Expression And Functional Study Of RANTES Receptors In Human Epididymis

ObjectiveThe different kinds of epididymal epithelial cells work in a concerted manner to maintain the acidic luminal environment,which helps to keep sperm quiescent and to avoid premature activation of sperm.Our previous study has shown that the chemokine Regulated upon activation normal T cell expressed and secreted(RANTES)was mainly expressed in the epithelial macrophages of human epididymis.In order to further study the expression of RANTES receptors in epididymis and whether RANTES participates in the interaction among epididymal cells via its receptors,we examined the distribution of RANTES receptors CCR1?CCR3 and CCR5 in human epididymis,and evaluated the cellular localization of RANTES in rat epididymal epithelium.Moreover,we explored the role of RANTES and its receptors in the establishment and maintenance of the acidic luminal environment of rat epididymis by using an in vivo perfusion model.Method(s)1.Reverse transcription-polymerase chain reaction(RT-PCR),immunohistochemistry(IHC)and double immunofluorescence labelling were used to examine the expression and localization of RANTES receptors in human epididymis,Furthermore,double immunofluorescence labeling was employed to observe the cellular localization of RANTES in rat epididymal epithelium.2.Rat caudal epididymides were perfused luminally in vivo with phosphate-buffered saline(PBS)adjusted to either pH values 6.8 or 7.8,for 30 min.The expression changes of RANTES were examined by means of real-time PCR and Western blot.The distribution changes of V-ATPase in clear cells were detected by confocal laser scanning microscopy(CLSM).3.Rat caudal epididymides were perfused luminally in vivo with recombinant RANTES proteins for 30 min.Real-time PCR,Western blot and CLSM were applied to investigate the expression changes of V-ATPase and iNOS.4.Rat caudal epididymides were perfused luminally in vivo with Met-RANTES,a competitive receptor antagonist of RANTES,for 30 min.Real-time PCR,Western blot and CLSM were employed to examine the expression changes of V-ATPase and iNOS.Result(s)1.RT-PCR analysis demonstrated that RANTES receptors CCR1 and CCR5 were present in human epididymis,but CCR3 was undetectable.Immunohistochemical assay showed that CCR1-positive signals were accumulated in the ciliated cells of the efferent ducts as well as in the apical cells and basal region cells of the epididymal ducts,whereas CCR5 was widely distributed in the epididymis.Double-labelling analysis showed that RANTES colocalized with CCR1 in the ciliated cells of the efferent ducts,and in the cells in the basal compartment of caput,corpus and cauda epididymis.The localization of RANTES was restricted to the epithelial macrophages in rat epididymis.2.Real-time PCR and Western blot revealed that upon alkaline treatment(pH of 7.8),the expression of RANTES was significantly increased compared with that in the control group(pH of 6.8).The results of CLSM showed that the alkaline pH also induced the apical accumulation of V-ATPase in the clear cells.3.The effects of recombinant RANTES on V-ATPase and iNOS were then examined in the in vivo perfusion model.Real-time PCR and Western blot demonstrated that both mRNA and protein levels of V-ATPase and iNOS were up-regulated by the addition of RANTES compared to the control group(pH of 6.8).Meanwhile,the immunofluorescent staining showed a stronger signal of iNOS in the macrophages,along with a notable accumulation of V-ATPase in the apical membrane of clear cells.4.By using real-time PCR and Westernblot,we analyzed the effects of Met-RANTES on the expression levels of V-ATPase and iNOS,the results revealed that the expression levels of V-ATPase and iNOS were decreased with Met-RANTES replenishment compared with those in the RANTES perfusion group.Correspondingly,the immunofluorescent staining indicated that the signal intensity of iNOS in macrophages weakened and the accumulation of V-ATPase in the apical membrane of clear cell disappeared.Conclusion(s)1.CCR1 and CCR5 were expressed in human epididymal epithelium and they co-localized with RANTE in the cells within the basal compartment.RANTES was expressed in the macrophages of rat epididymal epithelium,these results suggest that RANTES may play a role in epididymal macrophages through its specific receptors CCR1 and CCR5.RANTES was expressed in the macrophages of rat epididymal epithelium.2.Alkaline luminal pH induced the expression of RANTES and the accumulation of V-ATPase in the apical membrane of clear cells.These results suggest that RANTES expression is asscoaited with the homeostasis of the acidic environment in epididymal lumen.3.RANTES perfusion significantly induced the accumulation of V-ATPase in the apical membrane of clear cells and the expression of iNOS in the macrophages.These results further demonstrated that RANTES might stimulate proton secretion by epididymal clear cells via activation of the NO release from macrophages.On this basis,RANTES may directly take part in the regulation of the acidic environment of epididymal lumen.4.Met-RANTES perfusion led to a complete abrogation of the increased expression of V-ATPase and iNOS in the epididymal epithelium.These results revealed that RANTES may induce the NO release from macrophages by interacting with its receptors to maintain the acidic luminal environment.