Adriamycin (ADR), an anticancer drug which induces testicular toxicity, was administrated to Slc:SD male rats at doses of 0.5, 1.0, and 2.0 mg/kg intravenously once a week for 4 weeks. The males treated with ADR were mated with untreated females, and sperm analyses (motion, count, and morphology) were performed. Sperm motion was analyzed by Hamilton-Thorne Sperm analyzer (HTM-IVOS) to investigate the useful parameters. Copulated females were necropsied at Day 13 of gestation, and reproductive status was evaluated.In ADR-treated groups, the testicular weight was dose-dependently decreased. Associated with this decrease was a depletion of the number of spermatogonia noted histopathologically at all dosage levels. Sperm morphological abnormalities, which were classified as tailless sperm and/or no-hook head sperm, were increased in both the 1.0 and 2.0 mg/kg groups. The males treated with ADR at 1.0 and 2.0 mg/kg had a decreased number of sperms per cauda epididymis. In sperm motion analysis, decreases in the percentage of motile sperm, percentage of progressive sperm, and sperm velocity (straight line velocity and curvilinear velocity) were noted at 2.0 mg/kg. Impaired fertility was noted at 2.0 mg/kg in the form of decreased numbers of implantations and live embryos, and an increased number of pre-implantation losses.In conclusion, ADR induced deterioration of sperm motion and sperm content, which were responsible for the adverse effect on male fertility. The most sensitive indicators to detect male reproductive toxicity induced by ADR were testicular weight and histopathological findings in the testis. Among the parameters generated by HTM-IVOS, the percentage of motile sperm, the percentage of progressive sperm, and sperm velocity are useful for assessing male fertility.

Dual fluorescent staining (DFS) with calcein acetoxy methyl ester (CAM), which labels the cellular esterase activity that is a major component of energy metabolism in cellular mitochondria, and with ethidium homodimer-1 (EthD-1) was used to evaluate mitochondrial function and membrane integrity in rat spermatozoa. The spermatozoa stained by DFS could be classified into three different populations microscopically when excited at 490 nm after 60 min incubation. 1) Spermatozoa, which were stained with CAM alone and had maintained either mitochondrial function or membrane integrity, were identified as live during incubation. 2) Spermatozoa, which were stained with EthD-1 alone and had lost either mitochondrial function or membrane integrity, were identified as already dead at the beginning of incubation. 3) Spermatozoa, which were stained with both CAM and EthD-1 and had maintained mitochondrial function with membrane breached, were identified as having died during incubation. Two toxicological tests, an in vitro triton X-100 experiment and an in vivo nitrobenzene experiment, were done. All spermatozoa were immobilized and lost either mitochondrial function or membrane integrity by 1.0% triton X-100 treatment. Almost no motile sperm were found at 0.1% in the triton X-100 group and in the groups treated with 60 and 40 mg/kg/day of nitrobenzene, and these spermatozoa maintained their mitochondrial function but had their membrane breached. In conclusion, the DFS procedure, which uses CAM and EthD-1, can clearly and visually identify the population of viable and dead spermatozoa simultaneously by fluorescence microscopy in rats. This is a useful technique to characterize sperm status, which is determined by the mitochondrial function assessed by CAM and membrane integrity evaluated by EthD-1.

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 FITCConA 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.