J Vet Sci.  2008 Sep;9(3):233-240. 10.4142/jvs.2008.9.3.233.

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

• Yon JM
• Baek IJ
• Lee SR
• Kim MR
• Lee BJ
• Yun YW
• Nam SY

• KMID: 1718575
• DOI: http://doi.org/10.4142/jvs.2008.9.3.233

Abstract

Cytoplasmic Cu/Zn superoxide dismutase (SOD1) is an antioxidant enzyme that converts superoxide to hydrogen peroxide in cells. Its spatial distribution matches that of superoxide production, allowing it to protect cells from oxidative stress. SOD1 deficiencies result in embryonic lethality and a wide range of pathologies in mice, but little is known about normal SOD1 protein expression in developing embryos. In this study, the expression pattern of SOD1 was investigated in post-implantation mouse embryos and extraembryonic tissues, including placenta, using Western blotting and immunohistochemical analyses. SOD1 was detected in embryos and extraembryonic tissues from embryonic day (ED) 8.5 to 18.5. The signal in embryos was observed at the lowest level on ED 9.5-11.5, and the highest level on ED 17.5-18.5, while levels remained constant in the surrounding extraembryonic tissues during all developmental stages examined. Immunohistochemical analysis of SOD1 expression on ED 13.5-18.5 revealed its ubiquitous distribution throughout developing organs. In particular, high levels of SOD1 expression were observed in the ependymal epithelium of the choroid plexus, ganglia, sensory cells of the olfactory and vestibulocochlear epithelia, blood cells and vessels, hepatocytes and hematopoietic cells of the liver, lymph nodes, osteogenic tissues, and skin. Thus, SOD1 is highly expressed at late stages of embryonic development in a cell- and tissue-specific manner, and can function as an important antioxidant enzyme during organogenesis in mouse embryos.

Keyword

antioxidant enzyme; extraembryonic tissue; mouse embryos; superoxide dismutase

MeSH Terms

Animals
Cerebral Cortex/embryology/enzymology
Copulation
Cytoplasm/*enzymology
Embryonic Development/*physiology
Female
Immunohistochemistry
Lung/embryology/enzymology
Male
Mice
Mice, Inbred ICR
Organogenesis/*physiology
Pregnancy
Stomach/embryology/enzymology
Superoxide Dismutase/deficiency/genetics/*metabolism

Figure

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  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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  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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