ACTL9 Antibody

What is ACTL9 and where is it primarily expressed?

ACTL9 (Actin-Like 9) is a testis-specific actin-like protein that shows abundant expression primarily in the testis, as confirmed through both database analyses and real-time PCR verification. Expression studies have demonstrated minimal presence in other human adult tissues, highlighting its tissue-specific nature. In normal sperm, ACTL9 is principally localized in the equatorial segment of the head (approximately 84.4% ± 11.1% of sperm) and neck regions (100.0% ± 0.0%), with a low proportion exhibiting ACTL9 in the acrosomal segment of the head (11.6% ± 10.7%) . This specialized distribution pattern suggests a critical role in sperm function and fertilization processes rather than having a general cytoskeletal function across multiple tissues.

What types of ACTL9 antibodies are commercially available for research?

Several types of ACTL9 antibodies are available for research applications. These include polyclonal antibodies that target different amino acid regions of the ACTL9 protein. Specific examples include antibodies targeting amino acids 1-416 (full-length protein), 1-160, 23-50 (N-terminal region), and 143-192 . These antibodies come in various formats including unconjugated forms and conjugated versions with FITC, HRP, or Biotin for different experimental applications . Most commercially available ACTL9 antibodies show reactivity with human samples, though some (such as antibodies targeting AA 143-192) also demonstrate cross-reactivity with mouse, guinea pig, monkey, and rat samples . This variety allows researchers to select antibodies appropriate for their specific experimental needs.

What applications are ACTL9 antibodies suitable for?

ACTL9 antibodies are suitable for multiple laboratory applications in reproductive biology research. Common applications include Western Blotting (WB) for protein expression analysis, Enzyme-Linked Immunosorbent Assay (ELISA) for quantitative detection, Immunohistochemistry (IHC) for tissue localization studies, and Immunofluorescence (IF) for cellular localization analysis . These antibodies have been successfully utilized in co-immunoprecipitation (co-IP) assays to study protein-protein interactions, particularly the interaction between ACTL9 and ACTL7A . The diversity of applications makes these antibodies versatile tools for investigating ACTL9's role in sperm function and fertilization. Researchers should note that optimal working dilutions should be determined experimentally for each specific application and antibody lot .

How can ACTL9 antibodies be used to investigate sperm fertility problems?

ACTL9 antibodies provide valuable tools for investigating certain types of male infertility, particularly total fertilization failure (TFF). Immunostaining protocols using these antibodies can reveal abnormal localization or absence of ACTL9 in the equatorial segment of sperm heads, which has been associated with fertilization failure . Research protocols should include:

• Sperm sample preparation with proper fixation and permeabilization

• Immunostaining with anti-ACTL9 antibodies (preferably those targeting the full-length protein)

• Co-staining with markers for acrosomal structures (such as PNA) and nuclear DNA (DAPI)

• Confocal microscopy analysis with line-intensity profiling to precisely determine ACTL9 localization

• Quantification of the proportion of sperm showing normal versus abnormal ACTL9 localization patterns

This approach can help identify potential molecular causes of unexplained fertilization failure in cases where conventional sperm parameters appear normal but fertilization does not occur even with intracytoplasmic sperm injection (ICSI) .

What experimental approaches can be used to study ACTL9 interactions with other proteins?

To investigate ACTL9's interactions with other proteins, particularly ACTL7A, researchers can employ the following methodological approaches:

• Co-immunoprecipitation (co-IP): Transfect HEK293T cells with expression constructs for wild-type or mutant ACTL9 (with His-tag) and potential interacting proteins like ACTL7A (with cMYC-tag). After 48 hours of culture, harvest cells and lyse in NP-40 Lysis Buffer supplemented with PMSF. Incubate protein lysates with protein A/G magnetic beads cross-linked with anti-His-tag antibodies for 4 hours at 4°C. After washing, elute captured protein complexes and analyze by immunoblotting with appropriate antibodies .

• Co-localization studies: Perform dual immunofluorescence staining of sperm samples using antibodies against ACTL9 and potential interacting proteins. Analyze co-localization using confocal microscopy and line-intensity profiles. This approach has successfully demonstrated that ACTL9 and ACTL7A co-localize in the subacrosomal layer of the perinuclear theca (PT) in normal sperm .

• Functional analysis of mutant proteins: Generate expression constructs with specific mutations (such as p.Ser345Leu, p.Val380Leu, or p.Tyr403Ter) to determine how these affect protein-protein interactions. This approach has revealed that certain mutations weaken or completely abolish the interaction between ACTL9 and ACTL7A .

These methodologies provide mechanistic insights into how ACTL9 functions within protein complexes to maintain normal sperm structure and function.

How should researchers optimize immunolocalization studies using ACTL9 antibodies?

For optimal immunolocalization of ACTL9 in sperm samples, researchers should consider the following methodological considerations:

• Sample preparation:

  • Use freshly collected or properly cryopreserved samples

  • For capacitated sperm studies, incubate samples in appropriate capacitation media

  • Optimize fixation protocols (typically 4% paraformaldehyde for 30 minutes)

  • Use gentle permeabilization methods to maintain structural integrity

• Antibody selection and validation:

  • Choose antibodies targeting the most relevant epitopes (full-length antibodies may provide the most comprehensive localization)

  • Validate antibody specificity using appropriate positive and negative controls

  • Determine optimal antibody concentration through titration experiments

• Co-staining approach:

  • Include acrosomal markers like peanut agglutinin (PNA) and nuclear stains (DAPI)

  • Consider co-staining with ACTL7A antibodies to assess potential interactions

  • For studying fertilization failure, include PLCζ staining to evaluate its localization

• Imaging and analysis:

  • Use confocal microscopy for precise localization

  • Employ line-intensity profiles across different sperm regions

  • Quantify the percentage of sperm showing specific localization patterns

  • Compare staining patterns between normal and abnormal samples

This methodological approach has successfully demonstrated that in normal sperm, ACTL9 localizes to the equatorial segment and neck regions, while abnormal localization patterns are associated with fertilization failure.

What is known about the functional domains of ACTL9 and how might antibodies targeting different regions vary in their applications?

Research on ACTL9's functional domains is still emerging, but current evidence suggests important functional regions particularly in the C-terminal portion of the protein. The functional significance of different domains can be inferred from mutation studies and antibody targeting:

• C-terminal region (beyond amino acid 403): This appears critical for interaction with ACTL7A, as the nonsense variant p.Tyr403Ter completely loses the ability to interact with ACTL7A. This suggests that the main interaction site may be located downstream of the 403rd amino residue . Antibodies targeting this region may be particularly useful for studying protein-protein interactions.

• Mid-region (around amino acids 345-380): Mutations in this region (p.Ser345Leu, p.Val380Leu) weaken but don't completely abolish interactions with ACTL7A, suggesting this area contributes to but isn't solely responsible for protein interactions . Antibodies targeting this region might be valuable for structural studies.

• N-terminal region (amino acids 1-160): Several available antibodies target this region, suggesting its accessibility and potential utility for general detection purposes . These antibodies may be particularly suitable for applications like Western blotting and immunofluorescence.

When selecting antibodies for specific applications, researchers should consider:

• For protein interaction studies: antibodies targeting the C-terminal region may provide the most specific information

• For general detection: N-terminal targeting antibodies appear widely available

• For comprehensive studies: full-length (AA 1-416) antibodies might offer the most complete picture of ACTL9 localization and function

Epitope mapping studies using various antibodies could further enhance our understanding of ACTL9's domain structure and function.

How do pathogenic variants in ACTL9 affect protein localization and function, and how can antibodies help study these effects?

Pathogenic variants in ACTL9 significantly alter both protein localization and function, which can be effectively studied using antibody-based approaches. Research has revealed several key alterations:

• Changes in sperm head localization: In normal sperm, ACTL9 localizes to the equatorial segment of the head, but sperm carrying mutations (p.Ser345Leu, p.Val380Leu, or p.Tyr403Ter) show no ACTL9 signal in this region. Interestingly, the neck region localization remains unaffected in mutant sperm . This differential effect suggests domain-specific functions in different subcellular locations.

• Altered protein interactions: Mutant ACTL9 proteins show weakened (p.Ser345Leu, p.Val380Leu) or completely lost (p.Tyr403Ter) interaction with ACTL7A, as demonstrated through co-IP studies . This disruption appears to be a critical mechanism underlying the functional defects.

• Impact on perinuclear theca (PT) structure: The PT, which anchors the acrosome to the nuclear envelope, shows abnormal ultrastructure in mutant sperm. This leads to detachment of the acrosome from the nuclear envelope, forming a loosened PT structure .

• Effects on PLCζ localization: PLCζ, a key oocyte activation factor, normally co-localizes with ACTL9 in the equatorial segment. In mutant sperm, PLCζ is either absent (in higher proportions than normal) or abnormally localized to the neck region .

To study these effects, antibody-based approaches should include:

• Comparative immunofluorescence analysis of normal versus mutant sperm

• Dual immunostaining with ACTL9 and ACTL7A antibodies

• Electron microscopy combined with immunogold labeling to visualize ultrastructural changes

• Functional assays to correlate structural changes with fertilization capacity

These methods can provide mechanistic insights into how ACTL9 mutations lead to fertilization failure and may guide the development of potential diagnostic tools.

What are the optimal storage and handling conditions for ACTL9 antibodies?

To maintain optimal antibody performance and extend shelf life, researchers should follow these specific storage and handling recommendations for ACTL9 antibodies:

• Storage temperature: Store antibodies at 4°C for short-term use (days to weeks) or at -20°C for long-term storage (months to years) . Avoid storing at room temperature for extended periods.

• Aliquoting strategy: To prevent repeated freeze-thaw cycles that can degrade antibody quality, aliquot antibodies into smaller volumes based on typical experimental needs. Each freeze-thaw cycle can reduce antibody activity .

• Buffer conditions: Most commercial ACTL9 antibodies are provided in PBS at pH 7.2 . If transferring or diluting antibodies, maintain similar buffer conditions to prevent denaturation.

• Protein stabilizers: Some antibody preparations contain stabilizers like BSA or glycerol. Do not remove these unless they interfere with specific applications.

• Contamination prevention: Use sterile technique when handling antibodies to prevent microbial contamination. Consider adding sodium azide (0.02%) for long-term storage, but note that azide can interfere with some applications like HRP-based detection.

• Transportation: When transporting between laboratories or facilities, maintain cold chain using ice packs or dry ice depending on distance and time.

• Documentation: Maintain records of antibody lot numbers, receipt dates, number of freeze-thaw cycles, and experimental performance to track potential degradation over time.

Following these protocols will help ensure consistent experimental results and maximize the useful life of ACTL9 antibody reagents.

What controls should be included when using ACTL9 antibodies for reproductive biology research?

When conducting experiments with ACTL9 antibodies, researchers should implement a comprehensive set of controls to ensure reliable and interpretable results:

• Positive controls:

  • Testis tissue sections (as ACTL9 is primarily expressed in testis)

  • Sperm samples from healthy donors with confirmed fertility

  • Cell lines transfected with ACTL9 expression constructs (e.g., HEK293T cells)

• Negative controls:

  • Tissues known not to express ACTL9 (e.g., non-reproductive tissues)

  • Primary antibody omission control

  • Isotype control antibodies at the same concentration as the ACTL9 antibody

  • Pre-adsorption of antibody with immunizing peptide/protein when available

• Technical validation controls:

  • Multiple antibodies targeting different epitopes of ACTL9 to confirm localization patterns

  • Multiple detection methods (e.g., IF, WB, IHC) to confirm expression

  • RNA expression data (RT-PCR) to correlate with protein detection

• Experimental comparison controls:

  • Paired fertility case-control samples when studying pathology

  • Comparison of different sperm fractions (e.g., capacitated vs. non-capacitated)

  • Wild-type vs. ACTL9 mutant models (mouse or cell models)

• Quantification controls:

  • Standardized image acquisition parameters

  • Blinded analysis of localization patterns

  • Clear criteria for categorizing normal vs. abnormal localization

Including these controls will significantly enhance the reliability of findings and allow for meaningful interpretation of results, particularly when investigating the role of ACTL9 in fertility-related research.

How might ACTL9 antibodies contribute to developing diagnostic tools for male infertility?

ACTL9 antibodies hold significant potential for developing diagnostic tools for specific types of male infertility, particularly cases involving fertilization failure despite normal conventional sperm parameters. Current research suggests several promising diagnostic applications:

• Immunodiagnostic sperm function assays: Development of standardized immunofluorescence protocols using ACTL9 antibodies could help identify men with abnormal ACTL9 localization or expression, which has been associated with fertilization failure . Such assays could be incorporated into advanced sperm function testing for cases of unexplained infertility or repeated failed IVF/ICSI.

• Combined protein marker panels: Creating diagnostic panels that include ACTL9 alongside other fertility-related proteins (ACTL7A, PLCζ) could increase diagnostic sensitivity and specificity. This multimarker approach might better predict fertilization potential than single protein assessment .

• Automated image analysis systems: Developing AI-assisted systems for analyzing ACTL9 immunostaining patterns could standardize interpretation and reduce subjective assessment. Such systems could quantify the percentage of sperm showing normal versus abnormal ACTL9 localization patterns.

• Genetic-protein correlation diagnostics: Combining genetic screening for ACTL9 variants with protein localization studies using antibodies could provide comprehensive diagnostic information. This approach could identify both genetic carriers and functional consequences.

• Point-of-care testing development: Adapting ACTL9 antibody-based assays to simplified formats suitable for andrology laboratories could make advanced diagnostics more accessible. This might include developing lateral flow assays or simplified immunofluorescence protocols.

For clinical implementation, these approaches would require validation in large cohorts with diverse fertility outcomes. Standardization of protocols, antibody specifications, and interpretation criteria would be essential for reliable diagnostics.

What is the potential for using ACTL9 antibodies in studying evolutionary aspects of fertilization mechanisms?

ACTL9 antibodies offer valuable tools for comparative evolutionary studies of fertilization mechanisms across species, potentially revealing important insights about reproductive biology evolution:

• Cross-species comparative studies: Some ACTL9 antibodies show cross-reactivity with multiple species including human, mouse, guinea pig, monkey, and rat . This allows for comparative studies of ACTL9 localization and function across evolutionarily diverse mammals, potentially revealing conserved versus species-specific aspects of fertilization.

• Evolutionary conservation analysis: By comparing ACTL9 localization patterns and interactions in different species, researchers can identify highly conserved functional domains that have remained stable throughout evolution, suggesting fundamental roles in fertilization. Antibodies targeting different epitopes are particularly valuable for such comparative studies.

• Reproductive isolation mechanisms: Studying ACTL9 variations across closely related species could provide insights into species-specific adaptations that contribute to reproductive isolation. Antibodies that recognize conserved epitopes while still detecting species-specific variations would be especially useful.

• Correlation with fertilization strategies: Different mammals employ varying fertilization strategies (e.g., different acrosome structures, sperm morphologies, etc.). Examining how ACTL9 localization and function correlate with these strategies using appropriate antibodies could reveal adaptive mechanisms.

• Evolutionary developmental biology: Investigating ACTL9 expression during spermatogenesis across species using stage-specific antibody labeling might reveal differences in developmental programs that contribute to species-specific sperm characteristics.

These evolutionary studies require careful selection of antibodies that recognize conserved epitopes while maintaining specificity. Validating antibody cross-reactivity for each species is essential, potentially requiring the development of species-specific antibodies for comprehensive comparative studies.

How does ACTL9 research contribute to our understanding of male infertility mechanisms?

Research using ACTL9 antibodies has significantly advanced our understanding of specific mechanisms underlying male infertility, particularly regarding fertilization failure. This research has revealed several important insights:

• Previously unrecognized causes of fertilization failure: The identification of ACTL9 mutations as causes of fertilization failure has highlighted a molecular mechanism previously not recognized in clinical practice . This expands our understanding beyond conventional sperm parameters.

• Structural basis for fertilization competence: ACTL9 research has demonstrated the critical importance of proper perinuclear theca (PT) structure and acrosomal anchoring for fertilization capacity . This structural aspect was previously underappreciated in infertility diagnoses.

• Protein interaction networks in fertilization: The interaction between ACTL9 and ACTL7A reveals complex protein networks required for normal sperm function . This systems biology perspective represents an advancement from single-gene or single-protein approaches to fertility.

• Connection between genetic variants and functional consequences: Studies using ACTL9 antibodies have established clear mechanisms linking genetic mutations to protein localization defects and ultimately to fertilization failure . This connects genotype to phenotype in a mechanistically informative way.

• Oocyte activation factor regulation: The relationship between ACTL9 mutations and abnormal PLCζ localization provides insights into the regulation of this critical oocyte activation factor . This connects sperm structure to the ability to trigger embryonic development.

These advancements contribute to a more comprehensive model of male fertility that integrates genetic, structural, and functional aspects. This integrated understanding may guide the development of more effective diagnostic approaches and potential therapeutic strategies for specific types of male infertility.

What are the most promising future research directions for ACTL9 antibody applications?

Several promising research directions could significantly advance our understanding of reproductive biology through ACTL9 antibody applications:

• Personalized fertility assessment: Developing standardized clinical protocols using ACTL9 antibodies for immunodiagnostic testing could enable personalized assessment of fertilization potential. This could guide treatment decisions in assisted reproduction, particularly for patients with unexplained fertilization failure.

• Therapeutic targeting: Understanding the structural relationships between ACTL9, ACTL7A, and PLCζ using antibody studies could potentially guide the development of interventions to address specific types of fertilization failure. This might include recombinant protein approaches or targeted drug development.

• Broader protein interaction network mapping: Using ACTL9 antibodies for high-throughput interaction studies (such as proximity labeling combined with mass spectrometry) could reveal the complete interactome of ACTL9 in sperm. This systems biology approach might identify additional proteins involved in fertilization.

• Developmental regulation studies: Investigating the expression and localization of ACTL9 during different stages of spermatogenesis using stage-specific antibody labeling could provide insights into the developmental regulation of this protein and potential points of vulnerability.

• Comparative reproductive biology: Expanding ACTL9 studies to diverse species using cross-reactive antibodies could reveal evolutionary adaptations in fertilization mechanisms, potentially informing conservation efforts for endangered species or providing insights into reproductive barriers.

• Integration with other "-omics" approaches: Combining antibody-based ACTL9 studies with proteomics, transcriptomics, and metabolomics could provide a comprehensive picture of the molecular networks governing fertilization capacity.