Role of Carbonic Anhydrase IV in the Bicarbonate-Mediated Activation of Murine and Human Sperm

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

Glucose is a pH-Dependent Motor for Sperm Beat Frequency during Early Activation

<div><p>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 HCO₃− 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 CO₂ hydration and thus the production of HCO₃− by intracellular CA isoforms. In summary, we propose that, in sperm, glycolysis is an additional pH_i-dependent way to produce HCO₃−_, thus enhancing sperm beat frequency and contributing to fertility.</p> </div

Glucose as energy substrate increases sperm beat frequency dramatically.

<p>Sperm were continuously perifused for 20 min with buffer HS containing different energy substrates (10 mM lactate, 10 mM pyruvate and 5 mM glucose) as indicated. Beat frequency of single sperm was analyzed at t  = 0 min (white bars), t  = 20 min (gray bars) and t  = 60 min (black bars). Shown are mean values±s.e. of 35 cells of 3 animals. With no energy substrate present, flagellar beat declines significantly compared to t  = 0 min (***p<0.001). With glucose as the mere substrate, beat acceleration is significant compared to t  = 20 min and 60 min (***p<0.001) of all other substrate compositions.</p

The enhancing effect of glucose on sperm beat frequency can be diminished with specific inhibitors against CAs, sAC and PKA. A,

<p>Sperm were initially perifused with 10 mM lactate. Then, perifusion was switched for 10 min to buffer HS either containing 0.1 mM EZA (solid line and filled circles) or AZA (dashed line and open squares) with the additional presence of 5 mM glucose. Subsequently, sperm were stimulated for 20 min with 5 mM glucose only. In the control measurements (gray plot), sperm were stimulated the same way except for omitting CA inhibitors. Significant changes were analyzed relating to t  = 0 min and accounted for ***p<0.001 at t ≥10 min under inhibiting and for ***p<0.001 at t ≥5 min in the control. B, Sperm were initially perifused with 10 mM lactate followed by application of 50 µM 2'OH-E for 10 min. For the next 20 min, sperm were stimulated with 50 µM 2'OH-E and 5 mM glucose. Subsequently, perifusion was switched for another 20 min to buffer HS containing 5 mM glucose only (black plot). Under inhibiting conditions, values were significantly increased (***p<0.001) at t ≥20 min and in the control at t ≥5 min. C, After perifusing with lactate, sperm were stimulated with 12.5 µM KH7 and 5 mM glucose for 20 min, and with 5 mM glucose only for another 20 min (black plot). In the respective control measurements (gray plots), sperm were treated the same way except for omitting sAC inhibitors. Significant changes were analyzed relating to t  = 0 min and averaged at **p<0.05 (t ≥10 min) and at ***p<0.001 (t ≥30 min) under inhibiting conditions. In the control, significance values of **p<0.05 at t  = 5 min and of ***p<0.001 at t ≥10 min were apparent. D, The same perifusion protocol was used as in A, whereupon 7.5 µM of the PKA inhibitor H89 was used instead of EZA and AZA (black plot). For control measurements (gray plot), sperm were perifused the same way except for omitting H89. Significant changes were analyzed relating to t  = 0 min of the respective plot with ***p<0.001 at t ≥10 min under inhibiting conditions and ***p<0.001 at t ≥10 min in the control. In each plot mean values±s.e. of 35 cells of 3 animals are shown.</p

2-deoxy-D-glucose (2DG) cannot mimic the effect of glucose on sperm beat frequency.

<p><b>A</b>, Sperm were initially perifused with buffer HS containing 10 mM lactate. During subsequent 20 min, perifusion was switched to buffer containing 5 mM glucose (filled circles) or 1.3 mM 2DG (open circles) as energy source. For control measurements, sperm remained in lactate-containing buffer (filled squares). Significant changes were analyzed relating to t  = 0. For clarity, statistical symbols are omitted in some of the following figures but explained in the respective figure legend. At t ≥5 min, values were significantly increased (***p<0.001) during application of glucose as well as at t ≥15 min during application of 2DG. <b>B</b>, Sperm were first perifused with buffer HS containing 10 mM lactate only. As indicated, sperm were then stimulated for 10 min with 1.3 mM 2DG followed by another 20-min treatment with 5 mM glucose. Significance was analyzed relating to t  = 0 min and values were significantly different at t ≥15 min (***p<0.001). Each plot represents mean values±s.e. of 36 cells of 3 animals.</p

Lactate and pyruvate reversibly prevent the enhancing effect of glucose on sperm beat frequency.

<p><b>A</b>, Sperm were exposed to 5 mM glucose for 40 min. During the first 5 min and the last 15 min, either 10 mM lactate (black plot) or 10 mM pyruvate (gray plot) were co-applicated by continuous perifusion. Significant changes were analyzed relating to t  = 0. At t  = 5, 10, 15, 20 and 25 min, values were significantly increased (***p<0.001) in both plots compared to t  = 0 min. <b>B</b>, Primarily, sperm were locally perifused with buffer HS containing 10 mM lactate. Then, stimulation occurred in the presence of 15 mM propionate (black plot) or 15 mM MA (gray plot) for 10 min, followed by another 20 min stimulation with 5 mM glucose. Compared to t  = 0 min, the values were significantly increased at t ≥15 min (***p<0.001) after removal of propionate or MA. The plots of <b>A</b> and <b>B</b> represent mean values±s.e. of 41 cells of 3 animals. <b>C</b>, Sperm were perifused for 100 s with buffer HS containing 10 mM lactate before being stimulated with 15 mM MA (dashed line) or 5 mM glucose (dotted line) for 150 s. After stimulation, cells were again bathed for 200 s in lactate-containing buffer solution. <b>D</b>, After a 100 s-treatment with 5 mM glucose, cells were stimulated for 200 s with 15 mM propionate (solid line), 10 mM pyruvate (dashed line) and 10 mM lactate (dotted line) before perifusion was switched back to lactate-containing buffer for another 200 s. The plots of <b>C</b> and <b>D</b> represent mean values of 25–33 cells of 3 animals. Mean slopes in <b>c</b> and <b>d</b> were analyzed during the first 50 s during application of the different stimulatory substrates and are presented as intracellular pH s-1.</p

Organization of the principle piece of the sperm tail and proposed model for the interplay between pH_i, glycolysis and production of HCO₃−.

<p><b>A</b>, 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>B</b>, Shown is an incomplete section of <b>A</b>, 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). <b>C</b>, 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), pH_i 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.</p

Methylamine (MA) hastens and propionic acid inhibits the enhancing effect of glucose on sperm beat frequency.

<p><b>A</b>, Sperm were exposed for 100 s to 10 mM lactate before being stimulated for 150 s with 5 mM glucose and 15 mM MA. Subsequently, cells were allowed to recover for 200 s in lactate-containing buffer. <b>B</b>, Cells were treated for 500 s with 5 mM glucose of which propionate was additionally present during 200 s as indicated. The plots of <b>A</b> and <b>B</b> show mean values of 25–33 cells of 3 animals. Mean slopes (in <b>a</b> and <b>b</b>) were analyzed as described in the previous figure legend. <b>C</b>, After primary perifusion with 10 mM lactate, sperm were perifused for 30 min with buffer HS containing 5 mM glucose of which 15 mM MA (black plot) or 15 mM propionate (gray plot) were concurrently present first 10 min (t  = 0 min to t = 10 min). Significant changes were analyzed relating to t  = 0 min and apparent at t ≥5 min (co-application of MA) and at t ≥15 min (after removal propionate) with ***p<0.001. Shown are mean values±s.e. of 40 cells of 3 animals.</p

ATP and NDH/NADPH production is dependent on the substrate and the pH_i.

<p><b>A</b>, Sperm prepared as described in the Materials and Methods section were transferred to buffer HS which was supplemented with 1.3 mM 2DG (filled triangles), 10 mM lactate (filled circles), 10 mM lactate +15 mM MA (filled squares), 5 mM glucose (open circles) or 5 mM glucose +15 mM MA (open triangles). The ATP content was determined at t  = 0, 5, 10, 20 and 30 min. Each line represents mean values±s.e. of 4 independent experiments. Significant changes were analyzed relating to t  = 0 min of the respective plot with **p<0.05 and ***p<0.001. <b>B</b>, At t  = 0, sperm were starved out and directly homogenized in HS buffer with pH 6.8 or pH 7.1. After starvation, sperm were further incubated in buffer containing energy substrates as indicated before being homogenized. Absorption at 340 nm was measured over 200 s. Each bar represents mean values±s.e. of 2 independent experiments. *p<0.1 and ***p<0.001.</p