Preliminary characterization of a factor in human follicular fluid that stimulates human spermatozoa motion

Follicular fluid alters the physiology and behaviour of spermatozoaby increasing acrosome reaction, accelerating capacitation,attracting the spermatozoon and enhancing vigorous motion ofthe cell. The objective of this study was to characterize thefactor(s) in human follicular fluid that causes vigorous spermatozoamotion. Follicular fluid and its fractions were tested for stimulationof spermatozoa motion using a standardized assay which employsa computerized digital imaging system. Our results show thatboth follicular fluid and its methanol extract stimulatevigorousspermatozoa motion. To determine the characteristics of theactive factor(s), the methanol extract was subjected to molecularweight fractionation, protease digestion, microcrystalline thinlayerchromatography (TLC) and C18 reverse-phase high-pressure liquidchromatography (HPLC). The spermatozoa motion stimulator inthe methanol extract was dialysable against a low molecularweight membrane (1000 Da), insensitive to boiling and low pH(3.5) and was largely inactivated by proteinase K digestion.The activity was detected near the solvent front on TLC. Usingreverse-phase HPLC monitored at 254 nm (UV), the activity elutedas a single peak of activity at low methanol concentration,indicating that the activity was relatively hydrophilic. Theactivity in the HPLC peak lost most of its motion-stimulatingability after digestion with proteinase K. The motion stimulatorcould be a peptide analogous to the egg-associated peptidescharacterized in echinoderms which stimulate spermatozoa motion,respiration and chemotaxis.