Human Sperm Remain Motile After a Temporary Energy Restriction but do Not Undergo Capacitation-Related Events

Abstract

To acquire fertilization competence, mammalian sperm must undergo several biochemical and physiological modifications known as capacitation. Despite its relevance, the metabolic pathways that regulate the capacitation-related events, including the development of hyperactivated motility, are still poorly described. Previous studies from our group have shown that temporary energy restriction in mouse sperm enhanced hyperactivation, in vitro fertilization, early embryo development and pregnancy rates after embryo transfer, and it improved intracytoplasmic sperm injection results in the bovine model. However, the effects of starvation and energy recovery protocols on human sperm function have not yet been established. In the present work, human sperm were incubated for different periods of time in medium containing glucose, pyruvate and lactate (NUTR) or devoid of nutrients for the starving condition (STRV). Sperm maintained in STRV displayed reduced percentages of motility and kinematic parameters compared to cells incubated in NUTR medium. Moreover, they did not undergo hyperactivation and showed reduced levels of ATP, cAMP and protein tyrosine phosphorylation. Similar to our results with mouse sperm, starvation induced increased intracellular Ca²⁺ concentrations. Starved human sperm were capable to continue moving for more than 27 h, but the incubation with a mitochondrial uncoupler or inhibitors of oxidative phosphorylation led to a complete motility loss. When exogenous nutrients were added back (sperm energy recovery (SER) treatment), hyperactivated motility was rescued and there was a rise in sperm ATP and cAMP levels in 1 min, with a decrease in intracellular Ca²⁺ concentration and no changes in sperm protein tyrosine phosphorylation. The finding that human sperm can remain motile for several hours under starvation due to mitochondrial use of endogenous metabolites implies that other metabolic pathways may play a role in sperm energy production. In addition, full recovery of motility and other capacitation parameters of human sperm after SER suggests that this treatment might be used to modulate human sperm fertilizing ability in vitro.

Keywords: ATP; capacitation; glucose; glycolysis; metabolism; oxidative phosphorylation; pyruvate/lactate; sperm motility.

FIGURE 1

Sperm motility in the presence or absence of exogenous nutrients. (A) Experimental design. Motile human sperm were selected by swim-up in BWW containing glucose, lactate and pyruvate (NUTR) or in medium devoid of glucose, pyruvate and lactate (STRV). Motility parameters were recorded by CASA immediately after swim-up (0 h) and after 3-h incubation in the same medium in capacitating conditions. (B) Total motility. (C) Progressive motility. (D) Curvilinear velocity (VCL). (E) Hyperactivated sperm. Results are expressed as mean ± SEM, n = 18 experiments, 8 donors (≤3 samples per donor). ^b p < 0.01, ^c p < 0.001, ^d p < 0.0001 vs. NUTR. Two-way ANOVA, and Bonferroni’s multiple comparison test.

FIGURE 2

Sperm motility after incubation for long periods of time in the presence or absence of exogenous nutrients. (A) Experimental design. Motile human sperm were selected and incubated in capacitating conditions in BWW containing glucose, lactate and pyruvate (NUTR) or in medium devoid of glucose, pyruvate and lactate (STRV). Motility parameters were recorded by CASA at 0, 1.5, 3, 6, 20, 27 and 48 h. (B) Total motility. (C) Progressive motility. (D) Curvilinear velocity (VCL). (E) Hyperactivated sperm. Results are expressed as mean ± SEM, n = 5 experiments. ^b p < 0.01, ^c p < 0.001, ^d p < 0.0001 for STRV vs. NUTR at each corresponding time. Two-way ANOVA, and Bonferroni’s multiple comparison test.

FIGURE 3

Effect of incubation with OXPHOS inhibitors on human sperm motility. (A) Experimental design. Motile human sperm were selected and incubated for 3 h in NUTR or STRV medium. The OXPHOS inhibitors (10 µM CCCP, 1 µM ROT or 0.3 µM AA) were added (indicated with arrows) and after 15 min, motility was recorded by CASA. (B) Motility parameters in sperm exposed to CCCP. (C) Motility parameters in sperm exposed to ROT. (D) Motility parameters in sperm exposed to AA. Results are expressed as mean ± SEM, n ≥ 4 experiments. ^a p < 0.05, ^b p < 0.01, ^c p < 0.001, ^d p < 0.0001. One-way ANOVA, and Tukey’s multiple comparison test.

FIGURE 4

Sperm energy restriction and recovery (SER) treatment after incubation for different periods of time. (A) Experimental design. Motile human sperm were selected and incubated in capacitating conditions in STRV medium for 1.5, 3 or 20 h. The energy recovery was done by adding the same volume of 2x NUTR. As a control, sperm were incubated for 1.5, 3 or 20 h in NUTR medium, and the same volume of NUTR medium was added at the time of recovery. Motility parameters were recorded by CASA before (time 0 min) and after 15, 30, 60 and 180 min of energy recovery. (B) Parameters for SER treatment after 1.5-h incubation in NUTR (full line) and STRV (dotted line). (C) Parameters for SER treatment after 3-h incubation in NUTR (full line) and STRV (dotted line). (D) Parameters for SER treatment after 20-h incubation in NUTR (full line) and STRV (dotted line). Results are expressed as mean ± SEM, n = 4 experiments. ^a p < 0.05, ^b p < 0.01, ^c p < 0.001, ^d p < 0.001 for STRV vs. NUTR at each corresponding time. Two-way ANOVA, and Bonferroni’s multiple comparison test.

FIGURE 5

Capacitation-related events in sperm incubated in starving condition. (A) Experimental design. Motile human sperm were selected by swim-up and incubated for 4 h in capacitating conditions in NUTR or STRV media. (B) Hyperactivated sperm. (C) ATP levels normalized against NUTR condition. (D) cAMP levels normalized against NUTR condition. (E) [Ca²⁺]_i levels. Top: Representative histograms of normalized frequency vs. Fluo-4 AM fluorescence of non-PI stained sperm (live) are shown. Bottom: Normalized Fluo-4 AM fluorescence compared to NUTR condition. (F) pPKAs and pY levels. Left: Representative Western immunoblotting results are shown. Right: Protein signals were quantified and expressed as relative to β-tubulin (Tub). Results are expressed as mean ± SEM, n ≥ 4 experiments. ^a p < 0.05, ^b p < 0.01, ^c p < 0.001 vs. NUTR. Student’s t test.

FIGURE 6

Capacitation-related events in sperm incubated in starving condition and subjected to recovery in NUTR medium for 1 min. (A) Experimental design. Motile human sperm were selected by swim-up and incubated for 4 h in capacitating conditions in NUTR or STRV, and then exposed to NUTR medium for 1 min. (B) Hyperactivated sperm. (C) ATP levels normalized against 4 h NUTR 1 min NUTR condition. (D) cAMP levels normalized against 4 h NUTR 1 min NUTR condition. (E) [Ca²⁺]_i levels. Top: Representative histograms of normalized frequency vs. Fluo-4 AM fluorescence of non-PI stained sperm (live) are shown. Bottom: Normalized Fluo-4 AM fluorescence compared to 4 h NUTR 1 min NUTR condition. (F) pPKAs and pY levels. Left: Representative Western immunoblotting results are shown. Right: Protein signals were quantified and expressed as relative to β-tubulin (Tub). Results are expressed as mean ± SEM, n ≥ 3 experiments. ^a p < 0.05 vs. 4 h NUTR 1 min NUTR. Student’s t test.

FIGURE 7

Simplified model of the molecular mechanisms underlying sperm incubation in the presence and absence of exogenous nutrients. 1) In NUTR condition, glucose is transported into sperm through GLUT and other active transporters, and enters the glycolytic pathway, producing ATP. The final product of glycolysis is pyruvate, which can be converted to lactate by LDH. 2) Exogenous pyruvate and lactate can also enter sperm plasma membrane by MCT and be transported to the mitochondria for further metabolization by the Krebs cycle and OXPHOS, which also generate ATP. 3) Influx of HCO₃ ⁻ stimulates Adcy10 with the production of cAMP and activation of PKA, which in turn, provokes the phosphorylation of substrates. 4) Under this condition, Ca²⁺ enters sperm mainly through CatSper channels and [Ca²⁺]_i levels are regulated by ATP-driven pumps (PMCA4 and SERCA). As a result, sperm can maintain motility and develop hyperactivation. 5) In the absence of exogenous substrates, ATP would be produced by fatty acid oxidation and/or by amino acid metabolism. Such ATP allows the production of sufficient levels of cAMP to stimulate pPKAs. 6) Decreased ATP concentrations inhibit the normal operation of Ca²⁺ pumps and transporters, increasing the [Ca²⁺]_i. All these events allow the development of motility but not hyperactivation. 7) In STRV, the addition of CCCP, ROT and AA blocks OXPHOS, resulting in motility loss. To simplify this model, transporters and some enzymes are named by generic terms. Filled arrows indicate activation; dotted arrows show hypothetic pathways. NUTR, medium supplemented with glucose, pyruvate and lactate; GLUT, glucose transporter; LDH, lactate dehydrogenase; MCT, monocarboxylate transporter; PMCA4, plasma membrane Calcium ATPase 4; SERCA, sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases; Acetyl-CoA, acetyl coenzyme A; OXPHOS, oxidative phosphorylation; ETC, mitochondrial electron transport chain; Adcy10, atypical soluble adenylyl cyclase; PKA, protein kinase A; pPKAs, phosphorylation in PKA substrates; pY, phosphorylation on tyrosine residues; STRV, starving conditions; CTP1, carnitine palmitoyl transferase I; CCCP, carbonyl cyanide p-chlorophenylhydrazone (a mitochondrial uncoupler); ROT, rotenone (inhibitor of ETC complex I); AA, antimycin A (inhibitor of ETC complex III).