Iwabuchi J et al. (2013), Expression profile of the aromatase enzyme in t...

XB-ART-47843

Gen Comp Endocrinol 2013 Dec 01;194:286-94. doi: 10.1016/j.ygcen.2013.09.024.

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Expression profile of the aromatase enzyme in the Xenopus brain and localization of estradiol and estrogen receptors in each tissue.

Iwabuchi J , Koshimizu K , Nakagawa T .

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Estradiol (E2) with the strongest bioactivity of the estrogens, is synthesized by the cytochrome p450 aromatase enzyme and plays a key role in sex differentiation of the vertebrate's gonads. In Xenopus, aromatase mRNA is highly expressed in the brain rather than in the gonad during sex differentiation. In this study, we analyzed the stage change, tissue specificity, and localization of the aromatase expression in the Xenopus brain. Regardless of the sex difference, expression level of aromatase was remarkably higher in the brain than in other tissues during the early stages of brain morphogenesis and was observed in the formation regions of the choroid plexus of cerebral ventricle and the paleocortex and olfactory bulb of the prosencephalon. However, E2 concentrations in each tissue indicated a different localization of aromatase and were seen in the heart at almost double the level as seen in the brain. In addition, while aromatase expression level in the brain was increasing, E2 in the whole body began to increase at the same stage. Since the expression level of estrogen receptor α also corresponded to localization of E2, these results may imply that the E2 synthesized by the high aromatase expression in the choroid plexus, which generates cerebrospinal fluid, circulates to the heart and acts through ERα.

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Species referenced: Xenopus laevis
Genes referenced: cyp19a1 msc

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	Fig. 1. Expression patterns of aromatase mRNA in Xenopus at the larval stage. Aromatase mRNA levels were quantified in the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and in the ZZ or ZW brain, gonads, liver, and heart at stages 50 and 56 (B). The relative transcription levels of aromatase mRNA were normalized to the transcription level of EF-1αs mRNA. The expression level in brains at stage 50 was set at 100%. Each real-time RT-PCR analysis was performed at least in triplicate. Error bars represent the standard error (SE) of means. An asterisk indicates a significant difference of mRNA expression level between ZZ and ZW using a Student t-test (∗P < 0.05).
	Fig. 2. Morphological change of the Xenopus brain at stages 42–54. Vertical section of the hemisphere (A–D) and cross section of the prosencephalon (E–H) for stage 42 (A and E), stage 46 (B and F), stage 50 (C and G), and stage 54 (D and H) were stained with hematoxylin–eosin (HE). Arrowheads show the formation region at the stage. PSC, prosencephalon; DC, diencephalon; MSC, mesencephalon; MTC, metencephalon; ID, infundibulum; PG, pituitary gland; LV, lateral ventricle; PC, paleocortex; CP; choroid plexus, OB, olfactory bulb; OL, optic lobe. Scale bars; 200 μm.
	Fig. 3. Expression patterns of aromatase protein in Xenopus at the larval stage. Western blotting analysis was performed using anti-aromatase antibody and anti-actin antibody as an internal standard. Mix of nuclear and cytosolic extracts from the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and brain, gonads, liver, and heart at stages 50 and 56 (B) were examined by immunoblotting with each primary antibody followed by an HRP-conjugated anti-mouse or rabbit IgG antibody.
	Fig. 4. Localization of aromatase protein in Xenopus brain at stage 50. The brain at stage 50 from the dorsal viewpoint to show orientation of sections in B-P (A); the vertical section (B–H), and the cross section (I–P). Immunohistochemistry (IHC) staining was performed using anti-aromatase antibody followed by an HRP-conjugated anti-mouse IgG antibody. Nuclei were counterstained by hematoxylin. Red arrowheads show parts with strong staining. PSC, prosencephalon; DC, diencephalon; MSC, mesencephalon; PC, paleocortex; CP; choroid plexus, OB, olfactory bulb. Scale bars, 200 μm. (For interpretation of color in this Figure, the reader is referred to the web version of this article.)
	Fig. 5. Time course and tissue distribution of estradiol (E2) concentration in Xenopus at the larval stage. E2 concentrations were quantified in the mixture of ZZ and ZW brains or the whole body at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and brain, gonads, liver, and heart at stage 50 (B). Each point presents mean ± SE (n ⩾ 3) of E2 concentration in pg per brain or individual (A) or tissue (B).
	Fig. 6. Expression characteristics of estrogen receptors (ERs) in Xenopus at the larval stage. ER mRNA levels were quantified in the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (A) and in ZZ or ZW brain, gonads, liver, and heart at stage 50 (B and C) for ERα (A and B) and ERβ (C). The relative transcription levels of ER mRNA were normalized to the transcription level of EF-1αs mRNA. The expression level in the stage-50 brains was set at 100%. Each real-time RT-PCR analysis was performed at least in triplicate. Error bars represent the standard error (SE) of means. ∗P < 0.05. Western blotting analysis was performed using anti-ERα antibody and anti-actin antibody as an internal standard. Mix of nuclear and cytosolic extracts from the mixture of ZZ and ZW brains at stages 42, 44, 46, 48, 50, 52, 54, and 56 (D) and brain, gonads, liver, and heart at stage 50 (E) was examined by immunoblotting with each primary antibody followed by an HRP-conjugated anti-rabbit IgG antibody.