Fig. 4. Final purification of the sperm chemoattractant by size-exclusion chromatography. (A) The bioactive fraction from RP-HPLC was chromatographed on a size-calibrated Bio-Sep S-2000 column, eluted at 1 ml min^-1 in water and absorbance monitored at 220 nm. Fractions corresponding to detected peaks were pooled, lyophilized, resuspended in sea water, and bioassayed with freshly spawned sperm. Numbers indicate the elution times of size standards used to calibrate the column. (B) Sperm swimming response to isolated fractions. Values are means + S.E.M. Fresh egg-conditioned sea water (ESW) and filtered sea water (FSW) were used as positive and negative controls, respectively. ^*Means significantly greater than negative controls (P<0.0001). (C) Chemotactic response of abalone sperm to isolated size fractions. Values are means + S.E.M.