MiR-181c/d Regulates Testicular Sertoli Cell Growth And Barrier Function By Targeting Pafah1b1 Gene

In the mammalian seminiferous epithelium,the blood-testis barrier(BTB)is constituted by tight junction proteins between adjacent SCs,and physically separates the seminiferous epithelium into basal compartments and apical compartments,creating an immune microenvironment for spermatogenesis.Increasing studies have shown that miRNAs play essential roles in the mammalian testicular development,cell growth and spermatogenesis.Our previous study has shown that miR-181 c or miR-181d(miR-181c/d)is higher expressed in testes from boars at the age of 60 days compared with boars at the age of 180 days.We hypothesize that miR-181c/d might have an important regulatory role in the spermatogenesis.In this study,we explored the functions of miR-181c/d in cell survival and barrier function of Sertoli cells.The main findings are as follows:1.The role of miR-181c/d in the Sertoli cell barrier function in mice(1)A mouse model of intratesticular overexpression of miR-181c/d was successfully constructed via injecting lentivirus-delivered miR-181c/d(LV-miR-181c/d)into the testes of 15-16 days old Kunming mice.Two weeks after the final LV-miR-181c/d administration,the phenotypes of mice were analyzed.LV-miR-181c/d treated mice showed similarities in testis size or testis weight/body weight ratio with the LV-control mice.Haematoxylin and eosin-stained sections showed no histological abnormalities in the testis or epididymis from the LV-control and LV-miR-181c/d mice.Additionally,we evaluated the quality of semen collected from the cauda epididymides of LV-control and LV-miR-181c/d treated mice.Although sperm count was not statistically different,the abnormal sperm rate significantly increased in LV-miR-181c/d mice(P<0.01).In addition,TUNEL/WT1 double-label staining and Ki67 staining of mouse testicular tissue sections revealed that the number of TUNEL-positive cells and TUNEL-positive Sertoli cells(TUNEL and WT1 doublepositive cells)was increased(P<0.05)in the LV-miR-181c/d murine testes compared with the LV-control testes,but there was no significant difference in the number of Ki67-positive cells.These findings indicate that LV-miR-181c/d administration in testes increases the abnormal sperm rate and Sertoli cell apoptosis.(2)The distribution of BTB-associated proteins(e.g.,TJ proteins ZO-1,Occludin and basal ES proteins N-cadherin,β-catenin)was detected in the seminiferous tubules of LVmiR-181c/d treated mice by immunofluorescence staining.In LV-control mice,these proteins were tightly localized at the BTB,whereas in LV-miR-181c/d-treated mice,these proteins were diffusely localized at the BTB near the basement membrane.Western blot analysis of TJ proteins and basal ES proteins in testes revealed that LV-miR-181c/d failed to induce any remarkable changes in the expression level of multiple BTB-associated proteins.F-actin staining revealed that LV-miR-181c/d disturbed F-actin organization across the seminiferous epithelium.In LV-miR-181c/d mice,the fluorescence distribution of F-actin is no longer lined up properly along the BTB as found in the LV-control mice.Furthermore,transmission electron microscopy(TEM)showed there were intercellular spaces between adjacent murine SC contact at the BTB,coupled with TJ structure fractures in LV-miR-181c/d-delivered testis.Finally,LV-miR-181c/d administration in testes effectively disturbed BTB integrity,making biotin tracer penetrate into certain seminiferous tubules.Surprisingly,six weeks post the final administration,the localization of BTBassociated proteins at the BTB was virtually indistinguishable between the LV-control mice and the LV-miR-181c/d mice.Moreover,the damaged BTB integrity and the sperm quality were restored in LV-miR-181c/d mice.The above results suggest that the in vivo LV-miR-181c/d treatment causes short-term BTB dysfunction in mice,which can be restored after a period of time.(3)Primary murine SCs cultured in vitro for 2 to 3 days can establish a functional TJ permeability barrier that mimics the BTB in vivo.miR-181c/d mimics were transfected into murine SCs,and TER and Na-F permeability were examined at different time points.Overexpression of miR-181c/d in murine SCs resulted in decreased TER value and increased Na-F permeability,indicating miR-181c/d destroys the integrity of Sertoli cell barrier.Furthermore,even though the levels of TJ proteins and basal ES proteins remained unchanged,the distributions of TJ proteins and basal ES proteins at the Sertoli cell-cell interface were disturbed in miR-181c/d mimics treated murine SCs.These proteins were diffusively localized at the Sertoli cell-cell interface.According to the TEM results,overexpression of miR-181c/d led to several breaks and vacuoles at cell-cell contact.Phalloidin staining showed that the F-actin structure become disorganized,irregularly arranged,and microfilaments appear crossed in miR-181c/d mimics treated murine SCs.The above results indicate that miR-181c/d alters the cytoskeletal F-actin structure,disrupts the distribution of BTB-associated proteins on F-actin,destabilizes cell junctions at the Sertoli cell-cell interface and ultimately perturbs the Sertoli cell barrier.(4)The dual luciferase reporter system verified that miR-181c/d could target the 3’untranslated region(3’UTR)of platelet-activating factor acetylhydrolase 1b regulatory subunit 1(PAFAH1B1).And miR-181c/d can negatively regulate the protein expression of PAFAH1B1.Subsequently,Knockdown of Pafah1b1 downregulated TER value and increased Na-F permeability in murine SCs.Further,immunofluorescence and Western blot assays showed that inhibition of Pafah1b1 expression altered the localization of BTBassociated proteins at the Sertoli cell-cell interface.However,the levels of BTB-associated proteins remained unchanged.TEM analysis revealed that knockdown of Pafah1b1 led to some fractures and vacuoles of TJ structures between adjacent murine SC contact.Furthermore,phalloidin staining results showed that the F-actin structure become disordered and microfilaments appear crossed in Pafah1b1 siRNA transfected murine SCs.The above results indicate that knockdown of Pafah1b1 disrupts the F-actin structure,alters the localization of BTB-associated proteins on cytoskeletal F-actin,destabilizes cell junctions at the Sertoli cell-cell interface and ultimately perturbs Sertoli cell barrier.(5)Western blot assay reveals that overexpression of miR-181c/d or inhibition of Pafah1b1 downregulated the levels of CDC42,PAK1,LIMK1,and p-Cofilin.And knockdown of miR-181c/d increased actin-regulatory proteins expression,whereas transfection of Pafah1b1 siRNA or Cdc42 siRNA partially restored the elevated actinregulatory proteins levels.The interaction between PAFAH1B1 and IQ motif-containing GTPase activating protein 1(IQGAP1)was predicted by ZDOCK software.Coimmunoprecipitation assay in murine SCs further demonstrated the interaction between PAFAH1B1 and IQGAP1.In addition,overexpression of miR-181c/d or knockdown of Pafah1b1 in murine SCs resulted in a reduction of PAFAH1B1-IQGAP1 complex.2.The function of miR-181c/d on the survival of testicular Sertoli cells(1)The effects of miR-181c/d on the survival of Sertoli cells(swine testicular cells and murine SCs)were examined using CCK-8,Ki67 staining,Annexin V-FITC/PI and flow cytometry,and Western blot assays.The results exhibited that overexpression of miR-181c/d significantly inhibited Sertoli cell proliferation and increased Sertoli cell apoptosis.Conversely,suppression of miR-181c/d repressed Sertoli cell apoptosis and induced Sertoli cell proliferation.The above results demonstrate miR-181c/d increases apoptosis and inhibits proliferation in Sertoli cells.(2)CCK-8,Ki67 staining,Annexin V-FITC/PI and flow cytometry,and Western blot assays showed that overexpression of Pafah1b1 increased Sertoli cell proliferation and decreased Sertoli cell apoptosis.And the opposite result was observed with inhibition of Pafah1b1 gene,which inhibited Sertoli cell proliferation and promoted Sertoli cell apoptosis.Meanwhile,we found that knockdown of Pafah1b1 partially suppressed the cell proliferation that increased by miR-181c/d inhibitors.Accordingly,the decreased Pafah1b1 level antagonized the inhibition effects on cell apoptosis by miR-181c/d inhibitors.These results demonstrate that miR-181c/d affects Sertoli cell proliferation and apoptosis by targeting Pafah1b1 gene.