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Inhibiting bridge integrator 2 phosphorylation leads to improved oocyte quality, ovarian health and fertility in aging and after chemotherapy in mice

Nature Aging volume 1, pages 1010–1023 (2021)Cite this article

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Abstract

Female ovaries degenerate about 20 years earlier than testes leading to reduced primordial follicle reserve and a reduction in oocyte quality. Here we found that bridge integrator 2 (BIN2) is enriched in mouse ovaries and oocytes and that global knockout of this protein improves both female fertility and oocyte quality. Quantitative ovarian proteomics and phosphoproteomics showed that Bin2 knockout led to a decrease in phosphorylated ribosomal protein S6 (p-RPS6), a component of the mammalian target of rapamycin pathway and greatly increased nicotinamide nucleotide transhydrogenase (NNT), the free-radical detoxifier. Mechanistically, we find that phosphorylation of BIN2 at Thr423 and Ser424 leads to its translocation from the membrane to the cytoplasm, subsequent phosphorylation of RPS6 and inhibition of Nnt translation. We synthesized a BIN2-penetrating peptide (BPP) designed to inhibit BIN2 phosphorylation and found that a 3-week BPP treatment improved primordial follicle reserve and oocyte quality in aging and after chemotherapy-induced premature ovarian failure without discernible side effects.

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Fig. 1: BIN2 is essential for primordial follicle activation.
Fig. 2: Bin2 knockout specifically reduced p-RPS6 of mTOR pathway.
Fig. 3: Phosphorylation at Thr423 and Ser424 activates BIN2.
Fig. 4: BIN2 phosphorylates RPS6 in vitro.
Fig. 5: Bin2 KO or RPS6 knockdown upregulates Nnt translation.
Fig. 6: p-RPS6 binds to Nnt 5′ UTR to inhibit Nnt translation.
Fig. 7: Inhibiting p-BIN2 prolongs ovarian lifespan of aging mice.
Fig. 8: Inhibiting p-BIN2 protects ovaries from CPA-induced POF.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Supplementary Datasets 1–5 have been deposited into Zenodo (https://doi.org/10.5281/zenodo.4006605). Raw data and extracted text files for quantitative proteomics and phosphoproteomics have been deposited into PRIDE, under accession nos. PXD028776 and PXD028777, respectively.

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Acknowledgements

This work was financially supported by the National Key Research and Development Program of China (grant nos. 2018YFC1003400 and 2017YFC1001503 to D.Z. and 2018YFA0107701 to Q.-Y.S.), the Youth Innovation Promotion Association CAS (grant no. 2017114) to Z.-B.W. and National Natural Science Foundation of China (grant nos. 32070840 to D.Z. and 31871504 to Q.-Y.S.). Funding for data collection and analysis was from grant nos. 2018YFC1003400, 2017YFC1001503, 2018YFA0107701 and 32070840; and funding for conceptualization, design, decision to publish and preparation of the manuscript was from grant nos. 2018YFC1003400, 2017YFC1001503, 2018YFA0107701, 2017114, 32070840 and 31871504.

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Author notes

  1. These authors contributed equally: Feng-Yu Zhu, Li-Li Wang, Tie-Gang Meng, Ruo-Lei Wang, Zhi-Xia Yang.

Authors and Affiliations

  1. State Key Lab of Reproductive Medicine, Nanjing Medical University, Nanjing, China

    Feng-Yu Zhu, Li-Li Wang, Ruo-Lei Wang, Zhi-Xia Yang, Ying Cao, Gang-Yi Zhu, Zhen Jin, Lei-Lei Gao & Dong Zhang

  2. Fertility Preservation Lab and Guangdong-Hong Kong Metabolism & Reproduction Joint Laboratory, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China

    Tie-Gang Meng & Qing-Yuan Sun

  3. State Key Lab of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

    Tie-Gang Meng & Zhen-Bo Wang

  4. Animal Core Facility, Nanjing Medical University, Nanjing, China

    Wen-Tao Zeng & Dong Zhang

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Contributions

D.Z., Q.-Y.S. and Z.-B.W. designed the research. T.-G.M. developed the Bin2-KO mice under the direction of Q.-Y.S. and Z.-B.W.; F.-Y.Z., L.-L.W. and R.-L.W. performed most of the experiments. Z.-X.Y. coordinated between authors. Y.C. assisted during the experiments. G.-Y.Z., Z.J., L.-L.G. and W.-T.Z. conducted preliminary studies of BIN2. F.-Y.Z. and L.-L.W. conducted all data analysis and prepared figures under the direction of D.Z., Q.-Y.S. and Z.-B.W. D.Z. and Z.-X.Y. wrote the manuscript. Q.-Y.S. and Z.-B.W. proofread the manuscript and gave advice.

Corresponding authors

Correspondence to Zhen-Bo Wang, Qing-Yuan Sun or Dong Zhang.

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

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Legends for Supplementary Figs. 1–10; legends for Supplementary Datasets 1–5; Supplementary Tables 1–7 and unprocessed blot images for Supplementary Fig. 2.

Reporting Summary

Supplementary Data 1

Related to all fertility assays. WT mating male mice were rotated on a monthly basis between cages according to this random allocation table.

Supplementary Data 2

Related to Fig. 2a–c. This Excel file contains three sheets: the ‘All identified phospho sites’ sheet includes all site-phosphorylation values and related identified information from two repeats of WT and Bin2-KO PND-21 ovaries. The ‘Bin2-KO vs WT ≥ 1.2 up’ sheet includes all differential site-phosphorylation values and related information with 1.2-fold upregulation. The ‘Bin2-KO vs WT ≤ 0.83 down’ sheet includes all differential site-phosphorylation values and related information with 0.83-fold downregulation.

Supplementary Data 3

Related to Figs. 2g and 5a,b. This Excel file contains three sheets: the ‘All identified proteins’ sheet includes all protein expression values and related identified information from two repeats of WT and Bin2-KO PND-21 ovaries. The ‘Bin2-KO vs WT ≥ 1.2 up’ sheet includes all differentially expressed protein (DEP) values and related information with 1.2-fold upregulation. The ‘Bin2-KO vs WT ≤ 0.83 down’ sheet includes all DEP values and related information with 0.83-fold downregulation.

Supplementary Data 4

Related to Fig. 2h. This Excel file contains one sheet: ‘All gene expression-FPKM & log2’ includes FPKM and log2 values of all identified genes and related information from three repeats of PND-21 WT and Bin2-KO mouse ovaries. To avoid the illegal calculation of value ‘0,’ we added a minimal value ‘0.001’ to all original values (and verified that this did not alter any differential trends).

Supplementary Data 5

Related to Fig. 6n–p. This Excel file contains four sheets: The ‘All gene expression-FPKM & log2’ sheet includes FPKM and log2 values for all identified genes and related information from three repeats of the 2-month (2 M), 9-month (9 M) and 9-month + BPP (9M-BPP) groups. The ‘|log2(Ave-2M)vs(Ave-9M)|≥2’ sheet includes all genes and related information with twofold differential expression between the 2 M and 9 M groups. The ‘|log2(Ave-9M-BPP)vs(Ave-9M)|≥2’ sheet includes all genes and related information with twofold differential expression between the 9M-BPP and 9 M groups. The ‘2Mvs9M-BPPvs9M overlap’ includes all genes and related information that overlapped between ‘|log2(Ave-2M)vs(Ave-9M)|≥2’ and ‘|log2(Ave-9M-BPP)vs(Ave-9M)|≥2’. To avoid the illegal calculation of value ‘0,’ we added a minimal value ‘0.001’ to all original values (and verified that this did not alter any differential trends).

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Zhu, FY., Wang, LL., Meng, TG. et al. Inhibiting bridge integrator 2 phosphorylation leads to improved oocyte quality, ovarian health and fertility in aging and after chemotherapy in mice. Nat Aging 1, 1010–1023 (2021). https://doi.org/10.1038/s43587-021-00133-4

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