Sperm in the cauda epididymis are immotile and quickly become motile when diluted in physiological media, but little is known about the changes that occur with activation of sperm motility. We predicted that one change would be an increase in ATP production by glycolysis. Although oxidative phosphorylation is the main source and glycolysis is a minor source of ATP in most cell types, fertilization was not prevented by pharmacological inhibition of oxidative phosphorylation [22
] or disruption of the testis-specific cytochrome C (Cyct
) gene [25
]. However, in vitro fertilization failed if the medium lacked glucose [24
], suggesting that glycolysis produces most of the ATP needed for sperm function. This was shown convincingly by using gene targeting to disrupt the sperm-specific Gapdhs
gene. Sperm from mice lacking the GAPDHS glycolytic enzyme had very low ATP levels and severely compromised motility, causing the male mice to be infertile [9
The rate-limiting enzyme for glycolysis in brain and red blood cells is HK1 [30
], suggesting that sperm activation might involve an increase in HK1S activity that enhances the rate of glycolysis and ATP production. Our study showed that HK1S activity and ATP levels are significantly higher in activated than in quiescent sperm, findings consistent with this idea. A clue to the regulation of this change was the observation that HK1S is present mainly as a dimer in quiescent sperm and a monomer in activated sperm. At the low concentrations used for kinetic studies, HK1 is a monomer, but dimerization can occur at higher concentrations in the presence of glucose and glucose 6-phosphate (G6P) [32
] and during crystallization [33
]. Interaction between the N- and C-terminal domains of HK1S is believed necessary for the monomer to function [36
], but this interaction is constrained in the dimer form [32
], suggesting that dimerization of HK1S subunits causes HK enzymatic activity to be lower in quiescent than in activated sperm.
A key observation for understanding the dimer-to-monomer conversion occurred when sperm lysates were analyzed by PAGE and dimers were found only under nonreducing conditions. In addition, treatment of quiescent sperm lysates with diamide to block disulfide-bond reduction inhibited the dimer-to-monomer conversion catalyzed by TXN1. Although these observations indicated that HK1S dimers probably are stabilized by disulfide bonds, x-ray crystallographic studies found that HK1 dimers were maintained by hydrogen bonds and not intramolecular disulfide bonds [37
]. However, the conformation of HK1 dimers in crystals presumably is different than their conformation in solution and not necessarily that of HK1S in the sperm flagellum. Furthermore, a cysteine residue in the SSR domain of HK1S (and not present in the PBD domain of HK1) is available for intramolecular disulfide bond formation between HK1S subunits.
Although the conversion of HK1S dimers to monomers in vitro occurred by disulfide bond reduction, it remained to be determined if the same occurs during activation of live sperm. We found that treating sperm with diamide to inhibit disulfide bond cleavage resulted in the reduction of both HK1S activity and sperm motility. The ATP levels remained unchanged for several minutes in the presence of diamide, suggesting that its effect was not due to cytotoxicity. However, the structure and activity of another glycolytic enzyme, muscle phosphofructokinase (PFKM), was reported to be sensitive to the oxidation state of protein sulfhydryl groups [38
]. Thus, at least some of the effect of diamide on HK1S activity might be indirect. If diamide inhibits PFKM or other glycolytic enzymes, this might result in the accumulation of G6P and lead to product inhibition of HK1S activity. Further studies will be needed to examine the possible role of sulfhydryl reduction in other glycolytic enzymes during sperm activation.
Cysteine residues of proteins in the principal piece were minimally detected by mBBr in quiescent sperm but were readily detected in activated sperm. The majority of HK1S is present in the principal piece region of the flagellum, and presumably some of the thiol groups labeled by mBBr were on HK1S monomers. It is not unusual for redox-sensitive proteins to form transient disulfide bonds under oxidizing conditions, and the presence of HK1S as a dimer in the epididymis is consistent with the previously reported redox status of sperm proteins in the epididymis [16
]. However, our studies strongly suggest that HK1S disulfide bond reduction contributes significantly to the increase in ATP levels and the initiation of motility that occur when mouse sperm undergo conversion from the quiescent to activated state.
Sperm are generators of reactive oxygen species (ROS) [39
], and redox-sensitive proteins form transient disulfide bonds under oxidizing conditions. This suggests that a redox reaction is responsible for the conversion of HK1S from a dimer to a monomer during sperm activation [42
], which is consistent with other studies indicating that redox reactions are involved in the induction of progressive sperm motility [43
Cells have two major redox-regulated systems, glutathiones (GSH) and thioredoxins (TRX) [44
]. Sperm are known to have an active glutathione reductase and glutathione (GSH) system for protecting proteins from oxidative damage [47
]. They also have a thioredoxin system for regulation of redox, and spermatogenic cell-specific thioredoxins TXNDC2, TXNDC3, and TXNDC8 (formerly called SPTRX-1, SPTRX-2, and SPTRX-3) [46
] have been identified recently. One or both of these systems might be responsible for catalyzing the HK1S dimer-to-monomer conversion during sperm activation, and current studies are evaluating this possibility.
The present studies found that HK1S undergoes dephosphorylation during sperm activation, which seems contrary to precious reports that HK1S is tyrosine phosphorylated in sperm [11
]. However, these results typically were determined after isolation and wash steps taking 15–20 min. This suggests that the tyrosine-phosphorylated HK1S observed in previous studies resulted from rephosphorylation of monomers dephosphorylated during sperm activation.
These studies indicate that a series of molecular and functional changes occur when sperm undergo conversion from quiescent to activated (). These include 1) reduction of disulfide bonds associated with HK1S dimers, 2) conversion of HK1S dimers to monomers, 3) HK1S dephosphorylation of HK1S, 4) an increase in HK activity, and 5) sperm becoming motile. Treatment with diamide impedes 1) reduction of disulfide bonds, 2) dimer-to-monomer conversion, and 3) increases in sperm motility. Dephosphorylation of HK1S occurs independently of disulfide bond reduction and is not required for dimer-to-monomer conversion. However, the molecular mechanisms responsible for the disulfide bond reduction and dephosphorylation that occur during sperm activation remain to be defined.
FIG. 6. Correlation during sperm activation of changes in conformation of HK1S, HK1S activity, redox status, and sperm motility. The HK1S in quiescent sperm is present mainly as a dimer. During 5 min of incubation in M2 medium, there is a reduction in disulfide (more ...)