Glycolysis and Respiration in Mouse Ldhc -/- Sperm.

Abstract

Targeted mutation of the lactate dehydrogenase type C gene (Ldhc) reduced considerably glucose consumption in sperm, resulting in decreased ATP production and loss of sperm motility. Upon backcrossing the mutation onto different genetic backgrounds for ten generations (KOF10), we observed significant fertility differences between C57Bl/6N (KOF10/C57) and 129SvEv (KOF10/129) strain males. While KOF10/C57 males were subfertile, KOF10/129 males were infertile. Sperm analyses revealed that KOF10/129 sperm had significantly lower ATP levels and progressive motility than KOF10/C57 sperm. Inhibition of glycolysis with sodium iodoacetate further lowered ATP levels and motility in KOF10/C57, but not in KOF10/129 sperm. These results suggest that KOF10/C57 sperm continue to produce significantly more ATP by glycolysis than KOF10/129 sperm, presumably explaining why a few pups were sired by KOF10/C57 but not by KO10/129 male mice. This difference between KOF10/C57 and KOF10/129 might be explained by a metabolic difference found by NMR spectroscopy between 129SvEv and C57Bl/6N WT sperm: glucose utilization was approximately 4 times lower in 129SvEv WT sperm (3.84±1.15 nmol glucose consumed/hour/106 spermatozoa) compared to C57Bl/6N WT sperm (12.93±3.48). However, there were no significant differences in fertility of WT 129SvEv and C57Bl/6N males, indicating that while glycolysis is essential for sperm function, a higher rate does not necessarily result in higher sperm fertilizing ability. A positive correlation has been reported between sperm quality and the efficiency of respiration, and this implies that sperm rely on respiration to support high levels of ATP production. Because of the extremely low level of ATP produced by glycolysis in KOF10/129 sperm, they provide a good model for examining the role of respiration in sperm motility. Sperm from WT 129SvEv and KOF10/129 mice were incubated with lactate or pyruvate as mitochondrial substrates, glucose as a glycolytic substrate, or a mix of lactate plus glucose. High levels of ATP and increases in progressive motility were observed in KOF10/129 and WT sperm incubated with mitochondrial substrates, and these effects were abrogated by the mitochondrial respiratory uncoupler CCCP. As expected, ATP levels and motility were extremely low when KOF10/129 sperm were incubated with glucose. However, the presence of lactate, as a mitochondrial substrate in medium containing glucose, did not rescue the KOF10/129 phenotype: respiration as measured by ATP production was inactive in the presence of glucose. The respiration rate was determined by measuring oxygen (O2) consumption, and was high when WT or KOF10/129 sperm were incubated with lactate alone, and lowered by alpha-cyano-4-hydroxycinnamic acid (inhibitor of mitochondrial lactate and pyruvate transport). However glucose or deoxy-glucose in combination with lactate caused a reduction in O2 consumption (putative Crabtree effect). These results strongly suggest that in mouse sperm the presence of glucose induces a shift to the glycolytic pathway. This also occurs when glycolysis is inhibited in KOF10/129 sperm. Further studies on the mechanism of this regulation and its physiological role are in progress. (poster)