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.
|Number of pages||11|
|State||Published - 2002|
Bibliographical noteFunding Information:
This work was supported by the Kenneth A. Scott Charitable Trust, a Keybank Trust. K Foristall was supported by the Geraldine Dodge foundation. The authors thank the VMTH at UC, Davis for providing canine testes, Anthony Vo for technical assistance and Dr. Paul Primakoff for providing the α-PH-20 antibody.
Copyright 2008 Elsevier B.V., All rights reserved.
- Substrate PAGE
ASJC Scopus subject areas
- Small Animals
- Food Animals
- Animal Science and Zoology