Characterization of PH-20 in canine spermatozoa and testis.
The purpose of this study was to characterize the sperm membrane protein PH-20 in the dog. Canine spermatozoa were extracted with Triton X- 100 and the presence of PH-20 was determined by immunoblot with an antibody against recombinant macaque PH-20. The hyaluronidase activity of canine PH-20 was determined with substrate gel electrophoresis based upon digestion of hyaluronic acid (HA) incorporated into the separating gels. Hyaluronidase activity was also quantified using a microplate assay. Sperm extracts were incubated at pH 4 or 7 in wells containing agarose and HA. For immunolabeling of PH-20 on canine sperm membranes, canine sperm were fixed and incubated with R-10 primary antibody, and an anti-rabbit IgG-FITC secondary antibody. Samples were visualized by fluorescence microscopy. Non-reducing SDS-PAGE and Western blot of detergent-extracted canine sperm revealed a major band at 50 kDa, and three other bands at 42, 124, and >209 kDa. Substrate PAGE revealed translucent bands of hyaluronidase activity of similar size to bovine testicular hyaluronidase. These bands were markedly more pronounced at pH 4 than at pH 7. The microplate assay also demonstrated that hyaluronidase activity was over four times greater at the acidic pH. Immunolabeling of canine spermatozoa demonstrated that PH-20 is localized to the anterior head region and appeared in the Golgi area of round spermatids as detected by the immunohistochemical staining of the testis. This study provides evidence that PH-20 is present on the membrane of canine spermatozoa and in round spermatids. Canine PH-20 exhibits hyaluronidase activity that is markedly more pronounced at acidic pH.
Sabeur
K
K
Department of Population Health and Reproduction, University of California, Davis 95616, USA.
Foristall
K
K
Ball
B A
BA
D000818:Animals; D015153:Blotting, Western; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D008297:Male; D013094:Spermatozoa / Q000737:chemistry*; D013737:Testis / Q000737:chemistry*
D015815:Cell Adhesion Molecules; D006821:Hyaluronoglucosaminidase; C066852:hyaluronidase PH-20