Assessment of the human sperm acrosome reaction using concanavalin a lectin

Abstract

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.

Lectin-Binding Sites on Human Sperm During Acrosome Reaction: Modifications Judged by Electron Microscopy/Flow Cytometry

“…In UV light microscopy the incidence of human spermatozoa with a highly fluorescent anterior region of the sperm head increases by about 3.5-fold between 1 and 5 h of capacitation [6]. Electron microscopy studies have revealed gold-conjugated Con-A bound specifically to the surface of the inner acrosomal membrane of acrosome-reacted spermatozoa after 1 h of incubation with calcium ionophore A-23 187 [9]. This is in accordance with our results, since at 30 min of incubation a weak label increased predominantly in the inner acrosome membrane after an hour of incubation with the ionophore.…”

Proteases and Proteases Inhibitors of Semen - A Review

“…This preliminary binding will boost-up the acrosome reaction which further releases acrosomal enzymes and its activation. This in association with the obtained hyper activated motility will trigger the sperm to penetrate zona pellucida (Holden et al, 1990). Premature acrosome reactions figure the sperm infertile, and therefore, assessment of acrosome integrity is very important for fertilization to occur.…”

Methods of Assessment of Cryopreserved Semen

“…For human sperm concanavalin A lectins (ConA) is used as well (Holden et al, 1990). The PNA labelling is specific for the outer acrosomal membrane and it binds to -galactose moieties.…”