Inhibition of the sea urchin sperm acrosome reaction by a lignin-derived macromolecule

The major organic components of effluents from commercial pulping processes are lignin-derived macromolecules (LDMs), which have recently been shown to inhibit fertilization and embryonic development in a variety of marine organisms, as well as to exhibit immunostimulating activity in mammalian cells. We conducted studies on the effects of an isolated LDM from bleached kraft mill effluent (BKME), and its sub-components, at the cellular level utilizing the purple sea urchin (Strongylocentrotus purpuratus) sperm acrosome reaction (AR) as an experimental system. The AR is an event that is induced by the egg's jelly coat and is prerequisite for successful fertilization. Sperm were preincubated with increasing concentrations of isolated LDM or electrophoretically purified LDM sub-components, followed by addition of isolated egg jelly to induce the AR in vitro. These LDM preparations significantly inhibited the AR as assessed by fluorescence (utilizing the rhodamine-conjugated phallicidin) and transmission electron microscopy. Preincubation of sperm with LDM did not have any effect on sperm motility. The level of AR inhibition was comparable to that observed in experiments assessing successful fertilization. The ability of LDM to inhibit jelly induced AR was overcome by the calcium ionophores A23187 and ionomycin. In addition, LDM was shown to inhibit the normal increase in intracellular calcium (Ca⁺⁺) associated with induction of the AR. When eggs were preincubated with LDM prior to addition of unexposed sperm, no effect on fertilization was observed, indicating that LDM specifically affects sperm function during fertilization. Fine structural studies, utilizing biotinylated LDM, confirmed LDM's specificity and revealed that its binding was restricted to the plasma membrane domain of the sperm head. The present observations on the inhibition of the AR by LDM is consistent with our hypothesis that this macromolecule inhibits the AR by blocking egg jelly interaction with the sperm surface, thus inhibiting ionic events such as increases in intracellular calcium. Our present approach also demonstrates that echinoderm sperm functions can be used as a model system for the investigation of the mode of action of toxicants at the sub-cellular level.