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Glucose is a pH-dependent motor for sperm beat frequency during early activation.

Mannowetz N, Wandernoth PM, Wennemuth G - PLoS ONE (2012)

Bottom Line: This evokes changes in the lactate content of sperm as well as in the intracellular pH (pH(i)) since sperm possess lactate/proton co-transporters.We show that the glucose-mediated acceleration of flagellar beat and ATP production are hastened by a pH(i) ≥7.1, whereas a pH(i) ≤7.1 leaves both parameters unchanged.Since we observed a diminished rise in beat frequency in the presence of specific inhibitors against carbonic anhydrases, soluble adenylyl cyclase and protein kinase, we suggest that the glucose-mediated effect is linked to CO(2) hydration and thus the production of HCO(3)- by intracellular CA isoforms.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Saarland University, Homburg/Saar, Germany.

ABSTRACT
To reach the egg in the ampulla, sperm have to travel along the female genital tract, thereby being dependent on external energy sources and substances to maintain and raise the flagellar beat. The vaginal fluid is rich in lactate, whereas in the uterine fluid glucose is the predominant substrate. This evokes changes in the lactate content of sperm as well as in the intracellular pH (pH(i)) since sperm possess lactate/proton co-transporters. It is well documented that glycolysis yields ATP and that HCO(3)- is a potent factor in the increase of beat frequency. We here show for the first time a pathway that connects both parts. We demonstrate a doubling of beat frequency in the mere presence of glucose. This effect can reversibly be blocked by 2-deoxy-D-glucose, dichloroacetate and aminooxyacetate, strongly suggesting that it requires both glycolysis and mitochondrial oxidation of glycolytic end products. We show that the glucose-mediated acceleration of flagellar beat and ATP production are hastened by a pH(i) ≥7.1, whereas a pH(i) ≤7.1 leaves both parameters unchanged. Since we observed a diminished rise in beat frequency in the presence of specific inhibitors against carbonic anhydrases, soluble adenylyl cyclase and protein kinase, we suggest that the glucose-mediated effect is linked to CO(2) hydration and thus the production of HCO(3)- by intracellular CA isoforms. In summary, we propose that, in sperm, glycolysis is an additional pH(i)-dependent way to produce HCO(3)-, thus enhancing sperm beat frequency and contributing to fertility.

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Related in: MedlinePlus

Organization of the principle piece of the sperm tail and proposed model for the interplay between pHi, glycolysis and production of HCO3−.A, Shown is a scheme of a cross section through the principle piece. Nine microtubule doublets - each carrying dynein arms - are connected via radial spokes to the central pair (CP), constituting the classical 9×2+2 core structure of the ciliar axoneme. Each microtubule doublet, in turn, is connected to two main longitudinal cytoskeletal structures – doublets 3 and 8 are fastened to the so-called longitudinal columns (LC), whereas doublets 1 and 2 and 4–7 are bound to the outer dense fibers (ODF). The outward facing area of each LC and ODF is tightly connected to the fibrous sheath (FS), a layer located right underneath the plasma membrane. B, Shown is an incomplete section of A, focusing on enzymes located to the FS, in close proximity to dynein ATPase. Glycolytic enzymes are hexokinase (HK), phosphokinase (PK), sperm specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) and lactate dehydrogenase A (LDHA). Proteins involved in the cAMP/HCO3- metabolism are A-kinase anchoring protein 3 and 4 (AKAP3/4) and testis A-kinase anchoring protein (TAKAP) with binding sites for protein kinase A (PKA). C, This drawing illustrates our working hypothesis. Glucose enters spermatozoa via glucose transporters (GLUT). Once lactate leaves the cell together with protons via monocarboxylate transporters (MCT), pHi rises and glycolysis proceeds intensified. GLUT and MCT transport is bidirectional and the solid arrows indicate the proposed route of transport. The glycolytic end product pyruvate is metabolized during the mitochondrial citrate cycle yielding CO2 which will be hydrated to HCO3− by intracellular CA. HCO3−, in turn, directly activates the sperm specific sAC thereby stimulating PKA and leading to an increase in sperm beat frequency.
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pone-0041030-g001: Organization of the principle piece of the sperm tail and proposed model for the interplay between pHi, glycolysis and production of HCO3−.A, Shown is a scheme of a cross section through the principle piece. Nine microtubule doublets - each carrying dynein arms - are connected via radial spokes to the central pair (CP), constituting the classical 9×2+2 core structure of the ciliar axoneme. Each microtubule doublet, in turn, is connected to two main longitudinal cytoskeletal structures – doublets 3 and 8 are fastened to the so-called longitudinal columns (LC), whereas doublets 1 and 2 and 4–7 are bound to the outer dense fibers (ODF). The outward facing area of each LC and ODF is tightly connected to the fibrous sheath (FS), a layer located right underneath the plasma membrane. B, Shown is an incomplete section of A, focusing on enzymes located to the FS, in close proximity to dynein ATPase. Glycolytic enzymes are hexokinase (HK), phosphokinase (PK), sperm specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) and lactate dehydrogenase A (LDHA). Proteins involved in the cAMP/HCO3- metabolism are A-kinase anchoring protein 3 and 4 (AKAP3/4) and testis A-kinase anchoring protein (TAKAP) with binding sites for protein kinase A (PKA). C, This drawing illustrates our working hypothesis. Glucose enters spermatozoa via glucose transporters (GLUT). Once lactate leaves the cell together with protons via monocarboxylate transporters (MCT), pHi rises and glycolysis proceeds intensified. GLUT and MCT transport is bidirectional and the solid arrows indicate the proposed route of transport. The glycolytic end product pyruvate is metabolized during the mitochondrial citrate cycle yielding CO2 which will be hydrated to HCO3− by intracellular CA. HCO3−, in turn, directly activates the sperm specific sAC thereby stimulating PKA and leading to an increase in sperm beat frequency.

Mentions: Once deposited in the vaginal fluid, which is rich in lactic acid, sperm travel through the cervix to enter the uterus where they encounter a fluid which is poor in lactate but rich in glucose and other glycolysable substrates [1]. Besides mitochondrial respiration, glycolysis is a major pathway for ATP production in murine spermatozoa [2], [3], [4]. Glycolytic enzymes are located in the fibrous sheath of the sperm tail [5] and some of them exhibit sperm-specific properties, such as type1 hexokinase (HK1S) [6], pyruvate kinase (PKS) [7] and glyceraldehyde 3-phosphate dehydrogenase (GAPDS) [8]. The fibrous sheath covers the outer dense fibers which, in turn, are located in close proximity to the microtubules. This arrangement permits the ATPases located on the dynein arms of the microtubule doublets (Fig. 1 A and B) to directly utilize the ATP generated during glycolysis to maintain bending of the sperm tail [5]. For mouse spermatozoa, muscle cells, leukocytes, erythrocytes and several other cell types it has been demonstrated that glycolytic key enzymes exhibit pH-dependency [6], [9], [10], [11]. We have previously shown that sperm possesses monocarboxylate/H+ co-transporters whose activity leads to intracellular acidification or alkalinization during application or removal of lactate and pyruvate [2]. With this work, we now investigate the way in which glycolysis is modulated in sperm by intracellular pH (pHi). Whereas the ATP generated during glycolysis helps sperm to maintain the resting beat stable, HCO3− is the only substance known so far in sperm to speed up their flagellar beat [10], [11], enabling them to travel along the uterus to reach the egg in the ampulla. Once inside the sperm cell, HCO3− directly activates the sperm-specific adenylyl cyclase (sAC) [12], [13] which, in turn, raises the intracellular level of cAMP, thereby stimulating protein kinase A (PKA) [14], [15], [16] and initiating the acceleration of beat frequency. Besides the above mentioned glycolytic enzymes the fibrous sheath is also a scaffold for A-kinase anchor protein 3 (AKAP3), A-kinase anchor protein 4 (AKAP4) and testis-specific A-kinase anchor protein 80 (TAKAP-80) (Fig. 1B), all of which contain binding sites for cAMP-dependent protein kinases [17]. Furthermore, AKAP3 of bovine sperm was shown to possess binding sites for phosphodiesterase 4A [18]. The near proximity of enzymes involved in both glycolysis and cAMP/HCO3− metabolism suggests that they together closely regulate sperm motility and beat frequency. HCO3− is available in the uterine fluid [19] and can enter sperm either directly by anion transporters as suggested by [20], [21], [22] or indirectly via carbonic anhydrases (CAs) [23], [24]. CAs catalyze the reversible hydration of CO2 to HCO3− and the majority of CA isoforms already identified is either membrane-bound proteins or existent in the cytoplasm. This raises the possibility that at least some portion of CO2 being produced during mitochondrial respiration is converted to HCO3−. With this work, we show evidence that glycolysis and cell respiration are interconnected for the production of HCO3− via CO2 as an additional way for sperm to regulate flagellar beat frequency pH-dependently during early activation.


Glucose is a pH-dependent motor for sperm beat frequency during early activation.

Mannowetz N, Wandernoth PM, Wennemuth G - PLoS ONE (2012)

Organization of the principle piece of the sperm tail and proposed model for the interplay between pHi, glycolysis and production of HCO3−.A, Shown is a scheme of a cross section through the principle piece. Nine microtubule doublets - each carrying dynein arms - are connected via radial spokes to the central pair (CP), constituting the classical 9×2+2 core structure of the ciliar axoneme. Each microtubule doublet, in turn, is connected to two main longitudinal cytoskeletal structures – doublets 3 and 8 are fastened to the so-called longitudinal columns (LC), whereas doublets 1 and 2 and 4–7 are bound to the outer dense fibers (ODF). The outward facing area of each LC and ODF is tightly connected to the fibrous sheath (FS), a layer located right underneath the plasma membrane. B, Shown is an incomplete section of A, focusing on enzymes located to the FS, in close proximity to dynein ATPase. Glycolytic enzymes are hexokinase (HK), phosphokinase (PK), sperm specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) and lactate dehydrogenase A (LDHA). Proteins involved in the cAMP/HCO3- metabolism are A-kinase anchoring protein 3 and 4 (AKAP3/4) and testis A-kinase anchoring protein (TAKAP) with binding sites for protein kinase A (PKA). C, This drawing illustrates our working hypothesis. Glucose enters spermatozoa via glucose transporters (GLUT). Once lactate leaves the cell together with protons via monocarboxylate transporters (MCT), pHi rises and glycolysis proceeds intensified. GLUT and MCT transport is bidirectional and the solid arrows indicate the proposed route of transport. The glycolytic end product pyruvate is metabolized during the mitochondrial citrate cycle yielding CO2 which will be hydrated to HCO3− by intracellular CA. HCO3−, in turn, directly activates the sperm specific sAC thereby stimulating PKA and leading to an increase in sperm beat frequency.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3401232&req=5

pone-0041030-g001: Organization of the principle piece of the sperm tail and proposed model for the interplay between pHi, glycolysis and production of HCO3−.A, Shown is a scheme of a cross section through the principle piece. Nine microtubule doublets - each carrying dynein arms - are connected via radial spokes to the central pair (CP), constituting the classical 9×2+2 core structure of the ciliar axoneme. Each microtubule doublet, in turn, is connected to two main longitudinal cytoskeletal structures – doublets 3 and 8 are fastened to the so-called longitudinal columns (LC), whereas doublets 1 and 2 and 4–7 are bound to the outer dense fibers (ODF). The outward facing area of each LC and ODF is tightly connected to the fibrous sheath (FS), a layer located right underneath the plasma membrane. B, Shown is an incomplete section of A, focusing on enzymes located to the FS, in close proximity to dynein ATPase. Glycolytic enzymes are hexokinase (HK), phosphokinase (PK), sperm specific glyceraldehyde 3-phosphate dehydrogenase (GAPDS) and lactate dehydrogenase A (LDHA). Proteins involved in the cAMP/HCO3- metabolism are A-kinase anchoring protein 3 and 4 (AKAP3/4) and testis A-kinase anchoring protein (TAKAP) with binding sites for protein kinase A (PKA). C, This drawing illustrates our working hypothesis. Glucose enters spermatozoa via glucose transporters (GLUT). Once lactate leaves the cell together with protons via monocarboxylate transporters (MCT), pHi rises and glycolysis proceeds intensified. GLUT and MCT transport is bidirectional and the solid arrows indicate the proposed route of transport. The glycolytic end product pyruvate is metabolized during the mitochondrial citrate cycle yielding CO2 which will be hydrated to HCO3− by intracellular CA. HCO3−, in turn, directly activates the sperm specific sAC thereby stimulating PKA and leading to an increase in sperm beat frequency.
Mentions: Once deposited in the vaginal fluid, which is rich in lactic acid, sperm travel through the cervix to enter the uterus where they encounter a fluid which is poor in lactate but rich in glucose and other glycolysable substrates [1]. Besides mitochondrial respiration, glycolysis is a major pathway for ATP production in murine spermatozoa [2], [3], [4]. Glycolytic enzymes are located in the fibrous sheath of the sperm tail [5] and some of them exhibit sperm-specific properties, such as type1 hexokinase (HK1S) [6], pyruvate kinase (PKS) [7] and glyceraldehyde 3-phosphate dehydrogenase (GAPDS) [8]. The fibrous sheath covers the outer dense fibers which, in turn, are located in close proximity to the microtubules. This arrangement permits the ATPases located on the dynein arms of the microtubule doublets (Fig. 1 A and B) to directly utilize the ATP generated during glycolysis to maintain bending of the sperm tail [5]. For mouse spermatozoa, muscle cells, leukocytes, erythrocytes and several other cell types it has been demonstrated that glycolytic key enzymes exhibit pH-dependency [6], [9], [10], [11]. We have previously shown that sperm possesses monocarboxylate/H+ co-transporters whose activity leads to intracellular acidification or alkalinization during application or removal of lactate and pyruvate [2]. With this work, we now investigate the way in which glycolysis is modulated in sperm by intracellular pH (pHi). Whereas the ATP generated during glycolysis helps sperm to maintain the resting beat stable, HCO3− is the only substance known so far in sperm to speed up their flagellar beat [10], [11], enabling them to travel along the uterus to reach the egg in the ampulla. Once inside the sperm cell, HCO3− directly activates the sperm-specific adenylyl cyclase (sAC) [12], [13] which, in turn, raises the intracellular level of cAMP, thereby stimulating protein kinase A (PKA) [14], [15], [16] and initiating the acceleration of beat frequency. Besides the above mentioned glycolytic enzymes the fibrous sheath is also a scaffold for A-kinase anchor protein 3 (AKAP3), A-kinase anchor protein 4 (AKAP4) and testis-specific A-kinase anchor protein 80 (TAKAP-80) (Fig. 1B), all of which contain binding sites for cAMP-dependent protein kinases [17]. Furthermore, AKAP3 of bovine sperm was shown to possess binding sites for phosphodiesterase 4A [18]. The near proximity of enzymes involved in both glycolysis and cAMP/HCO3− metabolism suggests that they together closely regulate sperm motility and beat frequency. HCO3− is available in the uterine fluid [19] and can enter sperm either directly by anion transporters as suggested by [20], [21], [22] or indirectly via carbonic anhydrases (CAs) [23], [24]. CAs catalyze the reversible hydration of CO2 to HCO3− and the majority of CA isoforms already identified is either membrane-bound proteins or existent in the cytoplasm. This raises the possibility that at least some portion of CO2 being produced during mitochondrial respiration is converted to HCO3−. With this work, we show evidence that glycolysis and cell respiration are interconnected for the production of HCO3− via CO2 as an additional way for sperm to regulate flagellar beat frequency pH-dependently during early activation.

Bottom Line: This evokes changes in the lactate content of sperm as well as in the intracellular pH (pH(i)) since sperm possess lactate/proton co-transporters.We show that the glucose-mediated acceleration of flagellar beat and ATP production are hastened by a pH(i) ≥7.1, whereas a pH(i) ≤7.1 leaves both parameters unchanged.Since we observed a diminished rise in beat frequency in the presence of specific inhibitors against carbonic anhydrases, soluble adenylyl cyclase and protein kinase, we suggest that the glucose-mediated effect is linked to CO(2) hydration and thus the production of HCO(3)- by intracellular CA isoforms.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Saarland University, Homburg/Saar, Germany.

ABSTRACT
To reach the egg in the ampulla, sperm have to travel along the female genital tract, thereby being dependent on external energy sources and substances to maintain and raise the flagellar beat. The vaginal fluid is rich in lactate, whereas in the uterine fluid glucose is the predominant substrate. This evokes changes in the lactate content of sperm as well as in the intracellular pH (pH(i)) since sperm possess lactate/proton co-transporters. It is well documented that glycolysis yields ATP and that HCO(3)- is a potent factor in the increase of beat frequency. We here show for the first time a pathway that connects both parts. We demonstrate a doubling of beat frequency in the mere presence of glucose. This effect can reversibly be blocked by 2-deoxy-D-glucose, dichloroacetate and aminooxyacetate, strongly suggesting that it requires both glycolysis and mitochondrial oxidation of glycolytic end products. We show that the glucose-mediated acceleration of flagellar beat and ATP production are hastened by a pH(i) ≥7.1, whereas a pH(i) ≤7.1 leaves both parameters unchanged. Since we observed a diminished rise in beat frequency in the presence of specific inhibitors against carbonic anhydrases, soluble adenylyl cyclase and protein kinase, we suggest that the glucose-mediated effect is linked to CO(2) hydration and thus the production of HCO(3)- by intracellular CA isoforms. In summary, we propose that, in sperm, glycolysis is an additional pH(i)-dependent way to produce HCO(3)-, thus enhancing sperm beat frequency and contributing to fertility.

Show MeSH
Related in: MedlinePlus