cyp19a1a Antibody

What is cyp19a1a and what distinguishes it functionally from related proteins?

Cyp19a1a is an ovarian-type aromatase enzyme that catalyzes the conversion of androgens to estrogens. In fish and other vertebrates, it represents one of two primary aromatase forms, with distinct tissue distribution patterns compared to its brain-type counterpart (cyp19a1b). Research indicates that cyp19a1a is predominantly expressed in gonads, particularly ovaries, while cyp19a1b shows higher expression in neural tissues including the pituitary, forebrain, and hypothalamus . Functionally, cyp19a1a plays critical roles in:

• Estrogen biosynthesis and reproductive development

• Ovarian differentiation and maturation processes

• Regulation of sex-specific immune responses

• Potential involvement in hormone-responsive cancer mechanisms

The protein typically has a molecular weight of approximately 75 kDa as detected in Western blot analyses of ovarian tissue . Understanding these basic characteristics is essential for proper experimental design when using cyp19a1a antibodies in research applications.

What validation approaches are recommended for cyp19a1a antibodies?

Proper validation of cyp19a1a antibodies is crucial for ensuring experimental reliability. Recommended approaches include:

• Western blot validation: Confirm antibody specificity by detecting the expected 75 kDa band in tissues known to express cyp19a1a (primarily ovarian tissue) . Include proper controls such as testicular tissue for comparison.

• Immunohistochemistry (IHC) correlation: Verify that IHC staining patterns match known expression profiles from transcriptomic data. For example, cyp19a1a protein detection should be predominantly localized to follicle cells in ovaries at stage V of development .

• Knockout/knockdown validation: When possible, use cyp19a1a knockout or knockdown models to confirm antibody specificity through the absence or reduction of signal.

• Cross-reactivity testing: Assess potential cross-reactivity with cyp19a1b or other related cytochrome P450 enzymes, particularly in species with high sequence homology.

• Recombinant protein controls: Use purified recombinant cyp19a1a as a positive control to establish detection sensitivity and specificity thresholds .

How are cyp19a1a antibodies used in cancer research?

Cyp19a1a antibodies serve as valuable tools in cancer research, particularly for investigating aromatase inhibitor (AI) resistance mechanisms in estrogen receptor-positive (ERα+) breast cancers. Key applications include:

• Detection of CYP19A1 amplification: Antibodies can help confirm gene amplification events at the protein level. Research has shown that approximately 21.5% of AI-treated breast cancer patients develop CYP19A1 amplification at first relapse, compared to only 4% of tamoxifen-treated patients .

• Investigation of resistance mechanisms: Studies have demonstrated that CYP19A1 amplification causes increased aromatase activity and estrogen-independent ERα binding to target genes, resulting in decreased sensitivity to AI treatment .

• Monitoring of treatment response: Cyp19a1a antibodies can help track changes in aromatase expression levels during treatment and progression.

• Patient-derived xenograft (PDX) models: Antibodies facilitate validation of appropriate PDX models by confirming maintenance of relevant CYP19A1 amplification characteristics from the original tumor .

Methodology for analyzing CYP19A1 amplification frequently combines DNA-based techniques (TaqMan CNA assay, FISH) with protein-level confirmation via antibody-based methods, providing a more comprehensive assessment of the amplification's functional consequences .

How can cyp19a1a antibodies be applied to developmental biology research?

In developmental biology, cyp19a1a antibodies provide critical insights into sexual differentiation, gonadal development, and steroidogenic pathways:

• Temporal expression analysis: Track cyp19a1a protein expression during key developmental stages, correlating with established transcriptional profiles. For example, qPCR shows that cyp19a1a is highly expressed from yolk plug stage to pre-hatching stage in early fish development .

• Cell-specific localization: IHC studies using cyp19a1a antibodies reveal that the protein is specifically expressed in follicle cells at stage V in ovaries and in spermatogonia from stage II to stage IV in testes .

• Functional studies with recombinant protein: Recombinant cyp19a1a (rCyp19a1a) can be produced and used in gonadal tissue culture experiments to investigate its effects on the expression of sex-related genes .

This methodological approach allows researchers to establish not just where and when cyp19a1a is expressed, but also how it functionally influences the expression of downstream genes involved in reproductive development.

What role does cyp19a1a play in immune responses and how can antibodies help investigate this function?

Recent research has revealed an unexpected role for cyp19a1a in immune function, particularly in antiviral responses. Studies show that:

• Sex-specific antiviral immunity: Female fish are more vulnerable to viral infections than males, with significantly weaker interferon (IFN) expression .

• Cyp19a1a as an immune regulator: The highly female-biased cyp19a1a expression correlates with this reduced antiviral response .

• Mechanistic pathways: Cyp19a1a targets the mediator of IRF3 activation (MITA) for autophagic degradation, thus dampening antiviral responses .

Methodological approaches using cyp19a1a antibodies to investigate these immune functions include:

• Co-immunoprecipitation assays: To detect Cyp19a1a interactions with MITA and autophagy factors like ATG14 .

• Immunofluorescence microscopy: To visualize co-localization of Cyp19a1a with autophagy-related proteins.

• Comparative immunoblotting: Between cyp19a1a+/+ and cyp19a1a-/- models to assess differences in interferon pathway activation.

This research area represents a novel intersection between reproductive biology and immunology, highlighting how cyp19a1a antibodies can contribute to understanding complex biological interactions beyond traditional steroidogenic pathways.

What are the recommended protocols for Western blotting with cyp19a1a antibodies?

Based on published research, the following protocol optimizations are recommended for Western blotting with cyp19a1a antibodies:

• Sample preparation:

  • Use fresh tissue samples from ovaries (primary source of cyp19a1a) or recombinant protein as positive control

  • Include appropriate negative controls (tissues with low expression)

  • Standard protein extraction with RIPA buffer containing protease inhibitors

• Gel electrophoresis and transfer:

  • 10-12% SDS-PAGE is typically sufficient for separating the ~75 kDa cyp19a1a protein

  • Use wet transfer methods for optimal large protein transfer efficiency

• Antibody incubation:

  • Primary antibody dilutions typically range from 1:500 to 1:2000

  • Include anti-β-Actin (42 kDa) as loading control

  • Overnight incubation at 4°C often yields best results for specific detection

• Detection considerations:

  • ECL detection systems with appropriate sensitivity for the expected expression level

  • Exposure times may need optimization depending on expression levels across different samples

• Special considerations:

  • When analyzing samples from different species, select antibodies raised against conserved epitopes

  • For experiments involving both cyp19a1a and cyp19a1b, verify antibody specificity to avoid cross-reactivity

Successful detection of cyp19a1a has been demonstrated in studies showing the expected 75 kDa band in ovarian tissue but not in other tissues with low expression .

What are the optimal conditions for immunohistochemistry with cyp19a1a antibodies?

Effective immunohistochemistry (IHC) with cyp19a1a antibodies requires careful optimization:

• Tissue fixation and processing:

  • 4% paraformaldehyde fixation for 24 hours followed by paraffin embedding is commonly effective

  • Section thickness of 5-7 μm is recommended for optimal antibody penetration and signal clarity

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective

  • Pressure cooking for 2-3 minutes followed by 20 minutes of cooling improves antigen accessibility

• Blocking and antibody incubation:

  • 5-10% normal serum (matching secondary antibody host) with 0.1-0.3% Triton X-100

  • Primary antibody incubation at 4°C overnight with dilutions typically between 1:100 and 1:500

  • Secondary antibody incubation for 1-2 hours at room temperature

• Signal development and analysis:

  • DAB (3,3′-diaminobenzidine) or fluorescent-conjugated secondary antibodies depending on experimental needs

  • For fluorescence, include DAPI nuclear counterstain for cellular context

• Expected patterns:

  • Signal should be detected specifically in follicle cells at stage V in ovary and in spermatogonia from stage II to stage IV in testis

  • Negative controls should include primary antibody omission and ideally tissue from knockout/knockdown models

This methodology has successfully demonstrated Cyp19a1a localization in gonadal tissues, with expression patterns consistent with qPCR data .

How can recombinant cyp19a1a be produced and validated for research purposes?

Production of recombinant Cyp19a1a (rCyp19a1a) is valuable for functional studies and antibody validation. Based on published methodologies:

• Cloning strategy:

  • Clone into appropriate expression vector with affinity tag (His, GST, etc.)

• Expression systems:

  • Prokaryotic: E. coli BL21(DE3) for high yield but potential folding issues

  • Eukaryotic: Insect cells or mammalian expression systems for better post-translational modifications

• Purification approach:

  • Affinity chromatography using tag-specific resins

  • Further purification via size exclusion or ion exchange chromatography as needed

• Validation methods:

  • Western blot analysis using anti-Cyp19a1a antibodies to confirm ~75 kDa product

  • Mass spectrometry for protein identification

  • Enzymatic activity assays measuring conversion of androgens to estrogens

• Functional validation:

  • In vitro tissue culture experiments to assess effects on gene expression

  • Examples show rCyp19a1a upregulates expression of cyp19a1a itself and estrogen receptor 2 alpha (esr2a) in cultured ovarian tissues

This methodological approach has been successfully employed to produce functional rCyp19a1a that significantly affects the expression of sex-related genes in gonadal tissue cultures .

How can researchers investigate cyp19a1a's role in aromatase inhibitor resistance?

Investigating cyp19a1a's role in aromatase inhibitor (AI) resistance requires multifaceted approaches:

• Copy number analysis:

  • TaqMan CNA assay comparing metastatic with matched normal breast tissue

  • DNA-FISH (Fluorescence In Situ Hybridization) for direct visualization of gene amplification

  • Validation across multiple patient cohorts to establish clinical relevance

• Expression correlation studies:

  • Assess relationship between CYP19A1 amplification and protein levels using validated antibodies

  • Compare expression in AI-resistant versus sensitive tumors

  • Correlate with clinical outcomes and treatment history

• Functional validation in cell models:

  • Generate Long-Term Estrogen Deprived (LTED) cell lines to model AI resistance

  • Measure cyp19a1a expression and activity in resistant versus sensitive cells

  • Assess letrozole (AI) sensitivity in cells with manipulated cyp19a1a levels

• Mechanistic investigation:

  • siRNA targeting CYP19A1 to assess impact on AI sensitivity (IC50 measurements)

  • Analysis of downstream estrogen-responsive gene activation

  • Investigation of estrogen-independent ERα binding to target genes

Research has demonstrated that CYP19A1 amplification is detected in 21.5% of AI-treated patients versus 4% of tamoxifen-treated patients at first relapse, with amplification-positive cells showing significantly reduced sensitivity to AI treatment (IC50=80μM) .

What methodology is recommended for studying cyp19a1a's interaction with autophagy pathways?

Recent discoveries linking cyp19a1a to autophagy-mediated immune regulation require specialized methodological approaches:

• Protein-protein interaction analysis:

  • Co-immunoprecipitation of Cyp19a1a with autophagy components (e.g., ATG14)

  • Proximity ligation assays to visualize interactions in situ

  • Yeast two-hybrid or mammalian two-hybrid screening to identify novel interactors

• Autophagic flux assessment:

  • LC3-II conversion assays with and without lysosomal inhibitors

  • p62/SQSTM1 accumulation measurements

  • Tandem fluorescent-tagged LC3 (mRFP-GFP-LC3) to distinguish autophagosome formation from degradation

• Target degradation studies:

  • Compare MITA stability in cyp19a1a+/+ versus cyp19a1a-/- models

  • Cycloheximide chase assays to measure protein half-life differences

  • Proteasome inhibitors versus autophagy inhibitors to determine degradation pathway

• Functional consequences measurement:

  • Interferon expression analysis following viral challenge

  • Viral replication assays in the presence/absence of cyp19a1a

  • Cell-based reporter systems for IRF3 activation

Research has established that Cyp19a1a targets MITA for autophagic degradation, thereby diminishing antiviral responses, with ATG14 playing a crucial role in this process by either promoting or attenuating Cyp19a1a-mediated MITA degradation when overexpressed or knocked down, respectively .

How should researchers approach the study of cyp19a1a regulation by estrogen feedback loops?

Investigation of the complex feedback relationships between cyp19a1a and estrogen requires specialized methodological approaches:

• In vitro culture systems:

  • Gonadal tissue culture with various hormonal treatments:

    • Estradiol (E2)

    • Aromatase inhibitors (AI)

    • Methyltestosterone (MT)

    • Combinations (E2+AI, E2+MT)

  • Measure cyp19a1a expression changes via qPCR following treatments

• Analysis of regulatory elements:

  • Identification of estrogen response elements (EREs) in cyp19a1a promoter

  • Chromatin immunoprecipitation (ChIP) to assess ERα/ERβ binding

  • Luciferase reporter assays with wild-type and mutated promoter constructs

• Receptor specificity studies:

  • Selective estrogen receptor modulators (SERMs) to distinguish ERα versus ERβ effects

  • Correlation between esr1 (ERα), esr2a (ERβ) and cyp19a1a expression

  • siRNA knockdown of specific estrogen receptors to determine their role in regulation

• In vivo validation:

  • Analysis of cyp19a1a expression in different hormonal states or following treatments

  • Developmental time course studies correlating hormone levels with expression

Research has demonstrated that E2 treatment significantly increases cyp19a1a expression in both ovarian and testicular tissue cultures, suggesting a positive feedback loop between estrogen and aromatase expression .

How should researchers address potential cross-reactivity between cyp19a1a and cyp19a1b antibodies?

Cross-reactivity between cyp19a1a and cyp19a1b antibodies presents a significant challenge due to sequence homology. Recommended approaches include:

• Antibody selection considerations:

  • Choose antibodies raised against unique, non-conserved epitopes

  • Review sequence alignments between cyp19a1a and cyp19a1b to identify divergent regions

  • Consider custom antibody development targeting specific peptide sequences

• Validation with multiple methodologies:

  • Correlate protein detection with RNA expression profiles across tissues

  • cyp19a1a is predominantly expressed in ovaries while cyp19a1b shows highest expression in neural tissues (pituitary, forebrain, hypothalamus)

  • Use both Western blotting and immunohistochemistry to confirm tissue-specific patterns

• Controls for specificity:

  • Include positive controls from tissues with known predominant expression of one isoform

  • When possible, use samples from knockout/knockdown models

  • Pre-absorption controls with specific peptide antigens

• Differential expression verification:

  • During gonadal development, cyp19a1a expression is significantly higher in ovaries than in testes

  • cyp19a1b expression is typically higher in males than females at specific developmental stages

  • These established patterns can help determine antibody specificity

• Recombinant protein standards:

  • Use purified recombinant cyp19a1a and cyp19a1b as controls

  • Compare antibody binding affinity and specificity against each protein

These approaches collectively allow researchers to confidently distinguish between the two aromatase forms despite their structural similarities.

What factors might cause inconsistent results in cyp19a1a amplification studies?

Researchers investigating cyp19a1a amplification may encounter inconsistent results due to several factors:

• Methodological considerations:

  • Different detection methods (TaqMan CNA vs. FISH) may yield varying results

  • DNA-FISH analysis shows cyp19a1a amplification as cluster amplification signals in >90% of nuclei from metastatic samples, while being undetectable in primary samples

  • Ensure standardized protocols across samples and replicates

• Tumor heterogeneity:

  • CYP19A1 amplification may not be uniform throughout a tumor

  • Multiple sampling from different tumor regions may be necessary

  • Single-cell approaches might reveal subpopulations with different amplification status

• Technical artifacts:

  • Low-quality DNA/RNA from FFPE samples can affect amplification detection

  • Reference gene selection for normalization can influence relative quantification

  • Aneuploidy can confound amplification results unless proper controls are used

• Treatment effects:

  • Prior exposure to aromatase inhibitors affects amplification frequencies

  • Document complete treatment history when interpreting results

  • Consider time-course studies to track amplification emergence

• Validation approaches:

  • Confirm gene amplification results at the protein level using validated antibodies

  • Correlate with functional outcomes (e.g., aromatase activity, AI resistance)

  • Employ multiple techniques to cross-validate findings

Research shows dramatic differences in CYP19A1 amplification rates between AI-treated (21.5%) and tamoxifen-treated (4%) patient cohorts, highlighting the importance of treatment history in data interpretation .

How can researchers effectively interpret cyp19a1a expression data across different developmental stages?

Interpreting cyp19a1a expression across developmental stages requires careful consideration of multiple factors:

• Temporal expression patterns:

  • cyp19a1a shows high expression from yolk plug stage to pre-hatching stage in early development

  • During gonadal development, highest expression occurs at stage V in ovaries

  • Expression patterns differ between males and females, particularly at later stages

• Tissue-specific localization:

  • In ovaries, cyp19a1a protein is primarily detected in follicle cells at stage V

  • In testes, expression is observed in spermatogonia from stages II to IV

  • These localization patterns should correlate with gene expression data

• Methodological considerations:

  • For qPCR data, selection of appropriate reference genes is critical

  • Ef1α is commonly used as an internal control for normalization

  • Primer specificity should be verified to avoid amplification of related genes

• Experimental design factors:

  • Sample collection timing requires standardization relative to developmental milestones

  • Statistical analysis should account for biological variation within developmental stages

  • Consider pooled samples for early stages with limited tissue availability

• Integration of protein and RNA data:

  • Correlate qPCR results with protein detection via Western blot or IHC

  • Time lags between transcription and translation may result in temporal discrepancies

  • Post-transcriptional regulation may affect correlation between RNA and protein levels

By systematically addressing these factors, researchers can generate reliable interpretations of developmental expression data for cyp19a1a.

What emerging technologies might enhance cyp19a1a research?

Several cutting-edge technologies show promise for advancing cyp19a1a research:

• CRISPR-Cas9 gene editing:

  • Generation of precise cyp19a1a knockout or knock-in models

  • Introduction of specific mutations corresponding to human polymorphisms

  • Creation of reporter lines with fluorescently tagged endogenous cyp19a1a

• Single-cell technologies:

  • Single-cell RNA-seq to identify cell populations with differential cyp19a1a expression

  • Spatial transcriptomics to map cyp19a1a expression in intact tissues

  • CyTOF or spectral flow cytometry for protein-level analysis in heterogeneous samples

• Organoid and 3D culture systems:

  • Development of ovarian or testicular organoids to study cyp19a1a in more physiologically relevant contexts

  • Co-culture systems to investigate cell-cell interactions affecting cyp19a1a function

  • Microfluidic "organ-on-a-chip" platforms for dynamic hormone regulation studies

• Computational approaches:

  • Systems biology modeling of estrogen synthesis pathways

  • Machine learning algorithms to predict cyp19a1a expression patterns from multi-omics data

  • Molecular dynamics simulations of aromatase inhibitor interactions

• In vivo imaging:

  • Development of specific probes for non-invasive imaging of aromatase activity

  • Longitudinal studies tracking cyp19a1a expression during development or disease progression

  • Correlation of imaging data with functional outcomes

These technological advances will likely provide deeper insights into cyp19a1a biology and its roles in development, reproduction, and disease processes.

How might evolutionary studies of cyp19a1a inform its functional understanding?

Evolutionary analyses of cyp19a1a offer valuable insights for functional research:

• Comparative genomics approaches:

  • Analysis of cyp19a1a and cyp19a1b across vertebrate species

  • Identification of conserved versus divergent domains suggesting functional importance

  • Study of regulatory element evolution to understand expression pattern differences

• Evolutionary pressures:

  • The divergence between cyp19a1a and cyp19a1b likely resulted from genome duplication under selective pressure

  • Functional specialization of these paralogs provides insights into tissue-specific roles

  • Correlation between evolutionary conservation and functional domains

• Ancestral reconstruction:

  • Identification of cyp19-like genes in basal chordates like amphioxus (identified as cyp19-like1 and cyp19-like2)

  • Comparison of these ancestral forms with specialized vertebrate aromatases

  • Functional testing of ancestral versus derived forms to understand evolutionary innovations

• Structure-function relationships:

  • Use of comparative sequence analysis to identify critical residues for substrate specificity

  • Three-dimensional structural comparisons between species to understand mechanistic differences

  • Correlation between structural conservation and enzymatic activity

• Ecological and behavioral correlations:

  • Investigation of cyp19a1a evolution in species with diverse reproductive strategies

  • Correlation between environmental factors and aromatase function/regulation

  • Understanding how sexual selection may have shaped aromatase evolution

Evolutionary perspectives provide a broader context for understanding cyp19a1a function beyond individual model organisms, potentially revealing novel functional aspects that might otherwise be overlooked.