Article

Aromatase immunoreactivity in fetal ovine neuronal cell cultures exposed to oxidative injury

Department of Animal Biology, University of Sassari, Italy.
European journal of histochemistry: EJH (Impact Factor: 2.04). 12/2009; 53(4):e28. DOI: 10.4081/ejh.2009.e28
Source: PubMed

ABSTRACT

A lot of evidence testifies that aromatase is expressed in the central nervous system where it has been detected not only in hypothalamic and limbic regions but also in the cerebral cortex and spinal cord. In physiological conditions, aromatase is expressed exclusively by neurons, where it has been mainly found in cell bodies, processes and synaptic terminals. Moreover, primary cultured cortical astrocytes from female rats are more resistant to oxidant cell death than those from males, suggesting a protective role of estradiol. The aim of this study was to evaluate changes in aromatase expression in response to 3-nitro-L-tyrosine, a marker of oxidative stress, in primary neuronal cell cultures from brains of 60-day old sheep fetuses. Cells were identified as neurons by using class III β-tubulin, a marker of neuronal cells. Two morphological types were consistently recognizable: i) bipolar cells with an oval cell body; ii) multipolar cells whose processes formed a wide net with those of adjacent cells. In situ hybridization technique performed on 60-day old fetal neurons revealed that in baseline conditions aromatase gene expression occurs. Importantly, cells exposed to 360 µM 3-nitro-L-tyrosine were fewer and showed more globular shape and shorter cytoplasmic processes in comparison to control cells. The immunocytochemical study with anti-aromatase antibody revealed that cells exposed to 360 µM 3-nitro-L-tyrosine were significantly more immunoreactive than control cells. Thus, it can be postulated that the oxidant effects of the amino acid analogue 3-nitro-L-tyrosine could be counterbalanced by an increase in aromatase expression that in turn can lead to the formation of neuroprotective estradiol via aromatization of testosterone.

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Available from: Marco Zedda, Sep 17, 2014
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    • "The sheep was chosen as an experimental model on account of two considerations. The former is that sheep fetuses allow considerable amounts of brain tissue to be rapidly collected (Lepore et al. 2009). The latter is the interest elicited by sheep in the investigations of neurodegenerative processes as that species is susceptible of Scrapie and Maedi-Visna diseases. "
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    ABSTRACT: Abstract OBJECTIVE: This research reports the expression of topoisomerase βII in fetal sheep neuronal cells. The β isoform of DNA topoisomerase II plays a role in DNA repair process in non proliferating cells as neurons and its expression tends to be downregulated with senescence. METHODS: Cortical neurons from 60-day-old sheep embryos underwent two protocols: the former based on rising time of culture (10, 20 and 30 days); the latter based on the 72hrs exposure to 3-nitro-L-tyrosine (oxidative/nitrosative stressor) and/or testosterone. RESULTS: Our results showed an increase in β-galactosidase activity and, in contrast, a reduction in topoisomerase βII expression with time (first protocol). The exposure of sheep primary neurons to 3-nitro-L-tyrosine led to an upregulation of βII topoisomerase expression to be likely seen as a reaction to nitrosative stress. Testosterone addition to 3-nitro-L-tyrosine-exposed cells results in topoisomerase βII decrease possibly due to the neuroprotective properties of testosterone (second protocol). No significant variations in the marker of aging β-galactosidase were observed in the cells exposed to 3-nitro-L-tyrosine and testosterone. CONCLUSION: The protocol based on time could be of some interest as a model of neuronal senescence in vitro. Topoisomerase βII decrease with aging likely indicates a reduced ability to repair DNA during neuronal senescence. In contrast, the second protocol may not be seen as a reliable model of aging since 3-nitro-L-tyrosine does not lead to a topoisomerase βII decrease. Testosterone was able to cope with oxidative/nitrosative damage, allowing cells to reduce their needs in DNA repair which in turn leads to a downregulation of topoisomerase IIβ expression.
    Full-text · Article · Jan 2013
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    • "Oxidative stress is known as a powerful factor inducing pathological processes in cells by modifying their redox state: alterations in some enzyme activities have been investigated with special attention to those occurring as a consequence of hypoxia-reoxygenation.54–57 The evidence that hypoxia and aging in the myocardial tissue are controlled by p53 and telomerase activity57 confirms that molecular events modifying nuclear structure and function often play a pivotal role in pathogenesis; this is apparently confirmed by several articles in which nuclear histochemistry has been profitably used to elucidate basic mechanisms leading to the pathological cell phenotype.23–27,58–60 "
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    ABSTRACT: In recent years, omic analyses have been proposed as possible approaches to diagnosis, in particular for tumours, as they should be able to provide quantitative tools to detect and measure abnormalities in gene and protein expression, through the evaluation of transcription and translation products in the abnormal vs normal tissues. Unfortunately, this approach proved to be much less powerful than expected, due to both intrinsic technical limits and the nature itself of the pathological tissues to be investigated, the heterogeneity deriving from polyclonality and tissue phenotype variability between patients being a major limiting factor in the search for unique omic biomarkers. Especially in the last few years, the application of refined techniques for investigating gene expression in situ has greatly increased the diagnostic/prognostic potential of histochemistry, while the progress in light microscopy technology and in the methods for imaging molecules in vivo have provided valuable tools for elucidating the molecular events and the basic mechanisms leading to a pathological condition. Histochemical techniques thus remain irreplaceable in pathologist's armamentarium, and it may be expected that even in the future histochemistry will keep a leading position among the methodological approaches for clinical pathology.
    Full-text · Article · Oct 2011 · European journal of histochemistry: EJH
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    • "ison with glial cells. Like another study carried out at our laboratories (Lepore et al. 2009), the present investigation points out that primary cultures of sheep glial cells are less susceptible to injury mediated by 3-nitro-L-tyrosine than are neurons. Indeed, in the central nervous system, neurons seem to be more vulnerable than astrocytes to nitric oxide and peroxynitrite-mediated damage (Bolaños et al. 1995, 2008). "
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    ABSTRACT: A lot of evidence testifies that aromatase is expressed in the central nervous system where it has been detected not only in hypothalamic and limbic regions but also in the cerebral cortex and spinal cord. In physiological conditions, aromatase is expressed exclusively by neurons, where it has been mainly found in cell bodies, processes and synaptic terminals. Moreover, primary cultured cortical astrocytes from female rats are more resistant to oxidant cell death than those from males, suggesting a protective role of estradiol. The aim of this study was to evaluate changes in aromatase expression in response to 3-nitro-L-tyrosine, a marker of oxidative stress, in primary neuronal cell cultures from brains of 60-day old sheep fetuses. Cells were identified as neurons by using class III β-tubulin, a marker of neuronal cells. Two morphological types were consistently recognizable: i) bipolar cells with an oval cell body; ii) multipolar cells whose processes formed a wide net with those of adjacent cells. In situ hybridization technique performed on 60-day old fetal neurons revealed that in baseline conditions aromatase gene expression occurs. Importantly, cells exposed to 360 µM 3-nitro-L-tyrosine were fewer and showed more globular shape and shorter cytoplasmic processes in comparison to control cells. The immunocytochemical study with anti-aromatase antibody revealed that cells exposed to 360 µM 3-nitro-L-tyrosine were significantly more immunoreactive than control cells. Thus, it can be postulated that the oxidant effects of the amino acid analogue 3-nitro-L-tyrosine could be counterbalanced by an increase in aromatase expression that in turn can lead to the formation of neuroprotective estradiol via aromatization of testosterone.
    Full-text · Article · Dec 2009 · European journal of histochemistry: EJH
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