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

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.

. These results suggest that CAII and CAIV are required for optimal fertilization.
Post-testicular sperm undergo a multitude of maturation processes to acquire fertility in terms of penetrating the egg and generating a new unique individual. Sperm are transcriptionally and translationally silent. Therefore, the multiple physiological and biochemical modifications (1-5) must result from their interaction with the different environments through which they migrate (6 -8). In the epididymis, including the caput, corpus, and cauda epididymides, a low bicarbonate (HCO 3 Ϫ ) concentration and an acidic pH of the luminal fluid are important for sperm maturation, storage, and fertility (9 -11). Segment-specific gene expression patterns of acid base transport proteins in epithelial cells (12)(13)(14) modulate the distinct fluid composition, which undergoes considerable changes along the epididymal duct (15). Several ion channels and transporters, such as cystic fibrosis transmembrane conductance regulator, SLC26A3/A6, and sodium bicarbonate exchanger, as well as V-ATPases have been proposed to produce the acidic and low HCO 3 Ϫ -concentrated fluid composition (7,(15)(16)(17). Carbonic anhydrases (CAs) 2 have also been identified for HCO 3 Ϫ resorption in the epididymis (18 -21). Extracellular CA isoforms convert HCO 3 Ϫ into CO 2 which can then diffuse into the epididymal epithelial cells. Upon entry, CO 2 can be reconverted into HCO 3 Ϫ by intracellular CAs (22,23). At the basolateral membrane, HCO 3 Ϫ is then extruded by either AE2 and or sodium bicarbonate cotransporters (24,25). Disturbance in the epididymal endothelium of the acid base homeostasis impairs sperm maturation processes and potentially causes male infertility (26).
Ejaculated sperm are not able to penetrate and fertilize the egg in vivo. They have to mature in the female genital tract. Chang (27) and Austin (28) discovered this essential process, termed capacitation, which includes strictly regulated and complex biochemical changes (4,5). Female genital tract fluids are rich in HCO 3 Ϫ and Ca 2ϩ and exhibit an alkaline pH, supporting capacitation (8,29). With regard to sperm motility, the capacitation process comprises major changes of the sperm beating pattern (30). Early HCO 3 Ϫ -mediated events (31) produce a fast, symmetrical flagellar beat and rapid progressive movement that allows sperm to travel the long distance through the uterus and the oviduct. Later events include the hyperactivated motility pattern characterized by high amplitude and asymmetrical flagellar beating, representing an increased torsional force (11,(32)(33)(34). Earlier work has proposed that hyperactivation may be the decisive factor in the release of sperm from the oviductal reservoir through detachment from the epithelium (35,36). Our own findings indicate that sperm attachment and release may be independent of hyperactivation (37). * This study was supported by Deutsche Forschungsgemeinschaft Grant WE The pathway for HCO 3 Ϫ -evoked signaling in sperm is relatively well understood. This work focuses on the early HCO 3 Ϫinduced signaling pathway. The downstream effects of HCO 3 Ϫ have already been well described. Intracellular HCO 3 Ϫ directly activates the soluble adenylyl cyclase, therefore increasing cAMP levels and activating PKA. This leads to increased protein tyrosine phosphorylation and an accelerated beat frequency (38 -41). The mechanisms that initiate responses to HCO 3 Ϫ in sperm have remained elusive. It has been proposed, but not shown, that the Na ϩ /HCO 3 Ϫ co-transporter NBC and the anion transporter SLC26A3/A6 are involved in the import of HCO 3 Ϫ into sperm (42)(43)(44). It has also been suggested that CAs are involved in HCO 3 Ϫ homeostasis because these enzymes catalyze the reversible reaction of CO 2 to HCO 3 Ϫ (45,46). This work focuses on the earliest events in HCO 3 Ϫ signaling in sperm.
So far, 16 CA isoforms have been identified (47), of which CAII and CAIV have been shown to be up-regulated during spermatogenesis (12,48). CAIV is an extracellular glycosylphosphatidylinositol-anchored protein (49) whose involvement in the generation of HCO 3 Ϫ in murine spermatozoa we have already established (50). That work showed that sperm of CAIV knockout mice display an HCO 3 Ϫ disequilibrium at the cell surface and show a delayed and reduced increase of beat frequency in response to stimulation with HCO 3 Ϫ and CO 2 . In this work, we determine the distribution and functional aspects of the CAII isoform as an intracellular counterpart to CAIV.
We show the presence of CAII in the epithelium lining, the epididymal duct, and the sperm tail. Specifically, we show that, in sperm lacking CAII, acceleration of the flagellar beat by HCO 3 Ϫ and CO 2 is delayed and reduced in magnitude. CAII and CAIV together contribute to almost 100% of total CA activity in sperm. CAII/CAIV double knockout mice display subfertility as well as reduced sperm motility. Our studies further reveal that, to uphold the HCO 3 Ϫ -induced rise in beat frequency, genetic double knockout sperm develop a compensatory mechanism. In conclusion, CAII and CAIV are key enzymes in the regulation of sperm motility and, therefore, essential for male fertility.
Animals, Phenotyping, and Fertility Analysis of CAII CAIV Double Knockout Mice-WT C57BL6/J and CAII knockout B6.D2-Car2 n /J (CAII Ϫ/Ϫ ) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). CAIV knockout B6.129S1-Car4 tm1Sly/J (CAIV Ϫ/Ϫ ) animals were provided by the laboratory of William S. Sly (Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, MO). Because of different chromosomal locations of the CAII (chromosome 8) and CAIV (chromosome 17) genes (51), CAII/CAIV double knockout (CAII Ϫ/Ϫ CAIV Ϫ/Ϫ ) animals were generated in accordance with approved protocols (no. 02/2011) by intercrossing individual heterozygous mice. According to Mendelian law, the probability of obtaining double knockout offspring is 6.25% at the F2 generation. For phenotype analysis of double knockout offspring, mutant mice were weighed once per week from day 21 on, body size was measured at the adult life stage, and organ weight of kidney and testis was determined and compared with WT mice. For further analysis, WT and double knockout testes were combined, embedded in paraffin, and used to study germ cell epithelia. For hematoxylin and eosin-stained testis, slices were examined with a bright-field microscope (Diaphot 300, Zeiss, Jena, Germany), and individual tubuli seminiferi contorti were documented. The thickness of germ cell epithelia was determined with Adobe Photoshop CS4 (Adobe Systems, San Jose, CA), whereby one tubule was calibrated orthogonally four times from the basal membrane to the tubule lumen, and advanced pixel lengths were converted into micrometer units. Results from three independently embedded testes for double knockout and WT mice with a total tubulus count Ն130 are shown as mean Ϯ S.E.
The fertility of double knock-out mice was studied in longterm mating experiments. Double knockout mice were housed as individual mating pairs for 16 weeks. For comparison, other pairs included double knockout mice with a WT partner. The numbers and sizes of litters were recorded, and offspring per week of mating was calculated. Pure WT matings served as a control.
Sperm Preparation and Motility Analysis-Sperm were isolated from the cauda epididymidis and vasa deferentia after animals were sedated with isoflurane (Baxter, Unterschleißheim, Germany), followed by a cervical dislocation as described before (50). Sperm were allowed to swim out in HS buffer for 20 min at 37°C and 5% CO 2 . Released sperm were washed twice with HS buffer (3 min at 300 ϫ g) and resuspended in a final concentration of 1-2 ϫ 10 7 cells/ml in HS buffer. The sperm samples, washed and stored in HS, were used for all subsequent experiments. For further analysis of the effects of phospholipase C (PLC, Life Technologies) on sperm, 1 ϫ 10 6 cells/ml were incubated in 500 l of HS buffer containing 2 units of PLC for 90 min at 37°C in a shaking water bath. Sperm were sedimented (3 min at 300 ϫ g) and resuspended in 250 l of fresh HS buffer.
CA Activity in Sperm-CA enzyme activity experiments were performed on a quadrupole mass spectrometer (OmniStar GSD 320, Pfeiffer Vaccum, Asslar, Germany). Analysis was carried out as described previously (50). The loss of double-labeled 13 (74). From this definition, 1 unit corresponds to 100% stimulation of the noncatalyzed 18 O depletion of doubly labeled 13 C 18 O 2 . For the experiment, 6 ml of HS buffer was filled into a cuvette, and a non-catalyzed reaction was started by adding 6 l of the double-labeled 13 C 18 O 2 for 8 min. 4 ϫ 10 6 sperm cells were added to measure the CAcatalyzed reaction for 10 min. Results are shown as mean Ϯ S.E. of three independent experiments.
Immunoblotting-For CAII and CAIV protein detection, we prepared WT and knockout tissues from mice in the same way as described before (50). 60 g of total protein and 30 l of sperm sample were separated in a NuPAGE 4 -12% BisTris gel (Invitrogen) and blotted on nitrocellulose membranes (Invitrogen). Air-dried membranes were blocked with TBS/5% Slim Fast TM for 1 h before primary antibody (rb anti-CAII IgG or/and gt anti-CAIV IgG, 1:1000, in 10% Roti Block (Roth, Karlsruhe, Germany)) incubation overnight at 4°C. Membranes were washed twice and incubated first with HRP-conjugated anti-gt IgG (1:1000 in TBS-T) and then with HRP-conjugated anti-rb IgG (1:1000 in TBS-T), each for 1 h at room temperature. CAII and CAIV detection was carried out with ECL reagent (GE Healthcare) on a Chemie-Doc TM XRS apparatus (Bio Rad).
To assess capacitation of sperm from WT and CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice, sperm from the cauda epididymidis and vasa deferentia were incubated for 180 min either in HS medium (37°C, air atmosphere) or in capacitation medium (37°C, 5% CO 2 ). Protein extraction and blotting were carried out according to a protocol published previously (31). Membranes were incubated with anti-phosphotyrosine IgG (diluted 1:1000 in Roti-Block) overnight at 4°C. After washing three times with TBS-T, the membranes were incubated with HRP-conjugated anti-mouse (diluted 1:10,000 in TBS-T) for 1 h at room temperature. Protein bands were detected with ECL reagent (GE Healthcare) on a Chemie-Doc TM XRS apparatus (Bio Rad).
Immunohistochemistry-Organs were isolated from mice, fixed, and cut as described previously (50). Isolated sperm were air-dried and fixed in methanol for 15 min at room temperature.
qRT-PCR-Tissue homogenization, RNA isolation, and cDNA synthesis were performed as described previously (49). cDNA served as a template for the following RT-PCR, and RNA expression of CAII, CAIV, and CAXIV in tissues from the male reproductive tract was assessed by relative quantification with the ⌬⌬Ct method (54). 18S rRNA served as the endogenous standard and kidney as the reference tissue. Amplification and detection were carried out according to an Applied Biosystems protocol, each cDNA template as triplicate with a respective TaqMan gene expression assay (Applied Biosystems, Darmstadt, Germany) on a StepOnePlus TM cycler and software (Applied Biosystems). Results are presented as mean real-time quantitative values or relative amounts Ϯ S.E., each from three independent experiments.
Determination of Flagellar Beat Frequency-Beat frequency was determined as described previously (50). In brief, flagellar beat frequency was observed on an inverted microscope (Diaphot 300, Nikon, Tokyo, Japan) and recorded at 300 Hz in a 1200 ϫ 1400 pixel region with an IDT M3 high-speed camera (IDT Inc., Tallahassee, FL) and Motion Studio 64 software (Imaging Solutions, Regensburg, Germany). Determination of single sperm beat frequency was performed as described previously (55). Single sperm sequences were cut, arranged, and contrasted by ImageJ v1.37 software. Images of maximum amplitudes were merged into one sum file with MetaMorph v7.1 (Molecular Devices, Sunnyvale, CA) and analyzed by a semiautomated algorithm written in Igor Pro TM v6.04 (Wavemetrics, Lake Oswego, OR). Data are shown as mean Ϯ S.E., calculated with SigmaPlot v11.0 (Systat Software) and with a minimum of 17 single sperm for each waveform experiment (for exact sperm numbers, see the figure legends).
Dye Loading and pH i Measurements-To measure the steady-state pH i , sperm were loaded and measured as described previously (53). In brief, 250 l of HS buffer was spiked with 0.1 M Pluronic-Fl27 (Invitrogen) and 0.1 M pH-sensitive 2Ј,7Јbis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (Invitrogen) and mixed with 250 l of HS-stored sperm (3 ϫ 10 6 cells/ml) suspension. After three washing steps, cells were measured on a Nikon Eclipse TE2000-U microscope equipped with a monochromator (Till Photonics, Munich, Germany). An intracellular calibration was performed by suspension of the dye-loaded sperm in K ϩ -based medium variously buffered at pH 5.0, 7.0, or 9.0 and treatment with the K ϩ -selective ionophore nigericin (Sigma Chemicals). For pH i equilibration, the fluorescence ratio of 436/488 was transferred to a cellspecific pH i (56). To measure the kinetics of changes in the pH i dye loading, the experimental procedures were carried out the same way as described above, with the following exceptions. 250 l of the sperm solution (3 ϫ 10 6 cells/ml) was mixed with an equal volume of HS buffer containing 0.1% PowerLoad TM and 0.5 M pHrhodo TM Red acetoxymethyl ester (Invitrogen). Cells were incubated for 30 min in the dark at room temperature, washed two times with fresh HS buffer, and subsequently used to measure the pH i . Fluorescence was sampled during 50 ms of excitation, applied at 1 Hz. Changes in fluorescence were normalized to resting fluorescence (F/F0) with SigmaPlot 11.0 (Systat Software).

CAII and CAIV Distribution in the Male Reproductive
Tract and in Sperm-Double immunostaining (Fig. 1A) was performed to localize CAII and CAIV in the testis, epididymis, and sperm. CAII was detected with DAB (brown) and CAIV with Texas Red (red). WT testis (Fig. 1A, a) shows a specific CAII signal in elongated sperm, whereas early germ cell states are CAII-negative. In the epididymis, CAII is present in single epithelial cells of the caput (Fig. 1A, c), the cauda epididymidis ( Fig.   1A, g), and in nearly all cells of the corpus epididymidis (Fig. 1A, e). CAIV signals are not detectable in WT testis (Fig 1A, a) and the caput epididymidis (Fig. 1A, c). However, a specific immunoreaction is visible in the apically located stereocilia network of epithelial cells in the corpus epididymidis (Fig. 1A, e) as well as in the cauda epididymidis (Fig. 1A, g). Luminally located sperm show a specific CAII signal in the testis and in all parts of the epididymis. In contrast to CAII, CAIV is not detectable in luminal sperm of the testis and caput epididymidis. CAIV is only present in sperm from the corpus and cauda epididymidis. Tissue from CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice, which served as a negative control, does not show any specific CAII and CAIV immunoreaction (Fig. 1A, b, d, f, and h). The same results were 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 ϭ 50 m (a-h) and 10 m (insets). obtained from Western blot analyses, as shown in Fig. 1B. Protein extracts from WT and CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice were stained for the presence of CAII and CAIV. In the WT testis and caput epididymidis, only a CAII signal (28 kDa) is detectable, whereas the WT corpus and cauda epididymidis display signals for CAII and CAIV (38 kDa). Protein extracts from isolated WT sperm show a prominent immunoreactive CAII and a weaker CAIV band. No signal was detected in any tissue or sperm of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ animals (Fig. 1B, Ϫ/Ϫ). To analyze development-dependent protein expression in the male genital tract, we stained WT tissues (ϩ/ϩ) of prepubertal (3-week-old) and pubescent (5-week-old) mice with antibodies against CAII ( Fig.  2A) and CAIV (Fig. 2B). CAII is not detectable in 3-week-old WT testis ( Fig. 2A, a) and the distinct parts of the epididymis ( Fig. 2A, c, e, and g). Puberty leads to significant changes in CAII distribution. In tissues from 5-week-old animals, CAII is localized throughout the entire genital tract with similar but weaker signals ( Fig. 2A, b, d, f, and h) compared with the tissues from adult animals in Fig. 1A. In contrast to CAII, a specific CAIV signal is already present in prepubertal (3-week-old) WT tissue from the corpus epididymidis (Fig. 2B, e). No CAIV signal is present in the 5-week-old testis (Fig. 2B, b) and caput (Fig. 2B,  d). Intense staining occurs in the apically located stereocilia network of the corpus epididymidis (Fig. 2B, f) and the cauda epididymidis (Fig. 2B, h) of 5-week-old mice.
To localize CAII and CAIV more systematically in epididymal sperm, we performed double immunofluorescence staining (Fig. 3). CAII signals (green) are detectable in the cytoplasm of the principal piece of sperm tail. CAIV signals (red) are localized in parts of the acrosome and in the plasma membrane of the entire sperm tail, predominantly in the mid-piece. In comparison with WT sperm, sperm from double knockout mice are negative for CAII and CAIV (data not shown).
CAII and CAIV Are the Most Abundant Isoforms in Sperm-To determine CAII and CAIV enzyme activity, mass spectrometry was performed. A comparison of total CA activity between the sperm of WT and CAII or CAIV knockout animals provides information about the relative activity of these CA isoforms in sperm. The results illustrated in Fig. 4A indicate a total CA enzyme activity in WT sperm of 5.20 Ϯ 0.2 units/ml. In comparison with WT sperm, CAIV Ϫ/Ϫ leads to a significant reduction of 31.4% (3.57 Ϯ 0.25 units/ml) and CAII Ϫ/Ϫ of 63.2% (1.84 Ϯ 0.07 units/ml). We consequently measured the CA activity in sperm of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ animals. The detected activity of 0.7 units/ml (Ϯ 0.02) equals a reduction of 86.9% compared with WT sperm. This activity is less than the activity measured in native Xenopus oocytes (1.5 units/ml), which do not express CA at all (57) Because CAs are involved in generating HCO 3 Ϫ , we examined the influence of CAII and CAIV on the HCO 3 Ϫ -mediated early activation of sperm. For this, we determined the beat frequency of single sperm as a function of time by application of 15 mM HCO 3 Ϫ (Fig. 4B). WT sperm respond to HCO 3 Ϫ with an acceleration of their beat frequency from 3.3 Ϯ 0.09 Hz to a maximum of 7.04 Ϯ 0.18 Hz within the first 20 s. Sperm from CAIV, as well as from CAII knockout mice, show a delayed and reduced HCO 3 Ϫ response compared with WT sperm. The sperm beat frequency of CAIV Ϫ/Ϫ animals rises from 2.78 Ϯ 0.09 Hz to 5.47 Ϯ 0.25 Hz within 20 s and displays a maximum of 5.88 Ϯ 0.26 Hz (t ϭ 60 s). Sperm from CAII Ϫ/Ϫ animals accelerate from 2.89 Ϯ 0.1 Hz to 3.9 Ϯ 0.18 Hz in the first 20 s, reaching a maximum of 5.58 Ϯ 0.19 Hz after 60 s of stimulation. Similar beat frequency results were obtained by stimulating sperm with 2% CO 2 (Fig. 8B). These findings confirm the direct involvement of both enzymes in the HCO 3 Ϫmediated pathway.
Compensatory Expression of CAII and CAIV in Tissues of Single Knockout Mice-qRT-PCR analyses were performed to examine the expression of CAII and CAIV mRNA in tissues of the male reproductive tract. First, the expression in WT tissue was determined (Fig. 4C). In relation to the kidney, which served as the internal standard (relative expression value, 1.0), CAIV mRNA is only expressed in the corpus epididymidis (1.66 Ϯ 0.49). CAII mRNA is present in all parts of the reproductive tract, with the highest expression values of 0.99 Ϯ 0.23 in the testis. Next, CAIV mRNA expression was determined in CAII Ϫ/Ϫ animals and vice versa to verify a potential compensatory overexpression. Fig. 4D shows the relative CAIV expression in CAII Ϫ/Ϫ knockout animals. In these experiments, WT tissue served as internal standards. An overexpression of CAIV in CAII knockout tissues can be detected in the caput (5.70 Ϯ 5.48), corpus (7.06 Ϯ 6.78), cauda epididymidis (9.65 Ϯ 7.18), and vas deferens (1.89 Ϯ 1.32). In contrast, CAIV Ϫ/Ϫ animals show (Fig. 4E) DECEMBER 4, 2015 • VOLUME 290 • NUMBER 49

Carbonic Anhydrase in Sperm
reproductive tract do not show any difference compared with WT organs (data not shown). However, a closer examination after weaning reveals a significant reduction in animal and testis weights compared with WT mice. The mutant offspring was weighed once a week from day 21 on, and a reduced weight in all life stages was detected. Fig. 5A shows the body weight of 10-week-old male mice. CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice have an average weight of 22.13 Ϯ 0.61 g, whereas WT mice of the same age weigh 26.82 Ϯ 0.78 g. Adult testes of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice reach an average weight of 72.33 Ϯ 3.51 mg, which is a reduc- 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 Ϫ/Ϫ CAIV Ϫ/Ϫ mice is 0.7 units/ml (Ϯ 0.02), which equals a reduction of 86.9%. Data are mean Ϯ S.E. of six or more measurements from three independent experiments. The asterisks refer to the values of the bar for WT. *, p Յ 0.05; **, p Յ 0.01; ***, p Յ 0.001. B, analysis of the single-sperm beat frequency response to 15 mM HCO 3 Ϫ . CAII Ϫ/Ϫ and CAIV Ϫ/Ϫ sperm display a delayed and reduced acceleration of beat frequency. Data are mean Ϯ S.E. of Ն40 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 Ϫ/Ϫ mice (D), whereas CAII expression is highly increased in the cauda epididymidis and vas deferens (vas def.) of CAIV Ϫ/Ϫ animals (E) (n ϭ 3). tion of 32.2% compared with WT testes (106.75 Ϯ 6.5 mg) (Fig.  5A). Because CAs are essential for the regulation of pH i , we determined the steady-state pH i of the sperm of double knockout mice (Fig. 5B). Mean values of pH i in CAII Ϫ/Ϫ CAIV Ϫ/Ϫ cells (pH i ϭ 6.75 Ϯ 0.07) do not show any significant change compared with the sperm of WT animals (pH i ϭ 6.78 Ϯ 0.05). Through morphological studies of the testes (Fig. 5C), we observed a thinner germ epithelium in the seminiferous tubules, which might be an explanation for the significant lower testis weight. By measuring 130 tubuli of three different mice, an average germ cell epithelium of 60.97 Ϯ 0.93 m in CAII Ϫ/Ϫ CAIV Ϫ/Ϫ animals and 76.00 Ϯ 0.95 m in WT testes was detected (Fig. 5D). We used capacitating conditions to compare the sperm of WT and CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice. Sperm of both animals were able to show tyrosin phosphorylation as an indicator of late effects of bicarbonate (Fig. 6A). Because no differences in the steady-state pH i of WT and double knockout sperm were observed, we next investigated whether there was a difference in the kinetics of intracellular alkalization upon stimulation with ammonium chloride. Fig. 6B shows the response of WT sperm (black line) and CAII Ϫ/Ϫ CAIV Ϫ/Ϫ sperm (gray line) to ammonium chloride. The decrease of fluorescence is 25.5% in WT and 22.6% in CAII Ϫ/Ϫ CAIV Ϫ/Ϫ sperm. To verify the compensatory expression of CAII in CAIV Ϫ/Ϫ mice and vice versa, we used Western blot analysis. Sperm of CAII Ϫ/Ϫ animals show a reduced CAIV protein level whereas the CAII protein level in sperm of CAIV Ϫ/Ϫ mice corresponds to that of WT mice (Fig. 6C).
CAII/CAIV Deletion Drastically Affects Sperm Motility and Fertility-To assess different motility patterns, we performed CASA analyses with sperm populations from CAII Ϫ/Ϫ and CAII Ϫ/Ϫ CAIV Ϫ/Ϫ animals (Fig. 7A)  determined by offspring analysis from three independent mating experiments. As indicated in Fig. 7B, WT males were mated with WT females (type 1) or CAII Ϫ/Ϫ CAIV Ϫ/Ϫ females (type 2). Furthermore, WT females were mated with CAII Ϫ/Ϫ CAIV Ϫ/Ϫ males (type 3). Type 4 indicates the mating of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ males with CAII Ϫ/Ϫ CAIV Ϫ/Ϫ females. Each pair type was mated over a period of 16 weeks, and the numbers of litters and pups were recorded. WT mating (type 1) produced an average offspring per week of 2.02 Ϯ 0.19. Smaller litter sizes resulted from matings of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ females with WT males (type 2). In this case, four individual matings produced nine living litters with a total number of offspring of 29, which equals 0.45 Ϯ 0. 16  Ϫ -mediated pathway in early sperm activation, we performed waveform analyses with single sperm. Fig.  8, A and B, shows the beat frequency of sperm from WT (solid line), CAII Ϫ/Ϫ (dashed line), and CAII Ϫ/Ϫ CAIV Ϫ/Ϫ (dotted line) mice over time by perfusion with HCO 3 Ϫ (Fig. 8A) and CO 2 (Fig. 8B). The WT sperm beat frequency accelerates within 20 s from 3.34 Ϯ 0.09 Hz to 7.04 Ϯ 0.18 Hz with HCO 3 Ϫ and from 3.53 Ϯ 0.08 Hz to 6.51 Ϯ 0.19 Hz with CO 2 . CAII Ϫ/Ϫ sperm show an increase in beat frequency within the first 20 s from 2.90 Ϯ 0.10 Hz to 3.90 Ϯ 0.18 Hz by HCO 3 Ϫ and from 2.77 Ϯ 0.09 Hz to 3.72 Ϯ 0.14 Hz by CO 2 application. The respective Ϫ ) and 6.57 Ϯ 0.21 (CO 2 ). These results reveal that the additional CAIV gene loss of CAII Ϫ/Ϫ mice does not potentiate the adverse effects of the mutant. A residual CA activity in the double KO sperm preparations could be due to contamination by somatic cells.
To confirm the cleavage of CAIV, WT sperm were first treated with PLC to produce biochemical CAIV knockout sperm (CAIV BCϪ ). These PLC-incubated sperm show a reduction of the immunoreaction with the CAIV antibody in the entire sperm tail compared with WT sperm (Fig. 8C). The same results can be obtained by Western blot analysis (Fig. 8D). The PLC-treated sperm fraction displays a diminished band intensity in comparison with non-treated WT sperm (Fig. 8D, left  panel). Additionally, the CAIV protein is present in the corresponding supernatant of PLC-incubated sperm, whereas no CAIV-specific band is detectable in the supernatant of nontreated WT sperm (Fig. 8D, right panel). To exclude that the treatment with PLC has an effect on the HCO 3 Ϫ -mediated beat frequency acceleration, we incubated sperm from genetic CAIV Ϫ/Ϫ mice with PLC (CAIV Ϫ/Ϫ / BCϪ ). The response of such cells to HCO 3 Ϫ compared with CAIV BCϪ sperm is not significantly different (Fig. 8E). The beat frequency of CAIV Ϫ/Ϫ / BCϪ sperm increases during 180 s from 3.06 Ϯ 0.19 Hz to 4.93 Ϯ 0.24 Hz after stimulation with HCO 3 Ϫ . Similar values were obtained with CAIV BCϪ sperm (3.09 Ϯ 0.21 Hz to 4.90 Ϯ 0.25 Hz). The fact that sperm of CAIV Ϫ/Ϫ / BCϪ mice respond similar to HCO 3 Ϫ as CAIV BCϪ cells confirms that treatment with PLC does not affect the response to HCO 3 Ϫ in early sperm activation because of a loss of other glycosylphosphatidylinositol-anchored proteins.

Loss of CAII and CAIV Substantially Affects Sperm Motility
and Murine Fertility-Our immunohistochemical and PCR studies show the presence of CAII in epithelial cells of all parts of the epididymis. These results concur with studies in rats (19,59) and humans (15). Because of its enzymatic activity, CAII is presumed to be involved in the acidification process of the epididymal fluid. The strongest CAII protein signal was detected in epithelial cells of the corpus epididymidis. This result indicates a dominant regulatory function of CAII in this section, where pH-dependent sperm maturation processes like protein transfer, lipid remodeling, and protein modifications take place (1, 60 -63). Furthermore, sperm storage in a quiescent state in the cauda epididymidis is a pH-and HCO 3 Ϫ -dependent process in which CAII is likely to be involved (64).
In contrast to CAII, this study and a previous study (50) demonstrate the presence of the membrane-bound CAIV only in the stereocilia network of the corpus epididymidis. Here, the transfer of CAIV from the stereocilia of the epithelial cells to the sperm plasma membrane takes place (50,65), which may explain the exclusive and high expression levels in this part of the epididymis. The high CAIV protein content in the cauda epididymidis we detected with Western blot analysis is the result of the luminal CAIV-positive sperm, which were not flushed out prior to protein isolation. An additional function of CAIV, together with the co-localized CAII in corpus epithelial cells, could be HCO 3 Ϫ resorption combined with parallel intracellular H ϩ generation (22,66). This hypothesis is supported by the work of Au and Wong (20), who showed that the intravenous application of the nonspecific CA inhibitor acetazolamide blocks luminal acidification in the rat epididymis. The analysis of prepubertal (3-week-old) and pubescent (5-week-old) mice show that the CAII protein is detectable during puberty, pointing to specifically regulated expression. This could be a hormonally regulated mechanism that has already been described in rats for CAs (67)(68)(69). We propose that CAII enzyme activity plays a major role during puberty to achieve fertility. In contrast to CAII, CAIV is already prepubertally expressed in the corpus epididymidis. However, in the cauda epididymidis, CAIV is initially present in 5-weekold mice. The specific tissue expression might also reflect different functions of CAIV in the male reproductive tract, such as CAIV protein transfer in the corpus epididymidis and sperm storage in the cauda epididymidis.
Generating CAII/CAIV double knockout mice allowed us to demonstrate the importance of both enzymes in sperm for the achievement of fertility. Zhou et al. (70) have described macroscopic anomalies in the rete testis and ductuli efferentes of CAII-deficient mice. This study demonstrates a significantly reduced animal and testis weight in CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice. Detailed macroscopic analyses of the testes indicate that the reduced weight is accompanied by a thinner germinal epithelium. Interestingly, spermatogenesis and sperm storage are not disturbed in CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice. Sperm can be recovered in equal numbers from WT and CAII-and CAIV-null mutant mice despite the significantly reduced height of the germ cell epithelium. It is known that, in the stomach of CAII Ϫ/Ϫ animals, gene loss is compensated by overexpression of CAIX (71). Analogous results were observed with qRT-PCR analyses in tissue from CAII-as well as CAIV-deficient mice. In comparison with the expression levels in WT tissue, CAIV is overexpressed in all parts of the epididymidis from CAII Ϫ/Ϫ mice, and the expression of CAII is higher in the cauda epididymidis and vas deferens from CAIV Ϫ/Ϫ animals. These results were substantiated by relative quantification. RNA expression levels were calculated in relation to the kidney as a control and 18S RNA as an internal control. According to gene and protein expression studies (59), CAXIV, which is localized in the distal part of the epididymidis, might be a putative isoform to compensate for the loss of CAII or CAIV.
Another plausible explanation for the subfertility of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ animals is the direct regulatory function of CAII and CAIV in sperm. CAII is a constitutively expressed protein during spermatogenesis and is located in the principal piece of the sperm tail, which explains the high CAII RNA amounts in the testis tissue of WT mice. In contrast, CAIV is a protein that is transferred into the plasma membrane of the entire sperm tail in the corpus epididymidis (65). CAII Ϫ/Ϫ as well as CAIV Ϫ/Ϫ (50) mice each display reduced sperm motility and velocity. Using mass spectrometry, we show that CAII and CAIV are the two most important isoforms in murine sperm. The loss of almost all CA activity in sperm of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ animals suggests that CAII and CAIV account for most of total CA activity in sperm. As might be expected, CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice exhibit an additional reduction in sperm motility and velocity, and, also, the percentage of immotile sperm was more than doubled compared with WT animals. Two important factors for the regulation of sperm motility are pH and HCO 3 Ϫ (31, 72), both of which are regulated by CAs (66). An imbalanced pH and/or HCO 3 Ϫ homeostasis seems to be the mechanistic basis for the reduced motility. However, the regulatory mechanisms are complex, species-specific, and not yet well understood (73). Nonetheless, the sperm pH i of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice under steady-state conditions is not affected and remains unchanged in physiological buffer. Furthermore, sperm of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ animals do not show any defects in intracellular alkalization upon stimulation with ammonium chloride. It is still unclear whether motility dysfunction is caused by an altered fluid composition in the epididymidis or by an unbalanced internal proton regulation in sperm by the loss of CAII and CAIV.
Fortunately, in vivo mating experiments provide the most informative findings because CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice are subfertile. The mating of male CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice with WT females results in a drastic decrease in offspring and a reduced fertility of 90% in comparison with pure WT matings. The fertility of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ female mice is also affected. One possible cause of the reduced female fertility of 78% could be an altered composition of the uterine fluid (8,29). In this view, unsuccessful sperm capacitation decreases the fertility potential. The effect of subfertility was even more dramatic when CAII Ϫ/Ϫ CAIV Ϫ/Ϫ male and female mice were mated. Only one living litter with one living pub was born. We think that this effect points to the possibility that CAII and CAIV are not only responsible for bicarbonate regulation in sperm but also for effects in the female reproductive tract.

CAII and CAIV Are Key Enzymes in the HCO 3
Ϫ -mediated Beat Frequency Increase during Early Sperm Activation-To prove the involvement of CAII and CAIV in early HCO 3 Ϫ -me- Ϫ stimulation. F and G, shown is the response of genetic CAII Ϫ/Ϫ and CAII Ϫ/Ϫ CAIV BCϪ double knockout sperm to HCO 3 Ϫ (F) and CO 2 (G). The beat frequency of CAII Ϫ/Ϫ CAIV BCϪ sperm increases only to a maximum of 4.16 Ϯ 0.15 Hz with HCO 3 Ϫ and of 3.87 Ϯ 0.19 Hz with CO 2 application (t ϭ 80 s) and displays the weakest response. Data are mean Ϯ S.E. of Ն17 single sperm. diated events of capacitation, we analyzed sperm motility on a single-cell level. HCO 3 Ϫ induces an increase in sperm beat frequency within the first 30 s after application (31). This occurs physiologically when sperm enter the uterus, which is rich in HCO 3 Ϫ (29). We are able to increase the sperm beat frequency in vitro by stimulating the cells with 15 mM HCO 3 Ϫ and 2% CO 2 (31). In a previous study, we have shown the involvement of CAIV in the HCO 3 Ϫ -mediated pathway during early sperm activation (50). This study demonstrates that sperm of CAII Ϫ/Ϫ mice also display a reduced and delayed response to HCO 3 Ϫ . We conclude that the loss of CAII activity leads to an unbalanced intracellular HCO 3 Ϫ homeostasis, which is reflected in the delayed and decreased response to HCO 3 Ϫ . The findings that CAII and CAIV are the two most important CA isoforms in murine sperm led to the idea of generating CAII Ϫ/Ϫ CAIV Ϫ/Ϫ animals to study sperm behavior. To address the question of whether capacitation and late activation of sperm are altered by the loss of CAII and CAIV, we performed a Western blot analysis with sperm of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice using a phosphotyrosine antibody. As expected, we did not see any changes in tyrosin phosphorylation under capacitating conditions in CAII Ϫ/Ϫ CAIV Ϫ/Ϫ compared with sperm of WT animals.
CASA analysis, which tracks the swimming path of the whole sperm population, reveals additional effects on the reduction of motility and velocity in CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice compared with sperm from single CAII knockout mice. Surprisingly, singlesperm beat frequency experiments of motile CAII Ϫ/Ϫ CAIV Ϫ/Ϫ sperm do not reveal the expected reduction of response to HCO 3 Ϫ . In fact, sperm of CAII Ϫ/Ϫ animals show the greatest delay in response to HCO 3 Ϫ . One explanation for the counterbalancing of the loss of CAII and CAIV could be that transporters or exchangers adopt the absent CA function, like Na ϩ /HCO 3 Ϫ co-transporter and Cl Ϫ /HCO 3 Ϫ exchanger (52), or that sperm of CAII Ϫ/Ϫ CAIV Ϫ/Ϫ mice compensate for gene loss with the expression of another CA isoform. Such compensatory mechanisms have already been described for CAIX in the stomach of CAII-deficient mice (71). We did not find a compensatory mechanism of CAII and CAIV for each other using Western blot analysis. Post-testicular sperm are translatively inactive, and a possible overexpression of another CA isoform in CAII Ϫ/Ϫ CAIV Ϫ/Ϫ sperm is not detectable by qRT-PCR, as is the case in other reproductive tissues. To bypass such a possible genetically induced compensatory mechanism in sperm, the genetic CAII deficiency was combined with a biochemical loss of CAIV by treating sperm of CAII Ϫ/Ϫ animals with PLC. The response of such CAII Ϫ/Ϫ CAIV BCϪ sperm to HCO 3 Ϫ or CO 2 is more delayed and reduced in comparison with the response of the sperm of CAII Ϫ/Ϫ animals. Because of the fact that HCO 3 Ϫ can evolve spontaneously, a response to HCO 3 Ϫ without any CA presence is also possible. We conclude that developing sperm possess compensatory mechanisms that help to sustain the essential HCO 3 Ϫ -mediated pathway during early sperm activation.
In summary, the epididymal localization of CAII and CAIV suggests their involvement in the acidification mechanism of the luminal fluid. In sperm, the catalytic reaction of these two enzymes contributes to nearly 100% of the total CA activity in sperm. They are key enzymes in the regulation of sperm motil-ity and essential for the HCO 3 Ϫ -mediated beat frequency increase during early sperm activation. Therefore, double knockout of CAII and CAIV leads to subfertility in mice.