Figure 10. Capacitation and acrosome reaction in Tas1r1 null sperm from the Tas1r1/mCherry mouse line.

[A] Capacitation dependent efflux of cholesterol in Tas1r1-deficient mice. To quantify capacitation dependent cholesterol release in isolated epididymal sperm of wild-type and Tas1r1 null mutant animals, equal amounts of a homogeneous sperm suspension were incubated for different time periods (0 min, 30 min, 60 min, 90 min, 120 min) in HS/BSA/NaHCO₃ as described in Materials and Methods. At the indicated time points, aliquots of the supernatant were collected and used to measure cholesterol release using a fluorometric-based quantification kit. Obtained data were calculated as cholesterol efflux per cell after subtracting basal cholesterol content at the beginning of the incubation (0 min: [+/+]: 42±3 ng cholesterol/10⁶ sperm; [−/−]: 37±2 ng cholesterol/10⁶ sperm). Time-dependent sterol release in sperm of both genotypes increased over time and showed no significant difference (p≤0.05). Data, presented as mean values ± SEM, are the average of nine independent sperm preparations of C57BL/6wild-types and Tas1r1-deficient animals from the same colony. [B] A23187 and zona pellucida induced acrosomal secretion in Tas1r1 null sperm. To assess whether Tas1r1-deficient sperm show a defect in the acrosomal exocytotic machinery or in recognizing the egg's coat, respectively, in vitro capacitated spermatozoa of wild-type and Tas1r1 null mutant littermates were either treated with 10 µM A23187 [A23187] or alternatively with solubilised zona pellucida [ZP] at 37°C for 30 min. Subsequently, aliquots of sperm were stained with Commassie blue G.250 and acrosomal status was quantified by counting at least 200 cells for each condition. Data, calculated as absolute percentages of acrosome reacted sperm represent mean values ± SEM of independent experiments with different mouse sperm preparations ([A23187], n = 15; [ZP], n = 7). Spontaneously occurring secretion rates were determined incubating sperm in corresponding buffer used to dilute the stimulating compounds [buffer with DMSO: wild-type [+/+]: 28.1±2.2%; Tas1r1 [−/−]: 35.2±2.5%; ZP buffer alone: wild-type [+/+]: 33.1±3.5%; Tas1r1 [−/−]: 37.7±3.0%). Statistical analysis was done using a Student's t-test comparing acrosome reacted sperm of both genotypes.