Effect of High Viscosity on Energy Metabolism and Kinematics of Spermatozoa from Three Mouse Species Incubated under Capacitating Conditions

Abstract

In order to sustain motility and prepare for fertilization, sperm require energy. The characterization of sperm ATP production and usage in mouse species revealed substantial differences in metabolic pathways that can be differentially affected by capacitation. Moreover, spermatozoa encounter different environments with varying viscoelastic properties in the female reproductive tract. Here, we examine whether viscosity affects sperm ATP levels and kinematics during capacitation in vitro. Sperm from three mouse species (Mus musculus, M. spretus, M. spicilegus) were incubated under capacitating conditions in a modified Tyrode's medium containing bicarbonate, glucose, pyruvate, lactate, and bovine serum albumin (mT-BH) or in a bicarbonate-free medium as a non-capacitating control. Viscosity was increased with the inclusion of polyvinylpyrrolidone. ATP was measured with a bioluminescence kit, and kinematics were examined with a computer-aided sperm analysis system. In M. musculus sperm, ATP declined during capacitation, but no differences were found between non-capacitating and capacitating sperm. In contrast, in M. spretus and M. spicilegus, ATP levels decreased in capacitating sperm. Increasing viscosity in the medium did not modify the timing or proportion of cells undergoing capacitation but did result in additional time- and concentration-dependent decreases in ATP in M. spretus and M. spicilegus under capacitating conditions. Additionally, increased viscosity altered both velocity and trajectory descriptors. The limited impact of capacitation and higher viscosity on M. musculus sperm ATP and kinematics could be related to the low intensity of postcopulatory sexual selection in this species. Responses seen in the other two species could be linked to the ability of their sperm to perform better under enhanced selective pressures.

Keywords: ATP; bioenergetics; capacitation; hyperactivation; sperm motility.

Figure 1

Percentage of spermatozoa exhibiting pattern B after CTC staining in the spermatozoa of three mouse species (M. musculus, M. spretus, and M. spicilegus) over time when incubated in mT-H, mT-H + PVP, mT-BH, and mT-BH + PVP. Data are means ± SE (M. musculus: N = 4; M. spretus: N = 5; M. spicilegus: N = 4). (A), M. musculus. (B), M. spretus. (C), M. spicilegus. Red squares: non-capacitating conditions with low viscosity, mT−H medium under air. Pink squares and letters: capacitating conditions with high viscosity, mT−BH + PVP medium under air. Dark blue squares: non-capacitating conditions with low viscosity, mT−BH medium under 5% CO₂/air. Light blue squares and letters: capacitating conditions with high viscosity, mT−BH + PVP under 5% CO₂/air. Different letters indicate significant differences between times for the same treatment in a Fisher post-hoc test (p < 0.05). Asterisks indicate significant differences between non-capacitating and capacitating conditions (p < 0.0001). There were no differences associated with the viscosity of the medium in non-capacitating or capacitating conditions.

Figure 2

Variation in ATP levels and kinematics in spermatozoa from three mouse species incubated under capacitating conditions with increasing concentrations of PVP. Sperm were incubated in mT-BH medium in 5% CO₂/air with 0, 1, 2, or 4% PVP for 30 min. Data are means ± SE (M. musculus: N = 4; M. spretus: N = 7; M. spicilegus: N = 7). (A–C): Amount of ATP per sperm (amol spz⁻¹). (D–F): curvilinear velocity (VCL). (G–I): straight-line velocity (VSL). (J–L): amplitude of lateral head displacement (ALH). (A,D,G,J): Mus musculus. (B,E,H,K): Mus spretus. (C,F,I,L): Mus spicilegus. Letters indicate statistically significant differences (a: p < 0.05; b: p < 0.01; c: p < 0.001; d: p < 0.0001) between PVP concentrations and control (0% PVP) in Tukey’s post-hoc test.

Figure 3

Variation in the principal components of swimming: OSV, OTS 1, and OTS 2 over time in sperm incubated in non-capacitating conditions in low viscosity (mT−H medium under air) and high viscosity (mT−H + PVP medium under air). Data are means ± SE (M. musculus: N = 4; M. spretus: N = 5; M. spicilegus: N = 4). (A–C): OSV. (D–F): OTS 1. (G–I): OTS 2. (A,D,G): M. musculus. (B,E,H): M. spretus. (C,F,I): M. spicilegus. Red squares and letters: non-capacitating conditions in low viscosity (mT−H). Pink triangles and letters: non-capacitating conditions in high viscosity (mT−H + PVP). Asterisks indicate significant differences between viscosity treatments for the same time point in a Fisher post-hoc test (p < 0.05). Different letters indicate significant differences between time points for the same viscosity treatment in a Fisher post-hoc test (p < 0.05).

Figure 4

Variation in the principal components of swimming: OSV, OTS 1, and OTS 2 over time in sperm incubated in capacitating conditions in low viscosity (mT−BH medium under 5% CO₂/air) and high viscosity (mT−BH + PVP medium under 5% CO₂/air). Data are means ± SE (M. musculus: N = 4; M. spretus: N = 5; M. spicilegus: N = 4). (A–C): OSV1. (D–F): OTS 1. (G–I): OTS 2. (A,D,G): M. musculus. (B,E,H): M. spretus. (C,F,I): M. spicilegus. Dark blue squares and letters: capacitating conditions in low viscosity (mT−BH). Light blue triangles and letters: capacitating conditions in high viscosity (mT−BH + PVP). Asterisks indicate significant differences between viscosity treatments for the same time point in a Fisher post-hoc test (p < 0.05). Different letters indicate significant differences between time points for the same viscosity treatment in a Fisher post-hoc test (p < 0.05).

Figure 5

Variation in the amount of ATP per sperm over time. Data are means ± SE (M. musculus: N = 4; M. spretus: N = 5; M. spicilegus: N = 4). (A–C): Amount of ATP per sperm incubated in non-capacitating conditions in low viscosity (mT−H under air) and high viscosity (mT−H + PVP under air). Red squares and letters: non-capacitating conditions in low viscosity (mT−H), and pink triangles and letters: non-capacitating conditions in high viscosity (mT−H + PVP). (D–F): Amount of ATP per sperm in capacitating conditions in low viscosity (mT−BH under 5% CO₂/air) and high viscosity (mT−BH + PVP under 5% CO₂/air). Dark blue squares and letters: capacitating conditions in low viscosity (mT−BH), and light blue triangles and letters: capacitating conditions in high viscosity (mT-BH + PVP). (A,D): M. musculus. (B,E): M. spretus. (C,F): M. spicilegus. Asterisks indicate significant differences between viscosity treatments for the same time point in a Fisher post-hoc test (p < 0.05). Different letters indicate significant differences between time points for the same viscosity treatment in a Fisher post-hoc test (p < 0.05).