The Basic And Applied Research Of BIN2, The Dominant Oocyte Expressing Protein, In Female Reproduction

Commonly speaking,the termination of the ovary "life" is marked by number of follicle decreased to less than a thousand and the ovary has lost its endocrine and reproduction capacity.The extending of ovary life will not only prolong the potential fertility,but also the endocrine function to ensure the health situation.So far strategy that most researchers have tried is to use chemicals that could inhibit the over-activation and atresia of primordial follicles.However,these chemicals are toxic and usually caused some remarkable side effect on various organs.Herein we happened to find a new upstream protein that can be a new target to properly and specifically inhibit the MTOR pathway within the ovaries.Bridging integrator 2(Bin2)belong to Bin2 family,it contains a conserved dimerization motif that can bind and detect membrane curvature.The only study about Bin2 showed that it is important for the podosome formation,motility and phagocytosis in leucocytes.And till now there are no any studies about Bin2 in reproductive system.In the current study,we firstly found that Bin2 is very superior in ovaries and oocytes,indicating that its function may be mainly in ovaries and oocytes.Next we made Bin2 global-knockout mice by crisper-cas9 and conducted a-year-long breeding assay.Unexpectedly,we found that the reproducive capacity has been significantly prolonged and cumulative litter size is significantly larger;Accordingly,the numbers of primordial follicles and total follicles also significantly increased from 6-month-old,while the numbers of all developing follicles(primary,secondary,antral)didn't change,indicating that the prolonged fertility might be correlated with the slowdown of primordial follicle exhaustion,while the significant increase of litter size might be correlated with the improvement of oocyte quality.We are carrying out RNA seq,DNA methlation and quantitive proteomics studies between WT and knockout ovaries to explore the mechanism of Bin2.In the meanwhile,we characterized its potential interactors through Bin2 antibody immunoprecipitation and mass spec to establish a signal pathway model how Bin2 functions.We have testified that Bin2 knockout significantly elevated p-LKB1 p-AMPK,indicating that Bin2 might be the upstream regulator of LKB1?AMPK?MTOR pathway.Since phosphorylation is a common regulatory mechanism within all links of MTOR pathway,we characterized two adjacent phosphorylation site(T423,S424)and made phospho-specific antibody within the C-teminal DUF(domain of unknown function)region.From immunostaing we found that compared with the membrane enrichment of total Bin2,p-Bin2 mainly concentrated within spindles,which largely fited with the cytoplasmic localization of p-AMPK and p-MTOR.And we also primarily showed that inhibiting Lck(lymphocyte protein tyrosine kinase),a pro oncogene-encoded kinase,We are now testifying the change of indivial molecules within the MTOR pathway in vivo and in vitro.From above,we think that Bin2 knockout increased p-AMPK,which will inhibit MTOR activity and hence prolong the fertility and increase the litter size.And moreover,Bin2 is very predominant within ovaries and oocytes,so clinically,is inactivation or depletion of Bin2 a better specific and safe target for prolonging the fertilty of the ovary? To check this,we established two types of mouse model: Inhibiting Bin2 phosphorylation to prolong the ovary reproductive life of 8-month old retired mice;Inhibiting Bin2 phosphorylation to rescue the POF(premature ovarian failure)caused by cyclophosphamide chemotherapy.We used PEP1-fused Bin2 peptide around the Bin2 phosphorylation site and injected into mice intraperitoneally to compete with endogenous Bin2 to be phosphorylated(by Lck?).And we successfully testified that p-Bin2 significantly decreased.Currently we have finished the models and the following breeding assays have been undergoing for several months,and within both models the reproductivity were significantly elevated and the numbers of total follicles and primordial follicles significantly increased.Currently we are carrying out RNA seq,DNA methylation and quantitive proteomics to verify whether the rescue mechanism within these two models agree with that in the Bin2 knockout ovaries.In all,considering all sorts of disadvantages that current strategies that prolong ovary life or prevent POF,the Bin2 inhibition in our study might be a potent simple,cheap,safe strategy good for ovary-targeting.