In-Jeoung Baek

Expression pattern of cytosolic glutathione peroxidase (cGPx) mRNA during mouse embryogenesis

The selenoprotein cytosolic glutathione peroxidase (cGPx) is ubiquitously distributed in a variety of organs, and its primary function is to protect oxidative damage. To investigate the spatial and temporal expression pattern of cGPx mRNA in embryogenesis, as this has not been studied before, reverse transcription-polymerase chain reaction (RT-PCR) was carried out in a thermal cycler using mouse-specific cGPx primers, and in situ hybridization was performed in whole embryos or embryonic tissues using digoxigenin-labeled mouse cGPx riboprobes. Expression of cGPx mRNA was detected in all the embryos retrieved from embryonic days (EDs) 7.5 to 18.5. On EDs 10.5–12.5, cGPx mRNA was highly expressed in the margin of forelimb and hindlimb buds and dorsally in the cranial neural tube, including the telencephalon, diencephalon, and hindbrain neural tube. On ED 13.5, cGPx mRNA was accumulated especially in vibrissae, forelimb and hindlimb plates, tail, and spinal cord. On EDs 14.5–16.5, cGPx mRNA was found in the developing brain, Rathke’s pouch, thymus, lung, and liver. On ED 17.5, the expression of cGPx mRNA was apparent in various tissues such as brain, submandibular gland, vibrissae, heart, lung, liver, stomach, intestine, pancreas, skin, and kidney. In particular, cGPx mRNA was greatly expressed in epithelial linings and metabolically active sites such as whisker follicles, alveolar epithelium of lung, surface epithelium and glandular region of stomach, skin epithelium, and cortex and tubules of kidney. Overall results indicate that cGPx mRNA is expressed in developing embryos, cell-specifically and tissue-specifically, suggesting that cGPx may function to protect the embryo against reactive oxygen species and/or hydroperoxides massively produced by the intracellular or extracellular environment.

Black ginseng inhibits ethanol-induced teratogenesis in cultured mouse embryos through its effects on antioxidant activity.

Fetal alcohol syndrome is caused by excessive ethanol consumption during pregnancy. We investigated the effect of black ginseng (red ginseng that is subjected to 9 cycles of 95-100 degrees C for 2-3h) on ethanol-induced teratogenesis using an in vitro whole embryo culture system. Postimplantational mouse embryos at embryonic day 8.5 were exposed to ethanol (1 microl/ml) in the presence or absence of black ginseng (1, 10, and 100 microg/ml) for 2 days, and then morphological scoring and real-time PCR analysis were carried out. In ethanol-treated embryos, the total morphological score and individual scores for flexion, heart, fore-, mid-, and hindbrains, otic, optic, and olfactory systems, branchial bars, maxillary and mandibular processes, caudal neural tube, and somites were significantly lower than the control group (p<0.05). Treatment with black ginseng improved most of the morphological scores significantly as compared to ethanol-treated embryos (p<0.05). The mRNA levels of the antioxidant enzymes cytosolic glutathione peroxidase (GPx), phospholipid hydroperoxide GPx, and selenoprotein P were significantly decreased in ethanol-treated embryos, but co-treatment with black ginseng restored the mRNA levels to those of control embryos. These results indicate that black ginseng has a protective effect on ethanol-induced teratogenesis through the augmentation of antioxidative activity in embryos.

Korean red ginseng extract does not cause embryo‐fetal death or abnormalities in mice

BACKGROUND Ginseng has been used for a long time and is well tolerated in humans. However, recent studies have shown that ginsenosides Rb1, Rg1, and Re exert embryotoxicity in in vitro culture systems. We investigated the effects of Korean red ginseng extract (KRGE) on embryonic implantation and fetal development in mice. METHODS Mice were orally administered KRGE (20, 200, or 2,000 mg/kg/day) from 2 weeks before mating to gestational day (GD) 18, and implantation rate, fetal mortality, body weights, as well as external, visceral, and skeletal abnormalities were determined by Caesarean section on GD18. Ginsenosides in KRGE and in the blood of dams were identified and quantified by HPLC analysis. RESULTS KRGE did not affect embryonic implantation and mortality as well as fetal body weights up to 2,000 mg/kg/day (approximately 200 times clinical doses), the upper-limit dose recommended by the Korea Food and Drug Administration (KFDA). Although the prevalence of supernumerary ribs increased at the medium dose (200 mg/kg/day), no dose-dependent increases in external, visceral, and skeletal abnormalities were observed. Major ginsenosides such as Rb1, Rg1, and Re were not detected in the blood of dams based on their chromatographic profiles. CONCLUSIONS Considerable developmental toxicities of KRGE, even at the upper-limit dose, were not observed in mice. These results might be due to the negligible blood concentrations of ginsenosides in their original forms following oral administration, suggesting that in vitro experiments to assess the effects of ginsenosides on embryotoxicity may not reliably explain the risks of ginsenosides to in vivo embryo-fetal development.

The spatio-temporal expression pattern of cytoplasmic Cu/Zn superoxide dismutase (SOD1) mRNA during mouse embryogenesis

The cytoplasmic Cu/Zn-superoxide dismutase (SOD1) represents along with catalase and glutathione peroxidase at the first defense line against reactive oxygen species in all aerobic organisms, but little is known about its distribution in developing embryos. In this study, the expression patterns of SOD1 mRNA in mouse embryos were investigated using real-time RT-PCR and in situ hybridization analyses. Expression of SOD1 mRNA was detected in all embryos with embryonic days (EDs) 7.5–18.5. The signal showed the weakest level at ED 12.5, but the highest level at ED 15.5. SOD1 mRNA was expressed in chorion, allantois, amnion, and neural folds at ED 7.5 and in neural folds, notochord, neuromeres, gut, and primitive streak at ED 8.5. In central nervous system, SOD1 mRNA was expressed greatly in embryos of EDs 9.5–11.5, but weakly in embryos of ED 12.5. At EDs 9.5–12.5, the expression of SOD1 mRNA was high in sensory organs such as tongue, olfactory organ (nasal prominence) and eye (optic vesicle), while it was decreased in ear (otic vesicle) after ED 10.5. In developing limbs, SOD1 mRNA was greatly expressed in forelimbs at EDs 9.5–11.5 and in hindlimbs at EDs 10.5–11.5. The signal increased in liver, heart and genital tubercle after ED 11.5. In the sections of embryos after ED 13.5, SOD1 mRNA was expressed in various tissues and especially high in mucosa and metabolically active sites such as lung, kidney, stomach, and intestines and epithelial cells of skin, whisker follicles, and ear and nasal cavities. These results suggest that SOD1 may be related to organogenesis of embryos as an antioxidant enzyme.

Effects of endocrine disrupting chemicals on expression of phospholipid hydroperoxide glutathione peroxidase mRNA in rat testes

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), an antioxidative selenoprotein, is modulated by estrogen in the testis and oviduct. To examine whether potential endocrine disrupting chemicals (EDCs) affect the microenvironment of the testes, the expression patterns of PHGPx mRNA and histological changes were analyzed in 5-week-old Sprague-Dawley male rats exposed to several EDCs such as an androgenic compound [testosterone (50, 200, and 1,000 µg/kg)], anti-androgenic compounds [flutamide (1, 5, and 25 mg/kg), ketoconazole (0.2 and 1 mg/kg), and diethylhexyl phthalate (10, 50, and 250 mg/kg)], and estrogenic compounds [nonylphenol (10, 50, 100, and 250 mg/kg), octylphenol (10, 50, and 250 mg/kg), and diethylstilbestrol (10, 20, and 40 µg/kg)] daily for 3 weeks via oral administration. Mild proliferation of germ cells and hyperplasia of interstitial cells were observed in the testes of the flutamide-treated group and deletion of the germinal epithelium and sloughing of germ cells were observed in testes of the diethylstilbestrol-treated group. Treatment with testosterone was shown to slightly decrease PHGPx mRNA levels in testes by the reverse transcriptionpolymerase chain reaction. However, anti-androgenic compounds (flutamide, ketoconazole, and diethylhexyl phthalate) and estrogenic compounds (nonylphenol, octylphenol, and diethylstilbestrol) significantly upregulated PHGPx mRNA in the testes (p < 0.05). These findings indicate that the EDCs might have a detrimental effect on spermatogenesis via abnormal enhancement of PHGPx expression in testes and that PHGPx is useful as a biomarker for toxicity screening of estrogenic or antiandrogenic EDCs in testes.

Spatiotemporal expression of the selenoprotein P genein postimplantational mouse embryos

Selenoprotein P (Sepp) is an extracellular glycoprotein which functions principally as a selenium (Se) transporter and antioxidant. In order to assess the spatiotemporal expression of the Sepp gene during mouse embryogenesis, quantitative RT-PCR and in situ hybridization analyses were conducted in embryos and extraembryonic tissues, including placenta. Sepp mRNA expression was detected in all embryos and extraembryonic tissues on embryonic days (E) 7.5 to 18.5. Sepp mRNA levels were high in extraembryonic tissues, as compared to embryos, on E 7.5-13.5. However, the levels were higher in embryos than in extraembryonic tissues on E 14.5-15.5, but were similar in both tissues during the subsequent periods prior to birth. According to the results of in situ hybridization, Sepp mRNA was expressed principally in the ectoplacental cone and neural ectoderm, including the neural tubes and neural folds. In whole embryos, Sepp mRNA was expressed abundantly in nervous tissues on E 9.5-12.5. Sepp mRNA was also expressed in forelimb and hindlimb buds on E 10.5-12.5. In the sectioned embryos, on E 13.5-18.5, Sepp mRNA was expressed persistently in the developing limbs, gastrointestinal tract, nervous tissue, lung, kidney and liver. On E 16.5-18.5, Sepp mRNA expression in the submandibular gland, whisker follicles, pancreas, urinary bladder and skin was apparent. In particular, Sepp mRNA was detected abundantly in blood cells during all the observed developmental periods. These results show that Sepp may function as a transporter of selenium, as well as an antioxidant, during embryogenesis.

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

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.

Dynamic expression of manganese superoxide dismutase during mouse embryonic organogenesis

The balance between reactive oxygen species production and antioxidant defense enzymes in embryos is necessary for normal embryogenesis. To determine the dynamic expression profile of manganese superoxide dismutase (MnSOD) in embryos, which is an essential antioxidant enzyme in embryonic organogenesis, the expression level and distribution of MnSOD mRNA and protein were investigated in mouse embryos, as well as extraembryonic tissues on embryonic days (EDs) 7.5-18.5. MnSOD mRNA levels were remarkably high in extraembryonic tissues rather than in embryos during these periods. MnSOD protein levels were also higher in extraembryonic tissues than in embryos until ED 16.5, but the opposite trend was found after ED 17.5. MnSOD mRNA was observed in the chorion, allantois, amnion, ectoderm, ectoplacental cone and neural fold at ED 7.5 and in the neural fold, gut, ectoplacental cone, outer extraembryonic membranes and primitive heart at ED 8.5. After removing the extraembryonic tissues, the prominent expression of MnSOD mRNA in embryos was seen in the sensory organs, central nervous system and limbs on EDs 9.5-12.5 and in the ganglia, spinal cord, sensory organ epithelia, lung, blood cells and vessels, intestinal and skin epithelia, hepatocytes and thymus on EDs 13.5-18.5. Strong MnSOD immunoreactivity was observed in the choroid plexus, ganglia, myocardium, blood vessels, heapatocytes, pancreatic acinus, osteogenic tissues, brown adipose tissue, thymus and skin. These findings suggest that MnSOD is mainly produced from extraembryonic tissues and then may be utilized to protect the embryos against endogenous or exogenous oxidative stress during embryogenesis.

Capsaicin prevents ethanol-induced teratogenicity in cultured mouse whole embryos

Prenatal exposure to alcohol promotes the level of reactive oxygen species within embryos and results in developmental disorders. In this study, we investigated the effect of capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major pungent ingredient in red peppers, on ethanol-induced teratogenicity in mouse embryos (embryonic days 8.5-10.5). In response to ethanol administration (1.0 microl/ml), developmental parameters such as yolk sac circulation, allantois, heart, hindbrain, midbrain, forebrain, otic and optic systems, branchial bar, olfactory system, forelimb, hindlimb, and somites decreased significantly in comparison with those of control group (p<0.05). However, the concurrent administration of capsaicin (1 x 10(-8) microg/ml or 1 x 10(-7) microg/ml) and ethanol significantly ameliorated most of the morphological scores excepting yolk sac circulation and hindlimb scores (p<0.05). Furthermore, the levels of superoxide dismutase activity and cytoplasmic glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase mRNAs in the ethanol-treated embryos recovered to the levels observed in control embryos by capsaicin co-administration. These results indicate that capsaicin has a protective effect against ethanol-induced teratogenicity via an antioxidative activity.

Enhanced Protective Effects of Combined Treatment with β-Carotene and Curcumin against Hyperthermic Spermatogenic Disorders in Mice.

Scrotal hyperthermia leads to oxidative stress and apoptosis in spermatogenic cells, which subsequently causes male infertility. In this study, we examined the effects of β-carotene and/or curcumin on heat-stress- (HS-) induced testicular injuries in mice. ICR male mice (8 weeks old) were consecutively treated with β-carotene (10 mg/kg) and/or curcumin (20 mg/kg) orally once a day for 14 days and then subjected to single exposure with scrotal HS at 43°C for 15 min on day 7. HS induced a significant reduction in testicular weight, appearance of multinucleated giant cells, and desquamation of germ cells in destructive seminiferous tubules, as well as degenerative Leydig cells. Moreover, HS reduced the superoxide dismutase (SOD) activity and mRNA levels of mitochondrial SOD, phospholipid hydroperoxide glutathione peroxidase, B-cell lymphoma-extra-large, and 3β-hydroxysteroid dehydrogenase, with increases in lipid peroxidation levels and mRNA levels of BCL2-associated X protein and caspase-3 relative to those of the control group. However, these changes were significantly recovered by combined treatment with β-carotene and curcumin after HS. These findings indicate that the combined treatment with β-carotene and curcumin might be a valuable protective agent to ameliorate hyperthermic spermatogenic disorders via its potent antioxidative, antiapoptotic, and androgen synthetic effects.