Study Of MTA1/ERα Mediated Transcription In Regulating Microenvironment Of Epididymis

BackgroundThe appropriate microenvironment of the epididymal lumen fluid is critical for sperm transport,maturation,storage and fertilization.Studies have shown that the direct causes of the infertility of the estrogen receptor alpha ERα knockout mice were fluid reabsorption disorder of the efferent duct and destruction of the epdidymal luminal acid microenviroment.What’s more,the expression levels of genes relating with luminal microenviroment(like water channels AQP1/9,sodium/hydrogen exchanger N HE3,carbonic anhydrase CAП)were reduced.It was also reported that both NHE3 and AQP9 were estrogen response element ERE contained genes,so ERa may maintain epididymal luminal microenvironment through regulating ion channel or transporters like NHE3 and AQP9.The transcription activity of ERα is regulated by some coregulatory factors,and studies showed that metastasis associated protein 1(MTA1)could repress the ERa transactivation by recruiting the HDAC complex on ER target genes in breast cancers.At present,most of studies are concentrating on its role in tumor metastasis.However,its physiological functions are less studied.O ur preliminary findings demonstrated that MTA1 was expressed in the mouse epididymis,suggesting that MTA1 may regulate the ERa mediated gene transcription and thereby maintaining the acidic microenvironment of the epididymal lumen.Methods1.The expression pattern of MTA1 in rat and mouse epididymis was examined by PCR,Western blot and immunohistochemistry.The colocalization of MTA1 and ERα in epididymal tissue of rat and mouse was detected by immunofluorescence staining.And the expression of NHE3,which is an ERE contained gene,was detected by Realtime PCR to preliminarily verify MTA1 ’s effect on ERα mediated gene transcription.2.To further investigate if genes with ERE elements were regulated by MTA1/ ERα in mouse epididymal epithelial cells,immunofluorescence staining was used to detect the expression of AQP9,NHE3 and CA12 in the mouse epididymal epithelial cell lines PC1.In order to verify whether they can be regulated by ERa,different concentrations of MPP,which is an inhibitor of ERa,were added to PC1 cells,and the expression levels of ERα,AQP9,NHE3 and CA12 were detected by Western blot.After that,the expression vector of MTA1 was constructed and transfected into PC1 cells,and then the expression levels of ERα and its target genes were detected by Western blot and Immunofluorescence staining.3.After identifying the genotype of Mta1 KO mice,the p H value of the luminal contents in the epididymis was measured by Hydrion p H paper.In addition,Western blot and immunofluorescence staining were used to detect the expression of ERα and its target genes in the initial segment of epididymis where the luminal pH value was significantly changed.4.A vasectomized mouse model was constructed,in which the epididymal microenvironment was destroyed.C hoose the time point that epididymal luminal pH changed greatly to perform this operation on WT and Mta1 KO mice at the same time.After that,HE staining was used to display the changes of epididymal morphology and Hydrion pH paper was used to detect the luminal p H.At last,the expression of ERα and its target genes was detected by Realtime PCR,Western blot and immunofluorescence staining.Results1.MTA1 was expressed in the caput,corpus and cauda of rat epididymis,and mainly expressed in the caput and corpus epididymis.MTA1 and ERα were colacalized on the nuclei of rat and mouse epididymal epithelial cells.NHE3 was expressed in the whole rat epididymis,and with the highest expression in the cauda epididymis.2.AQP9,NHE3 and C A12 were expressed in PC1 cells and were regulated by ERa.Overexpression of MTA1 could inhibit the expression of ERa target genes like AQP9,NHE3 and CA12 in PC1 cells.3.The luminal p H value in the initial segment of the Mta1 KO mice was decreased,while the expression of ERa target genes like AQP9,NHE3 and C A12 were increased in the initial segment.4.After 8 weeks of vasectomy in the mice,the epididymal epithelium height was thinner,but changes in the Mta1 KO mice were lower than that in the wild type.Epididymal luminal pH was increased in different segments of the wild type;however,it only increased in the cauda epididymis of the Mta1 KO mice,and showed significant difference compared with the wild type.The expression levels of NHE3 and CA12 were decreased in the cauda epididymis,however,the expression level of NHE3 in the Mta1KO mice was higher than that in the wild type.Conclusions1.The higher expression of MTA1 in the caput and corpus epididymis and the colocalization of MTA1 and ERa in the nuclei of rat and mouse epididymal epithelial cells indicate that MTA1 may involve in the regulation of ERa mediated epididymal functions.2.In PC1 cells,the expression levels of AQP9,NHE3 and CA12 were decreased after overexpressing MTA1,suggesting that MTA1 may inhibit ERa mediated gene transcription.3.In the initial segment of the Mta1 KO mice,luminal p H were more acid than the controls,the expression of AQP9,NHE3 and CA12 was increased,suggesting that MTA1 may affect epididymal luminal microenviroment by regulating ERa mediated transcription.4.By eight weeks post vasectomy,the changes of epididymal epithelium height and luminal pH value in the Mta1 KO mice were less than that in the wild type,but the expression of NHE3 in the cauda epididymis of the Mta1 KO mice was higher than that in the wild type,futher confirmed that MTA1 may affect the epididymal microenvironment by regulating ERa mediated gene transcription.