Limits...
Role of carbonic anhydrase IV in the bicarbonate-mediated activation of murine and human sperm.

Wandernoth PM, Raubuch M, Mannowetz N, Becker HM, Deitmer JW, Sly WS, Wennemuth G - PLoS ONE (2010)

Bottom Line: We demonstrate murine and human sperm respond to CO(2) with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide.Comparing CA activity in sperm from wild-type and CA IV(-/-) mice we found a 32.13% reduction in total CA activity in the latter.The CA IV(-/-) sperm also have a reduced response to CO(2).

View Article: PubMed Central - PubMed

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

ABSTRACT
HCO(3) (-) is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO(3) (-) containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO(3) (-) stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO(3) (-) enters spermatozoa either directly by anion transport or indirectly via diffusion of CO(2) with subsequent hydration by intracellular carbonic anhydrase (CA). We here show that murine sperm possess extracellular CA IV that is transferred to the sperm surface as the sperm pass through the epididymis. Comparison of CA IV expression by qRT PCR analysis confirms that the transfer takes place in the corpus epididymidis. We demonstrate murine and human sperm respond to CO(2) with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide. Comparing CA activity in sperm from wild-type and CA IV(-/-) mice we found a 32.13% reduction in total CA activity in the latter. The CA IV(-/-) sperm also have a reduced response to CO(2). While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO(2) application, beat frequency of CA IV(-/-) sperm only increases from 3.06±0.20 Hz to 5.29±0.47 Hz. We show, for the first time, a physiological role of CA IV that supplies sperm with HCO(3) (-), which is necessary for stimulation of sAC and hence early activation of spermatozoa.

Show MeSH

Related in: MedlinePlus

Effect of CA-Inhibitors on sperm beat frequency and CA-activity.A, The effect of HCO3− und CO2 on sperm beat frequency. Values shown are mean ± S.E.M. Mouse sperm beat frequency was measured in HS buffer, HSB buffer (containing 15 mM HCO3−) and in HS buffer in the presence of 5% CO2. Mean values are 2.96±0.17 Hz of sperm in HS buffer, 8.48±0.17 Hz of sperm in HSB buffer and 7.94±0.31 Hz for sperm stimulated with CO2. (n = 10). B, Concentration-dependent inhibition of carbonic anhydrase activity was determined by mass spectrometry. The addition of varying EZA concentrations results in a decrease of enzymatic activity of between 35.0% (4.53±0.87 U/ml) for 50 nM EZA and 62.12% (2.72±0.12 U/ml) for 5 µM EZA (n = 6). C, Sperm beat frequency was measured in HS and HSB buffer in the absence or presence of different EZA or AZA concentrations. Resting beat frequency in HS is not influenced by EZA or AZA. In the presence of bicarbonate, the addition of 100 µM EZA decreases sperm beat frequency from 7.60±0.28 Hz to 4.26±0.21 Hz, whereas the addition of 100 µM AZA decreases beat frequency from 7.84±0.27 Hz to 5.13±0.31 Hz (n = 10). D, The cAMP analogon cBIMPS increases sperm beat frequency by acting downstream of carbonic anhydrases. Sperm measured in HS buffer including cBIMPS in the absence or presence of 10 µM ethoxyzolamide (EZA). (n = 10).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2991337&req=5

pone-0015061-g004: Effect of CA-Inhibitors on sperm beat frequency and CA-activity.A, The effect of HCO3− und CO2 on sperm beat frequency. Values shown are mean ± S.E.M. Mouse sperm beat frequency was measured in HS buffer, HSB buffer (containing 15 mM HCO3−) and in HS buffer in the presence of 5% CO2. Mean values are 2.96±0.17 Hz of sperm in HS buffer, 8.48±0.17 Hz of sperm in HSB buffer and 7.94±0.31 Hz for sperm stimulated with CO2. (n = 10). B, Concentration-dependent inhibition of carbonic anhydrase activity was determined by mass spectrometry. The addition of varying EZA concentrations results in a decrease of enzymatic activity of between 35.0% (4.53±0.87 U/ml) for 50 nM EZA and 62.12% (2.72±0.12 U/ml) for 5 µM EZA (n = 6). C, Sperm beat frequency was measured in HS and HSB buffer in the absence or presence of different EZA or AZA concentrations. Resting beat frequency in HS is not influenced by EZA or AZA. In the presence of bicarbonate, the addition of 100 µM EZA decreases sperm beat frequency from 7.60±0.28 Hz to 4.26±0.21 Hz, whereas the addition of 100 µM AZA decreases beat frequency from 7.84±0.27 Hz to 5.13±0.31 Hz (n = 10). D, The cAMP analogon cBIMPS increases sperm beat frequency by acting downstream of carbonic anhydrases. Sperm measured in HS buffer including cBIMPS in the absence or presence of 10 µM ethoxyzolamide (EZA). (n = 10).

Mentions: Figure 4A shows that both bicarbonate and CO2 increase flagellar beat of cauda sperm similarly. Sperm accelerate their beat frequency from 2.96±0.17 to 8.48±0.17 Hz when treated for 5 min with HS medium containing 15 mM bicarbonate. The application of 5% CO2 to sperm in HS medium alone increases the beat frequency within 5 minutes to 7.94±0.31 Hz. The total carbonic anhydrase activity of ∼4×106 cells was determined by mass spectrometry before and after the application of different concentrations of the carbonic anhydrase inhibitor EZA (Fig. 4B). The addition of 50 nM EZA significantly decreases enzymatic activity from 7.01±0.46 U/ml to 4.53±0.87 U/ml (35.38% reduction), 100 nM EZA leads to a highly significant decrease from 6.29±0.57 U/ml to 3.15±0.46 U/ml (49.92% reduction) and 5 µM EZA reduces enzymatic activity from 7.18±0.20 U/ml to 2.72±0.12 U/ml (62.12% reduction). EZA or AZA also produced a dose-dependent reduction in the action of HCO3− on the flagellar beat. Figure 4C shows that after treatment with 5 nM or 500 nM EZA, bicarbonate increases the beat frequency from 2.63±0.15 Hz to 5.71±0.23 Hz and from 2.61±0.15 Hz to 5.01±0.16 Hz. The addition of HCO3− in the presence of 100 µM EZA results in a <1.5-fold increase in sperm beat frequency (from 2.92±0.24 Hz to 4.26±0.21 Hz) only. Treatment with AZA in the same concentrations as EZA shows nearly the same inhibitory effect (5 nM AZA: from 2.63±0.10 Hz to 6.47±0.41 Hz; 500 nM AZA: from 2.68±0.10 Hz to 5.83±0.34 Hz; 100 µM AZA: from 3.01±0.25 Hz to 5.13±0.31 Hz). To examine if EZA has actions downstream of the action of HCO3−, we used cBIMPS to stimulate sperm both in the absence and in the presence of EZA. In the absence of EZA, sperm beat frequency is increased within 10 minutes from 2.32±0.10 Hz to 4.71±0.52 Hz after application of 50 µM cBIMPS (Fig. 4D). Sperm which were treated with 10 µM EZA increase their beat frequency to a similar extent (from 2.24±0.11 Hz to 5.12±0.35 Hz).


Role of carbonic anhydrase IV in the bicarbonate-mediated activation of murine and human sperm.

Wandernoth PM, Raubuch M, Mannowetz N, Becker HM, Deitmer JW, Sly WS, Wennemuth G - PLoS ONE (2010)

Effect of CA-Inhibitors on sperm beat frequency and CA-activity.A, The effect of HCO3− und CO2 on sperm beat frequency. Values shown are mean ± S.E.M. Mouse sperm beat frequency was measured in HS buffer, HSB buffer (containing 15 mM HCO3−) and in HS buffer in the presence of 5% CO2. Mean values are 2.96±0.17 Hz of sperm in HS buffer, 8.48±0.17 Hz of sperm in HSB buffer and 7.94±0.31 Hz for sperm stimulated with CO2. (n = 10). B, Concentration-dependent inhibition of carbonic anhydrase activity was determined by mass spectrometry. The addition of varying EZA concentrations results in a decrease of enzymatic activity of between 35.0% (4.53±0.87 U/ml) for 50 nM EZA and 62.12% (2.72±0.12 U/ml) for 5 µM EZA (n = 6). C, Sperm beat frequency was measured in HS and HSB buffer in the absence or presence of different EZA or AZA concentrations. Resting beat frequency in HS is not influenced by EZA or AZA. In the presence of bicarbonate, the addition of 100 µM EZA decreases sperm beat frequency from 7.60±0.28 Hz to 4.26±0.21 Hz, whereas the addition of 100 µM AZA decreases beat frequency from 7.84±0.27 Hz to 5.13±0.31 Hz (n = 10). D, The cAMP analogon cBIMPS increases sperm beat frequency by acting downstream of carbonic anhydrases. Sperm measured in HS buffer including cBIMPS in the absence or presence of 10 µM ethoxyzolamide (EZA). (n = 10).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991337&req=5

pone-0015061-g004: Effect of CA-Inhibitors on sperm beat frequency and CA-activity.A, The effect of HCO3− und CO2 on sperm beat frequency. Values shown are mean ± S.E.M. Mouse sperm beat frequency was measured in HS buffer, HSB buffer (containing 15 mM HCO3−) and in HS buffer in the presence of 5% CO2. Mean values are 2.96±0.17 Hz of sperm in HS buffer, 8.48±0.17 Hz of sperm in HSB buffer and 7.94±0.31 Hz for sperm stimulated with CO2. (n = 10). B, Concentration-dependent inhibition of carbonic anhydrase activity was determined by mass spectrometry. The addition of varying EZA concentrations results in a decrease of enzymatic activity of between 35.0% (4.53±0.87 U/ml) for 50 nM EZA and 62.12% (2.72±0.12 U/ml) for 5 µM EZA (n = 6). C, Sperm beat frequency was measured in HS and HSB buffer in the absence or presence of different EZA or AZA concentrations. Resting beat frequency in HS is not influenced by EZA or AZA. In the presence of bicarbonate, the addition of 100 µM EZA decreases sperm beat frequency from 7.60±0.28 Hz to 4.26±0.21 Hz, whereas the addition of 100 µM AZA decreases beat frequency from 7.84±0.27 Hz to 5.13±0.31 Hz (n = 10). D, The cAMP analogon cBIMPS increases sperm beat frequency by acting downstream of carbonic anhydrases. Sperm measured in HS buffer including cBIMPS in the absence or presence of 10 µM ethoxyzolamide (EZA). (n = 10).
Mentions: Figure 4A shows that both bicarbonate and CO2 increase flagellar beat of cauda sperm similarly. Sperm accelerate their beat frequency from 2.96±0.17 to 8.48±0.17 Hz when treated for 5 min with HS medium containing 15 mM bicarbonate. The application of 5% CO2 to sperm in HS medium alone increases the beat frequency within 5 minutes to 7.94±0.31 Hz. The total carbonic anhydrase activity of ∼4×106 cells was determined by mass spectrometry before and after the application of different concentrations of the carbonic anhydrase inhibitor EZA (Fig. 4B). The addition of 50 nM EZA significantly decreases enzymatic activity from 7.01±0.46 U/ml to 4.53±0.87 U/ml (35.38% reduction), 100 nM EZA leads to a highly significant decrease from 6.29±0.57 U/ml to 3.15±0.46 U/ml (49.92% reduction) and 5 µM EZA reduces enzymatic activity from 7.18±0.20 U/ml to 2.72±0.12 U/ml (62.12% reduction). EZA or AZA also produced a dose-dependent reduction in the action of HCO3− on the flagellar beat. Figure 4C shows that after treatment with 5 nM or 500 nM EZA, bicarbonate increases the beat frequency from 2.63±0.15 Hz to 5.71±0.23 Hz and from 2.61±0.15 Hz to 5.01±0.16 Hz. The addition of HCO3− in the presence of 100 µM EZA results in a <1.5-fold increase in sperm beat frequency (from 2.92±0.24 Hz to 4.26±0.21 Hz) only. Treatment with AZA in the same concentrations as EZA shows nearly the same inhibitory effect (5 nM AZA: from 2.63±0.10 Hz to 6.47±0.41 Hz; 500 nM AZA: from 2.68±0.10 Hz to 5.83±0.34 Hz; 100 µM AZA: from 3.01±0.25 Hz to 5.13±0.31 Hz). To examine if EZA has actions downstream of the action of HCO3−, we used cBIMPS to stimulate sperm both in the absence and in the presence of EZA. In the absence of EZA, sperm beat frequency is increased within 10 minutes from 2.32±0.10 Hz to 4.71±0.52 Hz after application of 50 µM cBIMPS (Fig. 4D). Sperm which were treated with 10 µM EZA increase their beat frequency to a similar extent (from 2.24±0.11 Hz to 5.12±0.35 Hz).

Bottom Line: We demonstrate murine and human sperm respond to CO(2) with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide.Comparing CA activity in sperm from wild-type and CA IV(-/-) mice we found a 32.13% reduction in total CA activity in the latter.The CA IV(-/-) sperm also have a reduced response to CO(2).

View Article: PubMed Central - PubMed

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

ABSTRACT
HCO(3) (-) is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO(3) (-) containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO(3) (-) stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO(3) (-) enters spermatozoa either directly by anion transport or indirectly via diffusion of CO(2) with subsequent hydration by intracellular carbonic anhydrase (CA). We here show that murine sperm possess extracellular CA IV that is transferred to the sperm surface as the sperm pass through the epididymis. Comparison of CA IV expression by qRT PCR analysis confirms that the transfer takes place in the corpus epididymidis. We demonstrate murine and human sperm respond to CO(2) with an increase in beat frequency, an effect that can be inhibited by ethoxyzolamide. Comparing CA activity in sperm from wild-type and CA IV(-/-) mice we found a 32.13% reduction in total CA activity in the latter. The CA IV(-/-) sperm also have a reduced response to CO(2). While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO(2) application, beat frequency of CA IV(-/-) sperm only increases from 3.06±0.20 Hz to 5.29±0.47 Hz. We show, for the first time, a physiological role of CA IV that supplies sperm with HCO(3) (-), which is necessary for stimulation of sAC and hence early activation of spermatozoa.

Show MeSH
Related in: MedlinePlus