Influence Of Estrogen Receptor ? Phosphorylation At Serine 309 On Microenvironment Of Mice Epididymis

ObjectiveSpermsacquire motility and fertilization capacity in the epididymis,and the appropriate microenvironment of the lumen fluid is critical to this process.Estrogen receptor ?(ER?)regulates the maturation of spermatozoa in epididymis mainly through ligand-independent pathway.ER?knockoutmale mice are infertile,with the increase in the pH value of the epididymal lumen fluid,the decrease in the sperm count and motility in the cauda of epididymis,and the reduction in the expression level of proteins that regulate the environment of efferent and epididymal duct.In human mammary epithelial cells,ER? can be phosphorylated at the 305 th amino acid in an estrogen-independent pathway,and then regulates the transcription of the target genes of ER?.The site of ER?309 in mouse is homologous to S305 in human.Hence,we hypothesized that the phosphorylation of ER? at serine 305 was involved in the maintenance of the luminal microenvironment of the epididymis.In the present study,we constructed ER?S309Aknock-in(S309AEKI)mice to investigate the effect of ER?309phosphorylation on microenvironment of mice epididymis and the possible mechanism.Methods1.The S309 AEKI mice were verified by gene-sequencing technology.Heterozygous male mice were mated with heterozygous females,and genome DNAwas extracted from the offspring.Then polymerase chain reaction(PCR)and restriction digestion were used to identify the genotype.2.Homozygous malemice were mated with wild type female mice to examine the male feritily(the changes of pregnant rate and average litter size),computer-assisted sperm analysis(CASA)was used to detect the sperm count and motility in the cauda epididymis of S309 AEKI mice.3.HE staining was used to observe the morphology of the testis,efferent duct and epididymis.Precise pH paper was employed to test the pH of epididymal fluid.Radioimmunoassay was used to detect the concentration of serum testosterone(T).Western blot and immunohistochemical staining(IHC)were applied to confirm the expression of ER? in the efferent duct and the epididymis of S309 AEKI mice.4.Western blot,Real-time PCR(RT-qPCR)and immunofluorescence labeling(IF)were used to investigate the expression changes of AQP9,CA12 and NHE3 in the efferent duct and the epididymis of S309 AEKI mice.Furthermore,ER?-WT,ER?-S309A(constitutively un-phosphorylated state)and ER?-S309E(constitutively phosphorylated state)expression vectors were constructed and transfected into 293 T cells,and Western blot was used to detect the expression level of AQP9 and CA12.Results1.Gene sequencing analysis demonstrated that the S309 AERKI mouse was constructed successfully.2.Mating study showed that S309 AERKI homozygote males were subfertile.The breed rate and the mean number of offspring were reduced by 40% and 48.7 % respectively,compared with that of wild type mice.The sperm numberwas normal inthe cauda epididymis of S309 AERKI adult mice,but the motility was reduced at fast,full and non-progressive grade.3.The testis and epididymis exhibited normal morphology,but efferent ducts weredilated and thinner in S309 AERKI mice than in the wild type mice.The fluid pH throughout the epididymis and the serum T level were elevated in S309 AERKI mice.The expression level of ER? in efferent duct and epididymis of S309 AERKI mice was unchanged compared with that of the wild type mice.4.In the efferent ductof S309 AERKI mice,the expression of CA12 and NHE3 remainedunchanged,but the expression of AQP9 was reducedcompared with that of the wild type mice.In the epididymisof S309 AERKI mice,the expression of AQP9 and NHE3 did not change,but the expression of CA12 was reduced.In vitro,the transfection of ER?S309A lowered the expression of AQP9 and CA12,however,the transfection of ER?S309E increased the expression of the two proteins.Conclusions1.The decline of fertility and the decrease of sperm motility suggested that ER?309 played a role in male reproduction.2.The morphology of efferent duct,the pH of epididymal fluid and the concentration of serum testosterone were changed in S309 AERKI mice,which implicated that ER?S09 might be involved in the fluid reabsorptionand luminal acidification in efferent duct and epididymis,which affected the sperm maturation.3.In S309 AERKI mice,the expression of AQP9 was reduced in efferent duct,and the expression of CA12 was reduced in epididymis.In vitro,the expression of AQP9 and CA12 were reduced or elevated by the transfection of ER?S309A or ER?S309E expression vectors,which suggested that ER?309 regulated the fluid reabsorption in efferent duct by targeting AQP9,and modulated the luminal acidification in epididymis by targeting CA12.