Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility

Abstract

Although glycolysis is highly conserved, it is remarkable that several unique isozymes in this central metabolic pathway are found in mammalian sperm. Glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) is the product of a mouse gene expressed only during spermatogenesis and, like its human ortholog (GAPD2), is the sole GAPDH isozyme in sperm. It is tightly bound to the fibrous sheath, a cytoskeletal structure that extends most of the length of the sperm flagellum. We disrupted Gapds expression by gene targeting to selectively block sperm glycolysis and assess its relative importance for in vivo sperm function. Gapds(-/-) males were infertile and had profound defects in sperm motility, exhibiting sluggish movement without forward progression. Although mitochondrial oxygen consumption was unchanged, sperm from Gapds(-/-) mice had ATP levels that were only 10.4% of those in sperm from WT mice. These results imply that most of the energy required for sperm motility is generated by glycolysis rather than oxidative phosphorylation. Furthermore, the critical role of glycolysis in sperm and its dependence on this sperm-specific enzyme suggest that GAPDS is a potential contraceptive target, and that mutations or environmental agents that disrupt its activity could lead to male infertility.

Fig. 1.

Targeted disruption of Gapds. (A) Abbreviated diagram of sperm glycolysis showing that GAPDS is required for all ATP production via this pathway. (B and C) Indirect immunofluorescence shows that GAPDS is localized in the principal piece of the flagellum in WT sperm (B) but is absent in sperm from Gapds^–/– mice (C). Sperm morphology is indistinguishable in these merged immunofluorescence and phase-contrast images. The arrow in B denotes the junction between the middle piece and principal piece of the flagellum. (D) Testis and sperm proteins from WT (+/+), heterozygous (+/–), and homozygous mutant (–/–) males were analyzed by Western blotting with an antibody specific for GAPDS. Similar results were obtained with antibodies that recognize peptide sequences either upstream (data not shown) or downstream of the deletion. The relative molecular weights (×10^–3) of protein standards are shown at right. (E) GAPDS/GAPDH enzyme activity was measured for sperm isolated from +/+, +/–, and –/– males. Values show mean activity ± SEM.

Fig. 2.

GAPDS is required for sperm motility. (A) Sperm were collected from WT (n = 6) and Gapds^–/– mice (n = 6) and incubated in M16 medium at 37°C in 5% CO₂ and air. Forward movement was scored by phase-contrast microscopy for >200 sperm per animal at each time point. Mean percentages of forward movement ± SEM are shown for WT (circles) and Gapds^–/– (squares) males. (B and C) Sperm tracks generated by CASA. Green tracks illustrate the movement of individual sperm in 1.5 sec and red dots indicate immotile or dead sperm. WT sperm (B) displayed vigorous progressive motility, whereas sperm from Gapds^–/– males (C) were motile but nonprogressive.

Fig. 3.

Fibrous sheath formation does not require GAPDS. Sperm ultrastructure was compared by transmission (A, B, E, and F) and scanning (C and D) electron microscopy. The ribs (R) and columns (C) of the fibrous sheath appear similar in cross sections of sperm from WT (A) and Gapds^–/– (B) mice. The outer dense fibers (O) and axonemes are also indistinguishable in these flagellar cross sections. Longitudinal sections of sperm from WT (C and E) and Gapds^–/– (D and F) mice show some variation in the spacing between the ribs of the fibrous sheath. (Bar: 0.1 μmin A, B, E, and F;1 μmin C and D.)

Fig. 4.

Sperm from Gapds^–/– mice have extremely low ATP levels. (A) Sperm ATP was extracted and measured by using a luciferase bioluminescence assay after incubation in M16 medium at 37°C in 5% CO₂ and air. Mean ATP levels ± SEM were ≈10-fold higher for sperm from WT mice (n = 6) compared with sperm from Gapds^–/– males (n = 6). (B) Oxygen consumption was determined with an oxygen probe. Sperm from WT (n = 4) and Gapds^–/– (n = 4) mice consumed similar amounts of oxygen, indicating comparable mitochondrial activity.