Carbonic anhydrase (CA) activity was localized in the late-term pregnant spiny dogfish uterus using the modified histochemical staining method of Hansson. Staining was found in the cytoplasm of red blood cells and in the membranes of the endometrial epithelial cells. Under the electron microscope, reaction products were seen in the basolateral membranes of the superficial cells, whereas the apical membranes were unstained. The cells of the underlying layer contacting the stromal capillaries showed staining in all their membranes. This staining pattern is similar to that found in other epithelia noted for active ion transport.
The pregnant late-term spiny dogfish, Squalus acanthias, is known to establish special environmental conditions for the pups lying in the uterus and completing the period of gestation. These are characterized by an acidic uterine fluid with high ammonia concentration and low CO2 content (Kormanik and Evans, 1986; Kormanik, 1988). The carbonic anhydrase (CA) inhibitor acetazolamide has been shown to prevent uterine seawater acidification and extraction of HCOa− (Kormanik and Kremer, 1986; Kormanik and Maren, 1988). However, it is not known which structures or cells are responsible for the acidification. During the sampling and investigation of developing embryos by Kormanik (1989) we were able to obtain uterine tissue from late-term pregnant spiny dogfish taken from Frenchman Bay at the Mount Desert Island Biological Laboratory (Salsbury Cove, Maine). We investigated the tissue histochemically to localize the carbonic anhydrase.
MATERIALS AND METHODS
Endometrial tissue from a late-term pregnant spiny dogfish uterus (about 6 kg, eighteenth month of gestation) was prepared by stripping the endometrium from the underlying muscle. The specimens also contained the branches of the posterior oviducal artery, which lie in the endometrial stroma between mucous membrane and muscle. The specimens were fixed for 3h in 2.5% glutaraldehyde solution buffered with 0.1 mol I−1 cacodylate buffer, pH7.4, then washed in the same buffer and embedded according to Ridderstråle (1976) in hydroxypropylmethacry-late (JB4, Polysciences, Pennsylvania). For the fight microscopical localization of CA, semithin sections 2 μm thick were stained according to the method of Hansson (1967) modified by Ridderstråle (1976, 1980). In the incubation medium the concentration of CoSO4 was 1.75 mmol I−1 and that of KH2PO4 was 11.7 mmol I−1. After being incubated for 1-9 min, the sections were washed in 0.9% NaCl, pH5.9, and floated in 0.5% (NH4)2S for 3min. The sections were washed three times for 1 min in distilled water, dried on glass slides and mounted in Eukitt (Kindler GmbH, Freiburg, FRG). Some of the sections were counterstained with Haematoxylin and Eosin before mounting in Eukitt.
Control sections were stained as described above, but with the incubation medium modified by the addition of 10μmoll−1 acetazolamide (Diamox; Cyana-mid-Novalis GmbH, Wolfratshausen, FRG). [For a discussion of the specificity of the method, see Lonnerholm (1974, 1980), Maren (1980) and Sugai and Ito (1980).] Other sections cut at 2 μm were stained with Richardson’s stain (Richardson et al. 1960) to compare them with the sections stained for CA.
For the electron microscopical demonstration of CA, 2μm thick methacrylate sections were stained as described above with periods of incubation ranging from 5 to 9 min. Those sections that showed clear staining without artificial precipitations were put on Thermanox™ tissue culture cover slips (Miles, Naperville, IL) and embedded in Epon. Ultrathin sections were cut and viewed with a Zeiss 902 EM either without counterstaining or after staining with uranyl acetate and lead citrate.
The mucosal epithelium consists of partly single-layered but mostly two-layered cuboidal cells (Figs 1A, 2A). The underlying stromal tissue is loosely arranged and contains many free cells. A large number of capillaries form a dense network lying directly underneath the mucosal epithelium. The epithelial cells are connected to the capillaries by foot-like cytoplasmic extensions (Figs 1A, 2A).
After staining for CA, reaction products are found only in the mucosal epithelial cells (Figs 1B, 2B). The staining appears after 4 min of incubation. No staining is seen in the endothelium of the capillaries or in any stromal cell (Fig. 1B). The red blood cells within the capillaries show faint cytoplasmic staining (Fig. 1B).
In the mucosal epithelial cells the lateral portions, including the foot-like extensions towards the stromal capillaries, are stained, whereas the luminal surface is not. With the electron microscope one can see that the reaction products are localized in the basolateral membranes, which have numerous invaginations. If two layers of epithelial cells are present, all the cell membranes of the round cells lying between the superficial cells and stromal cells are also stained. The cytoplasm near the stained membranes shows abundant mitochondria (Fig. 3).
The control sections treated with 10μmol I−1 acetazolamide in the incubation medium show total inhibition of staining.
In this study we demonstrate that the enzyme CA in the pregnant spiny dogfish uterus is localized not only in the red blood cells but also in the epithelium itself. In the surface epithelium, the basolateral cell membranes stain in the same way as epithelia performing active ion transport, e.g. intestine, choroid plexus, etc. (for a review, see Lütjen-Drecoll et al. 1985).
In places where the uterus epithelium consists of two cell layers, the abluminal cells also show membrane-bound staining. This staining pattern is similar to that of the stria vascularis of the inner ear (Hsu and Nomura, 1985; E. Lütjen-Drecoll, unpublished observations) and the ciliary epithelium in the eye (Hansson, 1968; Lütjen-Drecoll and Lonnerholm, 1981), where all the cells between the capillary and the lumen are stained.
The presence of abundant mitochondria adjacent to the stained membranes as well as the close contact of the stained foot-like extensions with the stromal capillaries support the suggestion that both cell layers are involved in active transport.
Cytoplasmic staining for CA was not observed in the epithelium. However, lack of staining does not prove the absence of an enzyme. It is well known that during the processing for histochemical staining some enzymes can be lost from the cytoplasm (Ridderstråle, 1980; Hyyppä, 1968). In contrast, the intense staining of the mucosal epithelial cells indicates real abundance of the enzyme in this location. Inhibition of the epithelial CA by acetazolamide could explain the changes in the uterine lumen described by Kormanik and Kremer (1986).
We are most grateful to J. Gehr and K. Gohler for their expert technical assistance and to M. GoBwein for his photographic assistance.