Effect Of LPS On Expression Of Epididymis-Specific Gene Bin1b And Its Mechanisms And Biological Significance

The mammalian epididymis is not only the site of sperm storage and transport,but also the site ofpost-testicular sperm maturation.Because of the absence of B lymphocytes, innate immunity plays a key role in epididymal defense system.There is such kind of molecules which have highly restricted expression patterns in epididymis.These molecules have both a defensin-liked trefoil motif and a capability of binding to sperms,and play a dual function in host defense and sperm maturation.Bin1b(Spaglle),an epididymis-specific protein found in rat,is cloned and reported by Zhang Yonglian firstly. Previous studies have found that rat Bin1b gene encodes a small peptide with 68 amino acids and belongs toβ-defensin super family in structure.Expression of Bin1b is found only in epithelia cells in the middle part of the caput epididymis and reaches a maximum expression during the sexually mature period,which suggests that the protein have temporally and spatially regulated expression pattern.Bin1b,as aβ-defensin-like molecule, can efficiently suppress E.coli colony growth in the medium collected from primary cultures of caput epididymal epithelia.At the same time,Bin1b can bind to the head of sperms in different regions of epididymidis and induce uptake of Ca²⁺ to promote sperm motility.Although the study in physiological functions of Bin1b progresses at a rapid rate, the expression of Bin1b in pathological conditions such as infections has been unknown yet.In this study,we observed expression patterns of Bin1b and other twelve epididymis-specificβ-defensins during lipopolysaccharide(LPS) - induced epididymitis in rat,and found LPS could down-regulate the expressions of Bin1b and some other epididymis-specificβ-defensins.Furthermore,we extended these findings to an analysis of the relationship with male infertility.Finally we investigated the possible mechanisms of down-regulated expression of Bin1b in primary cultured epithelial cells from caput epididymis of rat.1.LPS down-regulates expressions of Bin1b and other twelve epididymis-specificβ-defensins in caput epididymidis of rats. We made a model of LPS-induced epididymitis in rat by unilaterally intraparenchymal injection to the caput epididymis using an insulin syringe.H&E staining showed a pronounced interstitial changes including enlarged space between ducts of epididymis, white blood cells infiltration,and small blood vessels engorgement.The result of Northern blot analysis testifies expression of proinflammatory cytokine IL-1βmRNA was significantly increased in a dose- and time-dependent manner.It means the duplication of epididymitis model is successful.Then,we observed the effect of LPS treatment on Bin1b expression in caput epididymal tissues by Northern blot and Western blot analyses.Results showed LPS evidently down-regulated expression of Bin1b mRNA and protein.To determine whether the down-regulated expression induced by LPS is a common phenomenon in epididymis-specificβ-defensins,we also examined the responses of 12β-defensins present in caput epididymis to LPS challenge in the above rat model.The results showed that expressions of elevenβ-defensins mRNA(Defb12,17,21,25,27,39, 41,42,44,51 and 52) were decreased significantly,and only oneβ-defensin mRNA was not affected(Defb29).Though epididymis-specificβ-defensins belong toβ-defensin superfamily,they have comparative independences in both functions(such as a dual role in antibiosis and sperm maturation) and regulation of expression.Not inducible but inhibited expression was observed in Bin1b and some other epididymis-specificβ-defensins after LPS challenge suggests theseβ-defensins have a more important effect on sperm maturation.2.Decreased expression of Bin1b induced by LPS in caput of rat epididymis could weaken the motility of sperms from eauda of epididymis.Considering the ability of Bin1b in promoting the immature sperms of caput epididymis to gain progressive motility,we conjecture that the down-regulation may result in the impairment of sperms and even be responsible for the male infertility.The followed experiment by computer assisted sperm analysis(CASA) was to observe the influence of LPS treatment on sperms in different segments of epididymis.Results showed that with LPS treatment,the percentages of total motile and progressively motile spermatozoa in corpus and cauda segments of epididymis were significantly lower.Then we transiently transfected a Bin1b expression vector or empty vector into PC1 cells,an epididymal cell line which does not express Bin1b endogenously,and obtained culture medium containing recombination Bin1b protein.Interestingly,supplement of Bin1b protein in vitro could significantly reverse the decrease of sperm motility induced by LPS.Immunofluorescence and Flow cytometry showed similar results as CASA.These results suggest the impairment of sperms caused by epididymitis partly due to the reduced expression of Bin1b.3.LPS receptor expresses on epithelial cells of caput epididymis,but LPS can not directly down-regulate expression of Bin1b in epithelial cells.We have demonstrated that treatment with LPS significantly inhibited Bin1b mRNA expression in caput epididymis of rat.However many factors can influence on the expression of Bin1b after LPS treatment in vivo.It might be direct effect of LPS on the epithelial cells of epididymis,or indirect effect through mediation of other factors.To further investigate the mechanism of down-regulation,we isolated and cultured epithelial cells of caput epididymis and identified the responsibility of LPS pathway in primary cells. Results showed there was Toll-like receptor 4(TLR4) mRNA expression in primary cells and 2 days after 200μg LPS treatment could induce a 8-fold expression of IL-1βmRNA.It suggests that LPS is able to induce target genes expression in epididymidis mediated by LPS receptor and activated signal pathways.On the contrary,2 days after 200μg LPS treatment could not down-regulate the expression of Bin1b mRNA.These results demonstrate LPS can interact with epithelial cells of epididymis and activate intracellular downstream signals,but can not directly down-regulate the Bin1b expression by LPS pathways.Therefore the LPS-induced down-regulation of Bin1b might carry out via paracrine pathway,i.e.indirect mechanism mediated by some factors which induced by LPS.4.Not estrogen or glucocorticoid but androgen can up-regulates expression of Bin1b mRNA in primary cultured epithelial cells of caput epididymis.During the primary culture of epididymis epithelial cells,we also found a confusing phenomenon that expression of Bin1b mRNA revealed a gradually decreased inclination along with the culture and was almost undetectable after 5 d of culture.It suggests the stable expression of Bin1b in vivo is also maintained by some other factors.And these unknown factors may be absent or reduced in culture medium,which results in the decreased expression of Bin1b in vitro.It has been known that many epididymis-specific proteins are regulated by steroid hormone,so we observed the effect of different concentrations of androgen - dihydrotestosterone(DHT),estrogen - estradiol(E₂) or glucocorticoid - dexamethasone(Dex) on expression of Bin1b mRNA.The results showed that 10^-9-10^-7M DHT could all up-regulate expression of Bin1b mRNA in primary cells with DHT treatment was higher than control(p＜0.05),but neither E₂ nor Dex could regulate expression of Bin1b mRNA in primary cells.Even then,10^-9M - 10^-7M DHT, 10^-9M -10^-8M E₂ or 10^-8M-10^-7M Dex could not reverse the decreased expression of Bin1b in primary cells.We have not known which factors can regulate the expression of Bin1b and which factors play a major role in maintain the physiological expression of Bin1b in vivo.It should a further study to find these unknown factors.A similarly gradually decreased expression pattern was also observed in Defb21,but the expression of Defb29 was sustained all along with the culture.Coincidentally, expression of Defb21 was down-regulated by LPS in a time- and dose-dependent manner in vivo as well as Bin1b,whereas expression of Defb29 was not regulated by LPS in vivo as well as its sustained expression in primary cultured cells.Whether the LPS-induced down-regulated expression of Bin1b is due to the depletion of its physiological regulation factors in inflammation is still to be known.In summary,we have found although Bin1b plays an important role in epididymal host defense in physiological condition,its protection against pathogens may be weaken owing to the down-regulated expression in LPS-induced epididymitis.Moreover down-regulated expression could be a common expression pattern in epididymis-specificβ-defensins during infectious inflammation.The decreased expression of Bin1b leads to the decreased binding of Bin1b proteins to the head of sperms,which could depress the motility of sperms in caudal epididymis.On the other hand,epithelial cells of caput epididymis express TLR4 and have a responsibility to LPS.However,the main mechanism of down-regulation effect of LPS on Bin1b in vivo is not mediated by the direct interaction between LPS and epididymal epithelial cells.In addition,expression of Bin1b mRNA in primary cells is gradually decreased in a time-dependent manner.Though DHT can mildly up-regulate expression of Bin1b mRNA in vitro,DHT,E² or Dex can not reverse the decreased expression of Bin1b in primary cells.All above findings will help to understand pathophysiological functions of epididymis-specificβ-defensins in the progress of inflammation and sperm maturation,elucidate the relationship with epididymitis and male infertility,and find new medicine targets for prevention and therapy of epididymitis caused male infertility and sexually transmitted diseases.