Abstract
Rat cauda epididymal spermatozoa treated with α-chlorohydrin at concentrations of 0, 0.01, 0.1, and 1.0 under conditions that support in vitro fertilization were used to evaluate the effects of acrosomal status and sperm motility in predicting their fertilizing capacity. Acrosomal status was assessed by fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) lectin assay in combination with supravital stain using calcein acetoxy methyl ester (CAM) and ethidium homodimer-1 (EthD-1) at 1, 3, and 5 h of incubation. Sperm motility was also examined with a computer assisted sperm analysis (CASA) system at the same time points as acrosomal status. The movement of spermatozoa treated with α-chlorohydrin at 0.01 mM was similar to the control spermatozoa over 5 h of incubation. The 0.1 mM treated spermatozoa showed progressive movement at 1 h of incubation, and low vigorous movement was seen after 3 h of incubation. The 1.0 mM treated spermatozoa showed low progression and low vigorous movement over 5 h of incubation. The FITC-ConA patterns of rat spermatozoa undergoing acrosome reaction were marked by the appearance of bright green fluorescence from the acrosomal region on their head, and were clearly distinguished from the degenerative acrosome loss in dead sperm. A time-related increase in the percentage of live acrosome lost sperm was observed in the control and 0.01 mM α-chlorohydrin treated groups, but a low percentage and no time-related increase in live acrosome lost sperm without degenerative acrosome loss in dead sperm were observed in the 0.1 and 1.0 mM treated groups. On the basis of these results, rat spermatozoa labeled with FITC-ConA combined with CAM and EthD-1 can have their acrosomal status clearly distinguished. The α-chlorohydrin treated spermatozoa were suppressed not only vigorous movement but also acrosome reaction, and the results suggested that the present staining procedure for acrosomal status can be a useful indicator for predicting spermatozoal fertilizing capacity.
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