John F. Hartmann

Modulation of fertilization by the vitellus after interaction with peptides released by hamster sperm-zona pellucida contact.

Abstract Two to three minutes after hamster sperm make contact with and adhere to the surface of homologous zonae pellucidae in vitro , the first of several sets of peptides (S1 peptides) is released into the supernatant. This release occurs whether the zonae have or have not been mechanically separated from the vitellus (cellular part of the egg). Presence of the S1 peptides is detected by means of a sperm-egg assay, the premature binding assay. This assay is based on the ability of an aliquot of the medium, in which sperm are interacting with the zona surface, to induce early binding, upon addition of the aliquot to a second drop of interacting gametes. To determine if the vitellus affected the 2-min S1 peptides the ultrafiltrates of the supernatants containing them, released through sperm-egg and sperm-zona interactions, were fractionated on Biogel P-6 and their elution profiles were compared using the premature binding assay. The sperm-egg ultrafiltrates were resolved into two main domains of activity, while those of the sperm-zona formed three. The ultrafiltrates collected 2 min after the interaction of sperm with eggs or with isolated zonae were compared for their abilities to inhibit the penetration of the zona pellucida, a previously demonstrated capacity of the 2-min sperm-egg S1 peptides. The ultrafiltrate containing the sperm-zona peptides, except at a very low level, failed to inhibit penetration significantly. However, when the sperm-zona ultrafiltrate was preincubated with eggs then the resulting supernatant inhibited penetration in a dose-related manner, and the three-domain elution profile, characteristic of the sperm-zona ultrafiltrate, was converted to the egg-like two-domain profile. Taken together these data suggest that the 2-min S1 peptides consist of several subpopulations, at least one of which interacts with the vitellus. The resulting solution then acquires the ability to inhibit penetration of the egg by the sperm in a dose-related manner. Taken together these data indicate that by interacting with at least one of the components of the 2-min peptides, the vitellus is involved in regulating sperm-zona interactions.

Pig Bronchial Mucous Membrane: A Model System for Assessing Respiratory Mucus Release in Vitro

A convenient organ culture system is described in which fragments of mucous membrane isolated from bronchi of the pig were maintained in either screw-cap tubes or multiwell tissue culture plates. The mucous membrane of the pig bronchus, like that of the human, is rich in mucus-secreting submucosal glands and can respond to cholinergic stimulation in vitro by releasing either L-[3H]fucose- or L-[3H]serine-labeled acid-precipitable macromolecules. Reproducible cholinergic-mediated release of labeled macromolecules was attained by first washing the mucous membrane fragments in serum-free modified Earles medium (Dulbeccos) for 120 min at 4 degrees C. Maximum stimulation was obtained when the incubation medium was supplemented with 0.5-2.0% horse serum. Approximately 50% of L-[3H]fucose-labeled macromolecules were eluted in the void volume from a column of Sepharose CL-6B in 6 M urea. Cochromatography of L-[3H]- and L-[14C]fucose-labeled glycoproteins released by mucous membranes of control and methacholine-treated tissue fragments failed to reveal any significant difference in any specific population of fucose-labeled glycoproteins. It is concluded that, as a whole, many different labeled molecules are released in response to cholinergic stimulation. Taken together, these results suggest that the mucous membrane of the porcine bronchus is a useful in vitro model for studying respiratory mucus secretion.

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

Abstract 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 may disappear from the supernatant through an egg-mediated mechanism.

Proteinase activities of the golden hamster eggs and cells of the cumulus oophorus.

Abstract Proteinase activities of eggs and cells of the cumulus oophorous of the golden hamster were investigated with highly sensitive fluorogenic amide substrates. Eggs contain a neutral endopeptidase which hydrolyzed Suc-Ala-Ala-Phe-7-amino-4-methylcoumarin amide between the Ala and the Phe residues. Endopeptidase action on this substrate resulted in the accumulation of Phe-7-amino-4-methylcoumarin amide which was monitored by tlc identification. Hamster eggs also contained aminopeptidase and elastase-like activities but no detectable trypsin-like activity. Aminopeptidase, endopeptidase, trypsin-like, and elastase-like activities were detected in cumulus cells.