INHIBITION OF THE SEA-URCHIN SPERM ACROSOME REACTION BY A LIGNIN-DERIVED MACROMOLECULE

The major organic components of effluents from commercial pulping proc esses are lignin-derived macromolecules (LDMs), which have recently be en shown to inhibit fertilization and embryonic development in a varie ty of marine organisms, as well as to exhibit immunostimulating activi ty in mammalian cells. We conducted studies on the effects of an isola ted LDM from bleached kraft mill effluent (BKME), and its sub-componen ts, at the cellular level utilizing the purple sea urchin (Strongyloce ntrotus purpuratus) sperm acrosome reaction (AR) as an experimental sy stem. The AR is an event that is induced by the egg's jelly coat and i s prerequisite for successful fertilization. Sperm were preincubated w ith increasing concentrations of isolated LDM or electrophoretically p urified LDM sub-components, followed by addition of isolated egg jelly to induce the AR in vitro. These LDM preparations significantly inhib ited the AR as assessed by fluorescence (utilizing the rhodamine-conju gated phallicidin) and transmission electron microscopy. Preincubation of sperm with LDM did not have any effect on sperm motility. The leve l of AR inhibition was comparable to that observed in experiments asse ssing successful fertilization. The ability of LDM to inhibit jelly in duced AR was overcome by the calcium ionophores A23187 and ionomycin. In addition, LDM was shown to inhibit the normal increase in intracell ular calcium (Ca++) associated with induction of the AR. When eggs wer e preincubated with LDM prior to addition of unexposed sperm, no effec t on fertilization was observed, indicating that LDM specifically affe cts sperm function during fertilization. Fine structural studies, util izing 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 c onsistent with our hypothesis that this macromolecule inhibits the AR by blocking egg jelly interaction with the sperm surface, thus inhibit ing ionic events such as increases in intracellular calcium. Our prese nt approach also demonstrates that echinoderm sperm functions can be u sed as a model system for the investigation of the mode of action of t oxicants at the sub-cellular level.