BIOLOGY OF MALE AND FEMALE GAMETES
7:30 AM-10:00 AM
(171) ACQUISITION OF ARYLSULFATASE A (AS-A) ONTO THE MOUSE SPERM SURFACE DURING EPIDIDYMAL TRANSIT.
Xu, Hongbin1, Anupriwan, Araya1,2, Weerachatyanukul, Wattana1,2, Wade, Michael3, Carmona, Euridice1, Hermo, Louis4, Rippstein, Peter5, Sobhon, Prasert2, Tanphaichitr, Nongnuj1,6, 1 Department of Obstetrics/Gynecology, Ottawa, Canada2 Department of Anatomy, Bangkok, Thailand3 Environmental and Occupational Toxicology Division, Ottawa, Canada4 Department of Anatomy and Cell Biology, Kingston, Canada5 Anatomical Pathology, Ottawa, Canada6 Department of Biochemistry/Microbiology/Immunology, Ottawa, Canada
ABSTRACT- We have recently shown that arylsulfatase A (AS-A) exists on the surface of mature mouse sperm and participates in the sperm-zona pellucida binding event. Since AS-A is known as a lysosomal/acrosomal enzyme, it is pertinent to understand how it is targeted to the sperm surface. In this study, we first localized AS-A in testicular germ cells. Indirect immunofluorescence (IIF) of fixed dissociated testicular germ cells and immunoelectron microscopy of mouse testis sections revealed that AS-A was first detected in pachytene spermatocytes, localized to their Golgi bodies. Intracellular localization of AS-A was also observed in the developing acrosome granules in differentiating spermatids and in the dorsal region of the mature acrosome in testicular sperm. However, IIF of live testicular germ cells indicated the absence of AS-A on the plasma membrane of any testicular germ cell types. Using live epididymal sperm, we localized AS-A by IIF to the plasma membrane overlying the postacrosomal region and the head convex ridge in both caput and cauda epididymal sperm. Flow cytometry analysis showed higher amount of AS-A on the surface of cauda epididymal sperm than that of caput sperm. This was further supported by immunoblotting results, revealing a more intense AS-A band in cauda epididymal sperm. Interestingly, AS-A is present in the caput and cauda epididymal fluid as shown by immunoblotting and possession of the AS-A enzymatic activity, with a higher level in the cauda region. To determine the origin of AS-A in the epididymal fluid, localization of AS-A in the mouse epididymis was performed. Immunocytochemistry showed AS-A protein in the epithelial cells of the entire epididymis. In addition, the presence of AS-A transcript in the epididymis was demonstrated by RT-PCR and in situ hybridization. These results suggested that AS-A secreted into the epididymal lumen may be adsorbed onto the sperm surface during their migration through this organ. In fact, AS-A was detected on the sperm surface following incubation of testicular sperm with epididymal fluid. Collectively, our data strongly suggested that sperm acquired their surface AS-A from the epididymal fluid during their transit through the epididymis. (Supported by CIHR, NSTDA and TRF)
KEY WORDS: arylsulfatase A, sperm, epididymis