Transmembrane carbonic anhydrase isozymes IX and XII in the female mouse reproductive organs

Abstract

Background: Carbonic anhydrase (CA) classically catalyses the reversible hydration of dissolved CO2 to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems.

Methods: The present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract.

Results: The data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta.

Conclusion: The conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.

Figure 1

Immunohistochemical staining of CA XII (A), CA IX (B), CA II (C), and CA XIII (D) in the mouse endometrium. All the studied CA isozymes show positive immuostaining, although the staining intensity varies between different isozymes. CA XII shows stronger reaction in the deep endometrial glands compared to the surface epithelium. This pattern is inversed with CA II showing high reaction in the surface epithelium. Insert in panel A shows that the CA XII immunostaining is most abundant in the basolateral plasma membrane of the epithelial cells. Insert in panel C demonstrates that CA II immunoreactivity is also closely associated with the plasma membrane. CA IX and XIII show faint immunoreactions in both the surface and glandular epithelia. Arrows = endometrial glands, arrowheads = surface epithelium. Original magnifications: × 400.

Figure 4

Control immunostaining of mouse uterus, ovary and placenta with normal rabbit serum. No immunoreaction is seen. Oringinal magnifications: × 400.

Figure 2

Immunolocalization of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse corpus luteum (A,C,E,G) and follicle (B,D,F,H). Only faint positive reaction for CA XII can be seen in occasional cells of the corpus luteum that is indicated in the insert of the panel A (arrows). Original magnifications: × 200 (A,C,E,G), × 400 (B,D,F,H).

Figure 3

Immunohistochemical staining of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse placenta (A,C,E,G) and amnionic epithelium (B,D,F,H). CA II is located in the endothelium of the blood vessels and erythrocytes (arrows in the panel E). It is also expressed in the amnionic epithelium (arrows in the panel F). Insert of the panel B shows that CA XII is highly expressed in the decidual glands, while the amnionic epithelium is negative. DE = Decidua, P = placenta. Original magnifications: × 400.

Figure 5

Western blotting of total homogenate from mouse uterus, kidney and colon for CA II, IX, XII and XIII. Normal non-immune rabbit serum (NRS) was used instead of the first antibodies as a negative control. Both CA IX and XII show weak positive reactions for the uterine proteins (arrowheads). The molecular weights for these isozymes are 51 and 46 kDa, respectively. The signal for CA XII is weaker in the uterus than in the colon or kidney. Note that anti-CA XII serum cross-reacts with a 30-kDa polypeptide. This cross-reaction is evident only in western blotting conditions as pointed out in the Results section. CA IX shows the strongest signal in the colon. CA II and XIII are positive in all tissue specimens.