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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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Immunoblot and real-time PCR of CA IV.A, Immunoblot of CA IV. A CA IV signal in the range of 38 kDa is present in wild-type corpus and cauda epididymidis and vas deferens. No specific CA IV band is detectable in wild-type testis and caput epididymidis or in any of the CA IV−/− tissues. B, Analysis of sperm protein fractions isolated from the different sections of the epididymidis shows a positive signal in corpus and cauda sperm and sperm from vas deferens. No specific signal is present in wild-type caput sperm or in any of the CA IV−/− sperm. C, CA IV is present in the whole vas deferens tissue and not present in the flushed vas deferens. With the luminal content only a specific CA IV band can be seen. D, kidney and brain tissue were used as positive control. E, CA IV qRT PCR analysis of wild-type and CA IV−/− mice. The diagram shows mean RQ values ± s.e.m. of three independent experiments for each tissue. In relation to wild-type kidney (calibrator) the RQ value of wild-type corpus epididymidis averages at 0.54. No CA IV mRNA is detectable in the other wild-type or in any of the CA IV−/− tissues (n = 3).of wild-type and CA IV−/− mice.
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pone-0015061-g002: Immunoblot and real-time PCR of CA IV.A, Immunoblot of CA IV. A CA IV signal in the range of 38 kDa is present in wild-type corpus and cauda epididymidis and vas deferens. No specific CA IV band is detectable in wild-type testis and caput epididymidis or in any of the CA IV−/− tissues. B, Analysis of sperm protein fractions isolated from the different sections of the epididymidis shows a positive signal in corpus and cauda sperm and sperm from vas deferens. No specific signal is present in wild-type caput sperm or in any of the CA IV−/− sperm. C, CA IV is present in the whole vas deferens tissue and not present in the flushed vas deferens. With the luminal content only a specific CA IV band can be seen. D, kidney and brain tissue were used as positive control. E, CA IV qRT PCR analysis of wild-type and CA IV−/− mice. The diagram shows mean RQ values ± s.e.m. of three independent experiments for each tissue. In relation to wild-type kidney (calibrator) the RQ value of wild-type corpus epididymidis averages at 0.54. No CA IV mRNA is detectable in the other wild-type or in any of the CA IV−/− tissues (n = 3).of wild-type and CA IV−/− mice.

Mentions: Figure 2A shows immunoblots for protein extracts of wild type caput, corpus, and cauda epididymidis and the vas deferens. A single ∼38 kDa immunoreactive CA IV band is detectable in corpus, cauda epididymidis and the vas deferens but is absent in caput epididymidis and whole testis. No signal is detected in tissue from CA IV−/− mice. For wild type mice, extracts of corpus and cauda sperm and sperm from vas deferens show a prominent 38 kDa immunoreactive CA IV band (Fig. 2B). A small signal is detectable in caput sperm. CA IV−/− sperm do not show any CA IV signal. Tissue of flushed vas deferens and sperm were examined separately and the results demonstrate, that CA IV is localized in luminal sperm only (Fig. 2C). Kidney and brain were used as positive controls (Fig. 2D).


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)

Immunoblot and real-time PCR of CA IV.A, Immunoblot of CA IV. A CA IV signal in the range of 38 kDa is present in wild-type corpus and cauda epididymidis and vas deferens. No specific CA IV band is detectable in wild-type testis and caput epididymidis or in any of the CA IV−/− tissues. B, Analysis of sperm protein fractions isolated from the different sections of the epididymidis shows a positive signal in corpus and cauda sperm and sperm from vas deferens. No specific signal is present in wild-type caput sperm or in any of the CA IV−/− sperm. C, CA IV is present in the whole vas deferens tissue and not present in the flushed vas deferens. With the luminal content only a specific CA IV band can be seen. D, kidney and brain tissue were used as positive control. E, CA IV qRT PCR analysis of wild-type and CA IV−/− mice. The diagram shows mean RQ values ± s.e.m. of three independent experiments for each tissue. In relation to wild-type kidney (calibrator) the RQ value of wild-type corpus epididymidis averages at 0.54. No CA IV mRNA is detectable in the other wild-type or in any of the CA IV−/− tissues (n = 3).of wild-type and CA IV−/− mice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991337&req=5

pone-0015061-g002: Immunoblot and real-time PCR of CA IV.A, Immunoblot of CA IV. A CA IV signal in the range of 38 kDa is present in wild-type corpus and cauda epididymidis and vas deferens. No specific CA IV band is detectable in wild-type testis and caput epididymidis or in any of the CA IV−/− tissues. B, Analysis of sperm protein fractions isolated from the different sections of the epididymidis shows a positive signal in corpus and cauda sperm and sperm from vas deferens. No specific signal is present in wild-type caput sperm or in any of the CA IV−/− sperm. C, CA IV is present in the whole vas deferens tissue and not present in the flushed vas deferens. With the luminal content only a specific CA IV band can be seen. D, kidney and brain tissue were used as positive control. E, CA IV qRT PCR analysis of wild-type and CA IV−/− mice. The diagram shows mean RQ values ± s.e.m. of three independent experiments for each tissue. In relation to wild-type kidney (calibrator) the RQ value of wild-type corpus epididymidis averages at 0.54. No CA IV mRNA is detectable in the other wild-type or in any of the CA IV−/− tissues (n = 3).of wild-type and CA IV−/− mice.
Mentions: Figure 2A shows immunoblots for protein extracts of wild type caput, corpus, and cauda epididymidis and the vas deferens. A single ∼38 kDa immunoreactive CA IV band is detectable in corpus, cauda epididymidis and the vas deferens but is absent in caput epididymidis and whole testis. No signal is detected in tissue from CA IV−/− mice. For wild type mice, extracts of corpus and cauda sperm and sperm from vas deferens show a prominent 38 kDa immunoreactive CA IV band (Fig. 2B). A small signal is detectable in caput sperm. CA IV−/− sperm do not show any CA IV signal. Tissue of flushed vas deferens and sperm were examined separately and the results demonstrate, that CA IV is localized in luminal sperm only (Fig. 2C). Kidney and brain were used as positive controls (Fig. 2D).

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