Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Aromatase (Aro) expression in astrocytes in glia-enriched subcultures. Cells were isolated from P0–P5 newborn rats. Aro (red) is strongly expressed in GFAP-positive astrocytes (green) and colocalizes with the nuclear staining (DAPI, blue). Fibrous astrocytes with small somata and long, thin unbranched processes ( a – h ) show strong nuclear Aro expression ( a , c , d , white arrowheads). Cytoplasmic Aro expression is weaker, and the long thin processes do not show Aro immunopositivity. Protoplasmic astrocytes ( i – p ) with bigger somata and short, thick and frequently branched processes also display nuclear Aro expression ( i , k , l , white arrowheads). Weak immunopositivity is detectable in the processes. The nuclear Aro signal distributes evenly or in a dotted pattern ( c , k , white arrowheads) or strongly concentrates to certain parts of the nucleus ( g ) in both astrocyte subtypes. (White arrowheads indicate a selected representative cell and its nucleus with the Aro signal). Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Expressing, Isolation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Quantitative analysis of aromatase localization in astrocytes. Analysis of mean fluorescent intensity revealed that Aro intensity is significantly stronger in the nucleus than in the cytoplasm in different astrocyte subtypes. GFAP-positive fibrous astrocytes showed stronger nuclear Aro intensity (36.36 ± 7.7; n = 25) than GFAP-positive protoplasmic astrocytes (32.25 ± 9.18; n = 29). Nuclear Aro intensity in the S100b-positive subtype (21.72 ± 4.98; n = 24) was significantly lower than in the GFAP-positive subtype. Data are presented in mean ± SD. * p < 0.05, *** p < 0.001, ### p < 0.001. Asterisks indicate the significance between the nucleus and cytoplasm within each group. Pound (###) indicates the significantly lower intensity of nuclear Aro between GFAP and S100b positive astrocyte subtypes.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Localization of aromatase in astrocytes in adult rat brain sections. Nuclear (nucleus: blue) appearance of Aro enzyme (red; c , g , k , o ; yellow arrowheads) in GFAP-labelled (green) astrocytes ( a , b , e , f , i , j , m , n ; white arrowheads) is also detectable in frozen cortical tissue sections from male ( a – h ) and female ( i – p ) rats. Interestingly, the Aro immunopositive signal in the adult cortical astrocytes is abundant in the processes ( c , g , k , o ; green arrowheads), while astrocytes derived from newborn rats do not show an Aro signal in the branches. Quantitative analysis of total GFAP-positive astrocytes and nuclear Aro immunopositive astrocytes ( q ) in 10 randomly selected microscopic fields of different frozen sections from both sexes showed that the abundance of nuclear Aro immunopositive GFAP-labelled astrocytes is about 20% in both males (19.79%; n = 197) and females (20.13%; n = 154). Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Localization of aromatase in microglia cells. CD11b/c (Ox42)-labelled microglia cells (green) isolated from newborn rats show Aro (red) expression, but the signal is detectable exclusively in the cytoplasm. Microglia cells with typical ameboid/activated morphology ( a – h ) show stronger Aro expression compared with the resting/ramified forms ( i – p ), where Aro immunopositivity is much weaker. Aro signal is weak or undetectable in the branches of ramified microglia cells. Quantitative analysis of the fluorescent intensity confirmed that the Aro signal is significantly higher in the cytoplasm of ameboid cells (18.64 ± 2.81; n = 15) than in ramified cells (9.98 ± 1.35; n = 15) ( r ). Although Aro is not presented in the nucleus of microglia cells, a low level of fluorescent intensity was detectable in the nuclear area due to the overprojection of staining of neighbouring cells from the upper and lower cellular layers and/or the overlapping of cytoplasmic signal, but its level was significantly lower (9.71 ± 2.31; n = 45) than the cytoplasmic Aro intensity (14.67 ± 4.68; n = 45). The analysis of mean fluorescent intensity also revealed that the level of Aro immunopositivity in the cytoplasm is similar in astrocytes (14.20 ± 4.9; n = 54) and microglia cells ( q ). Data are presented in mean ± SD. ** p < 0.01, *** p < 0.001, ### p < 0.001. Asterisks indicate the significance between microglia cells. Pound (###) indicates the significance between astrocytes and microglia. Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Isolation, Expressing, Staining

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Nuclear aromatase expression overlaps with nuclear ERα signal. Double immunostaining with ERα (green) and Aro (red) in glia culture ( a – l ) shows that nuclear Aro signals colocalize with nuclear ERα ( a , e , i ; white arrowheads). Both Aro and ERα are strongly represented in the nucleus ( b , c , f , g , j,k ; yellow arrowheads) and also in the cytoplasm ( b , c , f , g , j , k ; blue arrowheads). ERα positive dots are also detectable in the processes ( b , f,j ; orange arrowheads). Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Expressing, Double Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: Localization of aromatase in the rat ovary and in human granulosa cells. Immunostaining of rat ovarian tissue sections is presented in images ( a – f ). Immunohistochemistry revealed that Aro (red) is strongly expressed in the whole ovarian tissue during the oestrus cycle but does not colocalize with the nuclear signal (blue). Results from Aro-stained human granulosa cells (red) corroborate the immunohistochemical findings as Aro immunopositivity is abundant in granulosa cells but only in the cytoplasm ( g – l ). Scale bar: 100 μm.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Immunostaining, Immunohistochemistry, Staining, Immunohistochemical staining

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: The simplified structure of human aromatase (Aro) protein with important conserved regions shown in red (based on Di Nardo et al. ). Blue lines indicate which parts of the human Aro protein were used to produce antibodies for Aro detection in CNS studies . Green line indicates a mouse Aro peptide to generate an anti-mouse Aro antibody. Co: commercially available antibodies; In-h: antibodies made and validated in-house; * the Novus Aro antibody used in this study.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System

doi: 10.3390/ijms23168946

Figure Lengend Snippet: List of primary antibodies used in this study. According to the technical information provided by the manufacturers, the commercial primary antibodies used in the study were verified by Knockdown or Relative expression to ensure that the antibody binds to the antigen stated.

Article Snippet: In spite of the limited availability of commercially available aromatase antibodies for studying its expression in the rat brain by immunohistochemistry, as highlighted by Krentzel et al. [ ], to date, the aromatase antibody from Novus Biologicals (B100-1596) was not applied for investigating Aro expression in the CNS.

Techniques: Knockdown, Expressing, Binding Assay, Recombinant

Journal: Animals : an Open Access Journal from MDPI

Article Title: NTRK2 Promotes Sheep Granulosa Cells Proliferation and Reproductive Hormone Secretion and Activates the PI3K/AKT Pathway

doi: 10.3390/ani14101465

Figure Lengend Snippet: Information on the antibodies used in this study.

Article Snippet: bs-1292R , anti-Aromatase , 1:1000 , Bioss, Beijing, China.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: High Doses of D-Chiro-Inositol Alone Induce a PCO-Like Syndrome and Other Alterations in Mouse Ovaries

doi: 10.3390/ijms22115691

Figure Lengend Snippet: Levels of aromatase in the ovaries of mice at the end of the 21-day treatment. ( A ) Representative Western blot of aromatase in protein extracts from the ovaries of mice under the experimental conditions indicated. ( B ) Densitometric analysis of aromatase/GAPDH. Values represent the mean value ± SD of extracts from three independent experiments. *, difference vs. negative control mice, p < 0.05.

Article Snippet: Identification of aromatase, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal standard, was performed by membrane incubation with polyclonal rabbit anti-aromatase (PA1-21398, Thermo Fisher Scientific, Waltham, MA, USA; 1:500) and mouse anti-GAPDH (TA802519, OriGene Technologies Inc., 1:750) antibodies, overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP) conjugated anti-rabbit (7074S, Cell Signaling Technologies, Danvers, MA, USA) 1:5000) or anti-mouse secondary antibody (Ab6728, Abcam, 1:5000), respectively, for 1 h at RT.

Techniques: Western Blot, Negative Control

Journal: Annals of Translational Medicine

Article Title: RAF1 mediates the FSH signaling pathway as a downstream molecule to stimulate estradiol synthesis and secretion in mouse ovarian granulosa cells

doi: 10.21037/atm-22-393

Figure Lengend Snippet: RAF1 involved in estradiol secretions by activating ERK phosphorylation. The primary ovarian GCs were treated with RAF709, 5 nM for 6 h then induced by FSH (100 ng/mL) for 24 h. (A-D) The protein expression ratios of FSHR, RAF1, ERK-P, and CYP19A1 were respectively detected by WB, and the protein ratios were analyzed in treatment groups relative to the GAPDH protein abundance. (E) Estradiol content was measured in each treatment group by RIA. The values are the means ± SEM of three independent experiments. Different letters indicate a significant difference between the compared groups (P<0.05). Different letters (a, b, c, and d) between two bars show a significant difference (a versus b, b versus c, and c versus d: P <0.05).

Article Snippet: Reagents and antibodies All reagents and antibodies were commercially available, and included RAF709 (HY-100510, MCE); anti-Raf1 (ab137435, Abcam); FSHR(1:1,000; sc-13935, Santa); GAPDH (1:2,000; Am4300, Ambion); CYP19A1 antibody (1:2,000; BA3704, Boster); P-ERK antibody (1:1,000; CST); goat anti-rabbit IgG (1:5,000, ZB-2301; Zhongshan, Beijing, China); ECL Western blotting substrate (32209; Thermo Scientific, Waltham, MA); DMEM/F12 (D2906; Sigma); fetal bovine serum (FBS, Gibco); streptomycin (Sigma); corn oil (Sigma); FSH (Boleide, Beijing, China).

Techniques: Phospho-proteomics, Expressing, Quantitative Proteomics

Journal: Annals of Translational Medicine

Article Title: RAF1 mediates the FSH signaling pathway as a downstream molecule to stimulate estradiol synthesis and secretion in mouse ovarian granulosa cells

doi: 10.21037/atm-22-393

Figure Lengend Snippet: RAF1 acts as a downstream molecule to mediate the FSH signaling pathway to stimulate E2 synthesis and secretion in vivo. Mice were given a single dose of FSH (10 IU/mouse) by intraperitoneal injection, and after 24 h, were injected with RAF709. After 24 h, blood samples of mice were collected for E2 content. Vegetable oils were used as RAF709 reference substance and PBS served as a comparison with FSH for first injections. (A-D) FSHR, RAF1, ERK-P, and CYP19A1 protein expression in each treatment group detected by WB. Data were analyzed by GraphPad Prism version 5. (E) Estradiol content in mouse serum was measured in each treatment group by RIA. The same letters indicate the difference is not significant, and different letters indicate the difference is significant (P<0.05). The values are the means ± SEM of three independent experiments. Different letters (a, b, c, and d) between two bars show a significant difference (a versus b, b versus c, and c versus d: P <0.05).

Article Snippet: Reagents and antibodies All reagents and antibodies were commercially available, and included RAF709 (HY-100510, MCE); anti-Raf1 (ab137435, Abcam); FSHR(1:1,000; sc-13935, Santa); GAPDH (1:2,000; Am4300, Ambion); CYP19A1 antibody (1:2,000; BA3704, Boster); P-ERK antibody (1:1,000; CST); goat anti-rabbit IgG (1:5,000, ZB-2301; Zhongshan, Beijing, China); ECL Western blotting substrate (32209; Thermo Scientific, Waltham, MA); DMEM/F12 (D2906; Sigma); fetal bovine serum (FBS, Gibco); streptomycin (Sigma); corn oil (Sigma); FSH (Boleide, Beijing, China).

Techniques: In Vivo, Injection, Comparison, Expressing

Journal: Gastro Hep Advances

Article Title: Sex Differences in Colonic Inflammation are Driven by Epithelial-Specific Expression of Estrogen Receptor Alpha

doi: 10.1016/j.gastha.2025.100624

Figure Lengend Snippet: Colonic epithelial cells express significant levels of ERα and ERβ. (A) Relative gene expression of Esr1 (ERα) and Esr2 (ERβ) in primary colonic epithelial cells obtained from healthy adult WT mice was determined by qPCR. Expression of target genes was normalized to that of Gapdh and fold change was calculated relative to male samples. (B) Ratio of Esr1:Esr2 mRNA expression was calculated for male and female samples. (C–H) Protein expression of ERα and ERβ were determined by western blot of nuclear (C–E) or cytoplasmic (F–H) protein lysates isolated from primary colonic epithelial cells of healthy adult WT mice. Expression of ERα and ERβ were normalized to levels of Lamin B1 (nuclear lysates, [D]) or β-actin (cytoplasmic lysates, [G]) using densitometry. Densitometry values were used to calculate ratios of ERα:ERβ expressed in nucleus (E) and cytoplasm (H). Subcellular localization of ERα (I) and ERβ (J) was calculated using densitometry values. (K) Expression of Cyp19A1 (aromatase) was determined by western blot of nuclear protein lysates isolated from primary colonic epithelial cells of healthy adult WT mice and (L) quantified by densitometry. For all figures, statistical analysis was performed with 2-way analysis of variance and Tukey post hoc test. Individual points represent individual animals.

Article Snippet: Antibodies used for western blots included α-Cyp19A1 (NSJ Bioreagents #RQ4643) α-ERα (Novus #NB300-560), α-ERβ (Novus #NB120-3577), α-GPER1 (Abcam #ab260033), α-Lamin B1 (Cell Signaling #12586), and α-β-Actin (Cell Signaling #12262).

Techniques: Gene Expression, Expressing, Western Blot, Isolation

Journal: ASN NEURO

Article Title: Sex-Dimorphic Octadecaneuropeptide (ODN) Regulation of Ventromedial Hypothalamic Nucleus Glucoregulatory Neuron Function and Counterregulatory Hormone Secretion

doi: 10.1177/17590914231167230

Figure Lengend Snippet: Description of Primary Antisera Used.

Article Snippet: Aromatase , Novus Biologicals LLC, Centennial CO, US , NB100-1596 , AB_10000919 , WB; 1:2000 (0.5 uL/mL) , Human Aromatase protein (between residues 400-502). [UniProt# P11511] , Giles et al., Breast Cancer Res. 2018; 20(1): 50 , 29898754.

Techniques: Recombinant, Sequencing

Journal: Frontiers in Endocrinology

Article Title: Steroidogenic differentiation of human amniotic membrane-derived mesenchymal stem cells into a progesterone-/androgen-producing cell lineage by SF-1 and an estrogen-producing cell lineage by WT1−KTS

doi: 10.3389/fendo.2024.1410433

Figure Lengend Snippet: Effects of SF-1 and WT1 on estrogen-producing cell lineage. (A) WT1−KTS significantly increased the mRNA expression of ALDH1A2 , AMHR2, LHX9, GPX3 , and NR5A2 in hAmMSCs. (B) WT1−KTS, but not WT1+KTS or SF-1, significantly increased the mRNA expression of CYP19A1 in hAmMSCs. SF-1 increased the mRNA level of HSD17B1 . (C) Immunocytochemical analysis confirmed that WT1−KTS increased aromatase expression in hAmMSCs. (D) WT1−KTS and SF-1 did not increase estrogen production by hAmMSCs even in the presence of testosterone. (E) Co-expression of SF-1 and WT1−KTS also did not alter the mRNA levels of estrogen-producing enzymes in hAmMSCs. Data are presented as the mean ± SD from three independent experiments. *, p < 0.05; **, p < 0.01; ****, p < 0.0001; N.S., not significant. hAmMSCs, human amniotic membrane-derived mesenchymal stem cells; WT1, Wilms’ tumor 1; SF-1, steroidogenic factor 1; KTS, lysine, threonine, and serine.

Article Snippet: The cells were then incubated with Blocking One (Nacalai Tesque, Inc, Kyoto, Japan) for 10 min at room temperature, after which they were immunolabeled with the primary antibody WT1 (1:100, NB110-60011; Novus Biologicals, Centennial, CO, USA) and anti-aromatase antibody (1:100, NBP2-61939; Novus Biologicals) for 1 h. After washing three times with PBS, the secondary antibody donkey anti-mouse Alexa 488 (1:1000, Invitrogen) was used for detection.

Techniques: Expressing, Membrane, Derivative Assay, Wilms Tumor Assay