Nature and fate of the factors released during early contact interactions between hamster sperm and egg prior to fertilization in vitro

Initial attempts have been made to characterize three factors (Hartmann and Hutchison, 1977, J. Cell Physiol., 93, 41) which are released in vitro at 2, 31, and 50 min after capacitated hamster sperm make contact with but prior to penetration of the zona pellucida. A fourth factor is known to be released at 20–25 min but in the experiments described no effort was made toward its characterization. The assay for the factors is based upon their ability to induce early binding between gametes. Because the release of each of the factors occurs at a different time they can be harvested by sampling the supernatant at the appropriate time. The short-lived activity of these factors, which normally disappears soon after their release, was stabilized by buffering the medium to pH 7.0–7.4 with Tris or TES and removing the cells. Under these conditions, the factors released at 31 and 50 min were stable when incubated at 37°C in the absence of cells for at least 60 min, but the activity of the factor released at 2 min was erratic under similar incubation conditions. The factors released at 31 and 50 min passed unimpeded through filters with molecular weight cutoffs of 2000, and both eluted off Bio-Gel P-2 columns as single peaks of activity in regions corresponding to molecular weights of at least 1800 (void volume) and approximately 1400, respectively. The 2-min factor passed unimpeded through a filter capable of excluding molecules of molecular weight larger than 5000, but 50–86% of the activity was recovered after passing through a filter with a molecular weight cutoff at 2000. The release of the 2- and 31-min factors was inhibited 48 and 74%, respectively, by macromolecular trypsin inhibitors at concentrations which also blocked penetration of the egg by the sperm; these inhibitors had little or no effect on the release of the 50-min factor. The activity of ultrafiltered and pH-stabilized 31- and 50-min factor was destroyed by the proteases subtilisin and leucine aminopeptidase but was unaffected by trypsin or glycosidases. The disappearance of the 31- and 50- min factors after their release was investigated by incubating each factor with each of the cell types present in the drop and it was found that activity only declined in the presence of the eggs. Taken together, these results provide evidence that (1) at least two populations of peptides are released in a time-dependent manner when capacitated sperm make contact with the zona pellucida and (2) these peptides