Abstract
HCO3 − is the signal for early activation of sperm motility. In vivo, this occurs when sperm come into contact with the HCO3 − containing fluids in the reproductive tract. The activated motility enables sperm to travel the long distance to the ovum. In spermatozoa HCO3 − stimulates the atypical sperm adenylyl cyclase (sAC) to promote the cAMP-mediated pathway that increases flagellar beat frequency. Stimulation of sAC may occur when HCO3 − enters spermatozoa either directly by anion transport or indirectly via diffusion of CO2 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 CO2 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 CO2. While the beat frequency of wild-type sperm increases from 2.86±0.12 Hz to 6.87±0.34 Hz after CO2 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 HCO3 −, which is necessary for stimulation of sAC and hence early activation of spermatozoa.
Highlights
Post-testicular maturation of sperm in the epidydimis and the female genital tract includes multiple changes in sperm membrane composition and signal transduction [1,2,3,4]
We show that extracellular carbonic anhydrase IV is a key enzyme in the early activation of sperm
In addition we found immunoreactions with carbonic anhydrase (CA) IV antibodies in both the stereocilia network and spermatozoa of cauda epididymids (Fig. 1M/m)
Summary
Post-testicular maturation of sperm in the epidydimis and the female genital tract includes multiple changes in sperm membrane composition and signal transduction [1,2,3,4]. After spermatogenesis sperm travel along the epididymal tract and the female genital tract where they undergo fundamental changes in their motility. Bicarbonate plays a major role in this early activation of spermatozoa and is necessary for successful fertilization [5,6,7]. The activation of soluble adenylyl cyclase (sAC) by bicarbonate induces the elevation of intracellular cAMP and subsequent phosphorylation of several proteins by protein kinase A. We have previously shown that bicarbonate in concentrations of 15 mM induces a reversible, robust acceleration of sperm resting beat frequency from 2–3 Hz to 7 Hz in less than 1 min. Acceleration of beat frequency is accompanied by facilitation of Ca2+ channels and beat symmetry [8]
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