Localization and cellular distribution of a unique hyaluronidase in stallion spermatozoa during epididymidal transit

Three protein bands with hyaluronidase activity and molecular masses of 87, 48 and 43 kDa were isolated from purified equine sperm plasma membranes. Indirect immunofluorescence was used to assess sperm labelling patterns using a polyclonal antibody to sperm hyaluronidase. In ejaculated spermatozoa, surface-associated hyaluronidase was localized to the posterior head region of 98 +/- 2% of spermatozoa (n=10). Epididymides were isolated from mature stallions (n=5) and divided into caput, corpus and cauda epididymides in separate Petri dishes. The epididymidal tubules were dissected and washed using Dulbecco's PBS on ice and spermatozoa were collected from each region in the separate Petri dishes. After fixation and washing, the cells were labelled using indirect immunofluorescence. Spermatozoa from the caput epididymidis displayed > 90% sperm head fluorescence over the anterior head region overlying the acrosome, whereas spermatozoa from the cauda epididymidis displayed fluorescence over the posterior head region only, which is similar to ejaculated spermatozoa. Spermatozoa from the corpus epididymidis displayed sperm head fluorescence similar to that of spermatozoa from the caput epididymidis. These data indicate that surface-associated hyaluronidase is redistributed during epididymidal transit and that this maturation-associated redistribution occurs during late transit. The results indicate that epididymidal sperm maturation is a dynamic event and that hyaluronidase could potentially be used as a novel marker for epididymal dysfunction in stallions.