Glycolytic Enzyme Activity Is Essential for Domestic Cat (Felis catus) and Cheetah (Acinonyx jubatus) Sperm Motility and Viability in a Sugar-Free Medium1 (original) (raw)
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Biology of Reproduction, 2011
We have previously reported a lack of glucose uptake in domestic cat and cheetah spermatozoa, despite observing that these cells produce lactate at rates that correlate positively with sperm function. To elucidate the role of glycolysis in felid sperm energy production, we conducted a comparative study in the domestic cat and cheetah, with the hypothesis that sperm motility and viability are maintained in both species in the absence of glycolytic metabolism and are fueled by endogenous substrates. Washed ejaculates were incubated in chemically defined medium in the presence/absence of glucose and pyruvate. A second set of ejaculates was exposed to a chemical inhibitor of either lactate dehydrogenase (sodium oxamate) or glyceraldehyde-3-phosphate dehydrogenase (alpha-chlorohydrin). Sperm function (motility and acrosomal integrity) and lactate production were assessed, and a subset of spermatozoa was assayed for intracellular glycogen. In both the cat and cheetah, sperm function was maintained without exogenous substrates and following lactate dehydrogenase inhibition. Lactate production occurred in the absence of exogenous hexoses, but only if pyruvate was present. Intracellular glycogen was not detected in spermatozoa from either species. Unexpectedly, glycolytic inhibition by alpha-chlorohydrin resulted in an immediate decline in sperm motility, particularly in the domestic cat. Collectively, our findings reveal an essential role of the glycolytic pathway in felid spermatozoa that is unrelated to hexose metabolism or lactate formation. Instead, glycolytic enzyme activity could be required for the metabolism of endogenous lipid-derived glycerol, with fatty acid oxidation providing the primary energy source in felid spermatozoa.
Biology of Reproduction, 2010
Cheetahs and certain other felids consistently ejaculate high proportions (!60%) of malformed spermatozoa, a condition known as teratospermia, which is prevalent in humans. Even seemingly normal spermatozoa from domestic cat teratospermic ejaculates have reduced fertilizing capacity. To understand the role of sperm metabolism in this phenomenon, we conducted a comparative study in the normospermic domestic cat versus the teratospermic cat and cheetah with the general hypothesis that sperm metabolic function is impaired in males producing predominantly pleiomorphic spermatozoa. Washed ejaculates were incubated in chemically defined medium containing glucose and pyruvate. Uptake of glucose and pyruvate and production of lactate were assessed using enzyme-linked fluorescence assays. Spermatozoa from domestic cats and cheetahs exhibited similar metabolic profiles, with minimal glucose metabolism and approximately equimolar rates of pyruvate uptake and lactate production. Compared to normospermic counterparts, pyruvate and lactate metabolism were reduced in teratospermic cat and cheetah ejaculates, even when controlling for sperm motility. Rates of pyruvate and lactate (but not glucose) metabolism were correlated positively with sperm motility, acrosomal integrity, and normal morphology. Collectively, our findings reveal that pyruvate uptake and lactate production are reliable, quantitative indicators of sperm quality in these two felid species and that metabolic function is impaired in teratospermic ejaculates. Furthermore, patterns of substrate utilization are conserved between these species, including the unexpected lack of exogenous glucose metabolism. Because glycolysis is required to support sperm motility and capacitation in certain other mammals (including dogs), the activity of this pathway in felid spermatozoa is a target for future investigation.
Biology of Reproduction, 2010
Cheetahs and certain other felids consistently ejaculate high proportions (!60%) of malformed spermatozoa, a condition known as teratospermia, which is prevalent in humans. Even seemingly normal spermatozoa from domestic cat teratospermic ejaculates have reduced fertilizing capacity. To understand the role of sperm metabolism in this phenomenon, we conducted a comparative study in the normospermic domestic cat versus the teratospermic cat and cheetah with the general hypothesis that sperm metabolic function is impaired in males producing predominantly pleiomorphic spermatozoa. Washed ejaculates were incubated in chemically defined medium containing glucose and pyruvate. Uptake of glucose and pyruvate and production of lactate were assessed using enzyme-linked fluorescence assays. Spermatozoa from domestic cats and cheetahs exhibited similar metabolic profiles, with minimal glucose metabolism and approximately equimolar rates of pyruvate uptake and lactate production. Compared to normospermic counterparts, pyruvate and lactate metabolism were reduced in teratospermic cat and cheetah ejaculates, even when controlling for sperm motility. Rates of pyruvate and lactate (but not glucose) metabolism were correlated positively with sperm motility, acrosomal integrity, and normal morphology. Collectively, our findings reveal that pyruvate uptake and lactate production are reliable, quantitative indicators of sperm quality in these two felid species and that metabolic function is impaired in teratospermic ejaculates. Furthermore, patterns of substrate utilization are conserved between these species, including the unexpected lack of exogenous glucose metabolism. Because glycolysis is required to support sperm motility and capacitation in certain other mammals (including dogs), the activity of this pathway in felid spermatozoa is a target for future investigation.
Biology of Reproduction, 2011
Compared with the normospermic domestic cat, sperm metabolic function is compromised in the teratospermic cat and cheetah, but the pathway(s) involved in this deficiency are unknown. Glycolysis is essential for sperm motility, yet it appears to function normally in spermatozoa of either species regardless of structural morphology. We conducted a comparative study to further understand the mechanisms of energy production in felid spermatozoa, with the hypothesis that oxidative phosphorylation is required for normal sperm function and is impaired in teratospermic ejaculates. Electroejaculates from both species were stained with MitoTracker to quantify mitochondrial membrane potential (MMP) or were incubated to assess changes in sperm function (motility, acrosomal integrity, and lactate production) after mitochondrial inhibition with myxothiazol. Sperm midpiece dimensions also were quantified. Sperm mitochondrial fluorescence (directly proportional to MMP) was ;95% lower in the cheetah compared with the normospermic and teratospermic cat, despite the cheetah having a 10% longer midpiece. In both species, MMP was increased 5-fold in spermatozoa with retained cytoplasm compared with structurally normal cells. Inhibition of oxidative phosphorylation impaired sperm function in both species, but a 100-fold higher inhibitor concentration was required in the cat compared with the cheetah. Collectively, findings revealed that oxidative phosphorylation was required for sperm function in the domestic cat and cheetah. This pathway of energy production appeared markedly less active in the cheetah, indicating a species-specific vulnerability to mitochondrial dysfunction. The unexpected, cross-species linkage between retained cytoplasmic droplets and elevated MMP may reflect increased concentrations of metabolic enzymes or substrates in these structures.
Evidence for a functional glycogen metabolism in mature mammalian spermatozoa
Molecular Reproduction and Development, 2000
The glycogen content in fresh raw dog spermatozoa was 0.22 Ϯ 0.03 mol/mg protein. This matched with the presence of a glycogenlike staining in the head and midpiece. Glycogen levels lowered to 0.05 mol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 mol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35 Ϯ 0.07, measured in the absence and the presence of glucose 6-P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25 Ϯ 0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10-20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo. Mol.
Biology of Reproduction, 2012
Although substantial evidence exists that sperm ATP production via glycolysis is required for mammalian sperm function and male fertility, conflicting reports involving multiple species have appeared regarding the ability of individual glycolytic or mitochondrial substrates to support the physiological changes that occur during capacitation. Several mouse models with defects in the signaling pathways required for capacitation exhibit reductions in sperm ATP levels, suggesting regulatory interactions between sperm metabolism and signal transduction cascades. To better understand these interactions, we conducted quantitative studies of mouse sperm throughout a 2-h in vitro capacitation period and compared the effects of single substrates assayed under identical conditions. Multiple glycolytic and nonglycolytic substrates maintained sperm ATP levels and comparable percentages of motility, but only glucose and mannose supported hyperactivation. These monosaccharides and fructose supported the full pattern of tyrosine phosphorylation, whereas nonglycolytic substrates supported at least partial tyrosine phosphorylation. Inhibition of glycolysis impaired motility in the presence of glucose, fructose, or pyruvate but not in the presence of hydroxybutyrate. Addition of an uncoupler of oxidative phosphorylation reduced motility with pyruvate or hydroxybutyrate as substrates but unexpectedly stimulated hyperactivation with fructose. Investigating differences between glucose and fructose in more detail, we demonstrated that hyperactivation results from the active metabolism of glucose. Differences between glucose and fructose appeared to be downstream of changes in intracellular pH, which rose to comparable levels during incubation with either substrate. Sperm redox pathways were differentially affected, with higher levels of associated metabolites and reactive oxygen species generated during incubations with fructose than during incubations with glucose.
Biology of Reproduction, 2004
In vitro capacitation of dog spermatozoa in a medium without sugars and with lactate as the metabolic substrate (l-CCM) was accompanied by a progressive increase of intracellular glycogen during the first 2 h of incubation, which was followed by a subsequent decrease of glycogen levels after up to 4 h of incubation. Lactate from the medium is the source for the observed glycogen synthesis, as the presence of [ 14 C]glycogen after the addition to l-CCM with [ 14 C]lactate was demonstrated. The existence of functional gluconeogenesis in dog sperm was also sustained by the presence of key enzymes of this metabolic pathway, such as fructose 1,6-bisphophatase and aldolase B. On the other hand, glycogen metabolism from gluconeogenic sources was important in the maintenance of a correct in vitro fertilization after incubation in the l-CCM. This was demonstrated after the addition of phenylacetic acid (PAA) to l-CCM. In the presence of PAA, in vitro capacitation of dog spermatozoa suffered alterations, which translated into changes in capacitation functional markers, like the increase in the percentage of altered acrosomes, a distinct motion pattern, decrease or even disappearance of capacitation-induced tyrosine phosphorylation, and increased heterogeneity of the chlorotetracycline pattern in capacitated cells. Thus, this is the first report indicating the existence of a functional glyconeogenesis in mammalian spermatozoa. Moreover, gluconeogenesis-linked glycogen metabolism seems to be of importance in the maintenance of a correct in vitro capacitation in dog sperm in the absence of hexoses in the medium.
Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa
Reproduction, 2002
Incubation of dog spermatozoa with 10 mmol l -1 glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of Llactate and CO 2 . In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l -1 ). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l -1 to 10 mmol l -1 , fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.
Metabolic changes in mouse sperm during capacitation†
Biology of Reproduction, 2020
Mammalian sperm are stored in the epididymis in a dormant state. Upon ejaculation, they must immediately start producing sufficient energy to maintain motility and support capacitation. While this increased energy demand during capacitation is well established, it remains unclear how mouse sperm modify their metabolism to meet this need. We now show that capacitating mouse sperm enhance glucose uptake, identifying glucose uptake as a functional marker of capacitation. Using an extracellular flux analyzer, we show that glycolysis and oxidative phosphorylation increase during capacitation. Furthermore, this increase in oxidative phosphorylation is dependent on glycolysis, providing experimental evidence for a link between glycolysis and oxidative phosphorylation in mouse sperm.