Figure 1.
Figure 1.

Mobility of washed, reactivated sperm as a function of incubation time. Sperm from pooled ejaculates were transferred into 300 mmol/kg TES-buffered potassium chloride, pH 7.4, by centrifugation through 12% (wt/vol) Accudenz containing 5 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA; n = 5). This treatment rendered sperm immobile at 20°C. Such sperm had a concentration of 5.3 ± 0.43 × 109 sperm/mL (mean ± SD) before assay. Sperm were reactivated at 41°C in TES-buffered sodium chloride containing 2 mM Ca2+. Each open circle represents a mean ± SD. The solid line denotes the linear function y(x) = 71– 0.00009(x). The slope did not differ from zero (P > 0.8558). This experiment demonstrated that random samples could be withdrawn from a pool of immobile sperm and then uniformly reactivated independent of time.

 


Figure 2.
Figure 2.

Sperm mobility as a function of cyanide-mediated cytochrome oxidase inhibition. Before assay, sperm were immobilized by transfer from seminal plasma into 300 mmol/kg TES-buffered potassium chloride, pH 7.4, containing 5 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). This was achieved by centrifugation through 12% (wt/vol) Accudenz. Washed sperm (n = 5 replicate pools) had a concentration of 5.5 ± 0.21 × 109 sperm/mL (mean ± SD). Washed sperm were held briefly at 20°C before assay. Sperm were reactivated at 41°C in TES-buffered sodium chloride containing 2 mM Ca2+. Data within replicates were normalized against the observation made with cyanide-free buffer. Thus, standard deviations are only shown for treatments that included cyanide. The solid line denotes the logistic function p(x) = 1/[1 + e-3.464 + 0.013(x)]. This experiment demonstrated that the sperm mobility assay could be used to define a dose response.

 


Figure 3.
Figure 3.

Sperm mobility as a function of extracellular pH. Before assay, sperm were immobilized by transfer from seminal plasma into 300 mmol/kg TES-buffered potassium chloride, pH 7.4, containing 5 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). This was achieved by centrifugation through 12% (wt/vol) Accudenz. Washed sperm (n = 5 replicate pools) had a concentration of 5.5 ± 0.34 × 109 sperm/mL (mean ± SD). Washed sperm were held briefly at 20°C before assay. Sperm were reactivated at 41°C with 2 mM Ca2+ in TES-buffered sodium chloride at varying pH. Each open circle represents a mean ± SD. The solid line denotes the parabolic function y(x) = –4.99 + (1.59)(x)– (0.11)(x2). The maximal value was at pH 7.2. This experiment demonstrated that the sperm mobility assay could be used to predict an optimum condition.

 


Figure 4.
Figure 4.

Sperm mobility as a function of extracellular osmolality. Before assay, sperm were immobilized by transfer from seminal plasma into 300 mmol/kg TES-buffered potassium chloride, pH 7.4, containing 5 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). This was achieved by centrifugation through 12% (wt/vol) Accudenz. Washed sperm (n = 5 replicate pools) had a concentration of 5.5 ± 0.21 × 109 sperm/mL (mean ± SD). Washed sperm were held briefly at 20°C before assay. Sperm were reactivated at 41°C with 2 mM Ca2+ in TES-buffered sodium chloride, pH 7.4, with varying osmolality. Each open circle represents a mean ± SD. The solid line denotes the parabolic function y(x) = –8.24 + (0.06576)(x)– (0.00012)(x2). The maximal value was 274 mmol/kg. This experiment further demonstrated the ability of the sperm mobility assay to predict an optimum condition.

 


Figure 5.
Figure 5.

Sperm reactivation as a function of extracellular osmolality. Before assay, sperm were immobilized by transfer from seminal plasma into 360 mmol/kg TES-buffered potassium chloride, pH 7.4, containing 5 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). This was achieved by centrifugation through 12% (wt/vol) Accudenz. Washed sperm (n = 5 replicate pools) had a concentration of 5.5 ± 0.20 × 109 sperm/mL (mean ± SD). Washed sperm were held briefly at 20°C before assay. Sperm were reactivated at 41°C with 2 mM Ca2+ in TES-buffered sodium chloride, pH 7.4, with varying osmolality. Each open circle is a mean ± SD. The solid line denotes the logistic function y(x) = [75]/[1 + e0.098 (339– x)], where 75% recovery denotes an asymptote. This experiment demonstrated that the sperm mobility assay could be used to define how sperm recover from an imposed, non-physiological condition.

 


Figure 6.
Figure 6.

Motile concentration of sperm as a function of time. Motile concentration is a time-averaged estimate of the number of motile sperm within a volume defined by sample chamber depth and the area of the analysis field of a Hobson SpermTracker. Sperm were collected from each of 10 semen donors. Sperm were diluted to 1.2 × 106 sperm/mL in a 12 × 75 mm polypropylene tube as outlined by Froman and Feltmann (2010). Each sperm suspension contained 5 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA) in TES-buffered NaCl, pH 7.4. Each tube was incubated at 30°C within a water bath. A series of 7-μL samples was withdrawn over a 90-min interval. Each sample was transferred to a 50-µm microcell prewarmed to 30°C, the temperature at which data were collected. Each open circle is a mean ± SD. The solid line denotes the parabolic function y(x) = 0.79 + (0.009)(x)– (0.0002)(x2). As motile concentration approached zero, Ca2+ was added to the residual sperm suspension to overcome the effect of BAPTA. Immediately thereafter, a 3.5-fold isothermal increase in motile concentration was observed. This experiment demonstrated that intracellular Ca2+ enabled motility while sperm were incubated in a Ca2+ sink.

 


Figure 7.
Figure 7.

Sperm cell linear velocity as a function of time. Sperm were incubated at 30°C in TES-buffered NaCl, pH 7.4, containing 5 mM 1,2-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Linear velocity is the quotient of the straight line distance between the first and last point on a track and track time, where track denotes a computer-generated path based on a framing rate of 60 Hz. Each open circle is a mean ± SD. The solid line denotes the parabolic function y(x) = 18.3 + (0.539)(x)– (0.0055)(x2). After 90 min of incubation, Ca2+ was added to the sperm suspension to overcome the effect of BAPTA. Immediately thereafter, a 1.6-fold isothermal increase in motile concentration was observed. This data set demonstrated that mitochondrial Ca2+ cycling modulates sperm cell energy generation.