Metabolic substrates exhibit differential effects on functional parameters of mouse sperm capacitation

Abstract

Although substantial evidence exists that sperm ATP production via glycolysis is required for mammalian sperm function and male fertility, conflicting reports involving multiple species have appeared regarding the ability of individual glycolytic or mitochondrial substrates to support the physiological changes that occur during capacitation. Several mouse models with defects in the signaling pathways required for capacitation exhibit reductions in sperm ATP levels, suggesting regulatory interactions between sperm metabolism and signal transduction cascades. To better understand these interactions, we conducted quantitative studies of mouse sperm throughout a 2-h in vitro capacitation period and compared the effects of single substrates assayed under identical conditions. Multiple glycolytic and nonglycolytic substrates maintained sperm ATP levels and comparable percentages of motility, but only glucose and mannose supported hyperactivation. These monosaccharides and fructose supported the full pattern of tyrosine phosphorylation, whereas nonglycolytic substrates supported at least partial tyrosine phosphorylation. Inhibition of glycolysis impaired motility in the presence of glucose, fructose, or pyruvate but not in the presence of hydroxybutyrate. Addition of an uncoupler of oxidative phosphorylation reduced motility with pyruvate or hydroxybutyrate as substrates but unexpectedly stimulated hyperactivation with fructose. Investigating differences between glucose and fructose in more detail, we demonstrated that hyperactivation results from the active metabolism of glucose. Differences between glucose and fructose appeared to be downstream of changes in intracellular pH, which rose to comparable levels during incubation with either substrate. Sperm redox pathways were differentially affected, with higher levels of associated metabolites and reactive oxygen species generated during incubations with fructose than during incubations with glucose.

FIG. 1.

ATP and motility parameters of sperm incubated in the presence or absence of energy substrates. Sperm were incubated in HTF complete medium (A–D) or in substrate-free HTF medium (A–C and E) and analyzed at 30-min time points over a 2-h in vitro capacitation period. A) Sperm ATP levels in the presence and absence of substrates. B) Total sperm motility assessed by CASA. C) The percentage of motile sperm displaying hyperactivated motility over the time course. D) Motility profiles of sperm incubated in HTF complete media. E) Motility profiles of sperm incubated without energy substrates. Data are represented as the mean ± SEM of sperm from seven mice. Differences between conditions at corresponding time points were analyzed using two-tailed, unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIG. 2.

Total motility, hyperactivation, and motility profiles of sperm incubated with glycolysable substrates. Sperm were incubated in HTF complete medium or HTF medium with 2.78 mM glucose (blue), 5 mM mannose (pink), 5 mM fructose (green), or 5 mM sorbitol (olive) as the sole energy substrate. A and B) At the indicated time points, sperm were analyzed for total motility (A) and percentage hyperactivation (B). C) Motility profiles of sperm incubated in HTF complete medium (open bars) or HTF medium with a glycolysable substrate (colored bars) for 90 min. Data are represented as the mean ± SEM of sperm from five or more mice. Differences between conditions at corresponding time points were analyzed using two-tailed, unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

FIG. 3.

Total motility, hyperactivation, and motility profiles of sperm incubated with nonglycolysable substrates. Sperm were incubated in HTF complete medium or HTF medium with 0.33 mM pyruvate (light blue), 21.4 mM lactate (purple), 5 mM hydroxybutyrate (orange), or 10 mM citrate (gray) as the sole energy substrate. A and B) At the indicated time points, sperm were analyzed for total motility (A) and percentage hyperactivation (B). C) Motility profiles of sperm incubated in HTF complete medium (open bars) or HTF medium with a glycolysable substrate (colored bars) for 90 min. Data are represented as the mean ± SEM of sperm from eight or more mice. Differences between conditions at corresponding time points were analyzed using two-tailed, unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIG. 4.

Capacitation-associated tyrosine phosphorylation of sperm incubated in glycolysable or nonglycolysable substrates. Pooled sperm from two mice were divided and incubated for 0 or 2 h in HTF complete medium, substrate-free medium, or HTF medium with individual glycolysable substrates (A) or non-glycolysable substrates (B and C). Sperm were lysed and subjected to SDS-PAGE, followed by Western blot analysis with 4G10. Experiments were repeated a minimum of seven times for each substrate. Blots in A and B are from the same pool of sperm, except sorbitol. The blot in C is from a different pool of sperm to illustrate the observed variability of tyrosine phosphorylation in the presence of nonglycolysable substrates. Arrows denote a phosphorylated doublet with apparent molecular weights of 84 000 and 88 000.

FIG. 5.

ATP levels, percentage motility, and motility profiles incubated in the presence of an inhibitor of glycolysis. Sperm were incubated in HTF medium containing 2.78 mM glucose, 5 mM fructose, 0.33 mM pyruvate, or 5 mM hydroxybutyrate in the presence of either 10 mM ACH or the vehicle control (DMSO). After 90 min of incubation, sperm were analyzed for ATP content (A), percentage motility (B) and motility profiles (C, glucose; D, fructose; E, pyruvate; and F, hydroxybutyrate). Data are represented as the mean ± SEM of sperm from three or more mice. Differences between conditions at corresponding time points were analyzed using two-tailed, unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIG. 6.

ATP levels, percentage motility, and motility profiles incubated in the presence of an uncoupler of oxidative phosphorylation. Sperm were incubated in HTF medium containing 2.78 mM glucose, 5 mM fructose, 0.33 mM pyruvate, or 5 mM hydroxybutyrate in the presence of either 10 μM CCCP or the vehicle control (DMSO). After 90 min of incubation, sperm were analyzed for ATP content (A), percentage motility (B), and motility profiles (C, glucose; D, fructose; E, pyruvate; F, hydroxybutyrate). Data are represented as the mean ± SEM of sperm from three or more mice. Differences between conditions at corresponding time points were analyzed using two-tailed, unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIG. 7.

Intracellular pH of sperm incubated in glucose or fructose-containing HTF medium. Sperm were loaded with 3 μM BCECF-AM and incubated in medium containing HTF with 2.78 mM glucose (blue, solid lines) or 5 mM fructose (green solid lines). Sperm were also incubated with these substrates in noncapacitating media (dashed lines). The pH_i was determined from BCECF fluorescence ratios at each time point and calculated against a standard curve of known pH values. Data are represented as the mean ± SEM of sperm from five mice.

FIG. 8.

Hyperactivation after preincubation in glucose or fructose-containing HTF medium. Sperm were preincubated in medium containing HTF with 2.78 mM glucose (A) or 5 mM fructose (B) for 30 min, washed, and then resuspended in either substrate-free HTF medium, HTF medium with 2.78 mM glucose, or HTF medium with 5 mM fructose. The percentage of the motile population displaying hyperactivated motility was determined 30 min (black bars), 60 min (open bars), or 90 min (gray bars) after resuspension. Data are represented as the mean ± SEM of sperm from four mice. Differences between conditions at corresponding time points were analyzed using one-way ANOVA. *P < 0.05, ***P < 0.001.

FIG. 9.

Luminol-dependent ROS production in sperm incubated in the presence of glucose or fructose. Sperm from two mice were pooled and incubated in medium containing either 2.78 mM glucose (blue) or 5 mM fructose (green) along with 50 μM luminol and 8.8 U of HRP. ROS levels were determined by measurement of luminol signal over a 20-sec integration period. The graph is representative of six experiments. Data are represented as mean ± SEM of triplicate measurements. Differences between conditions at corresponding time points were analyzed using two-tailed unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001.