Previous reports established that butylated hydroxy
toluene (
BHT) minimized cold-induced membrane
rupture in sperm from several species. No data regarding the specificity of its effect is available. In this study 25
BHT analogs were tested for their effect on bovine sperm membrane stability. Fourteen were membrane lytic at 25 degrees C and 6 were neither membrane lytic nor membrane stabilizing. The remaining 5 compounds, a family of 2,6-tert-butyl
phenols with substitutions at position 4 of
hydrogen, methyl (
BHT), ethyl, butyl, hexyl, or octyl, afforded effective membrane protection to cold shock. Since membrane protection is a function of both the ability of a compound to partition into the membrane and a molecule's effectiveness once there, an analysis of each analog's membrane partitioning, assessed by measuring the cellular analog/
cholesterol ratio, showed the following extents of transfer for the analogs: ethyl = butyl greater than methyl =
hydrogen greater than hexyl greater than octyl. Thus, an optimum chain length exists for partitioning from micellar donors into cells. A separate experiment established that all analogs, when incorporated in equivalent amounts, protect equally plasma and mitochondrial membranes from cold shock. No effect on acrosomal membrane stability was noted.
BHT, but not the other analogs, reduced sperm motility. Addition of egg yolk to extender containing
BHT analog protected sperm motility from cold shock but had little effect on membrane stabilization. Analysis of sperm membrane compartments revealed that little to no analog was partitioned into the outer acrosomal membrane or the plasma membrane overlying the acrosome, but rather was localized in other portions of the sperm. We conclude that (a) the effective
BHT analogs, if partitioned into the membrane, are indistinguishable with regard to their capacity to eliminate cold-induced membrane lysis; (b) membrane-linked events (e.g., motility) are uniquely disrupted by a subset of this analog family; and (c) when concentrations of egg yolk and
BHT analogs are carefully controlled, unique synergistic effects are noted.