Bridge Integrator 2, a New Specific Target for Improving Reproductive Life Span through RPS6 and NNT

Abstract Female ovary is the earliest degenerated organ and it faces distinct medical disadvantages that impair primordial follicle reserve and oocyte quality. Herein, we found that bridge integrator 2 (Bin2) was predominant within mouse ovaries and oocytes, and global-knockout of Bin2 improved both female fertility and oocyte quality with healthy physiology in mice. Ovarian quantitative proteomics and phosphomics showed that Bin2 knockout specifically decreased only p-RPS6 of mTOR pathway; meanwhile, it increased nicotinamide nucleotide transhydrogenase (NNT), the free-radical detoxifier, over 6-fold. Mechanically, phosphorylation at Thr423 & Ser424 translocated membrane Bin2 into cytoplasm to phosphorylate RPS6, while p-RPS6 bound 42-95 bp NNT UTR to inhibit NNT translation. We then synthesized a peptide (BPP) to mimic Bin2 inhibition, and found that 3-week BPP injection improved primordial follicle reserve and oocyte quality in aging or chemotherapeutics-treated mice. In all, Bin2 inhibition improve both primordial follicle reserve and oocyte quality without discernible side effects. Supplementary Dataset Legends Supplementary Dataset 1. Related to all fertility assays. WT mating male mice were monthly rotated between cages according to this random allocation table. Supplementary Dataset 2. Related to figure 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-knockout 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 up-regulation. The “Bin2-KO vs WT ≤ 0.83 down” sheet includes all differential site-phosphorylation values and related information with 0.83-fold down-regulation. Supplementary Dataset 3. Related to figures 2F, figure 5A and 5B. 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-knockout 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 up-regulation. The “Bin2-KO vs WT ≤ 0.83 down” sheet includes all differentially-expressed protein (DEP) values and related information with 0.83-fold down-regulation. Supplementary Dataset 4. Related to figure 2G. 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 (we have verified that this did not alter any differential trends). Supplementary Dataset 5. Related to figure 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 (2M), 9-month (9M), and 9-month + BPP (9M-BPP) groups. The “|Log2(Ave-2M)vs(Ave-9M)| ≥2” sheet includes all genes and related information with two-fold differential expression between the 2M and 9M groups. The “|Log2(Ave-9M-BPP)vs(Ave-9M)| ≥2” sheet includes all genes and related information with two-fold differential expression between the 9M-BPP and 9M 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 (we have verified that this did not alter any differential trends).