Identification of Sperm Associated Antigen 6 (SPAG6) on Canine Sperm.

Sperm associated antigen 6 (SPAG6), a sperm protein, has been linked with sperm flagellar motility and structural integrity of the axoneme central apparatus of mature sperm in mice and humans. Sperm proteins especially those associated with sperm motility are potential biochemical targets for therapeutic or contraceptive management. Detection of SPAG6 on canine sperm to date has not been reported. It was hypothesized that canine spermatozoa expressed SPAG6. The objectives of this experiment were to identify and determine the localization pattern of SPAG6 on fresh canine sperm. To test this hypothesis, the sperm-rich second fractions of four dogs were collected, pooled and SPAG6 localized by immunofluorescence detection. Sperm was incubated with a monoclonal antibody against SPAG6, washed and secondary Alexa Fluor IgG goat anti-mouse antibody was added. The controls consisted of immortalized cat T cells stained with anti-SPAG6 and labeled with an IgG goat anti-mouse secondary antibody. Visualizations were ascertained using a Leica TCS SP5 Laser Scanning Confocal microscope at 200-400X magnifications. The study was repeated three times. Sperm associated antigen 6 was identified on fresh canine sperm and was highly expressed in the acrosomal region of the spermatozoa. Furthermore, SPAG6 was identified with none to mild expression of the midpiece region of the spermatozoa. Controls were negative for SPAG6. In conclusion, we have shown that SPAG6 has strong expression localized at the acrosomal region of the sperm head. In addition, SPAG6 has none to mild expression localized at the midpiece of the sperm tail. The localization of SPAG6 to the midpiece, similar to that seen in mice and humans may indicate that SPAG6 in the canine has a similar role as that found in other species. However, the strong expression of SPAG6 localized to the canine acrosomal region of the sperm head has not been shown in mice and humans. These observations are important because the sperm protein SPAG6 can potentially be used as a method for infertility assessment in the canine. This research is supported by Floridian Branch of the American Kennel Club Association Endowment. (poster)