The Functions Of Foxo1 In The Development Of Mouse Sertoli Cells

Objective: To explore the role of Foxo1 in gonadal cell fate maintenance by overexpressing Foxo1 in sertoli cells.Methods : 1.Using CRISPR-Cas9 technology,a mouse model of Foxo1 persistently expressed in testis sertoli cells was constructed by inserting the phosphorylation site mutated Foxo1 gene into the Rosa26 region,divided into Control as well as Foxo1-7mu;Sf1-Cre group,with three biological replicates(n=6)set in each group.2.FOXO1 and WT1 immunofluorescence co-localization staining was performed on 4M mouse testicular tissue to verify that FOXO1 was activated in sertoli cells and the mouse model was successfully constructed.3.Basal data(mouse body weight and testicular weight)and testicular tissues of 6M,2M,3W,2W and1 W mice were collected for phenotypic analysis;the number of spermatozoa in the epididymis of 6M and 2M mice was detected,and fertility were also performed.4.The effect of sertoli cell overactivation of Foxo1 on germ cells and sertoli cells was examined by H&E and immunohistochemical for MVH and SOX9.5.Effect of persistent FOXO1 expression on apoptosis in testicular tissue of 6M and 2M mice by TUNEL assay.6.Testicular sertoli cells from Foxo1-7mu;Sf1-Cre group and control mice were cultured in vitro for 2W,treated with Tris-Hcl after 48 h of culture to remove excess spermatogenic cells,and digested with 0.25% trypsin after 24 h for cell counting,seeded at 20,000 cells per well into seven 24-well plates,and subjected to MTT assay at the same time each day to detect absorbance at 490 nm and plot cell proliferation curves,and detect any abnormalities in the cell cycle of sertoli cells from Foxo1-overactivated mice by Hoechst-stained nuclei combined with flow analysis.7.The total proteins of the sertoli cells were extracted,and the protein levels of FOXL2,SOX9,WT1 and GAPDH were detected by Western Blot;the expression of Foxl2,Wnt4,Dax-1,Sox9,Dmrt1 and Amh was detected by RT-PCR;Immunohistochemical detection of FOXL2 expression in testicular tissue of 2W and 3W Foxo1-7mu;Sf1-Cre mice.Results:1.Foxo1-7mu;Sf1-Cre mice can survive normally with no significant developmental abnormalities.In the immunofluorescence co-staining of FOXO1 with WT1,a clear FOXO1 nuclear localization signal was visible in WT1-positive sertoli cells,indicating that FOXO1 was activated in sertoli cells and the mouse model was successfully constructed.2.Compared with the control group,Foxo1-7mu;Sf1-Cre mice had abnormal testicular development,significantly reduced testicular morphology,massive loss of germ cells,severely reduced sperm count in the epididymis and low fertility.3.TUNEL assay of testicular tissue from Foxo1-7mu;Sf1-Cre mice revealed that persistent activation of FOXO1 in sertoli cells leads to increased apoptosis in testicular varicocele.4.MTT proliferation assays on sertoli cells cultured in vitro for 7 consecutive days revealed that sertoli cells from Foxo1-7mu;Sf1-Cre mice had slowed proliferation and cell cycle arrest in the G0/G1 phase.5.Foxo1-7mu;Sf1-Cre mice showed decreased expression of sertoli cell male-specific genes Dmrt1、Amh and started to express the granulosa cell-specific gene Foxl2.Conclusion: Persistent expression of FOXO1 in sertoli cells resulted in significantly smaller testes,significantly lower sperm counts and loss of support for spermatogenic cells in male mice,as well as the transformation of sertoli cells into granulocyte-like cells,thus FOXO1 plays an important role in the genealogical maintenance of sertoli cells.