eno4 Antibody

What is ENO4 and what biological functions does it serve?

ENO4, also known as Enolase 4 or C10orf134, is a protein that catalyzes the reversible conversion of 2-phosphoglycerate to phosphoenolpyruvate, an essential step in glycolysis critical for cellular energy production . Research indicates that ENO4 is primarily expressed in the testis and appears essential for sperm function and motility . Studies in mouse models demonstrate that ENO4 is the predominant enolase in sperm cells, where it contributes to proper assembly of the fibrous sheath, a cytoskeletal structure unique to sperm flagellum . Disruption of the ENO4 gene in mice results in significant fertility issues, including reduced sperm motility and structural abnormalities in sperm flagella .

What applications are ENO4 antibodies suitable for in laboratory research?

ENO4 antibodies have been validated for multiple research applications, enabling comprehensive protein characterization:

When designing experiments, researchers should perform antibody validation and optimization for their specific biological samples, as reactivity can vary based on sample preparation methods and the specific epitope targeted by the antibody .

How should ENO4 antibodies be stored and handled to maintain effectiveness?

To preserve antibody functionality and specificity, ENO4 antibodies should be stored as small aliquots at -20°C, avoiding repeated freeze-thaw cycles that can cause protein denaturation and reduced activity . Commercial ENO4 antibodies are typically supplied in PBS buffer containing 0.09% sodium azide as a preservative . When working with ENO4 antibodies:

• Thaw aliquots completely before use and maintain on ice during experimental procedures

• Centrifuge briefly before opening to collect solution at the bottom of the vial

• Return unused portions to storage promptly

• Use proper personal protective equipment when handling, as some preparations contain hazardous components like sodium azide

• Document lot numbers and maintain consistency within experimental series to minimize variation

What controls should be included when using ENO4 antibodies?

Proper experimental controls are essential for interpreting results with ENO4 antibodies:

• Positive controls: Tissues or cell lines with known ENO4 expression, such as testicular tissue, RT4 or U251 MG cell lysates for Western blot applications

• Negative controls: Samples with confirmed absence of ENO4 expression, or tissues from ENO4 knockout models

• Loading controls: Use established housekeeping proteins (β-actin, GAPDH) when quantifying ENO4 expression by Western blot

• Primary antibody omission: Samples processed identically but without primary antibody addition to identify non-specific binding of secondary antibodies

• Isotype controls: Use of irrelevant antibodies of the same isotype and concentration to identify non-specific interactions

How can ENO4 antibodies be used to investigate male infertility mechanisms?

ENO4 antibodies provide valuable tools for investigating male infertility through multiple methodological approaches:

• Comparative expression studies: ENO4 antibodies can be used to compare protein expression levels between fertile and infertile individuals through quantitative Western blotting and immunohistochemistry. Research demonstrated that ENO4 knockout mice exhibit significantly reduced sperm motility and structural abnormalities in the fibrous sheath, suggesting similar mechanisms may contribute to human male infertility .

• Subcellular localization analysis: Immunofluorescence microscopy with ENO4 antibodies reveals that the protein localizes primarily to the sperm flagellum, specifically associated with the fibrous sheath structure. Altered localization patterns may correlate with specific infertility phenotypes .

• Functional coupling studies: By pairing ENO4 immunoprecipitation with enzymatic activity assays, researchers can assess whether mutations or post-translational modifications affect the protein's catalytic function in converting 2-phosphoglycerate to phosphoenolpyruvate during glycolysis .

• Protein-protein interaction networks: ENO4 antibodies enable co-immunoprecipitation experiments to identify binding partners within the sperm flagellum, providing insights into the molecular architecture of the fibrous sheath and its role in sperm function .

When investigating infertility, researchers should consider combining ENO4 antibody-based approaches with genetic analysis, as gene trap experiments in mice have established a direct link between ENO4 disruption and male infertility .

What approaches can resolve cross-reactivity concerns with ENO4 antibodies?

Cross-reactivity with other enolase isoforms (ENO1, ENO2, ENO3) represents a significant challenge when working with ENO4 antibodies due to sequence homology among family members. To address this:

• Epitope selection: Choose antibodies targeting unique regions within ENO4. The amino acid sequence 334-353 (N-CLPPPKQETKKGHNGSKRAQP-COOH) has been successfully used to generate ENO4-specific antibodies with minimal cross-reactivity .

• Absorption controls: Pre-absorb ENO4 antibodies with recombinant ENO1, ENO2, and ENO3 proteins to deplete cross-reactive antibodies before experimental use.

• Knockout verification: Validate antibody specificity using tissues from ENO4 knockout models as negative controls. ENO4 Gt/Gt mouse models provide excellent specificity controls, as demonstrated in published research .

• Orthogonal detection methods: Confirm antibody specificity by correlating protein detection with mRNA expression using techniques like qRT-PCR or RNA-seq.

• Western blot analysis: Compare banding patterns with known molecular weights (ENO4: 69 kDa, ENO1: 47 kDa, ENO2: 47 kDa, ENO3: 47 kDa) to identify potential cross-reactivity .

Researchers studying ENO4 should note that the gene Gm5506 encodes a protein identical to ENO1 and is transcribed at low levels in testis, which may complicate interpretation of results if antibodies cross-react .

How can ENO4 antibodies be optimized for immunohistochemical detection in reproductive tissues?

Optimizing ENO4 antibody protocols for reproductive tissues requires addressing tissue-specific challenges:

• Fixation optimization: For testicular and epididymal tissues, 2% paraformaldehyde (PFA) fixation for 15 minutes at room temperature followed by 50 mM glycine in PBS for 30 minutes has been successfully employed to preserve ENO4 antigenicity while maintaining tissue architecture .

• Antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes

  • Enzymatic retrieval using proteinase K (20 μg/mL for 15 minutes at 37°C)

  • Comparison of retrieval methods by signal-to-noise ratio assessment

• Permeabilization protocol: Ice-cold methanol permeabilization for 1 minute provides optimal antibody access to ENO4 epitopes in sperm cells while preserving cellular structures .

• Signal amplification strategies: For low-abundance detection, implement tyramide signal amplification or quantum dot-based detection systems to enhance sensitivity while maintaining specificity.

• Multi-labeling approaches: Combine ENO4 detection with markers of sperm differentiation stages (ACR, PRM1, PRM2) or structural components (AKAP4) to contextualize ENO4 expression patterns during spermatogenesis .

Dilution optimization experiments indicate that 1:50 dilution of commercial ENO4 antibodies typically provides optimal staining in paraffin-embedded human testicular tissues, while maintaining acceptable background levels .

What strategies can address inconsistent results when working with ENO4 antibodies?

Researchers encountering variability in ENO4 antibody results should implement a systematic troubleshooting approach:

• Antibody characterization panel:

  • Test multiple lots of the same antibody

  • Compare monoclonal versus polyclonal antibodies targeting different ENO4 epitopes

  • Document reactivity patterns across experimental conditions

• Sample preparation standardization:

  • Establish consistent tissue harvesting protocols, particularly important for testicular tissues where ENO4 expression varies with the spermatogenic cycle

  • Implement standardized protein extraction methods optimized for membrane-associated proteins

  • Control for post-translational modifications by using phosphatase or deglycosylation treatments prior to analysis

• Quantitative validation:

  • Employ recombinant ENO4 protein standards in Western blotting

  • Use digital image analysis with calibrated exposure settings

  • Implement statistical analysis to distinguish biological from technical variation

• Complementary techniques:

  • Validate antibody-based findings with orthogonal methods such as mass spectrometry

  • Correlate protein detection with mRNA expression analysis

  • Consider CRISPR-Cas9 gene editing to generate control cell lines with modified ENO4 expression

Researchers should note that ENO4 expression is highly tissue-specific, with predominant expression in testis beginning around postnatal day 12 in mice, which coincides with the onset of meiosis during spermatogenesis .

How do different antibody preparation methods affect ENO4 detection?

The method of ENO4 antibody generation significantly influences detection characteristics:

Current evidence indicates that polyclonal antibodies generated against the central region (amino acids 388-414) or the 50-150 amino acid region of human ENO4 provide optimal detection sensitivity across multiple applications . Antibodies targeting the 334-353 amino acid region have demonstrated excellent specificity in mouse models .

What are the critical parameters for Western blot optimization when detecting ENO4?

Successful Western blot analysis of ENO4 requires optimization of several parameters:

• Sample preparation:

  • Use specialized extraction buffers containing 1% Triton X-100, 0.5% NP-40, and protease inhibitors to efficiently solubilize ENO4

  • Sonicate samples to ensure complete protein extraction

  • Determine optimal protein loading (typically 20-50 μg total protein)

• Electrophoresis conditions:

  • Employ 10% polyacrylamide gels for optimal resolution around the 69 kDa range where ENO4 migrates

  • Include molecular weight markers spanning 50-100 kDa range for accurate band identification

  • Consider gradient gels (4-15%) when analyzing multiple proteins simultaneously

• Transfer parameters:

  • Implement semi-dry transfer at 15V for 60 minutes or wet transfer at 100V for 60 minutes

  • Use PVDF membranes with 0.45 μm pore size for optimal protein retention

  • Verify transfer efficiency with reversible staining methods (Ponceau S)

• Antibody incubation:

  • Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with ENO4 antibody at 1:100 dilution overnight at 4°C

  • Perform 3 x 10-minute TBST washes followed by HRP-conjugated secondary antibody (1:5000) for 1 hour

• Detection optimization:

  • Employ enhanced chemiluminescence (ECL) for standard detection

  • Consider fluorescent secondary antibodies for multiplexing and quantitative analysis

  • Validate results with multiple exposure times to ensure linear range detection

Research indicates that ENO4 typically appears as a 69 kDa band in Western blots, though post-translational modifications may result in additional bands or mobility shifts in certain tissues or developmental stages .

How can ENO4 antibodies be utilized in comparative studies of normal versus pathological reproductive conditions?

ENO4 antibodies enable sophisticated comparative analyses in reproductive pathology research:

• Quantitative expression profiling:

  • Implement tissue microarray analysis with ENO4 antibodies to compare expression across multiple patient samples simultaneously

  • Utilize digital pathology tools to quantify staining intensity, subcellular localization, and cell-type specificity

  • Correlate ENO4 expression patterns with clinical parameters such as sperm count, motility, and morphology

• Functional correlation studies:

  • Pair ENO4 immunodetection with enzymatic activity assays to correlate protein levels with glycolytic function

  • Investigate whether ENO4 expression correlates with ATP production in sperm cells

  • Analyze ENO4 in conjunction with other glycolytic enzymes to identify metabolic bottlenecks in pathological samples

• Structural analysis approaches:

  • Combine ENO4 immunogold labeling with electron microscopy to assess ultrastructural localization in normal versus abnormal sperm

  • Implement super-resolution microscopy (STORM, STED) with ENO4 antibodies to map precise distribution within the fibrous sheath

  • Correlate ENO4 mislocalization with structural abnormalities in the sperm flagellum

• Temporal expression analysis:

  • Use ENO4 antibodies to track protein expression during spermatogenesis in normal versus pathological testes

  • Implement multiplex immunofluorescence to co-localize ENO4 with stage-specific markers

  • Document changes in ENO4 expression patterns during sperm maturation in the epididymis

Mouse models have demonstrated that ENO4 disruption results in a 2-fold reduction in epididymal sperm numbers and substantial decrease in sperm motility, suggesting that similar quantitative assessments may reveal correlations with human infertility phenotypes .

What considerations are important when selecting an ENO4 antibody for sperm metabolism research?

Selecting the optimal ENO4 antibody for sperm metabolism research requires careful consideration of several factors:

• Target epitope location:

  • Choose antibodies targeting conserved catalytic domains (amino acids 50-150) when investigating enzymatic function

  • Select antibodies recognizing C-terminal regions (amino acids 388-414) for studying protein-protein interactions

  • Consider antibodies against unique regions (amino acids 334-353) when specificity is paramount

• Species cross-reactivity:

  • Validate antibody reactivity in your experimental species (human, mouse, etc.)

  • Consider evolutionary conservation of the target epitope when working with non-model organisms

  • Test species cross-reactivity experimentally rather than relying solely on sequence homology predictions

• Application compatibility:

  • For enzymatic activity studies, select antibodies that don't interfere with catalytic sites

  • For co-localization studies, ensure antibodies are compatible with fixation methods

  • For immunoprecipitation, verify antibody works under native conditions

• Validation evidence:

  • Review published literature using the specific antibody clone

  • Assess manufacturer validation data across multiple applications

  • Consider antibodies validated in knockout/knockdown systems

• Technical considerations:

  • Host species (rabbit polyclonal antibodies show strong performance in ENO4 detection)

  • Clonality (monoclonal for reproducibility, polyclonal for sensitivity)

  • Conjugation status (unconjugated for flexibility, directly labeled for multiplexing)

Research has established that ENO4 provides most of the enolase activity in sperm, while ENO1 (and possibly the ENO1-identical Gm5506 gene product) contributes only minor activity, making ENO4-specific antibodies particularly valuable for sperm metabolism studies .

How can researchers address non-specific background in ENO4 immunostaining?

High background when using ENO4 antibodies can be systematically reduced through multiple approaches:

• Blocking optimization:

  • Test multiple blocking agents (BSA, normal serum, commercial blockers)

  • Implement dual blocking strategy with protein blocking followed by additional Fc receptor blocking

  • Extend blocking time to 2 hours at room temperature for challenging tissues

• Antibody dilution optimization:

  • Perform systematic titration series (1:25, 1:50, 1:100, 1:200, etc.)

  • Compare signal-to-noise ratios across dilutions

  • Consider reduced primary antibody incubation time with higher concentrations

• Tissue-specific considerations:

  • For testicular tissue, minimize endogenous peroxidase activity with 0.3% H₂O₂ treatment

  • Reduce autofluorescence with Sudan Black B (0.1% in 70% ethanol) treatment

  • Implement additional washing steps (5 x 5 minutes) with 0.1% Tween-20 in PBS

• Fixation adjustments:

  • Compare cross-linking fixatives (PFA, glutaraldehyde) with precipitating fixatives (methanol, acetone)

  • Optimize fixation duration to balance epitope preservation and tissue morphology

  • Consider post-fixation quenching of residual aldehydes with glycine buffer

• Technical controls:

  • Include absorption controls using immunizing peptide

  • Implement isotype controls at matching concentrations

  • Compare staining patterns between multiple antibodies targeting different ENO4 epitopes

Empirical data indicate that a 1:50 dilution for immunohistochemistry and 4 μg/mL for immunofluorescence provide optimal signal-to-noise ratios for ENO4 detection in most tissues, though this should be validated for each experimental system .

What approaches can resolve discrepancies in ENO4 molecular weight detection?

Researchers may encounter variations in ENO4's apparent molecular weight across different experimental systems. To address this:

• Sample preparation factors:

  • Compare reducing versus non-reducing conditions to assess potential disulfide bonding

  • Evaluate different lysis buffers (RIPA, NP-40, Triton X-100) for extraction efficiency

  • Test denaturation at different temperatures (37°C, 70°C, 95°C) and durations

• Post-translational modification analysis:

  • Treat samples with phosphatase to identify phosphorylation-dependent mobility shifts

  • Apply deglycosylation enzymes (PNGase F, O-glycosidase) to assess glycosylation status

  • Consider other modifications by comparing mobility with prediction algorithms

• Gel system optimization:

  • Compare Tris-glycine versus Bis-Tris gel systems

  • Test gradient gels versus fixed percentage gels

  • Calibrate with appropriate molecular weight markers flanking the expected 69 kDa range

• Isoform consideration:

  • Investigate potential alternative splicing through RT-PCR analysis

  • Compare migration patterns across different tissues and developmental stages

  • Validate observed bands through mass spectrometry

• Methodological validation:

  • Compare multiple antibodies targeting different epitopes

  • Correlate observations with recombinant protein standards

  • Verify results in knockout/knockdown systems

How can researchers ensure reproducibility in ENO4 antibody-based quantitative analyses?

Ensuring reproducible quantitative results with ENO4 antibodies requires implementing robust standardization protocols:

• Antibody validation and standardization:

  • Maintain detailed records of antibody source, lot number, and validation data

  • Prepare master aliquots of working dilutions to minimize freeze-thaw cycles

  • Include standard curves with recombinant ENO4 protein in quantitative assays

• Sample processing standardization:

  • Establish consistent timing between sample collection and processing

  • Implement standardized protein extraction methods with controlled buffer-to-sample ratios

  • Process comparative samples simultaneously to minimize batch effects

• Technical replication strategy:

  • Perform technical triplicates for each biological sample

  • Include inter-assay calibrators across experimental runs

  • Implement randomization strategies to distribute samples across multiple experiments

• Normalization approaches:

  • Select appropriate housekeeping proteins verified for stability in your experimental system

  • Consider multiple normalization strategies (GAPDH, β-actin, total protein staining)

  • Validate normalization methods through coefficient of variation analysis

• Data analysis standardization:

  • Establish consistent image acquisition parameters (exposure time, gain, offset)

  • Implement automated analysis workflows to reduce subjective interpretation

  • Document all analysis parameters and statistical approaches in detail

Research on ENO4 mouse models has demonstrated that quantitative analysis requires careful standardization, as disruption of ENO4 expression leads to approximately 50% reduction in enolase enzymatic activity in sperm, highlighting the protein's substantial contribution to sperm metabolism .

What are the critical considerations when interpreting ENO4 localization in sperm cells?

Accurate interpretation of ENO4 subcellular localization requires addressing several technical and biological complexities:

• Fixation and permeabilization effects:

  • Compare multiple fixation methods (2% PFA, methanol, acetone) to verify consistent localization patterns

  • Assess different permeabilization approaches (Triton X-100, saponin, digitonin) for their effect on membrane structures

  • Consider the impact of fixation duration on epitope accessibility and structural preservation

• Resolution limitations:

  • Distinguish between proximal and principal piece localization using high-resolution confocal microscopy

  • Implement super-resolution techniques (STED, STORM) to resolve fibrous sheath association

  • Correlate immunofluorescence with immunoelectron microscopy for ultrastructural precision

• Co-localization analysis:

  • Pair ENO4 detection with established markers of sperm compartments (mitochondrial sheath, fibrous sheath, axoneme)

  • Implement quantitative co-localization metrics (Pearson's correlation, Manders' coefficients)

  • Use spectral unmixing for multi-color imaging to minimize bleed-through artifacts

• Species-specific considerations:

  • Account for morphological differences between mouse and human sperm when interpreting localization

  • Compare staining patterns between species with antibodies targeting conserved epitopes

  • Validate localization findings across multiple antibodies targeting different ENO4 regions

• Maturation state analysis:

  • Track ENO4 localization changes during epididymal transit and capacitation

  • Compare localization in swimming versus non-motile sperm populations

  • Consider potential redistribution during physiological processes like hyperactivation

Research has established that ENO4 predominantly localizes to the fibrous sheath in the principal piece of the sperm flagellum, consistent with its role in providing localized ATP production for sperm motility. This localization is disrupted in ENO4 knockout mouse models, resulting in coiled flagella and disorganized fibrous sheath structures .

How might ENO4 antibodies contribute to developing male contraceptive approaches?

ENO4 antibodies offer several promising avenues for male contraceptive research:

• Target validation studies:

  • Use ENO4 antibodies to screen for correlations between protein expression/localization and fertility outcomes

  • Employ antibody-based approaches to identify accessible epitopes for contraceptive targeting

  • Validate the specificity of ENO4 disruption effects on sperm function versus other tissues

• Functional inhibition screening:

  • Develop assays using ENO4 antibodies to screen for small molecule inhibitors of ENO4 activity

  • Test antibody-based inhibition of ENO4 function in in vitro fertilization models

  • Assess reversibility of inhibition through longitudinal studies

• Mechanism elucidation:

  • Utilize ENO4 antibodies to track protein-protein interactions critical for sperm motility

  • Investigate how ENO4 integrates with other glycolytic enzymes in the fibrous sheath

  • Map the temporal sequence of ENO4 involvement during capacitation and hyperactivation

• Delivery system development:

  • Use fluorescently labeled ENO4 antibodies to assess penetration of potential delivery vehicles into sperm

  • Determine accessibility of ENO4 epitopes under physiological conditions

  • Test localized delivery approaches targeting the male reproductive tract

Mouse knockout studies demonstrate that disruption of ENO4 leads to male infertility through multiple mechanisms including reduced sperm numbers, impaired motility, and structural abnormalities in the sperm flagellum, suggesting that targeted inhibition could provide an effective contraceptive approach with multiple mechanisms of action .

What novel techniques could enhance ENO4 detection sensitivity and specificity?

Emerging technologies offer opportunities to advance ENO4 detection capabilities:

• Proximity ligation assays (PLA):

  • Implement PLA to detect ENO4 interactions with other fibrous sheath proteins

  • Achieve single-molecule sensitivity for low-abundance interactions

  • Visualize protein complexes in their native cellular context

• CRISPR-engineered reporter systems:

  • Develop knock-in fluorescent tags to monitor endogenous ENO4 dynamics

  • Create split-GFP complementation systems to study protein-protein interactions

  • Establish inducible degradation systems to study acute ENO4 loss

• Mass spectrometry applications:

  • Combine ENO4 immunoprecipitation with targeted mass spectrometry

  • Implement selective reaction monitoring for absolute quantification

  • Develop methods to detect post-translational modifications specific to ENO4

• Single-cell analysis technologies:

  • Apply ENO4 antibodies in single-cell Western blotting platforms

  • Implement imaging mass cytometry for multiplexed protein detection

  • Utilize microfluidic approaches for single sperm cell analysis

• Advanced imaging techniques:

  • Employ lattice light-sheet microscopy for dynamic imaging of ENO4 in living sperm

  • Implement expansion microscopy to resolve nanoscale distribution in the fibrous sheath

  • Utilize correlative light and electron microscopy to link function with ultrastructure

These emerging approaches could address current limitations in studying ENO4, particularly regarding the protein's dynamic behavior during sperm activation and the relationship between its enzymatic function and structural role in the fibrous sheath .

How can ENO4 antibodies contribute to understanding evolutionary aspects of sperm glycolysis?

ENO4 antibodies can facilitate comparative evolutionary studies across species:

• Cross-species reactivity assessment:

  • Test existing ENO4 antibodies against targeted epitopes in diverse mammalian species

  • Identify conserved versus divergent epitopes through epitope mapping

  • Develop pan-species antibodies targeting evolutionarily conserved regions

• Comparative localization studies:

  • Map ENO4 distribution patterns across reproductive strategies (internal vs. external fertilization)

  • Compare subcellular localization in species with different sperm morphologies

  • Correlate ENO4 compartmentalization with metabolic adaptations to diverse environments

• Functional conservation analysis:

  • Combine antibody detection with activity assays across species

  • Investigate whether ENO4 contribution to total enolase activity varies by species

  • Correlate structural differences with functional specialization

• Developmental expression profiling:

  • Track ENO4 expression timing across species with different reproductive maturation rates

  • Compare expression onset relative to spermatogenic milestones

  • Investigate regulatory mechanisms governing expression across evolutionary distances

• Paralog relationship studies:

  • Use specific antibodies to distinguish ENO4 from other enolase family members

  • Investigate potential functional redundancy between enolase paralogs

  • Trace the evolutionary history of functional specialization

Research has established that while ENO4 provides the majority of enolase activity in mouse sperm, ENO1 and potentially the ENO1-identical Gm5506 gene product contribute minor activity, suggesting evolutionary adaptations that could vary across species with different sperm energy requirements .

What potential role might ENO4 antibodies play in diagnosing specific forms of male infertility?

ENO4 antibodies hold promise for developing diagnostic approaches for male infertility:

• Diagnostic panel development:

  • Incorporate ENO4 antibodies into multiplexed immunoassays detecting multiple fertility biomarkers

  • Establish reference ranges for ENO4 expression and localization in normal sperm

  • Correlate ENO4 abnormalities with specific infertility phenotypes

• Morphological assessment enhancement:

  • Develop ENO4-based immunofluorescence protocols for clinical sperm analysis

  • Create automated image analysis algorithms to quantify ENO4 distribution patterns

  • Establish correlations between ENO4 localization defects and functional outcomes

• Non-invasive testing approaches:

  • Investigate ENO4 in seminal plasma as a potential biomarker for sperm dysfunction

  • Develop ELISA or lateral flow assays for point-of-care testing

  • Correlate extracellular ENO4 with intracellular expression patterns

• Personalized treatment stratification:

  • Use ENO4 antibody-based diagnostics to classify infertility subtypes

  • Guide selection of assisted reproductive technologies based on ENO4 status

  • Track treatment efficacy through ENO4-based monitoring

• Genetic counseling integration:

  • Correlate ENO4 protein abnormalities with genetic variants

  • Support interpretation of variants of unknown significance

  • Guide targeted genetic testing based on protein analysis

Mouse models demonstrate that ENO4 disruption creates a distinctive phenotype including coiled flagella and disorganized fibrous sheath, suggesting that similar structural abnormalities in human sperm might be linked to ENO4 dysfunction and could be detected using antibody-based approaches .