Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System
Abstract
:1. Introduction
2. Results
2.1. Nuclear Localization of Aromatase in Astrocytes
2.2. Microglia Cells Only Show Cytoplasmic Aromatase Positivity
2.3. Estrogen Receptor Alpha Is Strongly Expressed in Astrocytes
2.4. Colocalization of Nuclear Aromatase and Estrogen Receptor Alpha
2.5. Nuclear Aromatase Expression Is Not Present in the Ovarian Cells and Tissue
3. Discussion
4. Materials and Methods
4.1. Housing and Handling of the Animals
4.2. Preparation of Mixed Primary Cortical Cultures and Glia Enriched Subcultures
4.3. Preparation of Human Granulosa Cell Culture
4.4. Immunocytochemistry
4.5. Immunostaining of Frozen Brain Tissue Sections
4.6. Immunostaining of Formalin-Fixed, Paraffin-Embedded Ovary Tissue Sections
4.7. Image Analysis and Statistics
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Species | Cell Type/Area | Localization of Aro | Method | Reference |
---|---|---|---|---|
Rat (Sprague-Dawley, adult, male, female) | Neurons/hypothalamus Neurons/subcortical telencephalic areas | Granular immunoreactivity in the somata and axon-like processes, but no staining in dendrites in hypothalamic cells Homogeneous but patchy labelling in the somata and dendritic processes in the subcortical areas Glia cells were immunonegative! There was no sexual dimorphism in the distribution and intensity of the staining No indication of nuclear localization | Immunostaining (a purified polyclonal rabbit-anti human placental Aro; In-house antibody) | [13] |
Rat (Wistar, adult, male) | Pyramidal neurons/CA1-CA3 Granule cells/dentate gyrus Astrocytes/stratum radiatum and oriens Oligodendrocytes/hippocampus | Cell body, dendrites in CA3 pyramidal neurons, axon terminals and dendritic spines of principal cells, pre- and postsynaptic compartments Most of the glia cells were lacking P450arom immunoreactivity No indication of nuclear localization | Immunohistochemistry (a anti-P450arom IgG; In-house antibody) Postembedding immunogold labelling | [18] |
Rat (Wistar, adult, male) | Neurons/olfactory bulb | Immunoreactivity mainly in the somata and also in cellular processes in juxtaglomerular neurons Weaker immunoreactivity in the cytoplasm (surrounding the nuclei) of mitral/tufted cells No indication of nuclear localization | Immunohistochemistry (b rabbit anti-ARO polyclonal antibody-epitope corresponding to amino acids 209–503 mapping at the C-terminus of human CYP19. Commercial antibody, Santa Cruz Biotechnology; Antibody registry ID: AB_2088681) | [16] |
Rat (Sprague-Dawley newborn, adult, male, female) | Nucleus caudatus/putamen Nucleus accumbens core and shell | Immunopositivity in the cytoplasm but empty nucleus No sexual dimorphism in the Aro immunopositivity No indication of nuclear localization | Immunohistochemistry (c monoclonal anti-aromatase, Residue 376–390 human p450, clone H4, Commercial antibody, BioRad; Antibody registry ID: AB_566942) | [4] |
Human (male, female) | Neurons and glia cells/cholinergic basal forebrain nuclei, hypothalamic nuclei | Immunoreactivity in the processes and the soma in granular cytoplasmic pattern accumulating around the nucleus and near plasmalemma (staining the endoplasmic reticulum) in neurons Immunopositivity in glia cells (astrocytes, oligodendrocytes, ependymal and choroid plexus cells) but no data about the subcellular localization No indication of nuclear localization | Immunohistochemistry (d polyclonal rabbit antibody raised against the 20 amino acid peptide coupled to thyroglobulin by glutaraldehyde corresponding to the N-terminus of human Aro; In-house antibody) | [14] |
Human (male, female) | Neurons/lateral temporal neocortex Astrocytes/ lateral temporal neocortex | Immunoreactivity in the perikaryon and proximal processes Immunoreactivity located in the soma and processes No indication of nuclear localization | Immunohistochemistry (antibody A: rabbit polyclonal AB recognizes human, rat, bovine, mouse, chicken Aro. Commercial antibody, Acris Antibody) (e antibody B: rabbit polyclonal AB generated from 15 amino acid peptic corresponding to residues 488–502 of mouse Aro. In-house antibody) | [15] |
Human (male, female) | Whole brain | Distribution volume values followed the rank order: thalamus > amygdala > preoptic area > medulla > cortex > putamen > cerebellum > white matter No information about the subcellular distribution of aromatase No indication of nuclear localization | Positron emission tomography (PET) (with radiolabelled aromatase inhibitor[N-methyl-11C]vorozole) | [20] |
Japanese quail (adult, male) | Neurons/hindbrain | Immunoreactive perikarya with immunonegative nuclei in neurons Immunoreactive fibres with punctate structures were also observed in neurons No indication of nuclear localization | Immunohistochemistry (polyclonal affinity-purified rabbit-anti quail recombinant antibody; In-house antibody) | [32] |
Name/Order Number (Manufacturer) Antibody Registry ID | Host/Clonality | Immunogen | Reactivity | Dilution | Citation |
---|---|---|---|---|---|
Aromatase NB100-1596 (Novus Biologicals) AB_10000919 | Rabbit/Polyclonal | C-terminal portion of the human aromatase protein (between residues 400–502) | Human, Mouse, Rat, Primate, Bovine, Rabbit | 1:100 | [31] |
CD11b/c (OX42) MA1-90756 (Thermo Scientific/Invitrogen) AB_2280688 | Mouse/Monoclonal | Rat peritoneal macrophages | Rat | 1:100 | [44] |
Estrogen receptor alpha MA1-310 (Thermo Scientific/Invitrogen) AB_325422 | Mouse/Monoclonal | Synthetic peptide corresponding to the residues E(247) V G M M K G G I R K D R R G(261) of the ER alpha DNA binding domain | Bovine, Human, Human, Mouse, Primate, Rat | 1:100 | [45] |
Glial fibrillary acidic protein (GFAP) MA5-12023 (Thermo Scientific/Invitrogen) AB_10984338 | Mouse/Monoclonal | Glial fibrillary acidic protein | Chicken, Human, Pig, Rat | 1:100 | [46] |
Glial fibrillary acidic protein (GFAP) PA1-10019 (Thermo Scientific/Invitrogen) AB_1074611 | Rabbit/Polyclonal | Full-length recombinant human GFAP protein | Bovine, Horse, Human, Mouse, Pig, Rat | 1:1000 | [47] |
S100β 287 011 (Synaptic Systems) AB_2814881 | Mouse/Monoclonal | Recombinant protein corresponding to AA 1 to 92 from rat S100B | Mouse, Rat | 1:600 | [48] |
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Kata, D.; Gróf, I.; Hoyk, Z.; Ducza, E.; Deli, M.A.; Zupkó, I.; Földesi, I. Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System. Int. J. Mol. Sci. 2022, 23, 8946. https://doi.org/10.3390/ijms23168946
Kata D, Gróf I, Hoyk Z, Ducza E, Deli MA, Zupkó I, Földesi I. Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System. International Journal of Molecular Sciences. 2022; 23(16):8946. https://doi.org/10.3390/ijms23168946
Chicago/Turabian StyleKata, Diána, Ilona Gróf, Zsófia Hoyk, Eszter Ducza, Mária A. Deli, István Zupkó, and Imre Földesi. 2022. "Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System" International Journal of Molecular Sciences 23, no. 16: 8946. https://doi.org/10.3390/ijms23168946