Assessment of the human sperm acrosome reaction using concanavalin A lectin.
A method for assessment of the human sperm acrosome reaction is reported using fluorescein isothiocyanate (FITC)-conjugated Concanavalin A (ConA). The technique involved labelling prefixed spermatozoa, where only those spermatozoa that showed a complete loss of the acrosome bound FITC-ConA to the acrosomal region. Competitive sugar binding studies demonstrated that binding of ConA lectin to the acrosomal area of human spermatozoa was inhibited in the presence of 0.2 M D-mannose. Staining with the supravital stain Hoechst 33258 (H258) concomitantly with FITC-ConA allowed determination of only those spermatozoa that had undergone a true and not degenerative acrosomal loss. Incubation of human spermatozoa with 0, 1, 5, and 25 microM calcium ionophore, A23187, for 60 min demonstrated that changes in acrosomal status due to the different treatment protocols may be determined by the dual-staining method. Electron microscopy studies revealed that gold-conjugated ConA bound specifically to the surface of the inner acrosomal membrane of acrosome-reacted spermatozoa. A significant correlation (r = +.97) between transmission electron microscopy (TEM) and FITC-ConA labelling methods of acrosomal status assessment was achieved. The simple ConA labelling procedure reported here therefore provides a reliable method for quantitation of the physiological acrosome reaction of a population of human spermatozoa.
Holden
C A
CA
Centre for Early Human Development, Monash Medical Centre, Clayton, Victoria, Australia.
Hyne
R V
RV
Sathananthan
A H
AH
Trounson
A O
AO
D000177:Acrosome / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D001665:Binding Sites; D000001:Calcimycin / Q000494:pharmacology; D003208:Concanavalin A / Q000031:analogs & derivatives; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005452:Fluoresceins; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D013094:Spermatozoa / Q000502:physiology*
D005452:Fluoresceins; C042784:fluorescein isothiocyanate-concanavalin A; D003208:Concanavalin A; D000001:Calcimycin; D016650:Fluorescein-5-isothiocyanate