Metabolic changes in mouse sperm during capacitation†

Abstract

Mammalian sperm are stored in the epididymis in a dormant state. Upon ejaculation, they must immediately start producing sufficient energy to maintain motility and support capacitation. While this increased energy demand during capacitation is well established, it remains unclear how mouse sperm modify their metabolism to meet this need. We now show that capacitating mouse sperm enhance glucose uptake, identifying glucose uptake as a functional marker of capacitation. Using an extracellular flux analyzer, we show that glycolysis and oxidative phosphorylation increase during capacitation. Furthermore, this increase in oxidative phosphorylation is dependent on glycolysis, providing experimental evidence for a link between glycolysis and oxidative phosphorylation in mouse sperm.

Keywords: capacitation; energy production; glucose uptake; glycolysis; oxidative phosphorylation.

Figure 1

Capacitation is accompanied by an increase in glucose uptake. Fluorescence intensities of non-capacitated and capacitating CD1 and C57BL/6 sperm after incubation for 10 min in TYH with 5.6 mM 2-NBDG (green). The nucleus was co-stained with DAPI (blue), and images were taken with a confocal microscope; scale bar 30 μM.

Figure 2

Glycolysis is required for capacitation. (a) Phosphorylation of tyrosine residues of non-capacitated and capacitated CD1 (left) and C57BL/6 (right) sperm. Representative Western blot of tyrosine-phosphorylated proteins detected with an α-phosphotyrosine (pY) antibody after incubation for 90 min in TYH with glucose ±50 mM 2-deoxyglucose (2-DG) or 0.5 μM rotenone/antimycin A (Rot/AntA). (b, c) Acrosome reaction evoked by 50 isolated zonae pellucidae or 2 μM ionomycin in (b) CD1 or (c) C57BL/6 sperm incubated for 90 min in capacitating media ±50 mM 2-DG or 0.5 μM Rot/AntA; mean + SEM (n ≥ 5). (d, e) Quantitation of pY pattern from capacitated (d) CD1 or (e) C57BL/6 sperm after blocking glycolysis with 2-DG at indicated time points during capacitation, normalized to the hexokinase band and non-capacitated control; mean + SEM (n = 11). Differences between conditions were analyzed using two-tailed, unpaired t-test compared to capacitated control, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Figure 3

db-cAMP/IBMX in Seahorse TYH capacitate mouse sperm equally well as HCO₃⁻ in TYH. (a, b) pY of mouse sperm detected at different times during capacitation (0–90 min), normalized to phosphorylated hexokinase; mean + SEM (n ≥ 5). (a) CD1 and (b) C57BL/6 sperm were incubated in TYH supplemented with 25 mM HCO₃⁻, 3 mg/ml BSA and 10 mM HEPES or Seahorse TYH supplemented with 5 mM db-cAMP, 500 μM IBMX, 3 mg/ml BSA, and 1 mM HEPES. (c, d) Acrosome reaction evoked by 50 isolated zonae pellucidae or 2 μM ionomycin in (c) CD1 or (d) C57BL/6 sperm capacitated for 90 min in TYH supplemented with 25 mM HCO₃⁻, 3 mg/ml BSA, and 10 mM HEPES or Seahorse TYH supplemented with 5 mM db-cAMP, 500 μM IBMX, 3 mg/ml BSA, and 1 mM HEPES; mean + SEM (n = 4). Differences between conditions were analyzed using one-way ANOVA compared to non-capacitated (a, b) or untreated, capacitated (c, d) control, ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Figure 4

Capacitation is accompanied by increased glycolysis and oxphos. (a) Normalized ECAR (left) and OCR (right) of non-capacitated and capacitating CD1 sperm ±2-DG (50 mM); mean ± SEM (n ≥ 7). (b) Normalized ECAR (left) and OCR (right) of non-capacitated and capacitating CD1 sperm ± Rot/AntA (0.5 μM); mean ± SEM (n ≥ 7). (c) Normalized ECAR (left) and OCR (right) of non-capacitated and capacitating C57BL/6 sperm ±2-DG (50 mM); mean ± SEM (n ≥ 7). (d) Normalized ECAR (left) and OCR (right) of non-capacitated and capacitating C57BL/6 sperm ± Rot/AntA (0.5 μM); mean ± SEM (n ≥ 7). The arrow indicates addition of 5 mM db-cAMP/500 μM IBMX. (e) Change in ECAR (left) or OCR (right) of capacitated CD1 sperm normalized to non-capacitated control ±50 mM 2-DG or 0.5 μM Rot/AntA; mean + SEM (n ≥ 7). (f) Change in ECAR (left) or OCR (right) of capacitated C57BL/6 sperm normalized to non-capacitated control ±50 mM 2-DG or 0.5 μM Rot/AntA; mean + SEM (n ≥ 7). The average of the last three data points from each time course was used for normalization. Differences between conditions were analyzed using two-tailed, unpaired t-test compared to non-capacitated sperm at t = 34 min, 61 min, 99 min (a–d) or using one-way ANOVA compared to untreated, capacitated control (e, f), ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Figure 5

Capacitation of mouse sperm is supported by glycolytic substrates. (a, b) pY of non-capacitated and capacitated (a) CD1 or (b) C57BL/6 sperm after incubation for 90 min in TYH or TYH supplemented with 25 mM HCO₃⁻, 3 mg/ml BSA, and 10 mM HEPES in the presence of no exogenous energy source (−), glucose (5.6 mM), glucose (5.6 mM)/pyruvate (0.56 mM), fructose (5.6 mM), fructose (5.6 mM)/pyruvate (0.56 mM), pyruvate (0.56 mM), lactate (0.56 mM), or citrate (0.56 mM), normalized to phosphorylated hexokinase; mean + SEM (n = 10). Swim-out and additional washes were performed in the respective buffer. Differences between conditions were analyzed using two-tailed, unpaired t-test compared to non-capacitated control, ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001.

Figure 6

The capacitation-induced increase in glycolysis and oxphos is dependent on glycolytic substrates. (a–d) Normalized ECAR (left) and OCR (right) of non-capacitated and capacitating CD1 and C57BL/6 sperm in the presence of (a) glucose (5.6 mM), (b) fructose (5.6 mM), (c) pyruvate (0.56 mM), and (d) glucose (5.6 mM)/pyruvate (0.56 mM); mean ± SEM (n ≥ 7). Arrow indicates addition of 5 mM db-cAMP/500 μM IBMX. (e) Change in ECAR (left) or OCR (right) of capacitated CD1 sperm relative to non-capacitated control in the presence of no exogenous energy source (−), glucose (5.6 mM), glucose (5.6 mM)/pyruvate (0.6 mM), fructose (5.6 mM), fructose (5.6 mM)/pyruvate (0.56 mM), pyruvate (0.6 mM), lactate (0.56 mM), or citrate (0.56 mM); mean ± SEM (n ≥ 7). (f) Change in ECAR (left) or OCR (right) of capacitated C57BL/6 sperm normalized to non-capacitated control in the presence of no exogenous energy source (−), glucose (5.6 mM), glucose (5.6 mM)/pyruvate (0.56 mM), fructose (5.6 mM), fructose (5.6 mM)/pyruvate (0.56 mM), pyruvate (0.56 mM), lactate (0.56 mM), or citrate (0.56 mM); mean + SEM (n ≥ 7). The average of the last three data points from each time course was used for normalization. Differences between conditions were analyzed using two-tailed, unpaired t-test compared to non-capacitated control at t = 34 min, 61 min, 99 min (a–d) or using one-way ANOVA compared to sperm capacitated without energy source (−) (e, f), ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001.

Figure 7

Sperm capacitation and in vitro fertilization is independent from capacitation-induced increase in glycolysis. (a, b) Acrosome reaction evoked by 50 isolated zonae pellucidae or 2 μM ionomycin in (a) CD1 or (b) C57BL/6 sperm incubated for 90 min in capacitating media in the presence of glucose (5.6 mM), glucose (5.6 mM)/pyruvate (0.56 mM), or pyruvate (0.56 mM); mean + SEM (n = 5). (c, d) Rate of two-cell stage oocytes after incubation of (c) CD1 and (b) C57BL/6 oocytes with capacitated CD1 or C57BL/6 sperm in the presence of glucose (5.6 mM), glucose (5.6 mM)/pyruvate (0.56 mM), or pyruvate (0.56 mM); mean + SEM (n−3); numbers indicate total number of oocytes from three independent experiments. Differences between conditions were analyzed using one-way ANOVA compared to untreated, capacitated control (a, b) or sperm capacitated in glucose (c, d), ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001.