Immunohistochemical identification and quantitative analysis of cytoplasmic Cu/Zn superoxide dismutase in mouse organogenesis

Experimental animals

Male and female ICR mice (8 to 10 weeks old) were purchased from a commercial breeder, Biogenomics (Korea). All animals were housed in polycarbonate cages and acclimatized for 1 wk. The environmental conditions were controlled, with an ambient temperature of 21 ± 2℃, relative humidity of 55% ± 10%, air ventilation rate of 10 cycles per h, and a 12 : 12 h light : dark cycle. The animals were fed a standard mouse chow (Samyang, Korea) and tap water ad libitum throughout the experimental period. All experiments were approved and performed according to the "Guide for Care and Use of Animals" (Chungbuk National University Animal Care Committee, Korea).

Preparation of embryos and extraembryonic tissues

One male and two female mice were housed in cages for mating. Successful mating was confirmed by the presence of a copulation plug in the vagina. The day on which the vaginal plug was observed was designated ED 0.5. Pregnant mice were sacrificed under pentobarbital anesthesia on ED 8.5-18.5, and embryos and extraembryonic tissues, including the placenta, were dissected.

Protein extraction and Western blotting analysis

Total protein was extracted from mouse embryos and extraembryonic tissues taken on ED 8.5-18.5. Tissue samples were homogenized in lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 0.5 mM sodium orthovanadate, and 20 mM sodium pyrophosphate). The lysates were clarified by centrifugation at 14,000 rpm for 10 min and incubated at 95℃ in loading buffer (0.125 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.002% bromophenol blue) for 5 min before electrophoresis. Aliquots containing 60 µg of total protein were separated by SDS-PAGE (using 10% polyacrylamide gels) and transferred to nitrocellulose membranes (Immobilon; Millipore, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline [TBS; 25 mM Tris (pH 7.4), 200 mM NaCl] for 1 h at room temperature to eliminate nonspecific binding, and were then incubated with the primary antibodies (diluted 1 : 1,000 in 5% non-fat milk in TBST), polyclonal rabbit anti-SOD1 (Stressgen, Canada) and polyclonal rabbit anti-β-actin (Cell Signaling, USA). Next, membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (1 : 1,000; Cell Signaling, USA) for 1 h at room temperature, visualized using a Western Lightning Chemiluminescence reagent (Perkin-Elmer Life Sciences, USA) according to the manufacturer's protocol, and photographed (Gel Doc EQ; Bio-Rad, USA). Immunoreactive bands were quantified and normalized using PDQuest Image software (Bio-Rad, USA). Experiments were performed in triplicate, and data are presented as mean ± SD.

Immunohistochemistry

For immunohistochemical analysis, mouse embryos obtained on ED 13.5-18.5 were fixed in Bouin solution. Tissue sections were deparaffinized with xylene and rehydrated through an ethanol gradient. Endogenous peroxidase activity was quenched by 0.3% hydrogen peroxide in methanol for 15 min, and then sections were washed 4 times in PBS, for 5 min each time. Nonspecific binding was blocked with a 20 min incubation in diluted normal serum, and sections were incubated for 1 h at 37℃ with the SOD1 antibody. Next, sections were incubated for 30 min at room temperature with a biotinylated secondary antibody (Vector, USA) followed by 40 min at room temperature with the peroxidase-conjugated biotin-avidin complex (Vectastain ABC kit; Vector, USA). Finally, the bound peroxidase was revealed by immersing the sections in diaminobenzidine (Vector, USA). Sections were counterstained with hematoxylin, rehydrated for 15 min in deionized water to remove any precipitated Tris, and then dehydrated in a series of ethanol and xylene. Negative control experiments for the antibodies were performed as above with the omission of the primary antibody.

Expression of SOD1 in developing embryos and extraembryonic tissues

Using Western blot analysis with β-actin as an internal standard, SOD1 was detected in all embryos and extraembryonic tissues from ED 8.5 to 18.5. The signal in embryos was observed at the lowest levels on ED 9.5-11.5 and the highest levels on ED 17.5-18.5 (Fig. 1A). Extraembryonic expression of SOD1 remained constant throughout all stages of embryonic development (Fig. 1B).

Fig. 1
Relative expression levels of SOD1 protein in developing embryos (A) and extraembryonic tissues (B). Total protein was extracted from mouse embryos and extraembryonic tissues on embryonic day (ED) 8.5 to 18.5, and Western blotting was performed using an anti-SOD1 antibody. β-actin was used as an internal standard.

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Localization pattern of SOD1 during organogenesis

SOD1 immunoreactivity was detected in various embryonic tissues from ED 13.5 to 18.5. The distribution and quantitative expression of SOD1 in developing embryos are described in detail below (Figs. 2 and 3) and are summarized in Table 1. In developing brains, SOD1 immunoreactivity was detected at moderate levels in the external layers (Fig. 2A; asterisk) and pyramidal cells of the cerebral cortex on ED 13.5 to 18.5 (Fig. 2B) and in the cerebellum on ED 17.5 and 18.5 (Fig. 2C). A strong SOD1 signal was observed in the ependymal epithelium of the choroid plexus on ED 13.5 to 18.5 (Fig. 2D) and in Rathke's pouch (hypophysis) after ED 15.5 (Figs. 2E and F). The SOD1 signal was also strong in the developing spinal ganglia on ED 13.5 to 17.5, but was present at only a moderate level on ED 18.5 (Fig. 2G). Moderate SOD1 immunoreactivity was detected in the spinal cord on ED 13.5 to 16.5, and the signal was strong on ED 17.5 and 18.5 (Table 1).

Fig. 2
Immunohistochemistry using a SOD1 antibody on sagittal sections of developing mouse embryos. (A) Embryonic day (ED) 17.5 cerebral cortex (outer layer; asterisk) (×100). (B) ED 17.5 pyramidal cells of cerebral cortex (×400). (C) ED 17.5 cerebellum (×100). (D) ED 17.5 choroid plexus (×100). (E) ED 15.5 Rathke's pouch (×400). (F) ED 17.5 Rathke's pouch (×100). (G) ED 15.5 spinal ganglion (×400). (H) ED 16.5 inner ear in bony labyrinth (×40). (I) ED 16.5 sensory epithelium of inner ear (×400). (J) ED 17.5 nasal cavity (×100), Bowman's glands (arrow). (K) ED 17.5 olfactory epithelium (×400). (L) ED 18.5 eye (×40), iris (arrow) and pigment layer (arrowhead) of retina. (M) ED 16.5 lung (×40). (N) ED 16.5 lung (×200), terminal bronchial epithelia of lung (asterisk). (O) ED 17.5 heart (×100). (P) ED 13.5 blood cells (×400). (Q) ED 17.5 blood vessel (×200; asterisk). (R) ED 16.5 stomach (×200). (S) ED 16.5 goblet cell (arrow) and Paneth cell (arrowhead) of intestine (×400). (T) ED 17.5 intestine (×100).

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Fig. 3
Immunohistochemistry using a SOD1 antibody on sagittal sections of developing mouse embryos. (A) Embryonic day (ED) 16.5 liver (×100). (B) ED 16.5 liver (×400). (C) ED 17.5 pancreas (×400). (D) ED 18.5 submandibular gland (×200). (E) ED 16.5 kidney (×200). (F) ED 14.5 adrenal gland (×100). (G) ED 16.5 adrenal gland (×200). (H) ED 17.5 thymus (×200). (I) ED 16.5 lymph node (×40). (J) ED 16.5 bone (×200). (K) ED 18.5 bone (×100). (L) ED 14.5 skin (×200). (M) ED 17.5 skin (×100). (N) ED 18.5 collagen fiber-like tissue (×100). (O) ED 17.5 skin (×100); negative control.

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Table 1
Comparison of cytoplasmic Cu/Zn superoxide dismutase expression in developing embryonic organs following embryonic day

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After ED 15.5, SOD1 was strongly expressed in the sensory cells of the cochlear and vestibular epithelia of the inner ear (Figs. 2H and I) and in the olfactory cells and Bowman's glands in the olfactory epithelium (Figs. 2J and K). In developing eyes, strong SOD1 immunoreactivity was observed in the lens fiber and the retinal pigment epithelium (RPE) on ED 16.5, and in the iris and RPE on ED 18.5 (Fig. 2L).

In the parenchyma of the developing lung, weak expression of SOD1 was observed on EDs 13.5 to 15.5, becoming moderate on ED 16.5 to 18.5. Strong SOD1 immunoreactivity was observed in the bronchial and terminal bronchiolar epithelia after ED 15.5 (Figs. 2M and N). SOD1 was weakly expressed in the myocardium of the developing heart up until ED 16.5, and increased slightly after ED 17.5 (Fig. 2O). In contrast, strong SOD1 immunoreactivity was consistently observed in nucleated and enucleated blood cells and in blood vessels (Figs. 2P and Q).

SOD1 was strongly expressed in the basal cells and muscular layers of the stomach on ED 16.5 (Fig. 2R). In developing intestine, SOD1 expression was moderate in the intestinal villi and muscle layers after ED 15.5, but was strong in the goblet cells and basal cell part of the crypt of Lieberkuhn after ED 16.5 (Fig. 2S and T). In the developing liver, strong SOD1 immunoreactivity was observed in hepatocytes and hematopoietic cells during all periods examined (Figs. 3A and B). In the developing pancreas, moderate SOD1 immunoreactivity was observed in the acinus and islet on ED 14.5 to 16.5. After ED 17.5, the SOD1 signal was higher in the acinus than the islet of the pancreas (Fig. 3C). Strong SOD1 immunoreactivity was observed in secretory acini and ducts of the submandibular glands on EDs 17.5 to 18.5 (Fig. 3D).

In the developing kidney, SOD1 immunoreactivity was higher in the cortex than in the medulla, but the signal intensity was moderate in the metanephric corpuscles and tubules on ED 13.5 to 16.5 (Fig. 3E). On ED 17.5, a strong SOD1 signal was observed in the glomeruli, but moderate in the tubules. However, the signal intensity in tubules and glomeruli was reversed on ED 18.5 (Table 1). Moderate SOD1 immunoreactivity was observed in the luminal epithelium of the urinary bladder on EDs 15.5 and 16.5. On ED 18.5, SOD1 immunoreactivity was strong in the surrounding muscular layers, but was weak in the epithelium (Table 1).

In the developing adrenal glands, SOD1 expression was higher in the medulla than in the cortex on EDs 13.5 and 14.5 but was similar in both areas on ED 15.5. On ED 16.5 to 18.5, the SOD1 signal in the cortex had exceeded that observed in the medulla (Figs. 3F and G).

Strong SOD1 expression was observed in the thymus on ED 15.5 to 17.5 (Fig. 3H), whereas expression was moderate on ED 18.5. In the developing lymph nodes, SOD1 expression was weak on ED 14.5 and was high after ED 15.5 (Fig. 3I). In the developing bone and cartilage, a very strong SOD1 signal was observed in chondrocytes on ED 13.5 to 18.5 (Figs. 3J and K). Strong SOD1 immunoreactivity was observed in the surface epithelium, hair follicles, and muscle layers of the skin after ED 15.5 (Figs. 3L and M) and in a collagen fiber-like tissue on ED 18.5 (Fig. 3N).