Changes in intracellular Ca~(2+)of sperm fore-and-aft acrosome reaction in the swimming crab Portunus trituberculatus

The acrosome reaction process of sperm in the swimming crab Portunus trituberculatus(Miers)consists of four consecutive stages:(1)swelling of the apical cap;(2)eversion of the acrosomal vesicle;(3)forward elongation of the perforatorium and reversal of the acrosomal vesicle membrane;and(4)shedding of the acrosomal vesicle membrane and formation of the acrosomal filament.In the present study,Laser Scanning Confocal Microscope(LSCM)was used to image spermatozoa stained with Fluo-3/AM at different stages of the fore-and-aft acrosome reaction.Fluo-3/AM was used as the Ca~(2+)fluorescence indicator.The 488 nm argon laser was used for excitation and the emitted fluorescence was 526 nm.On account of the stability of sperm during the acrosome reaction,we selected sperm at three stages(pre-acrosome reaction,the 3rd stage and the 4th stage of the acrosome reaction)to analyze intracellular Ca~(2+)changes.The average fluorescence intensity of sperms in the above-chosen stages were 35.95±5.71,66.80±7.35 and 3.87±2.82 respectively.Average fluorescence intensities among these stages exhibited significant differences(P0.01).The intracellular calcium concentration([Ca~(2+)]_i)of sperm shows a tendency to rise first and then decrease during the acrosome reaction.The[Ca~(2+)]_i of the sperm in the 3rd stage of the acrosome reaction increased significantly compared with that of the sperm before acrosome reaction.In the 4th stage of the acrosome reaction,with the acrosomal vesicle membrane shed and the acrosomal filament formed,the[Ca~(2+)]_i of the sperm decreased sharply.The primary distribution sites of the intracellular Ca~(2+)are the nucleus,perforatorium and cytoplasmic membrane remnants.The physiological actions of the intracellular Ca~(2+)changes of sperm during the fore-and-aft acrosome reaction in P.trituberculatus are also discussed.