Action of metallic ions on the precocious development by rabbit sperm of motion patterns that are characteristic of hyperactivated motility.

An earlier study demonstrated that rabbit sperm incubated for 16 hr under capacitation conditions acquire motility patterns identical to those seen in rabbit sperm capacitated in vivo. We now show that similar motion patterns develop after 0.5 hr incubation in a Tris-buffered medium, medium M. Development and decline of the motion patterns occurred in three phases each recognized by the character of the biphasic motion patterns. Hyperactivated sperm were objectively identified and quantified by a previously developed computer-directed model. The percentage of motile sperm that acquired hyperactivated motility and the period they remained in this state varied among sperm from different rabbits. The decline in hyperactivated motility was paralleled by a decrease in the average sperm curvilinear velocity (VCL) and average amplitude of lateral head displacement (AALH), but was not accompanied by a concomitant decrease in percentage of motile sperm. Pb2+ and Cd2+, at concentrations that did not inhibit motility, prevented development of hyperactivated motility. Inhibition of hyperactivated motility by Pb2+ was time- and concentration-dependent; the average percentage of hyperactivated sperm decreased from approximately 30% to < 5% (n = 5) in 1 hr at a Pb2+ concentration of 25 microM. Cd2+ inhibition of hyperactivation was dependent only on concentration of the cation. At a concentration of 100 microM, the decrease in the percent of hyperactivated sperm was approximately 50% (n = 3). Hg2+, Zn2+, and Cr6+ at sublethal concentrations had no effect on hyperactivated motility development. These results suggest that Pb2+ and Cd2+, by virtue of their ability to prevent the wide curvature flagella beating that is characteristic of hyperactivation, can compromise fertilization at concentrations that do not inhibit sperm motility and act as a reproductive toxicant at a level other than spermatogenesis.

Development of computer-directed methods for the identification of hyperactivated motion using motion patterns developed by rabbit sperm during incubation under capacitation conditions.

Rabbit spermatozoa developed motions that mimicked hyperactivated motility during incubation for 16-20 hours under capacitation conditions and in several other commonly used media. Sperm from some rabbits failed to acquire this type of motility, and sperm from others failed to survive the long incubation time. Four motility patterns developed during incubation for 16-20 hours. Motility parameters measured by the CellSoft and CellTrak motion analysis systems were similar except for the average amplitude of lateral head displacement. Multivariate discriminant analysis with complementary regression analysis, and an unrelated tree structured classification method (CART), were used to derive rules, based on motility parameters, for the objective classification of sperm into the two motility classes: 1) nonhyperactivated motility and 2) hyperactivated motility or motility that mimicked hyperactivated motility. The motility parameter wobble (WOB) as superior to the commonly used parameter, linearity, as a classifier of motility types. It classified sperm into the two motility groups with 96.6% efficiency and, together with curvilinear velocity (VCL), attained classification efficiencies of 98%. The classification model produced by CART was preferred over the one obtained by discriminant analysis. The rule for motility classification was dependent on the motion analysis system used to measure the motion parameters. The rule for the CellSoft system, WOB < or = 0.78 and VCL > or = 51 microns/second, classified sperm with an efficiency of 98%, whereas the rule for the CellTrak system, WOB < or = 0.6 and VCL > or = 55 microns/second, achieved a classification efficiency of 97%. These rules should facilitate the study of sperm hyperactivation and its role in sperm function.

Automated analysis of rabbit sperm motility and the effect of chemicals on sperm motion parameters.

Appropriate software settings and optimum procedures were determined for the measurement of the motion parameters of rabbit spermatozoa by the CellSoft (Cryo Resources Ltd., Montgomery, NY) computer-assisted digital image analysis system. The system was used to follow motion parameter changes occurring in spermatozoa incubated for 6 hr with or without exposure to chemicals. Mean amplitude of lateral head displacement (AALH) increased over the 6 hr period, while curvilinear velocity (Vc) first increased and then decreased. Values for linearity (Lin), or beat cross frequency (BCF), were unchanged. The majority of spermatozoa progressed linearly, with rapid rotation of the sperm head, but subpopulations of spermatozoa with different swimming patterns appeared after 1-3 hr of incubation. Percentage motile sperm and Vc were most sensitive to the action of the compounds (pyrogallol, hydroquinone, ammonium oxalate, triethyl phosphite, and pinocolyl alcohol), while BCF was least affected. The decline in percentage of motile sperm was dependent on duration of exposure and chemical concentration. Mean Vc of the sperm population decreased rapidly upon chemical exposure and remained at a low value until motility ceased. The initial decrease in Vc was dependent on the concentration of the added compound. Motion-based indices--motility concentration (MCI50), motility time (MTI50), and velocity (VI)--were defined and used as toxicological endpoints. The rank order of these indices, the end point of the neutral red in vitro assay for cytotoxicity, and LD50 values for the five compounds were the same, suggesting that chemical inhibition of sperm motility may be useful as a method for the in vitro assessment of chemical cytotoxicity.