QUANTITATIVE ASSESSMENT OF HUMAN SPERM ACROSOME REACTION BY USING FLUORESCEIN ISOTHIOCYANATE CONJUGATED CONCANAVALIN-A - COMPARISON BETWEENHIGHLY PURIFIED ACROSOME-REACTED WITH NON-ACROSOME REACTED SPERM

In the present study, to achieve quantitative assessment of human sper m acrosome reaction by using fluorescein isothiocyanate conjugated Con canavalin A (FITC-Con A), the optimum conditions for microscopic obser vation were established by comparing the fluorescent images between th e purified human sperm with the intact acrosome (non-acrosome reacted sperm; non-AR sperm) and those with exposed inner acrosomal membrane ( acrosome reacted sperm; AR sperm). FITC-Con A stained not only the inn er acrosomal membrane but also the other portions. These non-specific bindings were excluded by the competitive dissociation with alpha-D-ma nnose (Man) solution, the specific labeling of the inner acrosomal mem brane with FITC-Con A was accomplished in the presence of 0.5 mg/mL Ma n. The influence of methanol fixation on the acrosome status were exam ined. The assessment of the acrosome reaction should evaluate whether the inner acrosomal membrane is exposed or masked by the plasma membra ne, and methanol fixation is often employed in the histochemical stain . By methanol fixation, the fluorescent profiles of AR sperm were not altered before and after the treatment, bright fluorescence were found in the acrosomal regions. On the other hand, non-AR sperm gave weak f luorescence on the sperm heads before the treatment, the acrosomal reg ions fluoresced brightly after the treatment similar to those of AR sp erm. Methanol fixation caused false positive results, it might denatur ate the plasma membranes and facilitate permeability of FITC-Con A. FI TC-Con A fluorescent stain and the modified triple stain (rose bengal stain) gave good positive correlation to evaluate AR sperm. This resul t concluded that the optimum conditions with FITC-Con A stain in the p resent procedure might be an useful tool for observation of human sper m acrosome reaction.