FGF2 (147), Bovine

What is FGF2 (147) and how does it differ structurally from full-length bovine FGF2?

FGF2 (147), also known as basic fibroblast growth factor (bFGF), is a truncated form of the full-length FGF2 protein that results from N-terminal degradation when isolated from biological sources. The full-length bovine FGF2 contains 154 amino acids, while the truncated version contains 147 amino acids. Importantly, this structural difference affects subcellular localization but does not alter biological activity. The N-terminus extensions in the full-length protein influence intracellular localization, but both versions maintain identical receptor binding and signaling capabilities .

The molecular structure of FGF2 (147) is characterized by:

• Monomeric form with molecular mass of 16.5 kDa

• The amino acid sequence: MPALPEDGGS GAFPPGHFKD PKRLYCKNGG FFLRIHPDGR VDGVREKSDP HIKLQLQAEE RGVVSIKGVC ANRYLAMKED GRLLASKCVT DECFFFERLE SNNYNTYRSR KYTSWYVALK RTGQYKLGSK TGPGQKAILF LPMSAKS

When working with recombinant FGF2 (147), researchers should note there are no detectable differences in biological activity between the full-length and truncated forms, making the 147aa version suitable for most experimental applications investigating FGF2 function .

What are the primary biological functions of bovine FGF2 in different tissue contexts?

Bovine FGF2 functions as a multifunctional growth factor with tissue-specific roles that vary throughout development and in response to injury:

• Angiogenesis regulation: FGF2 stimulates the proliferation of endothelial cells derived from bovine corpora lutea and mediates critical angiogenic processes . It serves as a key determinant of vascular sprouting, particularly during the early stages of vessel formation.

• Reproductive biology: FGF2 is expressed at particularly high levels during the bovine follicular–luteal transition . FGF2 concentration in bovine follicular fluid (0.629 ng/mL) significantly exceeds levels found in culture media (0.009-0.032 ng/mL), suggesting its importance in follicular development .

• Cell differentiation control: FGF2 is a critical component for maintaining embryonic stem cells in an undifferentiated state, making it essential for stem cell culture systems .

• Neural development: Though most extensively studied in non-bovine models, FGF2 promotes neuronal survival, proliferation, and neurite outgrowth across species, suggesting conserved functions in bovine neural development .

• Tissue protection: FGF2 exhibits cardioprotective functions during heart injury, facilitating repair processes and cellular survival .

Understanding these diverse functions is essential when designing experiments to investigate FGF2's role in specific bovine tissue contexts.

How should recombinant FGF2 (147) be properly reconstituted and stored for optimal experimental results?

For optimal experimental outcomes when working with recombinant FGF2 (147), researchers should follow these methodological guidelines:

Reconstitution protocol:

• Centrifuge the vial before opening to ensure all material is at the bottom

• Reconstitute by gently pipetting sterile water down the sides of the vial to achieve a 0.1 mg/mL concentration

• Never vortex the solution as this can denature the protein

• Allow several minutes for complete reconstitution

• If a precipitate forms, centrifuge thoroughly and use only the soluble fraction

Storage recommendations:

• For prolonged storage, dilute to working aliquots in a 0.1% BSA solution

• Store at -80°C to maintain stability

• Avoid repeated freeze-thaw cycles which significantly reduce activity

• A 10% overfill is typically included to compensate for any loss of protein in precipitation

Quality control considerations:
Verify protein quality before experiments by assessing:

• Purity (≥ 95% via reducing and non-reducing SDS-PAGE)

• Endotoxin levels (≤ 0.1 EU/μg via Kinetic LAL)

• Biological activity (ED50 ≤ 5 ng/mL; ≥ 2.0 x 105 units/mg via NR6R-3T3 cell proliferation assay)

These methodological details ensure experimental reproducibility and reliable results when investigating FGF2 functions in bovine systems.

What methodologies are most effective for studying FGF2-mediated angiogenesis in bovine models?

Investigation of FGF2-mediated angiogenesis in bovine models requires specialized methodologies to capture the complex, multi-stage process of blood vessel formation. Based on established research approaches, the following methodologies have proven most effective:

In vitro primary cell culture system:
A novel primary cell culture system derived from early bovine corpora lutea effectively simulates luteal angiogenesis . This system allows researchers to:

• Observe distinct phases of endothelial growth (days 0-3: island formation; days 3-6: vascular sprouting; days 6-9: network development)

• Evaluate the effects of growth factor inhibition during specific developmental windows

• Quantify endothelial cell network formation using immunostaining for specific markers

Time-specific inhibitor application:
Applying inhibitors like SU5402 (FGFR1 inhibitor) at specific time points (days 0-3, 3-6, or 6-9) has revealed that:

• Days 3-6 represent the period of maximum sensitivity to FGF2 regulation (81% reduction in VWF immunostaining)

• Days 0-3 show moderate sensitivity (64% reduction)

• Days 6-9 exhibit minimal sensitivity to FGF2 inhibition

Quantitative metrics:
To effectively analyze FGF2-mediated angiogenesis, researchers should measure:

• Total area of endothelial cell immunostaining

• Number of endothelial branch points

• Number of vascular sprouts per island

• Mean island area

• Complexity of endothelial networks

These methodologies collectively provide a comprehensive approach to studying the dynamic role of FGF2 in bovine angiogenesis, particularly highlighting its critical function in initiating endothelial cell sprouting during early network formation.

How do FGF2 concentrations correlate with developmental stages in bovine reproductive biology?

FGF2 concentrations exhibit significant dynamic changes across different developmental stages in bovine reproductive biology, providing critical insights for researchers designing stage-specific experiments:

Follicular-luteal transition:
FGF2 protein concentrations reach their peak in the collapsed follicle immediately post-ovulation, indicating a critical role during this transitional period . This suggests that experimental designs targeting this transition should account for naturally elevated FGF2 levels.

Follicular development profile:
In bovine follicular fluid, FGF2 concentration averages 0.629 ng/mL, significantly higher than concentrations found in subsequent developmental media . This concentration gradient suggests a natural dilution or consumption of FGF2 as development progresses.

Embryonic development concentration gradient:
A progressive decline in FGF2 concentration occurs during embryonic development as shown in the following data:

This progressive reduction (approximately 50-60% decrease between each stage) suggests stage-specific requirements for FGF2 signaling . The highest concentration in follicular fluid indicates potential importance in oocyte development within follicles.

When designing experiments investigating bovine reproductive biology, researchers should consider these stage-specific concentration differences to achieve physiologically relevant conditions. Supplementation strategies should aim to replicate these natural concentration gradients rather than maintaining constant FGF2 levels throughout development.

What receptor interactions govern FGF2 signaling specificity in bovine tissues?

The signaling specificity of FGF2 in bovine tissues is governed by complex receptor interactions that determine downstream biological outcomes. Understanding these interactions is crucial for experimental manipulation of FGF2 pathways:

Receptor preference hierarchy:
FGF2 exhibits differential binding affinity for various FGF receptors:

• Primary targets: FGFR1C, FGFR3C, and FGFR4 (highest affinity)

• Secondary targets: FGFR1B and FGFR2C (lower affinity)

Tissue-specific receptor expression:
Bovine corpus luteum (CL) expresses negligible levels of FGFR3C and FGFR4, suggesting that in luteal tissue, FGF2 likely signals primarily through variants of FGFR1 and FGFR2 . This receptor expression profile creates tissue-specific signaling environments.

Receptor isoform dynamics:
While FGFR2C mRNA expression remains relatively constant throughout bovine luteal development in vivo , the relative contribution of FGFR1 variants to angiogenic processes appears more dynamic and functionally significant.

Functional evidence of receptor specificity:
Inhibition of FGFR1 during days 3-6 of culture results in:

• 90% reduction in total endothelial branch points

• Dramatic reduction in vascular sprout formation

• Minimal effect on island area

These findings indicate that FGFR1 signaling specifically mediates the sprouting phase of angiogenesis rather than initial endothelial cell proliferation, demonstrating how receptor specificity creates distinct biological outcomes.

For researchers investigating FGF2 functions in bovine tissues, selective receptor inhibitors (like SU5402 for FGFR1) provide valuable tools for dissecting the specific contributions of different FGF receptor subtypes to complex biological processes. When designing experiments, the relative expression levels of different FGF receptors in the target tissue should be considered to accurately interpret FGF2 signaling outcomes.

How do dosage requirements of FGF2 vary across different experimental systems?

FGF2 dosage requirements exhibit significant variation across experimental systems, species, and target cell types. Understanding these differences is critical for experimental design and interpretation:

Dosage comparison across research models:

Key factors affecting dosage requirements:

• Species differences: Requirements vary significantly between mammals and non-mammals, with some non-mammalian systems (like Xenopus embryos) requiring dramatically higher concentrations (up to 10,000 ng/mL) .

• Developmental stage: Sensitivity to FGF2 varies with developmental stage, with early developmental stages often showing greater responsiveness to lower concentrations.

• Experimental endpoint: Different biological processes (proliferation vs. differentiation vs. survival) may require different FGF2 concentrations for optimal effect.

• Receptor expression profile: Target tissues with higher expression of preferred FGF2 receptors may respond to lower concentrations.

• Culture system complexity: Three-dimensional cultures and tissue explants may require higher concentrations than monolayer cultures due to diffusion limitations.

When designing FGF2 experiments, researchers should conduct preliminary dose-response studies specific to their model system rather than relying on dosages reported for different species or cell types. The striking variability in effective concentrations (0.1-10,000 ng/mL across systems) underscores the importance of system-specific optimization.

What methodological approaches can resolve contradictory findings in FGF2 research?

Contradictory findings in FGF2 research can arise from multiple methodological factors. Researchers can address these contradictions through several strategic approaches:

Species and strain standardization:
Contradictions may result from species or strain differences. For example, discrepancies in FGF2 expression have been observed between Wistar and Sprague-Dawley rats . To address this:

• Explicitly report species, strain, gender, and age in all publications

• Design comparative studies that directly assess species/strain differences using identical methodologies

• Avoid extrapolating findings across species without validation

Developmental timing considerations:
FGF2's effects can vary dramatically across developmental stages. For instance, only one study has explored FGF2 expression in embryonic stage rats, making comparison with mouse embryonic studies problematic . Researchers should:

• Carefully define and report developmental stages using standardized criteria

• Design longitudinal studies that track FGF2 expression and function across multiple developmental timepoints

• Consider the dynamic nature of FGF2 concentration gradients (as seen in the 50-60% decrease between embryonic developmental stages)

Methodological standardization:
Different analytical techniques can yield contradictory results. For example:

• Protein quantification: Standardize on methods like ELISA with validated antibodies specific to bovine FGF2

• Activity assays: Use the NR6R-3T3 cell proliferation assay with consistent ED50 thresholds (≤ 5 ng/mL; ≥ 2.0 x 105 units/mg)

• Receptor inhibition: When using inhibitors like SU5402, employ consistent concentrations and exposure timing protocols

Contextual interpretation:
Seemingly contradictory results may reflect true biological complexity. For example, FGF2 stimulates proliferation in some contexts but promotes differentiation in others. Researchers should:

• Comprehensively characterize the experimental microenvironment

• Consider co-factors and signaling modulators present in different systems

• Design factorial experiments that systematically vary multiple parameters to identify interaction effects

By employing these methodological approaches, researchers can reconcile contradictory findings and develop a more nuanced understanding of FGF2's context-dependent functions in bovine and comparative systems.

How can researchers effectively use FGF2 (147) in bovine embryonic stem cell cultures?

FGF2 (147) plays a critical role in bovine embryonic stem cell (ESC) culture systems, requiring specific technical considerations for optimal results:

Optimal concentration determination:
While the search results don't specify exact concentrations for bovine ESC cultures, FGF2 is described as "a critical component for embryonic stem cell culture systems and is necessary for maintaining cells in an undifferentiated state" . Based on comparative research, researchers should:

• Conduct titration experiments starting with concentrations between 4-20 ng/mL

• Assess pluripotency marker expression (e.g., OCT4, NANOG) at each concentration

• Monitor differentiation markers to ensure maintenance of the undifferentiated state

Application protocols:
For bovine ESC culture, implement the following methodological approach:

• Reconstitute lyophilized FGF2 (147) in sterile water to 0.1 mg/mL

• Prepare working solutions in 0.1% BSA to enhance stability

• Add fresh FGF2 every 24-48 hours, as the protein has limited stability in culture conditions

• Consider combining with other factors that synergize with FGF2 to maintain pluripotency

Quality assessment criteria:
Monitor culture quality using these parameters:

• Colony morphology (compact, defined edges)

• Growth rate (doubling time)

• Expression of pluripotency markers

• Differentiation potential upon factor withdrawal

• Karyotypic stability across multiple passages

Technical troubleshooting:
Address common issues by:

• Preventing protein adsorption to plasticware by using low-binding tubes/plates

• Adding heparin (1-10 μg/mL) to stabilize FGF2 and enhance receptor binding

• Adjusting cell density to optimize cell-cell communications that support FGF2 signaling

• Ensuring minimal lot-to-lot variability by measuring biological activity (ED50 ≤ 5 ng/mL)

These technical guidelines provide a methodological framework for effectively incorporating FGF2 (147) into bovine embryonic stem cell culture systems while maximizing pluripotency maintenance.

What are the critical technical factors when studying FGF2's role in bovine luteal angiogenesis?

When investigating FGF2's role in bovine luteal angiogenesis, several critical technical factors must be addressed to ensure experimental validity and reproducibility:

Temporal staging considerations:
Luteal angiogenesis proceeds through distinct phases with varying FGF2 sensitivity:

• Days 0-3: Endothelial cell proliferation in dense island formations (64% reduction with FGF2 inhibition)

• Days 3-6: Rapid reorganization with vascular sprouting (81% reduction, maximum sensitivity)

• Days 6-9: Endothelial network development (minimal sensitivity to FGF2 inhibition)

Researchers must design experiments that account for these temporal windows when applying treatments or collecting samples.

Cell culture system optimization:
The primary cell culture system should include:

• Fresh bovine early corpora lutea tissue collected within 24 hours of ovulation

• Careful enzymatic digestion protocols to maintain cellular integrity

• Defined media composition with controlled serum factors that might influence angiogenesis

• Appropriate extracellular matrix components to support vascular network formation

Quantification methodologies:
Reliable quantification requires:

• Immunostaining with endothelial-specific markers (e.g., VWF)

• Standardized image acquisition parameters (magnification, exposure, field selection)

• Objective quantification methods for:

  • Total area of VWF immunostaining

  • Number of endothelial branch points (90% reduction with FGFR1 inhibition)

  • Mean number of vascular sprouts per island

  • Mean island area

Experimental controls:
Include appropriate controls:

• Vehicle-only controls for inhibitor studies

• Time-matched controls for temporal experiments

• Positive controls using known angiogenic stimulators

• Negative controls with established angiogenesis inhibitors

Receptor specificity validation:
Given that bovine CL expresses negligible levels of FGFR3C and FGFR4 , experiments should:

• Confirm receptor expression profiles in your specific model

• Use selective inhibitors (e.g., SU5402 for FGFR1)

• Consider comparative studies with FGFR2C inhibition

• Validate receptor activation with phosphorylation-specific antibodies

Attention to these critical technical factors will enhance experimental rigor when studying FGF2's role in bovine luteal angiogenesis and enable meaningful comparison with other angiogenic models.

How do technical approaches for measuring FGF2 activity differ across bovine tissue types?

Measuring FGF2 activity across different bovine tissue types requires tailored technical approaches due to tissue-specific characteristics, receptor expression profiles, and functional outcomes. These methodological differences are crucial for accurate assessment:

Luteal tissue:
In bovine corpus luteum, FGF2 activity assessment focuses on vascular changes:

• Primary readout: Endothelial network formation with quantification of branch points and vascular sprouting

• Cellular markers: VWF immunostaining for endothelial cells

• Temporal consideration: Maximum sensitivity during days 3-6 in culture

• Receptor focus: FGFR1, as inhibition with SU5402 causes 90% reduction in branching

Reproductive tissues (follicular/embryonic):
For follicular fluid and embryo cultures, concentration measurements predominate:

• Quantification method: ELISA for direct measurement of FGF2 protein

• Sample preparation: Centrifugation of follicular fluid or culture media supernatant

• Sensitivity requirement: Detection limits must accommodate the natural gradient (0.629 ng/mL in follicular fluid to 0.009 ng/mL in late embryo culture)

• Functional correlation: Correlating FGF2 levels with developmental outcomes

Neural tissue:
Based on comparative studies in other species, neural tissue requires:

• Activity assays: Neurite outgrowth and neuronal survival quantification

• Signaling assessment: Phosphorylation of downstream effectors (MAPK pathway)

• Co-factor analysis: Interaction with other neurotrophic factors

• Receptor specificity: Focus on FGFR1 and potentially FGFR2

Technical challenges and solutions:

When designing studies involving multiple bovine tissue types, researchers should carefully select detection methods appropriate for each tissue's unique characteristics while ensuring standardization of fundamental parameters to allow meaningful cross-tissue comparisons.

What experimental designs best elucidate the relationship between FGF2 and other growth factors in bovine systems?

To effectively investigate the complex interplay between FGF2 and other growth factors in bovine systems, researchers should employ strategically designed experimental approaches:

Factorial experimental designs:
Implement factorial designs that systematically vary:

• FGF2 concentration (low, medium, high)

• Concentrations of interacting factors (e.g., EGF, which was measured alongside FGF2 in follicular fluid studies)

• Temporal administration sequences (simultaneous vs. sequential exposure)

• Receptor inhibition combinations

This approach enables statistical identification of synergistic, additive, or antagonistic interactions between factors.

Pathway cross-talk analysis:
Examine signaling pathway interactions using:

• Phospho-proteomics to quantify activation of shared downstream effectors

• Selective pathway inhibitors (e.g., MAPK inhibition for FGF2 signaling)

• Genetic approaches (siRNA) targeting specific pathway components

• Time-course studies to identify sequential activation patterns

Co-expression correlation studies:
In bovine developmental contexts, analyze:

• Parallel measurements of multiple growth factors across developmental stages

• Correlation analysis between FGF2 and other factors

• Spatial co-localization in tissue sections

• Single-cell analyses to identify co-expressing cell populations

Receptor competition assays:
Investigate receptor interactions through:

• Binding competition assays with labeled ligands

• Expression manipulation of shared receptor components

• Analysis of receptor heterodimerization

• Quantification of receptor internalization and recycling kinetics

Specific interacting factor pairs:
Based on available data, prioritize these FGF2 interactions:

• FGF2-EGF: Both measured in bovine follicular fluid and embryo culture

• FGF2-VEGF: Both implicated in bovine luteal angiogenesis

• FGF2-Sphingosine 1-phosphate: Relationship established in auditory neuroblasts, potentially relevant to other bovine tissues

These experimental designs collectively provide a comprehensive framework for elucidating the complex interrelationships between FGF2 and other growth factors in bovine systems, moving beyond simple single-factor studies to capture the multilayered regulation of developmental and physiological processes.

How can contradictory findings about FGF2 function be reconciled through experimental design?

Contradictory findings regarding FGF2 function can be systematically addressed through carefully designed experimental approaches that account for context-dependent effects and methodological variables:

Meta-analysis and systematic review:
Begin by conducting a comprehensive analysis of existing literature to:

• Identify specific contradictions in reported FGF2 functions

• Catalog methodological differences that might explain discrepancies

• Map contextual factors associated with divergent outcomes

• Generate testable hypotheses about the sources of contradiction

Standardized comparative studies:
Design experiments that directly compare:

• Different species/strains under identical conditions (addressing observations like differences between Wistar and Sprague-Dawley rats)

• Male vs. female subjects (gender differences noted in some studies)

• Various developmental stages using consistent analytical methods

• Different tissue types with standardized FGF2 concentrations

Concentration-response relationship mapping:
Develop detailed concentration-response curves across:

• Wide concentration ranges (from 0.1 ng/mL to 10,000 ng/mL, spanning the range reported in literature)

• Multiple endpoints (proliferation, differentiation, survival)

• Different tissue contexts

• Various developmental stages

This approach may reveal biphasic responses or threshold effects that explain apparent contradictions.

Temporal dynamics assessment:
Implement time-course studies that:

• Track FGF2 responses from minutes to days

• Examine how the timing of FGF2 exposure affects outcomes

• Assess whether contradictory findings reflect different temporal snapshots of the same process

• Evaluate time-dependent receptor desensitization or feedback regulation

Microenvironmental context manipulation:
Systematically vary culture conditions to examine how:

• Different extracellular matrix components modify FGF2 responses

• Oxygen tension affects FGF2 signaling outcomes

• Cell density influences paracrine regulation of FGF2 function

• Co-factors (e.g., heparan sulfate proteoglycans) modulate FGF2 activity

Through these approaches, researchers can transform seemingly contradictory findings into a more nuanced understanding of how FGF2 function is modulated by concentration, timing, microenvironmental context, and biological variables—ultimately developing a unified model of context-dependent FGF2 activity in bovine systems.

What are the emerging methodologies for studying FGF2 signaling dynamics in bovine tissues?

Emerging methodologies are transforming how researchers investigate FGF2 signaling dynamics in bovine tissues, enabling more precise temporal and spatial resolution of signaling events:

Live-cell imaging technologies:
Advanced imaging approaches include:

• FRET-based biosensors for real-time visualization of FGF2-receptor interactions

• Fluorescently tagged FGF2 proteins to track ligand trafficking and internalization

• Calcium imaging to monitor immediate signaling responses

• Optogenetic tools to activate FGF receptors with precise spatial and temporal control

These techniques allow researchers to observe FGF2 signaling dynamics in bovine cells as they unfold, rather than relying on endpoint measurements.

Single-cell analysis techniques:
Single-cell approaches reveal heterogeneity in FGF2 responses:

• Single-cell RNA sequencing to identify cell type-specific transcriptional responses

• Mass cytometry (CyTOF) for high-dimensional protein phosphorylation analysis at single-cell resolution

• Digital spatial profiling to map FGF2 response patterns within intact bovine tissues

• Single-cell Western blotting for protein-level validation of signaling events

These methods are particularly valuable when studying tissues with complex cellular composition, such as the bovine corpus luteum where endothelial cells represent just one component of a heterogeneous population .

3D tissue models:
Advanced culture systems more accurately recapitulate in vivo conditions:

• Bovine luteal organoids that develop characteristic vascular networks

• Microfluidic "organ-on-chip" models that incorporate flow dynamics

• Bioprinted 3D tissues with defined architecture and cellular organization

• Co-culture systems that maintain critical cell-cell interactions

These models bridge the gap between traditional 2D cultures and in vivo complexity, potentially resolving contradictions that arise from oversimplified experimental systems.

Computational modeling approaches:
Mathematical models integrate experimental data to predict system behavior:

• Ordinary differential equation models of FGF2 signaling pathways

• Agent-based models of endothelial cell sprouting and vascular network formation

• Multiscale models linking molecular signaling to tissue-level outcomes

• Machine learning approaches to identify patterns in complex datasets

These computational approaches can generate testable hypotheses about how FGF2 signaling dynamics lead to specific bovine tissue responses.

By adopting these emerging methodologies, researchers can achieve unprecedented insight into the complex signaling dynamics through which FGF2 regulates diverse processes in bovine tissues, particularly during critical developmental transitions and angiogenic events.

What are the key consensus findings about FGF2 (147) in bovine systems that researchers should incorporate into their work?

The truncated FGF2 (147) protein maintains full biological activity compared to the full-length (154aa) version, making it suitable for experimental applications investigating native FGF2 function . This equivalence in activity occurs despite differences in intracellular localization, suggesting that receptor binding and activation properties remain unchanged after N-terminal truncation.

FGF2 serves as a critical regulator of vascular development, with particularly strong effects during specific temporal windows of angiogenesis. The days 3-6 period in luteal angiogenesis represents a key phase of sensitivity to FGF2 regulation, corresponding to the initiation of vascular sprouting . This temporal specificity should be carefully considered when designing experiments involving vascular development.

A natural concentration gradient of FGF2 exists across developmental stages in bovine reproductive biology, with highest levels in follicular fluid (0.629 ng/mL) and progressively decreasing concentrations through embryonic development . This gradient likely reflects functional requirements at different developmental stages and should inform concentration choices in experimental designs.

FGF2 signaling in bovine tissues occurs primarily through FGFR1 and potentially FGFR2, as bovine corpus luteum expresses negligible levels of other FGF receptors . The dramatic effect of FGFR1 inhibition on endothelial branching (90% reduction) highlights this receptor's central role in mediating FGF2's angiogenic functions.