Hyaluronidase in ram semen. Quantitative determination, and isolation of multiple forms

Abstract

A study was made of hyaluronidase in ram semen. The end-group assay conditions used to determine activity quantitatively were chosen to ensure reliability as well as sensitivity [Gacesa, Savitsky, Dodgson & Olavesen (1981) Anal. Biochem. 118, 76-84]; they led to 1 W.H.O. Standard International Hyaluronidase Unit displaying 0.1263 EC munit (1 EC unit of activity releases 1 mumol equivalent of N-acetylglucosamine end groups/min at 37 degrees C). All the activity in the semen was shown to be sperm-derived, and intact spermatozoa were estimated to contain 1.23 EC units per 10(9) cells. In a low-ionic-strength medium, only some 20% of the hyaluronidase was extractable, although up to 80% of the activity could be extracted as the ionic strength was increased; further addition of detergent extracted the remainder. During purification of the enzyme, it was found that inclusion of poly(vinyl alcohol) in the media stabilized the activity; detergent inclusion also improved the yield, especially during early stages. As a consequence both of reliable quantitative determination and of stabilization, a number of forms of hyaluronidase could be isolated in high yield, by using anion-exchange chromatography, cation-exchange chromatography, affinity chromatography and gel filtration. The existence of all these forms was confirmed by electrophoresis and immunoblotting with the use of a monoclonal anti-(ram hyaluronidase) antibody, and their presence in very freshly prepared sperm extracts was demonstrated. The specific activity of the isolated major hyaluronidase form was 15.0 EC units/mg; this was equivalent to 119,000 W.H.O. units/mg, higher than any other previously reported values.

Multiple forms of ram and bull sperm hyaluronidase revealed by using monoclonal antibodies

“…The patterns of reactivity of the two antibodies were identical to those shown in Fig. 1 (Harrison, 1988a, (Kessler, 1975).…”

“…Reactivity with differentforms of ram hyaluronidase Figure 1 presents Coomassie-stained blots of a purified ram hyaluronidase preparation, showing the range of hyaluronidase forms found in ram spermatozoa (Harrison, 1988a); when run unreduced, the various oligomers are resolved from each other and from the monomer, whereas when run reduced, all the oligomers are dissociated to their component monomers and the two monomer species present (Harrison, 1988b) are clearly resolved. Figure 1 also shows parallel blots probed with the 1A4 or the 1D6 antibodies.…”

“…In the case of hyaluronidase, for example, there are reports which suggest that not all the sperm-associated activity is sequestered within the acrosome and that some of it may act before the onset of the acrosome reaction (Talbot & Franklin, 1974;Lewin et ai, 1982;Zao et ai, 1985). Moreover, although sperm hyaluronidase has generally been considered a soluble enzyme, further investigation has revealed that some of the activity may be bound to structural components (Stambaugh & Buckley, 1970;Brown, 1975;Harrison, 1988a).…”

“…Unfortunately, the enzyme is difficult to purify, and totally specific antibody preparations have rarely been obtained with conventional antisera. However, monoclonal antibodies are inevi¬ tably specific for a given epitope (provided the techniques for raising them are properly followed), and they can be obtained from impure protein preparations (see review by Vora, 1985 Harrison (1988a), using anion-exchange, cation-exchange, affinity and gel chromatography; in this instance, polyvinyl alcohol was not included in the Chromatographie media. After gel chromatography, the enzyme appeared as a single component on SDS-gel electrophoresis.…”

“…The material was concentrated, dialysed against phosphate-buffered saline (PBS: 125mM-NaCl, 10-4mM-disodium hydrogen orthophosphate, 3-2mM-potassium dihydrogen orthophosphate, pH 7-3) and, after ultrafiltration through a sterile Millipore filter, was stored frozen at -20°C until use. The prep¬ aration contained 0-28 mg protein per ml with a specific activity of 12-6 IUPAC units (99800 WHO International units) per mg (see Harrison, 1988a, Raising of monoclonal colonies. Details of the methodology are published elsewhere (Gaunt, 1982).…”