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

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.

Aromatase expression in cultured fetal sheep astrocytes after nitrosative/oxidative damage

“…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(Bolaños et al , 2008.…”

“…The methodology that we used to obtain astrocytic primary cultures was the same as that performed to obtain neuronal primary cultures in a previous investigation (Lepore et al 2009). Preliminary tests performed at our laboratories have ascertained that the most suitable period to obtain viable astrocytes is around day 90 of sheep pregnancy, whereas neurons can grow successfully from around day 60.…”

“…AP00001PU-N) diluted 1:200 and a monoclonal anti-GFAP antibody (Sigma) diluted 1:100 were used. The immunoreactivity of the aromatase antibody had been preliminarily validated in the sheep in two previous studies carried out in our laboratories (Lepore et al 2009;Mura et al 2010).…”

Autophagic processes increase during senescence in cultured sheep neurons and astrocytes

“…The procedure to obtain neural primary cultures was the same previously described 28 and the most suitable period to grow viable neurons and astrocytes is around day 40 of sheep gestation. 29 …”

Protective Actions of 17β-Estradiol and Progesterone on Oxidative Neuronal Injury Induced by Organometallic Compounds

“…The expression of cytochrome P450 aromatase (CYP19), which synthesizes 17 β -estradiol from testosterone, is increased by oxidative stress in neurons and astrocytes [ 76 , 77 ], indicating that oxidative stress could potentiate 17 β -estradiol production. Cultured astrocytes isolated from female rats with high expression levels of cytochrome P450 aromatase showed better tolerance to cellular injury induced by oxygen-glucose deprivation than male rats with low cytochrome P450 aromatase expression [ 78 ].…”