| Beta-defensins,a kind of antibacterial cationic peptide,play a role in the innate immunity of animals,which is expressed preferentially expressed in epididymis.It forms a special defense system that protects sperm from invading microbes,and it is also important for sperm maturation,storage and maintain the ability of fertilization.However,the types and expression patterns of goat beta-defensin(gBD)in reproductive organs are still unknown.Therefore,the aim of this study was to investigate the types and expression patterns of gBD in the epididymis in Taihang Black goat,and to explore its expression characteristics in each segements of caprine epididymis,to screene specifically high expression of gBD in caput of epididymis and determine the role played by high-expressed gBD in epididymal epithelial cells.The results are as follows.Experiment 1:The results showed that a total of seven,twelve and thirty-three β-defensins were discovered in testes of 7-day-old kids,testes and epididymal caput of adult bucks respectively,while gBD 123,gBD 119 and gBD 124 were the highest expression in the testes of 7-day-old kids,testes and epididymal caput of adult bucks respectively.gBD 33 was specifically expressed in testes of adult bucks.Twenty-one β-defensins were specifically expressed in epididymal caput of adult bucks.The molecular weight of β-defensins was from 6.89 ku to 13.85 kDa.The bioinformatics analysis showed that goat β-defensins protein were small molecular,polar,hydrophobicity and basic polypeptide with positive charge.A complete conservation in spacing pattern was six cysteines in peptides sequence of β-defensins,which consensus pattern was-C-X2-4,5-C-X3-6-C-X3-13-C-X4-7-CC-.However,the pattern of twenty-four β-defensins was-C-X5,6-C-X3,4-C-X9-C-X5,6-CC-.A total of sixteen β-defensins was secreted glycoprotein.The rest of thirty-two P-defensins had the phosphorylation sites except for gBD 126.The goat β-defensins family protein were also the antimicrobial cationic peptides.The rich specific-epididymisβ-defensins were expressed in epididymal caput,which could coat sperm plasma membrane with glycocalyx in sperm maturation and play a vital role in making sperm motion.And β-defensins maybe were cell signal in some pathways,It was speculated that it may played the role of signaling moleculesExperiment 2:The results showed that 8 412 new genes were found in the nine segements of caprine epididymis compared to the reference database,including 32 beta-defensins.Thirty-two beta-defensins were located on chromosome 8,13,23 and 27 in four clusters across the genome Complete conservation gene order was observed for four chromosome beta-defensin orthologs between cattle and goat.Moreover,the expression pattern of 32 beta-defensins was different in caprine epididymis.The results of RT-PCR showed that gBD 104-109tr2 on chromosome 27 specially expressed and gBD 110-114 on chromosome 23 highly expressed in reproductive organs Except gBD 123-126、128 and 131 on chromosome 13 expressed in 30 tissues,the others 10 genes on chromosome 13 specially expressed in reproductive organs.gBD 124 was the highest expressed in segment 1 of the epididymis head(EH1).These highly expressed beta-defensins in the epididymis may also played a role in sperm maturation and storage in addition to their immunoprotective functionExperiment 3:the results of qRT-PCR showed that gBD 124 mRNA was detected in testis,caput,corpus,cauda,heart,liver,spleen,lung and kidney of bucks at the age of 18 months except that wasn’t detected in the longissimus dorsi,and the expression level in caput of epididymis was extremely higher than that in other tissues(P<0.01).Moreover,gBD 124 mRNA expression level in testis of buck at the age of 6 months was significantly higher than those at other ages,and the expressions in caput,corpus,cauda of epididymis was increased with the increase of the age.There was no obvious difference in gBD 124 mRNA expression levels between testis and epididymis of 1-week-old bucks(P>0.05).gBD 124 mRNA expressions in caput of epididymis were extremely higher than those in testis,corpus,cauda,of epididymis of bucks at 3-6 and 18 months old(P<0.01).The result of immunofluorescence suggested that gBD 124 protein was detected in testis and epididymis,and mainly located in false stratified columnar epithelium cells of caput of epididymis.The order of the expression level was caput>corpus>tcauda>testis,which identified with expression pattern of gBD 124 mRNA in testis and epididymis.In conclusion,the expression of gBD 124 was in the spatio-temporal expression,This indicated that gBD 124 played an important role in epididymal head.Experiment 4:the results showed that over-expressed gBD 124 promoted the expression of IL-1β and CCL5(P<0.05)in epithelium cells of caput of epididymis.However,it had no significant effect on IL-la(P>0.05).It was speculated that it played an immune protection role in epididymal head.There were 32 beta-defensins expressed in caput of epididymis,located on chromosome 8,13,23 and 27 in four clusters across the genome.The structure of protein is-C-X2-4,5-C-X3-6-C-X3-13-C-X4-7-CC-.gBD 124 specially highly expressed in epididymal caput,which played a role in immune protection.The further study should be focused on the function of gBD 124 on sperm maturation. |
