg: n = 31) oocytes. ****P < 0.0001, P = 0.9842, **P = 0.0046, *P = 0.0135. ns, no significance. Data were expressed as mean percentage (mean ± SEM) of at least three independent experiments. Statistical significance was determined by two-tailed unpaired t-test. b, Representative images of follicles at different developmental stages in young mice. PmF, primordial follicle, small oocytes without attached cells or surrounded by several flattened pre-granulosa cells; PF, primary follicle, middle sized oocytes surrounded by a single layer of cubical granulosa cells; SF, secondary follicle, large oocytes surrounded by more than two layers of granulosa cells; PAF, pre-antral follicle, large oocytes surrounded by many layers of granulosa cells with several small cavities; AF, antral follicle, follicles with a single big cavity. Scale bars: a’, 10 μm; b’, 20 μm; c’, 25 μm; d’, 60 μm; e’, 150 μm.

Source data

Extended Data Fig. 2 Supplementary data related to Fig. 4.

a, Representative images of ovastacin in young, aged, and Spd+aged M II oocytes. Scale bar, 30 μm. b, The fluorescence intensity of ovastacin signals was measured in young (n = 29), aged (n = 25), and Spd+aged (n = 25) M II oocytes. ****P < 0.0001, ****P < 0.0001. Data were expressed as mean value (mean ± SD) of at least three independent experiments. Statistical significance was determined by two-tailed unpaired t-test. c, Co-staining of mitochondrion and spindle in young MII oocytes. Scale bars, 40 μm, 20 μm.

Source data

Extended Data Fig. 3 Effects of dietary supplementation of spermidine on the quality of aged oocytes.

a, A timeline scheme for dietary spermidine supplementation, hormone priming and subsequent analyses. b, Representative images of in vivo matured oocytes collected from young, aged, and Spd+aged mice. Scale bars: a’, 80 μm; b’, 20 μm. c, The number of ovulated oocytes was counted in young (n = 8), aged (n = 8), and Spd+aged (n = 8) mice. ***P = 0.0001, *P = 0.0176. d, The percentage of PB1 extrusion was quantified in young (n = 206), aged (n = 110), and Spd+aged (n = 147) oocytes. ****P < 0.0001, *P = 0.0264. e, The proportion of fragmentation was quantified in young (n = 206), aged (n = 110), and Spd+aged (n = 147) oocytes. ****P < 0.0001, **P = 0.0042. f, Representative images of the spindle morphology and chromosome alignment in young, aged, and Spd+aged oocytes at M II stage. Scale bars, 30 μm, 10 μm. g, The percentage of aberrant spindles was quantified in young (n = 47), aged (n = 51), and Spd+aged (n = 48) M II oocytes. **P = 0.0011, *P = 0.0252. h, The percentage of misaligned chromosomes was quantified in young (n = 47), aged (n = 51), and Spd+aged (n = 48) M II oocytes. ***P = 0.0004, *P = 0.0345. i, Representative images of chromosome numbers in young, aged, and Spd+aged M II oocytes. Scale bar, 2.5 μm. j, The proportion of aneuploidy was quantified in young (n = 64), aged (n = 68), and Spd+aged (n = 65) M II oocytes. ***P = 0.0004, *P = 0.0265. k, Representative images of 2-cell embryos and blastocysts developed from in vivo fertilized young, aged, and Spd+aged oocytes. Scale bar, 50 μm. l, The fertilization rate was quantified in the young (n = 135), aged (n = 96), and Spd+aged (n = 94) groups. ****P < 0.0001, *P = 0.0442. m, The proportion of blastocyst formation was quantified in the young (n = 135), aged (n = 96), and Spd+aged (n = 94) groups. ****P < 0.0001, *P = 0.0254. Data in c-e, g, h, j, l and m were expressed as mean value (mean ± SD) or mean percentage (mean ± SEM) of at least three independent experiments. Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 4 Effects of spermidine supplementation on the in vitro maturation and spindle/chromosome structure in aged oocytes.

a, The percentage of PB1 extrusion was quantified in young (n = 150), aged (n = 112), and Spd+aged (25 μM: n = 85, 50 μM: n = 136, 100 μM: n = 128) oocytes after in vitro maturation for 14 h. ***P = 0.0002, *P = 0.0223, **P = 0.0011, P = 0.4078. ns, no significance. b, Representative images of the spindle morphology and chromosome alignment in young, aged, and Spd+aged oocytes matured in vitro. Scale bars, 20 μm, 5 μm. c, The percentage of aberrant spindles was quantified in young (n = 56), aged (n = 50), and Spd+aged (n = 56) oocytes matured in vitro. ***P = 0.0002, **P = 0.0067. d, The percentage of misaligned chromosomes was quantified in young (n = 56), aged (n = 50), and Spd+aged (n = 56) oocytes matured in vitro. ***P = 0.0008, *P = 0.0499. Data in a, c and d were expressed as mean percentage (mean ± SEM) of at least three independent experiments. Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 5 Supplementary transcriptome data.

a, The number of DEGs that were upregulated (red) and downregulated (blue) in aged oocytes compared to young controls. b, The number of DEGs that were upregulated (red) and downregulated (blue) in Spd+aged oocytes compared to aged ones. c, Venn diagram displayed the number of overlapping DEGs between aged compared to young group and Spd+aged compared to aged group. d, Volcano plot showed DEGs (upregulated, red; downregulated, blue) in Spd+aged oocytes compared to young ones. Some highly different DEGs were listed. e, KEGG interaction network of DEGs in the autophagy pathway. f, KEGG interaction network of DEGs in the mitophagy pathway.

Extended Data Fig. 6 Co-localization of two fluorescent signals as assessed by Mander’s correlation coefficient.

a, The MCC-M1 of LC3 and lysosome signals was calculated in young (n = 20), aged (n = 17), and Spd+aged (n = 17) M II oocytes. ****P < 0.0001, ***P = 0.0001. b, The MCC-M2 of LC3 and lysosome signals was calculated in young (n = 20), aged (n = 17), and Spd+aged (n = 17) M II oocytes. ****P < 0.0001, ****P < 0.0001. c, The MCC-M1 of VDAC1 and LC3 signals was calculated in young (n = 21), aged (n = 21), and Spd+aged (n = 17) M II oocytes. ***P = 0.0001, **P = 0.0046. d, The MCC-M2 of VDAC1 and LC3 signals was calculated in young (n = 21), aged (n = 21), and Spd+aged (n = 17) M II oocytes. ***P = 0.0003, *P = 0.0106. e, The MCC-M1 of mitochondrion and lysosome signals was calculated in young (n = 21), aged (n = 19), and Spd+aged (n = 16) M II oocytes. ****P < 0.0001, ****P < 0.0001. f, The MCC-M2 of mitochondrion and lysosome signals was calculated in young (n = 21), aged (n = 19), and Spd+aged (n = 16) M II oocytes. ****P < 0.0001, ****P < 0.0001. Data were presented as mean value (mean ± SD) of at least three independent experiments. Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 7 Effects of spermidine supplementation on ROS levels, DNA damage accumulation and apoptosis in aged oocytes.

a, Representative images of ROS levels as detected by DCFH staining in young, aged, and Spd+aged M II oocytes. Scale bar, 80 μm. b, The fluorescence intensity of ROS signals was measured in young (n = 30), aged (n = 30), and Spd+aged (n = 32) M II oocytes. ****P < 0.0001, ***P = 0.0007. c, Representative images of DNA damage as stained with γH2AX antibody in young, aged, and Spd+aged M II oocytes. Scale bar, 10 μm. d, The fluorescence intensity of γH2AX signals was quantified in young (n = 25), aged (n = 19), and Spd+aged (n = 24) M II oocytes. **P = 0.0011, *P = 0.0175. e, Representative images of apoptotic oocytes at M II stage as assessed by Annexin-V staining in young, aged, and Spd+aged groups. Scale bar, 40 μm. f, The fluorescence intensity of Annexin-V signals was measured in young (n = 26), aged (n = 24), and Spd+aged (n = 29) M II oocytes. ***P = 0.0002, **P = 0.0074. Data in b, d and f were presented as mean value (mean ± SD) of at least three independent experiments. Statistical significance was determined by two-tailed unpaired t-test.

Source data

Extended Data Fig. 8 Effects of mitophagy inhibition on the improvement of aged oocytes by spermidine supplementation.

a, The percentage of PB1 extrusion was quantified in control (n = 87) and Mdivi1-treated (10 μM: n = 88, 20 μM: n = 90, 50 μM: n = 98, 100 μM: n = 88, 150 μM: n = 64, 200 μM: n = 93) young oocytes after in vitro maturation for 14 h. P = 0.446, P = 0.1149, P = 0.059, **P = 0.003, ****P < 0.0001, ****P < 0.0001. b, Representative images of autophagosomes as stained with LC3 antibody in control and Mdivi-1-treated young M II oocytes. Scale bar, 40 μm. c, The fluorescence intensity of LC3 signals was measured in control (n = 28) and Mdivi-1-treated (10: n = 28, 20: n = 25, 50: n = 29, 100: n = 32, 150: n = 15) young M II oocytes. P = 0.6328, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001, ****P < 0.0001. d, The percentage of PB1 extrusion was quantified in young (n = 94), aged (n = 83), Spd+aged (n = 67), and Spd+aged+Mdivi-1 (n = 86) oocytes after in vitro maturation for 14 h. **P = 0.0018, *P = 0.042, **P = 0.0041. e, Representative images of autophagosomes as stained with LC3 antibody in young, aged, Spd+aged, and Spd+aged+Mdivi-1 M II oocytes. Scale bar, 20 μm. f, The fluorescence intensity of LC3 signals was measured in young (n = 31), aged (n = 22), Spd+aged (n = 25), and Spd+aged+Mdivi-1 (n = 32) M II oocytes. ****P < 0.0001, **P = 0.0034, ***P = 0.0002. Data in a, c, d and f were presented as mean percentage (mean ± SEM) or mean value (mean ± SD) of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significance. Statistical significance was determined by two-tailed unpaired t-test.

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Extended Data Fig. 9 Effects of spermidine supplementation on the quality of porcine oocytes exposed to H2O2.

a, Representative images of in vitro matured porcine oocytes in control, H2O2-treated, and Spd-supplemented groups. Scale bars: a’, 400 μm; b’, 200 μm; c’, 40 μm. GV oocytes were treated with 100 μM H2O2 for 30 min and then cultured in the fresh medium with or without spermidine for in vitro maturation. b, The percentage of PB1 extrusion was recorded in control (n = 191), H2O2-treated (n = 203), and different concentrations of Spd-supplemented groups (50 μM: n = 196, 100 μM: n = 195) after in vitro culture for 44 h. **P = 0.002, P = 0.6526, **P = 0.0017. ns, no significance. c, Representative images of autophagosomes as stained with LC3 antibody in control, H2O2-treated, and Spd-supplemented oocytes at M II stage. Scale bar, 40 μm. d, The fluorescence intensity of LC3 signals was measured in control (n = 31), H2O2-treated (n = 36), and Spd-supplemented (n = 27) oocytes. ****P < 0.0001, ****P < 0.0001. e, Representative images of mitochondrial distribution in control, H2O2-treated, and Spd-supplemented oocytes. Scale bar, 40 μm. f, The fluorescence intensity of mitochondrial signals was measured in control (n = 31), H2O2-treated (n = 31), and Spd-supplemented (n = 29) oocytes. ****P < 0.0001, **P = 0.0023. g, Representative images of ROS levels detected by DCFH staining in control, H2O2-treated, and Spd-supplemented oocytes. Scale bar, 150 μm. h, The fluorescence intensity of ROS signals was measured in control (n = 30), H2O2-treated (n = 25), and Spd-supplemented (n = 31) oocytes. ****P < 0.0001, ****P < 0.0001. i, Representative images of apoptotic oocytes as assessed by Annexin-V staining in control, H2O2-treated, and Spd-supplemented groups. Scale bar, 40 μm. j, The fluorescence intensity of Annexin-V signals was measured in control (n = 26), H2O2-treated (n = 23), and Spd-supplemented (n = 30) oocytes. ****P < 0.0001, ****P < 0.0001. Data in b, d, f, h and j were presented as mean percentage or values (mean ± SEM or SD) of at least three independent experiments. Statistical significance was determined by two-tailed unpaired t-test.

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Zhang, Y., Bai, J., Cui, Z. et al. Polyamine metabolite spermidine rejuvenates oocyte quality by enhancing mitophagy during female reproductive aging. Nat Aging 3, 1372–1386 (2023). https://doi.org/10.1038/s43587-023-00498-8

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