Biochemical properties and localization of zonadhesin in equine spermatozoa

Reproduction in all manunals requires successful completion of recognition events between spermatozoa and the ovum. Failure of gamete recognition may underlie some forms of sub-fertility or sterility in the stallion. This study has focused on comparing biochemical properties and localization of zonadhesin, a sperm-specific adhesion molecule, in Equus cahallus (horse), Equus asinus (donkey) and Equus grevyi (Grevy's zebra). Sperm proteins that function as adhesion molecules are prime candidates for such markers because of their essential functions in fertilization.

One well-defined recognition event in mammalian fertilization is the species specific adhesion between spermatozoa and the egg's extracellular matrix (zona pellucida, ZP). Sperm proteins that may function in sperm-ZP adhesion include sp56, spermadhesins, RSA/Sp 17, PH-20, Sp38, and zonadhesin. The majority of these candidate adhesion molecules have been characterized in stallion spermatozoa. Among these proteins, only zonadhesin is capable of binding directly and in a species-specific manner to the zona pellucida. Zonadhesin is a high Mr protein evolutionarily related to von Willebrand factor, mucins, and other known cell adhesion molecules. Zonadhesin is expressed only in developing spermatozoa, and is localized to the apical head of spermatozoa. Zonadhesin has been most extensively characterized in pig spermatozoa.

Preliminary studies in our laboratory have detected zonadhesin in spermatozoa of eight mammals, including the horse. This study documents the detection and initial characterization of equine zonadhesm, with an emphasis on properties that may vary across the Equus species and with length of time extended semen is stored. In this study, semen was collected artificially from 23 stallions and one jack (mean age lOyr + .91). Breeds of horses included Quarter Horse, Quarter Horse cross. Thoroughbred and Arabian, in addition to a large standard donkey and a Grevy's zebra. Semen samples were processed, extracted sequentially using 1% Triton and 2% SDS, and evaluated for protein content via BCA protein assay. Western blots were prepared loading equal amounts of protein from each stallion and membranes were probed with domain specific, DODl and D3, antisera to zonadhesin protein. The D3 bands were consistently located at approximately 95 kD and 60 kD. This band pattern was similar between stallions and across the species. The DODl band pattern showed numerous proteins with molecular weights ranging from approximately 120 kD to 25 kD. In addition, variation in Western blot band patterns was detected in the only known sub-fertile stallion evaluated for this study. Zonadhesin localized by immunofluorescence to the apical head of spermatozoa, in agreement with other mammals that have been studied. The similarity of zonadhesin composition between Equus caballus, Equus sinus and Equus grevyi is consistent with the ability of these species to interbreed. This research represents the first molecular studies on sperm recognition proteins in three Equus species.