The effect of ammonium sulfate on the capacity of sera of pregnant animals to induce the expression of increased rosette inhibition titres in the rosette inhibition assay, that is, to express the so-called early pregnancy factor activity, was reinvestigated. The results show that the sera of pregnant mice contain low molecular mass (less than 1 kDa) moieties active in the rosette inhibition assay. Some of these moieties could be removed from the macromolecular components of sera by dialysis; however, most, or at least the most potent, of these molecules were shown to be associated with macromolecular components of the sera and were not removed by dialysis. Treatment of sera of pregnant mice with 40% ammonium sulfate released the bound low molecular mass moieties and these moieties partitioned into the supernatant fraction, whereas the macromolecular components to which they were bound partitioned into the pellet fraction. Extensive dialysis removed the low molecular mass active moieties from the supernatant fraction. The macromolecular components remaining in the supernatant retentate fraction obtained after extensive dialysis counteracted the action of the low molecular mass moieties in a dose-dependent manner in inducing increased rosette inhibition titres. However, macromolecular components in the extensively dialysed pellet fraction associated with the low molecular mass moieties in the absence of ammonium sulfate and modified their dose–response characteristics in the biological assay. The macromolecular components in the extensively dialysed pellet and supernatant retentate fractions alone could not increase rosette inhibition titres; however, when they were recombined, components in the extensively dialysed pellet retentate fraction cooperated with components in the extensively dialysed supernatant retentate fraction to allow for the expression of activity. These results are considered in the context of a new model of the system of components present in sera of pregnant mice which allow for the expression of so-called early pregnancy factor activity.

The supernatant and pellet retentate fractions obtained from sera from pregnant mice by ammonium sulfate fractionation and extensive dialysis cannot alone induce increased rosette inhibition titres in the rosette inhibition assay. In combination, however, the mixtures can induce increased rosette inhibition titres mimicking the activity of pregnancy sera previously ascribed to the early pregnancy factor. The studies described herein demonstrated that the supernatant retentate fractions derived from sera of pregnant mice were functionally equivalent to a platelet-activating factor (PAF) or a serum stimulus because they stimulated the spleen cells used in the assay to produce active moieties and cooperated with pure thioredoxin in allowing for expression of activity. Conversely, the pellet retentate fractions obtained from sera of pregnant mice were shown to be functionally equivalent to thioredoxin in that they cooperated with a PAF stimulus to allow for the expression of increased rosette inhibition titres. The supernatant retentate fractions obtained from sera from male mice were found to be functionally equivalent to the corresponding supernatant retentate fractions obtained from sera from pregnant mice in stimulating the production of active moieties and in cooperating with thioredoxin or the pellet fractions derived from sera from pregnant mice in allowing for increased rosette inhibition titres. The pellet retentate fractions obtained from male mouse sera, however, were not functionally equivalent to either thioredoxin or the corresponding pellet retentate fractions obtained from pregnancy sera. Consideration of these data led to a basic description of the system of components in pregnancy sera which is responsible for the expression of early pregnancy factor activity.