Expression Of Toll-like Receptor 2/4 In Chinese Soft-shelled Turtle Pelodicus Sinesis Spermatozoa And Epididymis,and Its Relationship With Sperm Storage

Male turtle due to special reproductive strategies,need to store sperm for a long time in the epididymis.Many factors affect sperm storage in vivo.Among these factors,immune regulation is crucial.Infection with foreign microorganisms can reduce the spermatozoa quantity and quality,which influence sperm storage in the epididymis and may ultimately reduce fertilization ability.TLRs act as a link between innate immunity and the specific immune response and can participate in innate immune recognition and initiate specific immunity.This study was carried out to confirm the presence of TLR2/4(Toll-like receptor 2/4)in epididymal spermatozoa and epididymis and to analyse whether TLRs play a role in sperm storage.1.Expression and localization of TLR2/4 in the spermatozoa of the soft-shelled turtle P.sinensisRT-PCR was used to analyse mRNA expression,while protein expression was determined via western blot.Immunofluorescence staining was used to confirm TLR2/4 localization in the spermatozoon.Results showed that TLR2/4 mRNA and proteins were all expressed in spermatozoa,and the expression of TLR2 protein in sperm during hibernation seasons increased significantly.While TLR2/4 were all localized in the midpiece and the posterior segment of the head of the spermatozoon.As TLRs play critical roles in detecting and responding to invading pathogens,this study provided molecular evidence that TLR2/4 might contribute to sperm storage in the epididymides.2.Expression and distribution of TLR2/4 in the epididymis of the soft-shelled turtle P.sinensisWestern blot was used to analyse TLR2/4 protein expression in epididymal caput,corpus and cauda during the hibernation and reproductive seasons.Immunohistochemical(IHC)method was used to examine the distribution of TLR2/4 in epididymal caput,corpus and cauda during the hibernation and reproduction seasons.Results showed that TLR2/4 proteins all present in the epididymis.The expression of TLR2 in hibernation seasons was similar to that in reproductive seasons.On the whole,TLR4 protein expression was higher in cauda than in caput and corpus in reproductive and hibernation seasons.TLR2/4 were all mainly expressed on the cellular membrane of epididymis epithelial cells and efferent duct also had a positive reaction.The distributions of TLR2/4 were similar during hibernation and reproductive seasons.And there was no significant difference between epididymis different parts.TLR2/4 expressed in eipdidymides and might contribute to sperm storage in the epididymides.