This study used an NMR approach to follow in real-time Gut
by sperm. In WT sperm, a high glucose metabolic flux in the direction of lactate was observed, in agreement with previous data from studies using rat [9
] and boar [22
] sperm in which a pentose phosphate pathway was undetectable or barely detectable. We confirmed that Lprod
in KO sperm was extremely low (around 30 times lower than in WT sperm) and that Gut
occurred at a very low level, indicating that the loss of LDHC directly perturbs the process of glycolysis [13
]. In addition, preliminary data suggest that LDHC was not essential for production of ATP by oxidative phosphorylation, but further studies will be necessary to confirm these results.
Our initial hypothesis was that the lack of LDHC led to an accumulation of pyruvate and a decrease in NAD+ renewal, thereby causing an imbalance in the NAD:NADH ratio that would inhibit glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS) function and concomitantly ATP production. Although there was a decrease in the NAD:NADH ratio in KO sperm compared to WT sperm after 4 h incubation, it was not statistically significant (P = 0.13). Moreover, no differences were observed in NAD+ and NADH levels between WT and KO sperm after incubation for 1.5 h, when ATP levels were already lower in KO sperm. However, treatment with methylene blue to oxidize NADH did not rescue the phenotype of KO sperm, suggesting that the down-regulation does not occur because of limitation of NAD+ renewal and inhibition of the GAPDHS reaction. Also, the low level of Lprod by KO sperm was not accompanied by an accumulation of pyruvate.
Following pyruvate utilization by NMR spectroscopy and using an enzymatic approach to assay the production of lactate from pyruvate, we found that KO sperm were able to convert pyruvate into lactate at the same rate as WT sperm, showing that sperm lacking LDHC still contain appreciable levels of LDH activity. We assumed previously that approximately 17.5% of the LDH activity in sperm was due to the action of the LDHA isozyme [13
] and our data suggest that LDHA is able to compensate partially for the absence of LDHC in terms of processing a high concentration of external pyruvate. Could the compartmentalization make the external pyruvate more accessible to LDHA than the internal pyruvate (produced during the glycolytic reactions)? Previous studies have shown that 1) the conversion of pyruvate to lactate under anaerobic conditions in skeletal muscle is performed by LDHA [23
]; 2) direct interaction between GAPDH and LDHA occurs in cardiac and skeletal muscle [24
] suggests that substrate channeling prevented the escape of NADH coenzyme into the bulk phase between both enzymes [25
]; 3) LDHC is loosely associated with the fibrous sheath (which makes LDHC more available to process external pyruvate) and both LDHA and GAPDHS are tightly bound [14
]; 4) of the five Ldha
splice variants in the mouse, LDHA_V2 has an N-terminal extension specific to the testis and is a strong candidate for the LDHA isozyme present in the fibrous sheath (NCBI accession number NP_001129541; http://www.ncbi.nlm.nih.gov/protein/257743039
). This led us to suggest that LDHA was responsible for some or most of the LDH activity in KO sperm associated with the renewal of NAD+
for the GAPDHS reaction [13
]. However, a conditional Ldha
KO in male germ cells will be needed to test this hypothesis.
We expected WT sperm treated with the LDH competitive inhibitor sodium oxamate to lack LDH activity and to have a phenotype similar to KO sperm. Indeed, the treated WT and KO sperm both failed to develop a hyperactivated motility pattern. However, whereas KO sperm had low ATP levels and no differences in the NAD:NADH ratio, the ATP level in treated WT sperm was normal and the NADH level was elevated, resulting in a decrease in the NAD:NADH ratio. These results strongly suggested that the molecular mechanism preventing hyperactivation was different between oxamate-treated WT sperm and sperm lacking LDHC. We suggested previously [13
] that the motility seen initially in KO sperm was due to ATP produced by LDHA. In that case, the inhibition of LDHA by sodium oxamate treatment should accentuate the phenotype of KO sperm. However, the lack of differences between treated and untreated KO sperm strongly suggested that glycolysis in KO sperm was not disrupted solely because of a lack of LDH activity.
KO sperm possessed the other enzymatic activities necessary for glucose metabolism, and glucose uptake was not different between WT, HET, and KO sperm. However, the NMR results indicated that glucose was consumed very slowly by KO sperm, with peaks detected only for glucose and lactate. The lack of accumulation of hexose phosphate intermediates suggests that the glycolytic flow was reduced considerably by a decrease in the first phase of glycolysis. However, it should be noted that the identification of other metabolites present at low concentrations is limited by the inherently low sensitivity of the NMR technique.
If the payoff phase of glycolysis is blocked, production of hexose phosphate should not only limit ATP production, but also waste ATP [28
]. Therefore, the ATP levels in KO sperm would be expected to decrease more rapidly in medium containing glucose than in medium lacking glucose. However, the opposite was observed. ATP levels and progressive motility decreased significantly in both KO and WT sperm in medium lacking glucose compared to when they were incubated in medium containing glucose. These data suggest that a low level of glycolysis in KO sperm is still present and needed to maintain a degree of motility. They also support our hypothesis that the inhibition of glycolysis occurs in earlier steps of glycolysis to avoid futile production of hexose phosphate and waste of ATP.
The efficient conversion of pyruvate to lactate by KO sperm was unexpected. One possible explanation is that the efficient conversion of pyruvate to lactate in KO sperm is related to differences in the kinetic characteristics of LDHA and LDHC. Earlier studies reported that LDHC had a higher affinity for pyruvate (lower Km
) than LDHA [29
]. In addition, LDHC was found to be susceptible to inhibition by pyruvate at lower concentrations than are optimal for LDHA. It is possible that at lower concentrations the pyruvate in WT sperm is reduced to lactate, whereas pyruvate would reach higher levels in KO sperm before being metabolized by LDHA. The NADH in KO sperm might also be shunted to other NADH-dependent enzymes, compensating for the changes in metabolism in KO sperm [30
]. However, the relationships between Km
and the saturation characteristics of purified enzymes in vitro [29
] and when they are present in intact sperm are unknown.
Another possibility was that LDHC has noncatalytic functions essential for regulation of glycolysis in sperm that other LDH isozymes are unable to provide. A systematic proteomic approach, combining coimmunoprecipitation of native protein complexes, separation on one-dimensional SDS-PAGE, and mass spectrometry, was used to identify proteins that directly or indirectly associated with LDHC. This led to the identification of 27 putative LDHC-interacting proteins, 13 of which were implicated in energy metabolism and 5 of which were cytoskeleton associated. Interestingly, four of the proteins associated with ATP metabolism are specifically expressed in male germ cells: ANT4 [31
], hexokinase-1 isoform S (HK1S) [34
], phosphoglycerate kinase 2 (PGK2) [35
], and pyruvate dehydrogenase E1 alpha 2 (PDHE1A2) [36
ANT subfamily proteins usually are associated with the inner mitochondrial membrane and involved in the exchange of ADP and ATP between cytosol and mitochondria, as well as with mitochondria-dependent apoptosis [37
]. ANT4 is expressed mainly in male germ cells and at highest levels in spermatocytes. A knockout of the gene (Slc25a31
) for ANT4 in mice led to disruption of spermatogenesis with an early meiotic arrest and an increase of apoptosis [32
]. However, ANT4 also was found in the sperm principal piece in most human sperm and only occasionally in the midpiece where the sperm mitochondria are located [33
]. This led to the suggestions that ANT4 acts as an ATP reservoir or ATP carrier in the sperm flagellum. HK1S and PGK2 are important sperm-specific glycolytic enzymes. HK1S uses ATP in the first phase of glycolysis to convert glucose into glucose-6-phosphate, whereas PGK2 catalyses the first reaction in the second phase of glycolysis to produce ATP. HK1S is the only hexokinase detected in mouse sperm, and a recent study demonstrated that the N-terminal 24-amino acid spermatogenic cell-specific region of HK1S binds to a testis-specific isoform of phosphofructokinase (PFKMS) [39
]. A lack of PGK2 induces male infertility and the phenotype of PGK2-null sperm is similar to the LDHC-null sperm [35
]. However, the HK1S and PFKMS enzymatic activities were not different in permeabilized LDHC-null sperm and WT sperm. PDHE1A2 is a testis-specific subunit of the PDH complex, which converts pyruvate into acetyl-coA. Like ANT proteins, the PDH complex usually is associated with mitochondria. However, studies in the hamster showed that a PDHA2 orthologue is located in the sperm flagellum, phosphorylated during sperm capacitation, and essential for sperm hyperactivation [36
]. However, cross-contamination with mitochondria is possible and further investigations will be needed in order to validate these preliminary data.
These observations suggest that the in vivo macromolecular organization of enzymes is crucial in the regulation of glycolysis in the sperm flagellum, with LDHC being an integral component of a complex containing an ATP carrier protein (ANT4) that redistributes ATP from PGK2 to HK1S and thereby regulates the initial phase of glycolysis. This is reminiscent of an intriguing model proposed for the compartmentalization of glycolysis in glycosomes of flagellated trypanosomes to protect them from toxic accumulation of intermediates [40
]. The glycosome model suggests that rather than allosteric events or feedback inhibition occurring if glycolysis is blocked downstream, compartmentalization keeps ATP from being made directly available to HK or to phosphofructokinase, thereby avoiding use of ATP in nonproductive glucose phosphorylation and an accumulation of hexose phosphates.
Additional studies will be necessary to determine if the results seen in this study are because LDHC is required for the integrity of a macromolecular complex. Nevertheless, LDHC has been conserved throughout evolution from marsupial to placental mammals, suggesting that its novel characteristics have conferred an advantage essential for the maintenance of glycolysis in mammalian sperm.