Expression of TLR2/4 on Epididymal Spermatozoa of the Chinese Soft-Shelled Turtle Pelodiscus sinensis During the Hibernation Season

Spermatozoa are known to be stored in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis for long periods during hibernation, but the mechanism that underlies the sperm storage is poorly understood. This study was carried out to confirm the presence of TLR2/4 (Toll-like receptor 2/4) in epididymal spermatozoa during the hibernation season and to analyze whether TLRs play a role in sperm storage. The structure and ultrastructure of a spermatozoon during the hibernation stage were investigated using light-and transmission electron-microscopy. RT-PCR was used to analyze mRNA expression, while protein expression was determined via Western blot. TLR2/4 mRNA and proteins were detected in spermatozoa. Immunofluorescence staining was used to confirm TLR2/4 localization in the spermatozoon, and TLR2/4 were localized in the mid-piece and the posterior segment of the head of the spermatozoon, which corresponded to the cytoplasmic droplets (CDs) of the turtle 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. (C) 2016 Wiley Periodicals, Inc.