Capacitation induces changes in metabolic pathways supporting motility of epididymal and ejaculated sperm

Abstract

Mammalian sperm require sufficient energy to support motility and capacitation for successful fertilization. Previous studies cataloging the changes to metabolism in sperm explored ejaculated human sperm or dormant mouse sperm surgically extracted from the cauda epididymis. Due to the differences in methods of collection, it remains unclear whether any observed differences between mouse and human sperm represent species differences or reflect the distinct maturation states of the sperm under study. Here we compare the metabolic changes during capacitation of epididymal versus ejaculated mouse sperm and relate these changes to ejaculated human sperm. Using extracellular flux analysis and targeted metabolic profiling, we show that capacitation-induced changes lead to increased flux through both glycolysis and oxidative phosphorylation in mouse and human sperm. Ejaculation leads to greater flexibility in the ability to use different carbon sources. While epididymal sperm are dependent upon glucose, ejaculated mouse and human sperm gain the ability to also leverage non-glycolytic energy sources such as pyruvate and citrate.

Keywords: capacitation; extracellular flux analysis; hyperactivation; metabolic profiling; sperm maturation.

FIGURE 1

In mouse and human sperm, hyperactivated motility is dependent on glycolysis and oxphos (A–C) Percentage of progressively motile (A) epididymal mouse sperm, (B) uterine mouse sperm, or (C) ejaculated human sperm incubated for 30 min (mouse) or 60 min (human) in non-capacitating (mouse sperm: TYH, human sperm: HTF) or capacitating media (mouse sperm: TYH + BSA + HCO₃ ⁻, human sperm: HTF + HSA + HCO₃ ⁻) with glucose (mouse) or glucose/pyruvate (human) in the presence or absence of 2-DG (mouse: 56 mM, human: 28 mM) and/or Rot/AntA (0.5 μM); mean +SEM (n ≥ 7). (D–F) Percentage of hyperactivated (D) epididymal mouse sperm, (E) uterine mouse sperm, or (F) ejaculated human sperm incubated for 30 min (mouse) or 60 min (human) in non-capacitating or capacitating media in the presence or absence of 2-DG or AntA/Rot; mean +SEM (n ≥ 7). Differences between conditions were analyzed using one-way ANOVA compared to the DMSO-treated non-capacitated (black asterisk) or capacitated (purple pound sign) control,*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 2

Capacitation of epididymal and ejaculated mouse and human sperm is accompanied by increased glycolysis and oxphos (A, E, I) Normalized ECAR of (A) epididymal mouse sperm, (E) uterine mouse sperm, or (I) ejaculated human sperm in non-capacitating or capacitating (db-cAMP + IBMX) media in the absence (DMSO) or presence of (left) 2-DG or (right) AntA/Rot; mean ± SEM (n ≥ 6). (B,F, J) Change in ECAR of (B) epididymal mouse sperm, (F) uterine mouse sperm, or (J) ejaculated human sperm in capacitating media relative to control in non-capacitating media in the absence or presence of 2-DG or AntA/Rot. The average of the last three data points was respectively used to calculate the fold change. (C,G, K) Normalized OCR of (C) epididymal mouse sperm, (G) uterine mouse sperm, or (K) ejaculated human sperm in non-capacitating or capacitating (db-cAMP + IBMX) media in the absence (DMSO) or presence of (left) 2-DG or (right) Rot/AntA; mean ± SEM (n ≥ 6) (D, H, L) Change in OCR of (D) epididymal mouse sperm, (H) uterine mouse sperm, or (L) ejaculated human sperm in capacitating media relative to control in non-capacitating media in the absence or presence of 2-DG or AntA/Rot; mean +SEM (n ≥ 6). The average of the last three data points was respectively used to calculate the fold change. Differences between conditions were analyzed using one-way ANOVA compared to the DMSO-treated capacitated control (B, D, F, H, J, L),*p < 0.05, **p < 0.01, ***p < 0.001. The arrow indicates addition of 5 mM db-cAMP/500 µM IBMX.

FIGURE 3

Sperm intracellular metabolite levels during capacitation are a product of production and consumption (A–C) Principal component analysis (PCA) plots of metabolites isolated from (A) epididymal mouse sperm (PC1 score p = ***, PC2 score p = n.s.), (B) uterine mouse sperm (PC1 score p = **, PC2 score p = n.s.), or (C) ejaculated human sperm (PC1 score p = **, PC2 score p = n.s.) incubated for 90 min in non-capacitating or capacitating media. To highlight the different cluster, sperm incubated in non-capacitating and capacitating conditions are surrounded by eclipses; representative plots are shown (D–F) Nucleotides detected in (D) epididymal mouse sperm, (E) uterine mouse sperm, or (F) ejaculated human sperm incubated for 90 min in capacitating conditions in glucose, fold change of integrated area under the peak (AUC) normalized to the non-capacitated control; mean +SEM (n = 3). Differences between conditions (A–C) were analyzed using two-tailed, paired t-test, **p < 0.01, ***p < 0.001, n. s = not significant.

FIGURE 4

Sperm intracellular ATP levels decrease during capacitation (A–C) Intracellular ATP levels in (A) epididymal mouse sperm, (B) uterine mouse sperm, or (C) ejaculated human sperm at different time points during incubation in non-capacitating or capacitating media in the presence or absence of 2-DG; mean ± SEM (n ≥ 5) (D–F) Intracellular ATP levels in (D) epididymal mouse sperm, (E) uterine mouse sperm, or (F) ejaculated human sperm incubated in non-capacitating or capacitating media for 90 min in the presence or absence of 2-DG; mean ± SEM (n ≥ 5). Differences between conditions (D–F) were analyzed using one-way ANOVA compared to the non-capacitated control in DMSO, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5

Metabolic profiling of mammalian sperm in non-capacitated and capacitating conditions (A,C, E) Glycolytic metabolites detected in (A) epididymal mouse sperm, (C) uterine mouse sperm, or (E) ejaculated human sperm incubated for 90 min in capacitating conditions in glucose, fold change AUC normalized to the non-capacitated control; mean +SEM (n = 3) (B, D, F) Citrate detected in (B) epididymal mouse sperm, (D) uterine mouse sperm, or (F) ejaculated human sperm incubated for 90 min in capacitating conditions, fold change AUC normalized to the non-capacitated control; mean +SEM (n = 3). G6P = glycerol-6-phosphate, F6P = fructose-6-phosphate, F-1,6-BP = fructose 1,6-bisphosphate.

FIGURE 6

Post ejaculation, non-glycolytic substrates can support the capacitation-induced changes in metabolism (A, B) Normalized OCR of epididymal mouse sperm in non-capacitating or capacitating media with (A) pyruvate and (B) citrate; mean ± SEM (n ≥ 7) (C, D) Normalized OCR of uterine mouse sperm in non-capacitating or capacitating media with (C) pyruvate and (D) citrate; mean ± SEM (n ≥ 7) (E, F) Normalized OCR of ejaculated human sperm in non-capacitating or capacitating media with (E) pyruvate and (F) citrate; mean ± SEM (n ≥ 7). Arrow indicates addition of 5 mM db-cAMP/500 µM IBMX. (G, I, K) Change in ECAR of capacitated (G) epididymal mouse sperm, (I) uterine mouse sperm, or (K) ejaculated human sperm relative to non-capacitated control in the presence of no exogenous energy source (−), glucose/pyruvate, glucose, pyruvate, or citrate; mean +SEM (n ≥ 7) (H, J, L) Change in OCR of capacitated (H) epididymal mouse sperm (J) uterine mouse sperm, or (L) ejaculated human sperm relative to non-capacitated control in the presence of no exogenous energy source (−), glucose/pyruvate, glucose, pyruvate, or citrate; mean +SEM (n ≥ 7). The average of the last three data points was respectively used to calculate the fold change. Differences between conditions were analyzed using one-way ANOVA compared to sperm incubated in glucose (G–L),*p < 0.05, **p < 0.01, ***p < 0.001. The arrow indicates addition of 5 mM db-cAMP/500 µM IBMX.

FIGURE 7

Capacitation induces an increase in oxphos independent from glycolysis (A, D) Normalized OCR of uterine mouse sperm in non-capacitating or capacitating media with pyruvate in the presence and absence of (A) 2-DG or (D) AntA/Rot; mean ± SEM (n ≥ 6). (B, E) Normalized OCR of ejaculated human sperm in non-capacitating or capacitating media with pyruvate in the presence and absence of (B) 2-DG or (E) AntA/Rot; mean ± SEM (n ≥ 6) (C, F) Normalized OCR of ejaculated human sperm in non-capacitating or capacitating media with citrate in the presence and absence of (C) 2-DG or (F) AntA/Rot; mean ± SEM (n ≥ 6). (G) Change in OCR of uterine mouse sperm in capacitating media containing pyruvate relative to control in non-capacitating media in the absence or presence of 2-DG or AntA/Rot (H, I) Change in OCR of ejaculated human sperm in capacitating media containing (H) pyruvate or (I) citrate relative to control in non-capacitating media in the absence or presence of 2-DG or AntA/Rot. The average of the last three data points was respectively used for normalization, mean +SEM (n ≥ 6). Differences between conditions were analyzed using one-way ANOVA compared to the DMSO-treated capacitated control (G–I),*p < 0.05, ***p < 0.001. The arrow indicates addition of 5 mM db-cAMP/500 µM IBMX.

FIGURE 8

Ejaculation provides higher flexibility in the energy requirements for capacitation (A–C) Intracellular cAMP levels in (A) epididymal mouse sperm, (B) uterine mouse sperm, or (C) ejaculated human sperm incubated for 10 min (epididymal mouse sperm), 15 min (uterine mouse sperm), or 30 min (human sperm) in non-capacitating or capacitating media in the presence of no energy source (−), glucose/pyruvate, glucose, pyruvate, or citrate quantified by cAMP Elisa; mean +SEM (n ≥ 8) (D–F) Percentage of progressively motile (D) epididymal mouse sperm, (E) uterine mouse sperm, or (F) ejaculated human sperm incubated for 30 min (mouse) or 60 min (human) in non-capacitating or capacitating media in the presence of no energy source (−), glucose/pyruvate, glucose, pyruvate, or citrate; mean +SEM (n ≥ 7) (G–I) Percentage of hyperactivated (G) epididymal mouse sperm, (H) uterine mouse sperm, or (I) ejaculated human sperm incubated for 30 min (mouse) or 60 min (human) in non-capacitating or capacitating media in the presence of no energy source (−), glucose/pyruvate, glucose, pyruvate, or citrate; mean +SEM (n ≥ 7). Differences between conditions were analyzed using one-way ANOVA compared to the non-capacitated control in glucose,*p < 0.05, **p < 0.01, ****p < 0.0001.