Normal Fertility Requires the Expression of Carbonic Anhydrases II and IV in Sperm

Abstract

HCO3 (-) is a key factor in the regulation of sperm motility. High concentrations of HCO3 (-) in the female genital tract induce an increase in sperm beat frequency, which speeds progress of the sperm through the female reproductive tract. Carbonic anhydrases (CA), which catalyze the reversible hydration of CO2 to HCO3 (-), represent potential candidates in the regulation of the HCO3 (-) homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms, CAII and CAIV, are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find that CAII is located in the principal piece, whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO3 (-) homeostasis, resulting in substantially reduced sperm motility, swimming speed, and HCO3 (-)-enhanced beat frequency. The CA activity remaining in the sperm of CAII- and CAIV-null mutants is 35% and 68% of that found in WT mice. Sperm of the double knockout mutant mice show responses to stimulus by HCO3 (-) or CO2 that were delayed in onset and reduced in magnitude. In comparison with sperm from CAII and CAIV double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO3 (-). These results suggest that CAII and CAIV are required for optimal fertilization.

Keywords: bicarbonate; carbon dioxide; carbonic anhydrase; fertilization; mutant; sperm; subfertility.

FIGURE 1.

Distribution of CAII and CAIV in the male reproductive tract. A and B, double immunohistochemical staining (A) and immunoblots (B) were performed with WT (+/+) and CAII^−/− CAIV^−/− (−/−) samples. Tissue slices were stained with DAB for CAII (brown) and HistoRed for CAIV (red) signals. CAII is present in elongated spermatids, epididymal spermatozoa, single epithelial cells of the caput and the cauda epididymidis, and nearly all epithelial cells of the corpus epididymidis. CAIV is localized in the stereocilia network of the corpus and the cauda epididymidis as well as in luminal sperm after passing the corpus region. In Western blot analyses, CAII (28 kDa) is detectable in the testis, all parts of the epididymidis, and cauda sperm. A specific CAIV band at 38 kDa is only present in the corpus and the cauda epididymidis as well as in sperm. Neither CAII nor CAIV are detectable in any of the double knockout tissues or in the protein samples, which served as control (Scale bars = 50 μm (a–h) and 10 μm (insets).

FIGURE 2.

CAII and CAIV protein detection changes with puberty in the male reproductive tract. A, CAII protein detection by immunohistochemical staining appears with the onset of puberty (5 weeks) in elongated spermatids, single epithelial cells, and the stereocilia network of the caput, corpus, and cauda epididymidis. B, CAIV immunoreactivity is localized in 3-week-old mice only in the stereocilia network of the corpus epididymidis but not detectable in the testis, caput, and cauda epididymidis. In tissues from pubescent mice, CAIV is present in the corpus and cauda epididymidis (Scale bars = 25 μm).

FIGURE 3.

CAII and CAIV have distinct localizations in murine spermatozoa. A–D, double immunofluorescence on fixed WT sperm indicates a CAII signal (green) in the principal piece of the sperm tail, whereas CAIV (red) is present in the acrosome and the plasma membrane of the entire sperm tail, predominantly in the mid-piece. CAII^−/− CAIV^−/− sperm did not show any signal (data not shown). Nuclei were stained with DAPI. Scale bar = 10 μm.

FIGURE 4.

CAII and CAIV activity in sperm and expression levels in the male reproductive tract of WT, CAII^−/−, and CAIV^−/− mice. A, CA activity was measured with mass spectrometry. In comparison with WT sperm, the deletion of CAII or CAIV leads to a reduction in activity of 32% and 65%, respectively. CA activity in CAII^−/− CAIV^−/− mice is 0.7 units/ml (± 0.02), which equals a reduction of 86.9%. Data are mean ± S.E. of six or more measurements from three independent experiments. The asterisks refer to the values of the bar for WT. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001. B, analysis of the single-sperm beat frequency response to 15 mm HCO₃⁻. CAII^−/− and CAIV^−/− sperm display a delayed and reduced acceleration of beat frequency. Data are mean ± S.E. of ≥40 single sperm. C, expression of CAII and CAIV mRNA in WT tissue detected by qRT-PCR. D and E, in relation to WT tissue, CAIV is overexpressed in the caput, corpus, and cauda epididymidis (epi.) of CAII^−/− mice (D), whereas CAII expression is highly increased in the cauda epididymidis and vas deferens (vas def.) of CAIV^−/− animals (E) (n = 3).

FIGURE 5.

Phenotype of CAII^−/− CAIV^−/− offspring. A, adult CAII^−/− CAIV^−/− mice show a significant reduction in body and testis weight in comparison with WT mice (n = 4). The asterisks refer to the values for animal and testis weight of the bar for WT. **, p ≤ 0.01. B, no change is detectable in the steady-state pH_i of isolated sperm from WT and double knockout animals (n ≥ 12). C, cross-sections through WT (top) and CAII^−/− CAIV^−/− (bottom) testis indicate a thinner germ epithelium in double knockout testis. Representative sections are shown. D, the analysis of 130 tubules of three different WT and double knockout testes displays a reduction of 20% of the germ cell epithelium in CAII^−/− CAIV^−/− mice.

FIGURE 6.

Basal pH level and ability to capacitate of CAII^−/− CAIV^−/− sperm is not altered. A, immunoblots were performed with protein extracts from untreated or capacitated sperm from WT (+/+) and CAII^−/− CAIV^−/− (−/−) mice. CAII^−/−CAIV^−/− sperm do not show any changes in the protein phosphorylation pattern induced by capacitation (Cap) compared with WT sperm. B, CAII^−/− CAIV^−/− sperm do not show any defects in intracellular alkalization upon stimulation with ammonium chloride. Sperm of both WT (black trace) and CAII^−/− CAIV^−/− (gray trace) mice were perfused continuously with HS buffer and 10 mm ammonium chloride as indicated. Shown are averaged normalized traces of 34 WT and 39 CAII^−/− CAIV^−/− sperm of two animals, respectively. A.U., arbitrary units. C, CAII^−/− sperm show a reduced CAIV protein level, whereas the amount of CAII protein in sperm from CAIV^−/− mice corresponds to that of WT mice.

FIGURE 7.

CAII^−/− CAIV^−/− mice are subfertile and display reduced sperm motility. A, CASA analyses were performed to characterize the sperm population of WT, CAII^−/−, and CAII^−/− CAIV^−/− mice. Velocity and motility are reduced in CAII^−/− and in CAII^−/− CAIV^−/− sperm, whereas the percentage of immotile sperm is increased significantly. Sperm concentrations (sperm conc.) are not affected. Data are mean ± S.E. of seven or more measurements from three independent experiments. *, p ≤ 0.05; ***, p ≤ 0.001. B, offspring analysis from mating pairs indicated as type 1, 2, 3, and 4. The offspring per week of mating were calculated from total mating time and number of live offspring. Both CAII/CAIV double knockout sexes indicate a significant reduced offspring per week.

FIGURE 8.

Analysis of sperm beat frequency on a single-cell level. A and B, sperm of WT, single CAII^−/−, and CAII^−/− CAIV^−/− animals were stimulated with 15 mm HCO₃⁻ (A) or 2% CO₂ (B). With both protocols, CAII^−/− sperm display the strongest delayed and reduced beat frequency increase compared with CAII^−/− CAIV^−/− and WT sperm. C and D, biochemical CAIV protein elimination by PLC treatment of WT sperm was tested successfully by DAB staining (C, scale bar = 4 μm) and Western blot analysis (D). E, in single-cell analysis, PLC-treated WT (CAIV^BC−) and genetic CAIV^−/− sperm (CAIV^{−/−/BC−}) display the same response upon HCO₃⁻ stimulation. F and G, shown is the response of genetic CAII^−/− and CAII^−/− CAIV^BC− double knockout sperm to HCO₃⁻ (F) and CO₂ (G). The beat frequency of CAII^−/− CAIV^BC− sperm increases only to a maximum of 4.16 ± 0.15 Hz with HCO₃⁻ and of 3.87 ± 0.19 Hz with CO₂ application (t = 80 s) and displays the weakest response. Data are mean ± S.E. of ≥17 single sperm.