PARENT SESSION

(40) CAPACITATION DEPENDENT CHANGES IN THE LOCALIZATION AND COMPOSITION OF ZONADHESIN IN MOUSE SPERMATOZOA.

Tardif, Steve¹, Hardy, Daniel¹, ¹ Texas Tech University Health Sciences Center, Lubbock, TX

ABSTRACT- Zonadhesin is a sperm-specific protein that binds in a species-specific manner to the zona pellucida (ZP) of the egg and may thereby contribute to the specificity of sperm-ZP adhesion. The zonadhesin precursor is a mosaic protein that is cleaved in the testis to produce functional polypeptide(s). The precursor in rodents includes numerous partial von Willebrand D (vWD) domains that arose by repeated duplication of the last two exons encoding the D3 domain. Because this expansion of partial vWD domains is absent in other species, we hypothesize that it confers unique functional properties on zonadhesin proteins in rodents. Accordingly, here we examined the localization and fate of these domains in mouse spermatozoa. Affinity purified antibodies to the D3p18 partial vWD domain labeled the apical acrosome of all (100%) acrosome intact, methanol permeabilized spermatozoa. Likewise, similarly prepared antibodies to the D3 domain labeled all permeabilized cells. When live spermatozoa were labeled in suspension, neither antibody labeled uncapacitated cells. Likewise, live, capacitated spermatozoa were not labeled with anti-D3. However, anti-D3p18 labeled 20-25% of live, capacitated spermatozoa. On Western blots, anti-D3 and anti-D3p18 both detected an Mr 340,000 zonadhesin polypeptide (p340) that was extractable with SDS. This polypeptide was only partially extractable with Triton X-100 at pH 4.5. However, disruption of sperm membranes with Triton X-100 at pH 5 or higher caused conversion of p340 to an Mr 250,000 and smaller polypeptides, and the release of these polypeptides from spermatozoa. These results suggest that capacitation dependent events, perhaps including membrane alterations and changes in acrosomal pH, convert mouse zonadhesin to a form with partial vWD domains exposed on the sperm cell's surface where they would be accessible for interaction with the ZP.

KEY WORDS: Zona pellucida, Adhesion, Spermatozoa, Fertilization