Sheep primary astrocytes under starvation conditions express higher amount of LC3 II autophagy marker than neurons.

Autophagy is a general term for the degradation of cytoplasmic components within lysosomes. Recent studies have clearly demonstrated that autophagy has a greater variety of physiological and pathophysiological roles than expected, such as starvation adaptation, intracellular protein and organelle clearance, development, anti-aging, elimination of microorganisms, cell death, tumor suppression and antigen presentation. MAP-LC3 is one of the most common markers  to evaluate autophagic processes. In our study, the autophagic activity in neurons and astrocytes from sheep brain under starving conditions was evaluated. In order to detect LC3 immunoreactivity, confocal analysis by double immunofluorescence was performed together with the cell type markers: GFAP to identify astrocytes, ß-III tubulin to identify neurons. The results show that astrocytes are characterized by LC3-positive areas, which increase in a time-dependent manner. In contrast, LC3 immunoreactivity was very weak in neurons. Therefore, it can be assumed that astrocytes show a higher capability than neurons to cope with stress and exhibit a stronger autophagic response.

Brain aging and testosterone-induced neuroprotection: studies on cultured sheep cortical neurons.

OBJECTIVE: This research reports the expression of topoisomerase ßII in fetal sheep neuronal cells. The ß 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 ß-galactosidase activity and, in contrast, a reduction in topoisomerase ßII expression with time (first protocol). The exposure of sheep primary neurons to 3-nitro-L-tyrosine led to an upregulation of ßII topoisomerase expression to be likely seen as a reaction to nitrosative stress. Testosterone addition to 3-nitro-L-tyrosine-exposed cells results in topoisomerase ßII decrease possibly due to the neuroprotective properties of testosterone (second protocol). No significant variations in the marker of aging ß-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 ßII 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 ßII 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 IIß expression.

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

Aromatase, the enzyme converting androgens into estrogens, is involved in many brain processes such as neural differentiation and plasticity or the prevention of cell death. We have previously observed an increase in aromatase immunoreactivity in sheep neurons exposed in vitro to the oxidant 3-nitro-L: -tyrosine. However, little is known regarding the way that sheep astrocytes cope with nitrosative stress, a condition occurring in sheep in the pathogenesis of neurodegenerative disorders such as scrapie and Maedi-Visna. Our aim has been to evaluate the effects of 3-nitro-L-tyrosine on astrocyte primary cultures from 90-day-old fetal sheep brain. Living cells were observed and characterized by immunofluorescence with a GFAP antibody, which indicated that the majority of the cells were astrocytes. A viability assay was performed on both untreated and treated cells. Reverse transcription with the polymerase chain reaction was undertaken to monitor time- and dose-dependent variations in aromatase gene expression. Stressed astrocytes showed signs of deterioration, were reduced in number, and appeared round with few short processes; the cell death rate was ∼30%. Aromatase expression was detected starting from a 24-h exposure to 1 mM 3-nitro-L-tyrosine and reached the highest levels at 72 h. Thus, oxidative damage probably results in the local production of neuroprotective estradiol by reactive astrocytes via the aromatization of testosterone.

Expression and distribution of P450-aromatase in the ovine hypothalamus at different stages of fetal development.

OBJECTIVES: An important step of sexual differentiation is the conversion of testosterone to estrogen by aromatase leading to masculinization and defeminization of the fetal brain areas crucial for normal sexual behavior and reproduction. Brain sexual differentiation occurs throughout a critical period starting from different prenatal stages depending on the species. Such period goes on from gestation day (GD) 30 to 100GD in the sheep. The fetal sheep brain is reported to aromatize androgens to estrogens at 64GD. The main goal of this work was to evaluate aromatase expression in sheep hypothalami during the whole period of sexual differentiation (35GD, 55GD, 80GD, 115GD) and whether differences may be observed depending on gestational stage and sex. METHODS: Sections at the hypothalamic level underwent immunoperoxidase technique employing anti-aromatase and anti-androgen receptor antibodies. Samples from 35GD and 55GD were also processed with in situ hybridization using aromatase cDNA probe. Blot analyses were performed to quantify possible aromatase immunoexpression differences between sexes. For sexing, samples at 35GD and 55GD underwent DNA extraction and SRY amplification. RESULTS: Our results revealed aromatase and androgen receptor immunoreactivity along the whole period of sexual differentiation. Both molecules were detected in many brain regions and markedly in the periventricular area. The highest aromatase and androgen receptor amounts were observed at 35GD and 55GD, when aromatase was more abundant in females than in males. CONCLUSIONS: In conclusion, the sheep can be included among the species where aromatase is highly expressed in the hypothalamus during the whole period of sexual differentiation.