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Am J Physiol Regul Integr Comp Physiol 280: R365-R375, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 2, R365-R375, February 2001

Expression of carbonic anhydrase IV in mouse placenta

Orna Rosen1, Carlos Suarez1, Victor L. Schuster2, and Luc P. Brion1

1 Department of Pediatrics, Division of Neonatology, and 2 Department of Medicine, Division of Nephrology, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York 10461


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Carbonic anhydrase (CA) facilitates acid-base transport in several tissues. Acidosis upregulates membrane-bound SDS-resistant hydratase activity in various tissues and CA IV mRNA in rabbit kidney. This study was designed to assess whether the expression of membrane-bound CA IV isozyme in mouse placenta is regulated developmentally and by maternal ammonium chloride loading at the end of pregnancy. For this purpose we used Northern blot analysis, Western blots of microsomal membranes, and immunocytochemistry. The expression of CA IV mRNA on Northern blots tripled from day 11 to day 15 and then remained stable until the end of pregnancy. Expression of CA IV immunoreactive protein on Western blot tripled from day 11 to day 15 and decreased almost to baseline by day 19. Strong staining for CA IV was detected by immunocytochemistry in labyrinthine trophoblast, in the endodermal layer of the yolk sac (both intra- and extraplacental) and in the uterine epithelium. Weak staining was observed in most fetal endothelial cells at 11 days but not later in gestation. Maternal acidosis did not upregulate the expression of CA IV mRNA or CA IV immunoreactive protein. Thus CA IV expression in mouse placenta is developmentally regulated. Maternal acidosis during the last quarter of pregnancy does not upregulate CA IV mRNA or CA IV immunoreactive protein.

ammonium chloride; cloning; immunocytochemistry; labyrinth; Northern blot analysis; Western blot analysis; yolk sac


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MAMMALIAN MEMBRANE-BOUND carbonic anhydrases (CA) include at least four different isozymes: CA IV, CA IX, CA XII, and CA XIV (25). Whereas both CA IV and CA XIV are expressed in lung, heart, muscle, liver, brain, and kidney, CA IV is also expressed in the gallbladder, distal intestine, and placenta and specialized capillaries, and cellular distribution of the two isozymes in the kidney is different (11-14, 25, 29, 35, 44, 48). CA XII is expressed in normal colon, kidney, pancreas, prostate, ovary, and testis (18). In contrast, the expression of CA IX in normal tissue is limited to gastric mucosa (21, 30). The abundance of CA IX and XII increases in various types of cancer (18, 30, 43).

Renal membrane-bound CA activity facilitates urinary acidification (16, 45) and may be rate limiting for acid-base transport (23). In the lung, however, only 5-8% of the total CO2 excretion in vivo is attributable to membrane-bound CA activity, whereas 60-70% is attributable to red blood cell CA II (3, 38). Although membrane-bound CA activity in normal epithelia has been classically attributed to CA IV, the relative contribution to acid-base transport of CA IV, XII, and XIV remains to be determined in specific tissues, including various nephron segments.

Chronic acidosis induced by ammonium chloride (NH4Cl) loading upregulates renal SDS-resistant hydratase activity both in rabbits and in mice (4-6) and CA IV mRNA expression in rabbits (42, 46). Nevertheless, in mice with the CA II deficiency (CAD) mutation (car-2n) (20), chronic acidosis is not associated with any change in CA IV immunoreactivity on Western blot analysis and immunocytochemistry (4, 5). Discrepancy between regulation of SDS-resistant hydratase activity and that of CA IV immunoreactivity in the mouse may have resulted from lack of specificity of SDS resistance (corresponding to both CA IV and CA XIV) (25), from low specific activity of rodent CA IV and its incomplete inactivation by SDS (40), and possibly from several other mechanisms (4).

Expression of membrane-bound CA in the placenta was initially suggested by inhibition of the uptake of [14C]bicarbonate-CO2 in guinea pig placenta after exposure of the fetal side (more than the maternal side) to the membrane-impermeant CA inhibitor aminothiazole (15). Membrane-bound CA activity was shown by histochemical studies of the placenta, although considerable variability was observed among species (32). Expression of CA IV in rat placenta was demonstrated by Fleming et al. (11) using Northern blot analysis.

We hypothesized that CA IV would be expressed in the zone of fetomaternal blood exchange, i.e., the labyrinth, and upregulated with acidosis. To assess the cellular localization and the regulation of CA IV expression, we selected mouse placenta. The latter, similar to human placenta, is hemochorial and discoid; nevertheless, mouse placenta has three trophoblast layers (hemotrichorial), whereas human placenta has only one (hemomonochorial) (27, 47).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. B6AF1/J mice were purchased from Jackson Laboratories (Bar Harbor, ME) and studied at an age of 3-5 mo. All mice had free access to fluid and food. Animals were mated overnight once; gestational age was confirmed by fetal maturity using established criteria (41).

We acid-loaded several mice by using a 3- to 6-day course of NH4Cl by daily orogastric feeding of 25 mmol/kg of NH4Cl (using a 5.6 mol/l solution) and by replacing drinking water with a solution of 280 mmol/l NH4Cl. This method of acid loading was needed to consistently induce severe metabolic acidosis, because simple replacement of the drinking water with NH4Cl minimally decreased average pH (7.28 ± 0.05, mean ± SD) and yielded metabolic acidosis in only one-third of the animals.

Animals were killed by intraperitoneal administration of 100 mg/kg pentobarbital sodium. Control animals were killed at either 11, 15, or 19 days of pregnancy. Acid-loaded animals were killed at 19 days of pregnancy, after 3-4 days of acid loading for Northern blot analysis and 3-4 days or 5-6 days for Western blot analysis. Some animals were used for blood gas analysis and Northern blot analysis. Other animals were used for Western blot analysis, and the last group was used for immunocytochemistry. All placentas from the same pregnant mouse were pooled as an n of one. For each procedure, samples for the same set of animals (controls vs. acid loaded, or at various gestational ages) were run in parallel. These protocols have been approved by the Animal Institute of the Albert Einstein College of Medicine, which is an American Association for Accreditation of Laboratory Animal Care-accredited facility.

Mouse CA IV cDNA. First strand cDNA was synthesized by incubating 20-30 µg of total mouse kidney RNA with 200 U of Superscript II, oligo(dT) [(dT)18], deoxy-nucleotides, and dithiothreitol at 45°C for 1 h, and then treating with RNAse H. PCR was done with 1 mmol/l magnesium at an annealing temperature of 57°C; the template for nested PCR was obtained by eluting and purifying DNA of the expected size from a slice of gel after electrophoresis of the first corresponding PCR reaction. The sequences of primers, selected from Fleming's sequence (11) with the McVector program (Oxford Molecular, Oxford, UK) were as follows: R4F1, 5'-GCC CCC TCT ACT GAA GAT TCA CAC (sense, bp 125-146); R4F3, 5'-TGA GAT TCA AGC CAA GGA GCC C (sense, bp 93-116); R4B11, 5'-CAG CTC TCA CTC ACT GTG GAG TTT G (antisense, bp 652-628).

The 520-bp product was cloned and then sequenced using the Taq DyeDeoxy Terminator Cycle Sequencing Kit on an Applied Biosystems model 373 DNA Sequencing System (Perkin Elmer, Norwalk, CT). The sequence, 70% identical to Fleming's rat CA IV cDNA, was used to design nested primers for rapid amplification of the cDNA ends (RACE). First-strand cDNA for 3'-RACE was obtained at 42°C by using Superscript II and the 3'-adapter primer (GIBCO BRL, Rockville, MD). First strand cDNA for 5'-RACE was obtained at 42°C by using Superscript II and the following antisense primer: MR5'9, 5'-CTC CTT GCT AGA TGT C.

The cDNA designed for the 5'-RACE was then purified and tailed with oligo(dT) at the 5'-end. We then did nested PCRs (3'-RACE and 5'-RACE) using a nonspecific primer provided in the corresponding kit (GIBCO BRL) and one of the following nested primers designed from the original 520-bp product: 3'-RACE: M4RF7, 5'-ACA AAG GTG AAC CCC AGA CTG AC; MR4F11, 5'-AGA CTG ACA CCC TTC ATC CTC GTC; 5'-RACE: M4RB6, 5'-TGT CTC CCA TCA ATA CTG TGC TCT G; MR4B4, 5'-CAA TAC TGT GCT CTG AAC CGT TGT C.

Nested 3'-RACE yielded a 1-kb product, whereas nested 5'-RACE yielded a 450-bp product (excluding the amplifier primer). These two products were combined by PCR using the universal amplifier primer (GIBCO BRL), yielding a final 1.2-kb product, which was cloned using the pAmp10 vector and DH5alpha competent cells. The consensus sequence from four separate CA IV cDNA clones (LPB) was 99% identical to the published full-length mouse CA IV cDNA obtained by library screening by Tamai et al. (39), except on the 5'-end.

Northern blot analysis. Placentae were frozen in liquid nitrogen and kept at -70°C until thawing. They were mechanically homogenized using a Teflon homogenizer. Total RNA was extracted using the single-step acid guanidium thiocyanate-phenolchloroform method (8, 9). Quality of the RNA was confirmed by a ratio of optical density at 280 nm (OD 280)/OD 260 >=  1.5 and by visualization of the 28S and 18S bands on a minigel electrophoresis using ethidium bromide staining and ultraviolet transillumination. Electrophoresis of 15 µg of total RNA was done in 4 h using a formaldehyde gel with 1× borate buffer. RNA was transferred overnight to a Hybond N membrane (Amersham Life Sciences, Arlington Heights, IL) and fixed to the membrane by ultraviolet cross-linking. The membrane was prehybridized in a rotatory oven for 1-3 h at 42° in 50% formamide, 5× sodium citrate-sodium chloride (SSC), 50 mmol/l sodium phosphate pH 6.8, 5% Denhardt solution, 2.5% denatured salmon sperm DNA, 0.1% SDS, and 10 mmol/l EDTA in DEPC H2O. The RNA was hybridized overnight at 42° with a cDNA probe that had been obtained by labeling the purified full-length CA IV cDNA with 32P using the New Megaprime kit (Amersham) (1.5 × 106 cpm per membrane). The membrane was washed successively at 55°C with 2× SSC-0.5% SDS, at 55° with 1× SSC/0.2% SDS and at 65° with 1× SSC-0.1% SDS; based on background radioactivity detected using a Geiger counter, some blots were then washed at 65°C using 0.3× SSC with 0.1% SDS. The membrane was then exposed to a radiosensitive film (Biomax MS, Eastman Kodak, Rochester, NY) and incubated at -70°C for 6-18 h. The membrane was then stripped and reprobed with a glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA probe prepared as described above, using a cDNA purchased from Ambion (Austin, TX).

After scanning the film using an HP Scanner IICX, we measured the density of the bands using two-dimension analysis with the Zero-Dscan Image Analysis System (Scanalytics, Billerica, MA) and corrected the value for adjacent background. The abundance of CA IV mRNA in each sample was normalized by calculating the ratio of the density of the CA IV band to that of the GAPDH band. To compare CA IV expression among samples run in different Northern blots, we normalized the ratio by running a sample from the same animal in all gels.

Western blot analysis. We used the same technique as described previously (4). Briefly, 5-10 µl of solubilized microsomal membranes were run on reducing SDS-PAGE and transferred overnight into nitrocellulose membranes. The amount and dilution of the sample were calculated to yield 30 µg of protein for microsomal membranes. Blocking and antisera incubations were all done in PBS with 3% albumin and 0.1% Tween 20. After blocking for 1 h, the membrane was incubated for 2 h with a 1:2,500 dilution of rabbit antiserum to mouse CA IV or to recombinant human CA IV (gifts from W. S. Sly). These antisera have been shown to be specific and sensitive to CA IV; the first antiserum recognizes two bands in mouse lung and kidney microsomal membranes (39 and 63 kDa), whereas the latter recognizes only the 39-kDa band (4). In one sample we used as negative control a random preimmune rabbit serum. The membrane was then incubated for 1 h with a 1:5,000 dilution of goat anti-rabbit IgG-antiserum conjugated to peroxidase. Bands were visualized using enhanced chemiluminescence (ECL; Amersham or Renaissance, NEN Life Science Products, Boston, MA). Chemiluminescence-sensitive film (Hyperfilm ECL, Amersham) was exposed to the membrane for 3-20 s. Densitometry is described above.

Immunocytochemistry. After anesthesia, the animals were perfused with a 25-gauge butterfly inserted into the left ventricle and connected to a peristaltic pump (Cole Parmer, Chicago, IL). We first infused ice-cold PBS (i.e., 10 mM sodium phosphate, 138 mM NaCl at pH 7.4) for 30 s, then 0.5% paraformaldehyde in PBS for 2 min, and then 18% sucrose in PBS for 2 min. The placenta was incubated in sucrose until sinking to preserve cell compartments (7). The tissue was then surrounded with embedding medium (OCT Compound, Sakura Finetech USA, Torrance, CA), snap-frozen in 2-methylbutane placed on dry ice, and kept at -70°C. Six- to ten-micrometer cryostat sections were prepared using L-lysin-coated slides, let to dry at room temperature, and kept at -70°C. Slides were stained using the Vectastain peroxidase ABC kit and the DAB substrate kit (yielding a brown color) (Vector Laboratories, Burlingame, CA). We used three modifications suggested by the manufacturer: incubating the ABC reagents in 0.5 M sodium chloride to reduce ionic interactions, incubating the primary and the secondary antisera in normal rabbit serum to reduce false-positive reactions, and incubating the slides in 0.3% H2O2 in 40% methanol in PBS overnight to block endogenous peroxidase activity. A coverslip was mounted in Mowiol (Calbiochem, La Jolla, CA), which was allowed to dry overnight at 4°C. Negative controls for immunocytochemistry were identical tissues simultaneously submitted to the exact same procedures, except that incubation with the primary antiserum was replaced by incubation with nonimmune rabbit serum. Tissues were counterstained with hematoxylin (Vector Laboratories).

Tissues were visualized with a Nikon Diaphot-inverted microscope equipped with a 75-W Xenon bulb, a ×4 or a ×50 water immersion lens, and a Nikon N70 35 mm camera with 400 ASA slide film. We used established nomenclature for the anatomy of mouse placenta (24, 31, 41, 47).

Statistical analysis and sample size. We compared continuous variables by unpaired Student t-test or by ANOVA followed if significant by Dunnet's C test or Tukey's test, as appropriate. A P < 0.05 was considered statistically significant. Values are expressed as means ± SD. For each experiment, sample size was calculated (using preliminary data) to reach a power of 0.8 of detecting an effect size of 2.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Description of the animals and physiological data. Acid-loaded animals received a mean of 66 ± 18 mmol · kg-1 · day-1 of NH4Cl. They had a significantly lower blood pH than control animals (7.06 ± 0.04, n = 4 vs. 7.36 ± 0.04, n = 11, P < 0.001) and a lower plasma bicarbonate concentration (11.0 ± 1.2 mmol/l, n = 4, vs. 21.3 ± 1.9 mmol/l, n = 11, P < 0.001). Values in controls were similar to those reported previously in nonpregnant adult mice (4).

Northern blot analysis. CA IV mRNA was detected as a single 1.2-kb band, as reported previously (39). Northern blot analysis showed a significant increase in the ratio of the density of CA IV mRNA to that of GAPDH from day 11 (1.00 ± 0.62 arbitrary units, n = 7) to day 15 (3.30 ± 0.63, n = 7) and day 19 (3.28 ± 0.76, n = 8) (P < 0.001 day 11 vs. day 15 and day 11 vs. day 19; Fig. 1A).


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  		Fig. 1.   Northern blot analysis. A: maturation; B: acid loading. Fifteen micrograms of total RNA were loaded in each lane and hybridized successively with a mouse carbonic anhydrase (CA) IV probe and a glyceraldehyde phosphate dehydrogenase (GAPDH) probe. CA IV mRNA was visualized as a single 1.2-kb band. The ratio of CA IV to GAPDH mRNA triples from 11 to 15 days and remains stable until day 19. A 3- to 4-day maternal acid loading does not affect the ratio of CA IV to GAPDH mRNA in 19-day-old placenta.

The ratio of the density of CA IV mRNA to that of GAPDH did not significantly change with a 3- to 4-day maternal acid loading (1.00 ± 0.12 arbitrary units in 9 controls vs. 1.22 ± 0.44 in 12 acid-loaded animals, P = 0.12; Fig. 1B).

Western blot analysis. The antiserum to mouse CA IV recognized one major band migrating at 39 kDa and one minor band at 63 kDa, which was visible only in some membranes (Fig. 2A); these were the same molecular weights as those reported previously for CA IV in mouse kidney (4). The antiserum to human CA IV recognized only the 39-kDa band (data not shown). Expression of the 39-kDa band in the whole placenta increased significantly from day 11 (1.00 ± 0.66 arbitrary units, n = 3) to day 15 (3.89 ± 0.84 units, n = 4, P = 0.02 vs. day 11) and decreased again by day 19 (1.27 ± 0.55 units, n = 4, P = 0.02 vs. day 15; Fig. 2B).


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  		Fig. 2.   Western blot analysis. A: maturation; B: 3- to 4-day acid loading; C: 5- to 6-day acid loading. Tissue microsomal membranes (30 µg protein) and recombinant human CA IV (gift from W. S. Sly), used as positive control, were run on SDS-PAGE, transferred into a nitrocellulose membrane, and incubated with rabbit antiserum to mouse CA IV (provided by W. S. Sly). Bands were visualized by enhanced chemiluminescence. The amount of CA IV immunoreactive protein triples from day 11 to day 15 and then decreases by day 19 (A). Only the major 39-kDa band is visible in this figure. A 3- to 4-day acid loading does not increase the amount of CA IV immunoreactive protein in 19-day-old placenta (B), and a 5- to 6-day acid loading results in a decrease in CA IV expression (C). These figures show 1 major band migrating at 39 kDa; the faint 63-kDa band is visible only in B.

The expression of CA IV (39-kDa band) on day 19 did not change significantly with 3-4 days of maternal acidosis (1.00 ± 0.33 units, n = 6 in controls vs. 1.15 ± 0.24 units, n = 7, in acid-loaded animals; Fig. 2A). In preliminary experiments (power 0.65), we observed a reduction of CA IV after a 5- to 6-day acid loading (1.00 ± 0.12 units, n = 4 controls, vs. 0.51 ± 0.33, n = 4 acid-loaded animals, P = 0.032).

Immunocytochemistry. CA IV immunoreactivity was found in the labyrinth, the endodermal layer of the yolk sac, the decidua, and the uterine epithelium, but not in the junctional zone (trophosphongium) and the zone of giant cells (Fig. 3). CA IV was expressed over the entire area of the labyrinth, which extended from the periumbilical area toward the periphery of the placenta from 11 to 19 days (Figs. 3 and 4). During gestation the proportion of the placenta occupied by the labyrinth increased both laterally and transversally. The rate of growth of the labyrinth was quantified using the labyrinth-to-placenta thickness ratio at the center of the placenta. The thickness ratio increased from 0.38 ± 0.14 (n = 5) at 11 days to 0.57 ± 0.07 (n = 5) at 15 days and 0.70 ± 0.12 (n = 5) at term (ANOVA, P = 0.003; day 11 vs. day 15, P = 0.046, day 11 vs. day 19, P = 0.002).


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  		Fig. 3.   CA IV immunocytochemistry. Chorionic plate and labyrinth, low magnification (×40). A and B: 11 days; C and D, 15 days; E and F, 19 days; A, C, E, CA IV; B, D, F, negative controls; labyrinth, long arrow; endodermal layer of the extraplacental yolk sac, short black arrow; endodermal layer of the intraplacental yolk sac, small arrowhead; junctional zone and zone of giant cells, triangle. CA IV appears as brown pigmentation, which is not visible in negative controls. Most CA IV immunoreactivity is found in the labyrinth (long arrow). Note a progressive increase in the area of expression of CA IV with gestational age, corresponding to the increase in size of the labyrinth. CA IV is also expressed on the endodermal layers of the extraplacental yolk sac (short black arrow) and of the intraplacental yolk sac (arrowhead).



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  		Fig. 4.   CA IV immunocytochemistry. Transverse cut at the middle of the placenta, low magnification (×40). A, 11 days; B, 15 days; C, 19 days; labyrinth, long arrow; endodermal layer of intraplacental yolk sac, small arrowhead; junctional zone and zone of giant cells, triangle; decidua, short open arrow. The labyrinth-to-placenta thickness ratio increased rapidly from day 11 to day 15 and slowly thereafter. See RESULTS for details and statistical analysis.

Perfusion-fixation removed most maternal red blood cells but fixed fetal red blood cells in situ, thereby facilitating the differentiation of fetal blood vessels from maternal spaces (Fig. 5).


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  		Fig. 5.   CA IV immunocytochemistry, labyrinth. High magnification (×500). A and B, 11 days; C and D, 15 days; E and F, 19 days; A, C, E, CA IV; B, D, F, negative controls; fetal endothelium, long arrow; fetal red cell line, short arrow; maternal red blood cells, arrowhead. CA IV is expressed in the trophoblast. In some cells, CA IV is mostly expressed at the apical surface, i.e., in direct contact with maternal spaces. Weak staining for CA IV is observed in fetal endothelium (long arrow) at 11 days but not later in pregnancy. Giant cells do not stain for CA IV. Note the progressive maturation of fetal red cell line (short arrow) with predominance of nucleated cells at 11 days and of mature erythrocytes at 19 days. Residual maternal erythrocytes (arrowhead) are smaller than fetal erythrocytes. Note progressive proliferation of fetal capillaries by the end of pregnancy.

In the labyrinth (Fig. 5), heavy CA IV staining was observed in the trophoblast, with, in some places, intense staining on the apical membrane of the superficial layer, i.e., in contact with maternal spaces. Giant cells did not express CA IV. Weak staining for CA IV was observed in many fetal endothelial cells at 11 days (Fig. 5A) but not later in gestation (Fig. 5, C and E).

In the intraplacental yolk sac (Fig. 6), CA IV was expressed in the visceral or endodermal layer, i.e., the columnar layer adjacent to large umbilical vessels (24). There was no CA IV expression in the parietal layer resting on Reichert's membrane; however, CA IV was expressed on all trophoblastic cells resting on the other side of Reichert's membrane. There was no CA IV expression in the periplacental parietal layer of the yolk sac (periplacental omphalopleure).


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  		Fig. 6.   CA IV immunocytochemistry, chorionic plate. High magnification (×500). A and B, 11 days; C and D, 15 days; E and F, 19 days; A, C, E, CA IV; B, D, F, negative controls; fetal endothelium, long arrow; fetal red cell line, short black arrow; endodermal layer of the extraplacental yolk sac, long arrowhead; endodermal layer of the intraplacental yolk sac, short open arrow; space of Duval, curved arrow; parietal layer of the intraplacental yolk sac, small arrowhead; Reichert membrane, triangle; maternal red blood cells, double-headed arrow. Endothelial cells stain for CA IV at 11 days but not later in pregnancy. CA IV is strongly expressed in the endodermal layer of the extraplacental yolk sac. There is also strong CA IV staining of the columnar epithelium surrounding the large umbilical vessels; at 15 and 19 days this epithelium is the visceral or endodermal layer of the intraplacental yolk sac. The space of Duval, situated between the endodermal layer and the parietal layer, does not contain any erythrocyte. CA IV is not expressed in the squamous epithelium or parietal layer of the intraplacental yolk sac, resting on the Reichert membrane; on the other side of the Reichert membrane is the maternal space.

In the endodermal layer of the extraplacental yolk sac (Fig.7), CA IV was expressed in the outermost cells of the villi, mainly at the upper half of the cells.


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  		Fig. 7.   CA IV immunocytochemistry, endodermal layer of the extraplacental yolk sac. High magnification (×500), 19-day placenta. A, CA IV; B, negative control; superficial cells of the endodermal layer, arrowhead. CA IV is expressed on the upper half of superficial cells on the villi, but not in the stroma.

Strong CA IV expression was also observed in the uterine epithelium (juxtauterine bilaminar omphalopleure), which is adjacent to the Reichert membrane before its disappearance at the end of gestation (Fig. 8).


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  		Fig. 8.   CA IV immunocytochemistry, uterine epithelium. High magnification (×500), 19-day placenta. A, CA IV; B, negative control; uterine epithelium, arrow. Strong staining is visible in the uterine epithelium (juxtauterine bilaminar omphalopleure).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our data showed strong CA IV immunoreactivity in mouse labyrinthine trophoblast and in the endodermal layer of the yolk sac (both intra- and extraplacental) and weak CA IV staining in fetal endothelial cells at 11 days. The area of strong expression of CA IV in mouse placenta increased with gestation, along with the increase in size of the labyrinthine area (41). In rat placenta the trophoblast accounts for 30% of the placenta by weight at 12 days of pregnancy and 60% at 20 days (10). In the mouse, most of the increase in size of the labyrinth occurs between day 11 and day 15, with less increment from day 15 to day 19. The thickness ratio (labyrinth-to-total placenta) at 11 days and 15 days was, respectively, 54 and 81% of term values. In addition, branching of major umbilical vessels and development of the intraplacental yolk sac occurs from day 10 to day 14, followed by further capillary development.

The abundance of CA IV mRNA and CA IV immunoreactive expression, measured, respectively, by Northern and Western blot analysis, tripled from day 11 to day 15, in parallel with the rapid labyrinthine growth. From day 15 to day 19, the abundance of CA IV mRNA remained stable, whereas that of CA IV immunoreactive protein decreased and labyrinthine growth slowed down. This discrepancy between CA IV mRNA, CA IV immunoreactive protein, and labyrinthine growth at the end of gestation could have occurred in part from fetal capillary growth into the trophoblast and possibly from changes in the rate of CA IV protein synthesis or catabolism.

Previous immunochemical studies have shown evidence for expression of CA II but not CA I and CA III in bovine placenta (28). Human placenta expresses CA I and II (1, 2, 26) in the syncytiotrophoblast and, especially CA II, in fetal villous endothelium of mature placenta (26). Ridderstråle and coauthors (32) showed considerable species heterogeneity of CA expression in placenta. Histochemistry of human placenta showed CA activity only in fetal capillaries but not in the trophoblast (32). In contrast, histochemistry of rat placenta showed CA activity in the trophoblast but not in fetal capillaries (19, 32). Thus our results using CA IV immunocytochemistry in mature mouse placenta match Ridderstråle's histochemical data in rat placenta.

In addition, our data show evidence for CA IV immunoreactive expression in the endodermal layers of the intrauterine and extrauterine yolk sac and in the uterine epithelium adjacent to Reichert's membrane. The role of CA IV expression at these locations remains to be determined; we hypothesize CA IV might have a role in epithelial transport. The endodermal layer of the yolk sac, whether intraplacental or extraplacental, expresses calbindin, a vitamin D-dependent calcium-binding protein implicated in calcium transfer (31). The endodermal layer of the extraplacental yolk sac selectively transfers proteins and is the major route of transport of intact maternal immunoglobulins toward fetus in rodents (47).

Fetal metabolism produces CO2, which is transferred across the placenta as dissolved CO2 rather than bicarbonate (17, 22). Under normal conditions, the rate of CO2 transfer depends on the fetomaternal PCO2 gradient, fetal and maternal blood flow rates, and the hemoglobin concentration and buffering capacity, but not on the placental membrane diffusion capacity or on usual levels of CA in fetal red blood cells (17). The differential role of maternal and fetal CA in acid-base regulation of the fetus has been studied in human pregnancies. Maternal administration of acetazolamide does not affect fetal blood pH or PCO2, regardless of whether controls are studied under baseline conditions or after NH4Cl administration (37). In contrast, injection of acetazolamide into lamb or human umbilical vein causes fetal acidosis and hypercapnia (22, 36) but does not decrease the fetal arteriovenous umbilical PCO2 gradient (22); fetal acidosis and hypercapnia could be due not only to inactivation of red blood cell CA but also to inactivation of CA localized on the fetal side of the placenta. The latter could decrease conversion of bicarbonate into CO2 and diffusion of CO2 across the placenta (15).

We did not observe any upregulation of CA IV mRNA or immunoreactive protein in response to a 3-day maternal acid loading. After 5-6 days, CA IV immunoreactive protein decreased to half the control value, possibly due to increased protein catabolism or decreased protein synthesis during severe acidosis. Previous data in our laboratory have shown lack of upregulation of CA IV immunoreactive protein in mouse kidney and central nervous system in the presence of chronic acidosis due to congenital CAD (5, 6). In contrast, in the rabbit, renal expression of CA IV (42) and CA II (34) mRNAs is upregulated by in vivo acidosis with a peak at 3 days, and CA II immunoreactive protein expression doubles in rat inner medullary collecting duct cells after a 2-day acidosis in vitro (33). In the present study, we did not assess possible changes in SDS-resistant hydratase activity in the placenta because of lack of sensitivity and specificity of that method (25, 40). It is possible that the upregulation of SDS-resistant hydratase activity we had previously observed with acidosis in rabbit and mouse kidney and in mouse central nervous system (4-6) might have in part resulted from upregulation of another membrane-bound CA isozyme, possibly CA XIV (in addition to or instead of CA IV) immunoreactive protein. Further studies are needed to assess the response of other membrane-bound CA isozymes in response to acidosis.

Further investigations are needed to determine the significance of the 63-kDa band on Western blot analysis. It is possible that this band might result from partial cross-reactivity with another or with other membrane-bound proteins (but not CA XII or CA XIV, which are smaller proteins). Indeed, the antiserum to recombinant human CA IV (provided by W. S. Sly), recognized the 39-kDa band but not the 63-kDa band, in both the kidney (4) and the placenta.

In summary, in mouse placenta, CA IV is mostly expressed in the labyrinth, at the interface between fetal capillaries and maternal blood. The expression of CA IV mRNA and that of CA IV immunoreactive protein in mouse placenta peak during the second third of gestation. Maternal acidosis during the last quarter of pregnancy does not upregulate CA IV mRNA or immunoreactive protein in mouse placenta.

Perspectives

Immunocytochemistry showed strong CA IV expression in mouse placenta in the labyrinth, where it is expected to facilitate acid-base transfer between maternal and fetal blood. Maternal acidosis did not upregulate the expression of CA IV mRNA or immunoreactive protein.

In addition, we found CA IV expression in the endodermal layer of the yolk sac. Further experiments are required to determine whether CA IV expression in this location may play a role in facilitating transport across this specialized layer.


    ACKNOWLEDGEMENTS

We thank W. S. Sly and W. Cammer for generous gifts of reagents. We thank Y. Bao, E. Herzberg, R. Lu, P. Mundel, L. Satlin, J. Satriano, S. Somlo, and B. Zavilowitz for expert help and advice, and the respiratory therapists of the Jacobi Medical Center for chemical analyses. We thank A. Waheed for reviewing the first version of the manuscript.


    FOOTNOTES

Preliminary results were presented at the Fourth International Conference on the Carbonic Anhydrases: Molecular Biology, Physiology and Clinical Applications, Oxford, UK (7/28/95); at the 31st Annual Meeting of American Society of Nephrologists, San Diego, CA (11/6 and 11/7/95); and at the Annual Meeting of the Society for Pediatric Research, San Francisco, CA (5/1/99). These results were published in abstract form: J Am Soc Nephrol 6: 306A, 1995 and Pediatr Res 45: 60A, 1999.

O. Rosen was supported by an internal grant from the Department of Pediatrics of the Albert Einstein College of Medicine-Montefiore Medical Center. V. L. Schuster was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant DK-49688. L. P. Brion was supported by NIDDK Clinical Investigator Award 5K08-DK-01984, by Grant-in-Aid 9650445N of the American Heart Association, and by Grants 9-526-4769, -0510, -0541, and -0640 of the Albert Einstein College of Medicine.

Address for reprint requests and other correspondence: L. P. Brion, Albert Einstein College of Medicine, Children's Hospital at Montefiore, Division of Neonatology, Weiler Hospital Rm. 725, 1825 Eastchester Rd., Bronx, NY 10461 (E-mail: brion{at}aecom.yu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 13 September 1999; accepted in final form 23 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aliakbar, S, Brown PR, Jauniaux E, Bidwell DE, and Nicolaides KH. Measurement of carbonic anhydrase isoenzymes in early human placental tissues. Biochem Soc Trans 18: 670, 1990[Web of Science][Medline].

2.   Aliakbar, S, Brown PR, and Nicolaides KH. Localization of CAI and CAII isoenzymes in normal term human placenta by immunofluorescence techniques. Placenta 11: 35-39, 1990[Web of Science][Medline].

3.   Bidani, A. Analysis of abnormalities of capillary CO2 exchange in vivo. J Appl Physiol 70: 1686-1699, 1991[Abstract/Free Full Text].

4.   Brion, LP, Cammer W, Satlin LM, Suarez C, Zavilowitz BJ, and Schuster VL. Expression of carbonic anhydrase IV in carbonic anhydrase II-deficient mice. Am J Physiol Renal Physiol 273: F234-F245, 1997[Abstract/Free Full Text].

5.   Brion, LP, Suarez C, Zhang H, and Cammer W. Up-regulation of carbonic anhydrase isozyme IV in CNS myelin of mice genetically deficient in carbonic anhydrase II. J Neurochem 63: 360-366, 1994[Web of Science][Medline] [correction: 70: 1777, 1998].

6.   Brion, LP, Zavilowitz BJ, Suarez C, and Schwartz GJ. Metabolic acidosis stimulates carbonic anhydrase activity in rabbit proximal tubule and medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 266: F185-F195, 1994[Abstract/Free Full Text].

7.   Brown, WJ, and Farquhar MG. The mannose 6 phosphate receptor for lysosomal enzymes is concentrated in cis Golgi cisternae. J Histochem Cytochem 22: 1077-1083, 1974[Abstract].

8.   Chomczynski, P. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. Biotechniques 15: 532-537, 1993[Web of Science][Medline].

9.   Chomczynski, P, and Sacchi N. Single-step method for the RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[Web of Science][Medline].

10.   Davies, J, and Glasser SR. Histological and fine structural observations on the placenta of the rat. Acta Anat (Basel) 69: 542-608, 1968[Web of Science][Medline].

11.   Fleming, RE, Crouch EC, Ruzicka CA, and Sly WS. Pulmonary carbonic anhydrase IV: developmental regulation and cell-specific expression in the capillary endothelium. Am J Physiol Lung Cell Mol Physiol 265: L627-L635, 1993[Abstract/Free Full Text].

12.   Fleming, RE, Parkkila S, Parkkila AK, Rajaniemi H, Waheed A, and Sly WS. Carbonic anhydrase IV expression in rat and human gastrointestinal tract regional, cellular, and subcellular localization. J Clin Invest 96: 2907-2913, 1995.

13.   Ghandour, MS, Langley OK, Zhu XL, Waheed A, and Sly WS. Carbonic anhydrase IV on brain capillary endothelial cells: a marker associated with the blood-brain barrier. Proc Natl Acad Sci USA 89: 6823-6827, 1992[Abstract/Free Full Text].

14.   Hageman, GS, Zhu XL, Waheed A, and Sly WS. Localization of carbonic anhydrase IV in a specific capillary bed of the human eye. Proc Natl Acad Sci USA 88: 2716-2720, 1991[Abstract/Free Full Text].

15.   Hatano, H, Leichtweiss HP, and Schroder H. Uptake of bicarbonate/CO2 in the isolated guinea pig placenta. Placenta 10: 213-221, 1989[Web of Science][Medline].

16.   Henry, RP. Multiple roles of carbonic anhydrase in cellular transport and metabolism. Annu Rev Physiol 58: 523-528, 1996[Web of Science][Medline].

17.   Hill, EP, Power GG, and Longo LD. A mathematical model of carbon dioxide transfer in the placenta and its interaction with oxygen. Am J Physiol 224: 283-299, 1973.

18.   Ivanov, SV, Kuzmin I, Wei MH, Pack S, Geil L, Johnson BE, Stanbridge EJ, and Lerman MI. Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes. Proc Natl Acad Sci USA 95: 12596-12601, 1998[Abstract/Free Full Text].

19.   Leder, O, and Groh R. Histochemische Untersuchungen über die Carboanhydrase-Aktivität der Rattenplacenta. Pfluegers Arch 285: 19-25, 1965.

20.   Lewis, SE, Erickson RP, Barnett LB, and Venta PJ. N-ethyl-N-nitroso-urea-induced null mutation at the mouse Car-2 locus: an animal model for human carbonic anhydrase II deficiency syndrome. Proc Natl Acad Sci USA 85: 1962-1966, 1988[Abstract/Free Full Text].

21.   Liao, SY, Brewer C, Zavada J, Pastorek S, Pastorekova S, Manetta A, Berman ML, DiSaia PJ, and Stanbridge EJ. Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial squamous and glandular neoplasia and cervical carcinomas. Am J Pathol 145: 598-609, 1994[Abstract].

22.   Longo, LD, Delivoria-Papadopoulos M, and Forster RE II. Placental CO2 transfer after fetal carbonic anhydrase inhibition. Am J Physiol 226: 703-710, 1974.

23.   Lucci, MD, Tinker JP, Weiner IM, and DuBose TD, Jr. Function of proximal tubule carbonic anhydrase defined by selective inhibition. Am J Physiol Renal Fluid Electrolyte Physiol 245: F443-F449, 1983[Abstract/Free Full Text].

24.   Matejevic, D, Grozdanovic Z, Gossrau R, Nakos G, and Graf R. Nitric oxide synthase I immunoreactivity and NOS-associated NADPHd histochemistry in the visceral epithelial cells of the intraplacental mouse yolk sac. Acta Histochem 98: 173-183, 1996[Web of Science][Medline].

25.   Mori, K, Ogawa Y, Ebihara K, Tamura N, Tashiro K, Kuwahara T, Mukoyama M, Sugawara A, Ozaki S, Tanaka I, and Nakao K. Isolation and characterization of CA XIV, a novel membrane-bond carbonic anhydrase from mouse kidney. J Biol Chem 274: 15701-15705, 1999[Abstract/Free Full Text].

26.   Mühlhauser, J, Crescimanno C, Rajaniemi H, Parkkila S, Milovanov AP, Castellucci M, and Kaufmann P. Immunohistochemistry of carbonic anhydrase in human placenta and fetal membranes. Histochemistry 101: 91-98, 1994[Web of Science][Medline].

27.   Müntener, M, and Hsu YC. Development of trophoblast and placenta of the mouse. A reinvestigation with regard to the in vitro culture of mouse trophoblast and placenta. Acta Anat (Basel) 98: 241-252, 1977[Web of Science][Medline].

28.   Nishita, T, Kinoshita C, Maegaki M, and Asari M. Immunohistochemical studies of the carbonic anhydrase isozymes in the bovine placenta. Placenta 11: 329-336, 1990[Web of Science][Medline].

29.   Parkkila, S, Parkkila AK, Juvonen T, Waheed A, Sly WS, Saarnio J, Kaunisto K, Kellokumpu S, and Rajaniemi H. Membrane-bound carbonic anhydrase IV is expressed in the luminal plasma membrane of the human gallbladder epithelium. Hepatology 24: 1104-1108, 1996[Web of Science][Medline].

30.   Pastorek, J, Patoreková S, Callebaut I, Mornon JP, Zelník V, Opavský R, Zat'ovicová M, Liao S, Portetelle D, Stanbridge EJ, Závada J, Burny A, and Kettmann R. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene 9: 2877-2888, 1994[Web of Science][Medline].

31.   Riad, NH, Bruns ME, Fares NH, Bruns DE, and Herr JC. Ultrastructural localization of the 9-kilodalton vitamin D-dependent calcium-binding protein in the murine intraplacental yolk sac. Anat Rec 222: 252-259, 1988[Medline].

32.   Ridderstråle, Y, Persson E, Dantzer V, and Leiser R. Carbonic anhydrase activity in different placenta types: a comparative study of pig, horse, cow, mink, rat, and human. Microsc Res Tech 38: 115-124, 1997[Web of Science][Medline].

33.   Schwartz, GJ, Brown D, Mankus R, Alexander EA, and Schwartz JH. Low pH enhances expression of carbonic anhydrase II by cultured rat inner medullary collecting duct cells. Am J Physiol Cell Physiol 266: C508-C514, 1994[Abstract/Free Full Text].

34.   Schwartz, GJ, Winkler CA, Zavilowitz BJ, and Bargiello T. Carbonic anhydrase II mRNA is induced in rabbit kidney cortex during chronic metabolic acidosis. Am J Physiol Renal Fluid Electrolyte Physiol 265: F764-F772, 1993[Abstract/Free Full Text].

35.   Sender, S, Gros G, Waheed A, Hageman GS, and Sly WS. Immunohistochemical localization of carbonic anhydrase IV in capillaries of rat and human skeletal muscle. J Histochem Cytochem 42: 1229-1236, 1994[Abstract].

36.   Simkovich, JW, Cefalo RC, Hellegers AE, and Bruns P. Effect of fetal hypercapnia on maternal and fetal cardiovascular and respiratory function. Eur J Obstet Reprod Biol 14: 311-315, 1983.

37.   Sismondi, P, Massobrio M, Coppo F, and Giardina G. L'influenza dell'anidrasi carbonica nel passaggio transplacentare degli idrogenioni. Minerva Ginecol 21: 1531-1539, 1969[Medline].

38.   Swenson, ER, Grønlund J, Ohlsson J, and Hlastal P. In vivo quantitation of carbonic anhydrase and band 3 protein contributions to pulmonary gas exchange. J Appl Physiol 74: 838-848, 1993[Abstract/Free Full Text].

39.   Tamai, S, Cody LB, and Sly WS. Molecular cloning of the mouse gene coding for carbonic anhydrase IV. Biochem Genet 34: 31-43, 1996[Web of Science][Medline].

40.   Tamai, S, Waheed A, Cody LB, and Sly WS. Gly-63right-arrowGln substitution adjacent to His-64 in rodent carbonic anhydrase IVS largely explains their reduced activity. Proc Natl Acad Sci USA 93: 13647-13652, 1996[Abstract/Free Full Text].

41.   Theiler, K. The Mouse House: Atlas of Embryonic Development. New York: Springer Verlag, 1989.

42.   Tsuruoka, S, Kittelberger AM, and Schwartz GJ. Carbonic anhydrase II and IV mRNA in rabbit nephron segments: stimulation during metabolic acidosis. Am J Physiol Renal Physiol 274: F259-F267, 1998[Abstract/Free Full Text].

43.   Türeci Ö, Sahin Y, Vollmar E, Siemer S, Göttert E, Seitz G, Parkkila AK, Shah GN, Grubb JH, Pfreundschuh M, and Sly WS. Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase that is overexpressed in some renal cell cancers. Proc Natl Acad Sci USA 95: 7608-7613, 1998[Abstract/Free Full Text].

44.   Waheed, A, Zhu XL, and Sly WS. Membrane-associated carbonic anhydrase from rat lung. Purification, characterization, tissue distribution, and comparison with carbonic anhydrase IVS of other mammals. J Biol Chem 267: 3308-3311, 1992[Abstract/Free Full Text].

45.   Wall, SM, Flessner MF, and Knepper MA. Distribution of luminal carbonic anhydrase activity along the rat inner medullary collecting duct. Am J Physiol Renal Fluid Electrolyte Physiol 260: F738-F748, 1991[Abstract/Free Full Text].

46.   Winkler, CA, Kittelberger AM, and Schwartz GJ. Expression of carbonic anhydrase IV mRNA in rabbit kidney: stimulation by metabolic acidosis. Am J Physiol Renal Physiol 272: F551-F560, 1997[Abstract/Free Full Text].

47.   Wooding, FBP, and Flint APF Placentation. In: Marshall's Physiology of Reproduction Part One. Ovulation and Early Pregnancy. Pregnancy and Lactation (4th ed.), edited by Lamming GE.. London: Chapman & Hall, 1994, vol. 3, p. 233-460.

48.   Zhu, XL, and Sly WS. Carbonic anhydrase IV from human lung. Purification, characterization, and comparison with membrane carbonic anhydrase from human kidney. J Biol Chem 265: 8795-8801, 1990[Abstract/Free Full Text].

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