This study evaluates the use of two fluorescein-labeled (FITC) plant lectins, Pisum sativum agglutinin (PSA) and Arachis hypogaea (peanut) agglutinin (PNA), to detect the acrosome in mouse spermatozoa in order to determine the most accurate and convenient method to experimentally assess acrosome reaction.

Labeling spermatozoa with PSA-FITC after methanol (Meth) or paraformaldehyde 4% (PAF) fixation showed an intense staining of the whole spermatozoa. The flagellum, the acrosomal cap and the equatorial segment were intensely marked. By contrast, PNA-FITC labeling was restricted to the head region and to the acrosomal cap, after either Meth or PAF fixation. Smear preparations of spermatozoa revealed an irregular surface of the acrosomal cap and a "bubble" of faint fluorescent staining beside the acrosomal cap. In acrosome reaction experiments (PNA-FITC labeling, Meth fixation), the percentage of acrosome reacted sperm (ARS) in control (DMSO) experiments was 50.12  2.58 % after 30 min incubation and 45.86  4.12 % after 60 min incubation. Incubation with Ionomycin (10 mM) for 30 min resulted in 66.05  2.67 % of ARS. A 60 min incubation with ionomycin 10 mM led to 90.88  2.22 % of ARS. In conclusion, PNA-FITC labeling was more accurate in terms of acrosome detection than PSA-FITC labeling. The staining was restricted to the acrosomal cap. The fixation method had no influence on the staining pattern. Ionophore challenge confirmed the existence of a calcium-dependent acrosome reaction in mouse spermatozoa and validated the use of PNA-FITC.