Lactic dehydrogenase (LDH), the enzyme which catalyses the final step in anaerobic glycolysis, exists in numerous molecular forms whose molecular nature and physiological significance are still the subject of controversy. Using paper electrophoresis or other fractionation methods with a limited capacity for resolving proteins, a maximum of five isozymes of LDH are found in most tissues (Appella & Markert, 1961; Cahn, Kaplan, Levine & Zwilling, 1962; Fritz & Jacobson, 1965; Vesell, 1961). These isozymes have been considered to be tetramers formed from two kinds of subunits; one called the H subunit, since this type predominates in heart tissue, and the other called the M subunit, since this type predominates in striated muscle (Cahn et al., 1962). Sorting these two subunits into all possible groups of four yields
R. STAMBAUGH and J. BUCKLEY
R. STAMBAUGH and M. SMITH
A variety of enzymes, lipids and polysaccharides have been described as `acrosomal' and implicated in the fertilization process (Hathaway & Hartree, 1963; Srivastava, Adams & Hartree, 1965; Hartree & Srivastava, 1965; Stambaugh & Buckley, 1968, 1969; Allison & Hartree, 1970; Zaneveld, Srivastava & Williams, 1969; Zaneveld, Polakoski & Williams, 1972). Several extraction procedures have been used specifically for removing the acrosomal contents, and it seemed appropriate to compare and reinvestigate their specificity. It was hoped that this comparison might provide some insight into the specificity of each procedure, and the need for additional criteria to label an extract or enzyme as `acrosomal'.
The first method examined involved a direct extraction of washed spermatozoa with detergents. By this procedure, epididymal spermatozoa were washed three times with isotonic saline,
R. Stambaugh and L. Mastroianni Jr
Summary. Pronase-resistant low molecular weight stimulators for the activation of proacrosin to acrosin were found in rhesus monkey oviduct fluid collected before, during and after ovulation, but the presence of high concentrations of acrosin inhibitors before and after ovulation partly masked the stimulation in unfractionated fluid. This low molecular weight fraction of oviduct fluid had no detectable esterase or amidase activity by itself, and the stimulating factors were sensitive to digestion by hyaluronidase and chondroitin ABC lyase and were presumed to be glycosaminoglycans. Heparin and hyaluronic acid had similar effects. The presence of soluble glycosaminoglycans at the site of fertilization suggests that they may have a role in capacitation and fertilization.
K. CONRAD, J. BUCKLEY and R. STAMBAUGH
Previously, we reported the isolation and identification of a trypsin-like enzyme from rabbit sperm acrosomes, which effects penetration of the zona pellucida by spermatozoa (Stambaugh & Buckley, 1968, 1969; Stambaugh, Brackett & Mastroianni, 1969; Stambaugh, 1971), and we now know that a similar enzyme exists in rhesus monkey, human and sea-urchin spermatozoa (Stambaugh & Buckley, 1970; Stambaugh, 1971). Since this enzyme has proved to be similar but not identical to pancreatic trypsin, we have given this enzyme the name `acrozonase' (Stambaugh, 1971).
Earlier observations, indicating that the zona pellucida of the unfertilized ovum is more readily dissolved by proteolytic enzymes than that of the fertilized ovum (Smithberg, 1953; Chang & Hunt, 1956; Austin, 1961; Gwatkin, 1964), suggested to us that the block to polyspermy may involve the
R. STAMBAUGH, C. NORIEGA and L. MASTROIANNI Jr
Experiments are described which identify the dialysable corona cell dispersing factor of rabbit oviduct fluid as the bicarbonate ion. In vitro dispersion of the corona cells with bicarbonate ion in media devoid of oviduct fluid or oviduct fluid extracts was also demonstrated. The dispersion process is initiated at bicarbonate ion concentrations of approximately 46 m-equiv/1, while concentrations of 66 m-equiv/1 effect complete corona cell dispersion within a 2-hr incubation period at 37·5° C with no mechanical agitation. Additional evidence that bicarbonate ion is indeed the in vivo corona cell dispersing factor of oviduct fluid was provided by in vivo inhibition of corona cell dispersion by acetazolamide, a carbonic anhydrase inhibitor. It was also observed that follicular ova, in contrast to mature tubal ova, are not invariably denuded with this sequential hyaluronidase and bicarbonate ion treatment.