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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.

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Sperm beat frequency and CA-activity in CA −/− mice.A, Enzymatic activity of CA measured by mass spectrometry. Mean value of enzymatic activity of wild type sperm, is 5.26±0.34 U/ml, whereas sperm of CA IV−/− mice show activity of 3.57±0.25 U/ml only (n = 6). B, Sperm of CA IV−/− mice show a decreased response to HCO3−. Within the first 20 seconds after the addition of bicarbonate, the beat frequency of wild-type sperm (solid line) increases from 2.81±0.17 Hz to 6.13±0.35 Hz and frequency of CA IV−/− sperm from 2.92±0.17 Hz to 5.57±0.18 Hz (n = 10). C, Sperm of CA IV−/− mice show a decreased response to CO2. Through the application of 2% CO2 in the measuring chamber and the manifolds wild-type sperm beat frequency (solid line) increase within the first 20 seconds from 2.86±0.12 Hz to 6.87±0.34 Hz, while the beat frequency of CA IV−/− sperm takes 1 min to rise from 3.06±0.20 Hz to 5.29±0.47 Hz. (n = 10). D, Sperm of CA IV−/− mice show the same response to the cAMP analogon cBIMPS as wild-type sperm. 20 seconds after stimulation with 5% CO2, the sperm of CA IV−/− mice show a 20.48% reduction in beat frequency compared to wild-type sperm. By contrast, after 10 minutes stimulation with 50 µM cBIMPS wild-type and CA IV−/− sperm speed their beat to comparable values (from 2.89±0.16 Hz to 5.71±0.24 Hz and from 2.80±0.15 Hz to 5.83±0.30 Hz, respectively)(n = 10).
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pone-0015061-g007: Sperm beat frequency and CA-activity in CA −/− mice.A, Enzymatic activity of CA measured by mass spectrometry. Mean value of enzymatic activity of wild type sperm, is 5.26±0.34 U/ml, whereas sperm of CA IV−/− mice show activity of 3.57±0.25 U/ml only (n = 6). B, Sperm of CA IV−/− mice show a decreased response to HCO3−. Within the first 20 seconds after the addition of bicarbonate, the beat frequency of wild-type sperm (solid line) increases from 2.81±0.17 Hz to 6.13±0.35 Hz and frequency of CA IV−/− sperm from 2.92±0.17 Hz to 5.57±0.18 Hz (n = 10). C, Sperm of CA IV−/− mice show a decreased response to CO2. Through the application of 2% CO2 in the measuring chamber and the manifolds wild-type sperm beat frequency (solid line) increase within the first 20 seconds from 2.86±0.12 Hz to 6.87±0.34 Hz, while the beat frequency of CA IV−/− sperm takes 1 min to rise from 3.06±0.20 Hz to 5.29±0.47 Hz. (n = 10). D, Sperm of CA IV−/− mice show the same response to the cAMP analogon cBIMPS as wild-type sperm. 20 seconds after stimulation with 5% CO2, the sperm of CA IV−/− mice show a 20.48% reduction in beat frequency compared to wild-type sperm. By contrast, after 10 minutes stimulation with 50 µM cBIMPS wild-type and CA IV−/− sperm speed their beat to comparable values (from 2.89±0.16 Hz to 5.71±0.24 Hz and from 2.80±0.15 Hz to 5.83±0.30 Hz, respectively)(n = 10).

Mentions: In comparison to wild type sperm, the enzymatic activity of CA IV−/− animals is decreased highly significantly by 32.13% (from 5.26±0.34 U/ml to 3.57±0.25 U/ml) (Fig. 7A). Waveform analysis was performed to analyze the effect of HCO3− and CO2 on the beat frequency of CA IV−/− sperm (Fig. 7B, C). Fig. 7B shows that there is only a slight difference in the first 20 seconds between the sperm of wild-type and CA IV−/− animals in the response to HCO3−. Perfusion of sperm of wild-type and CA IV−/− animals with buffer containing 15 mM HCO3− leads to an increase of sperm beat frequency within 20 seconds from 2.81±0.17 Hz to 6.13±0.35 Hz and from 2.92±0.17 Hz to 5.57±0.18 Hz, respectively (n = 10).


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)

Sperm beat frequency and CA-activity in CA −/− mice.A, Enzymatic activity of CA measured by mass spectrometry. Mean value of enzymatic activity of wild type sperm, is 5.26±0.34 U/ml, whereas sperm of CA IV−/− mice show activity of 3.57±0.25 U/ml only (n = 6). B, Sperm of CA IV−/− mice show a decreased response to HCO3−. Within the first 20 seconds after the addition of bicarbonate, the beat frequency of wild-type sperm (solid line) increases from 2.81±0.17 Hz to 6.13±0.35 Hz and frequency of CA IV−/− sperm from 2.92±0.17 Hz to 5.57±0.18 Hz (n = 10). C, Sperm of CA IV−/− mice show a decreased response to CO2. Through the application of 2% CO2 in the measuring chamber and the manifolds wild-type sperm beat frequency (solid line) increase within the first 20 seconds from 2.86±0.12 Hz to 6.87±0.34 Hz, while the beat frequency of CA IV−/− sperm takes 1 min to rise from 3.06±0.20 Hz to 5.29±0.47 Hz. (n = 10). D, Sperm of CA IV−/− mice show the same response to the cAMP analogon cBIMPS as wild-type sperm. 20 seconds after stimulation with 5% CO2, the sperm of CA IV−/− mice show a 20.48% reduction in beat frequency compared to wild-type sperm. By contrast, after 10 minutes stimulation with 50 µM cBIMPS wild-type and CA IV−/− sperm speed their beat to comparable values (from 2.89±0.16 Hz to 5.71±0.24 Hz and from 2.80±0.15 Hz to 5.83±0.30 Hz, respectively)(n = 10).
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pone-0015061-g007: Sperm beat frequency and CA-activity in CA −/− mice.A, Enzymatic activity of CA measured by mass spectrometry. Mean value of enzymatic activity of wild type sperm, is 5.26±0.34 U/ml, whereas sperm of CA IV−/− mice show activity of 3.57±0.25 U/ml only (n = 6). B, Sperm of CA IV−/− mice show a decreased response to HCO3−. Within the first 20 seconds after the addition of bicarbonate, the beat frequency of wild-type sperm (solid line) increases from 2.81±0.17 Hz to 6.13±0.35 Hz and frequency of CA IV−/− sperm from 2.92±0.17 Hz to 5.57±0.18 Hz (n = 10). C, Sperm of CA IV−/− mice show a decreased response to CO2. Through the application of 2% CO2 in the measuring chamber and the manifolds wild-type sperm beat frequency (solid line) increase within the first 20 seconds from 2.86±0.12 Hz to 6.87±0.34 Hz, while the beat frequency of CA IV−/− sperm takes 1 min to rise from 3.06±0.20 Hz to 5.29±0.47 Hz. (n = 10). D, Sperm of CA IV−/− mice show the same response to the cAMP analogon cBIMPS as wild-type sperm. 20 seconds after stimulation with 5% CO2, the sperm of CA IV−/− mice show a 20.48% reduction in beat frequency compared to wild-type sperm. By contrast, after 10 minutes stimulation with 50 µM cBIMPS wild-type and CA IV−/− sperm speed their beat to comparable values (from 2.89±0.16 Hz to 5.71±0.24 Hz and from 2.80±0.15 Hz to 5.83±0.30 Hz, respectively)(n = 10).
Mentions: In comparison to wild type sperm, the enzymatic activity of CA IV−/− animals is decreased highly significantly by 32.13% (from 5.26±0.34 U/ml to 3.57±0.25 U/ml) (Fig. 7A). Waveform analysis was performed to analyze the effect of HCO3− and CO2 on the beat frequency of CA IV−/− sperm (Fig. 7B, C). Fig. 7B shows that there is only a slight difference in the first 20 seconds between the sperm of wild-type and CA IV−/− animals in the response to HCO3−. Perfusion of sperm of wild-type and CA IV−/− animals with buffer containing 15 mM HCO3− leads to an increase of sperm beat frequency within 20 seconds from 2.81±0.17 Hz to 6.13±0.35 Hz and from 2.92±0.17 Hz to 5.57±0.18 Hz, respectively (n = 10).

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