ACTL7A Antibody

What is ACTL7A and why is it important in reproductive research?

ACTL7A belongs to the family of actin-related proteins (ARPs) that share significant amino acid sequence homology with conventional actins. Unlike ubiquitously expressed ARPs that function in cytoskeletal dynamics, motor transport, and chromatin remodeling, ACTL7A is specifically expressed in the testis. The protein contains a conserved actin domain and a unique intrinsically disordered N-terminal domain of approximately 70 amino acids, which is evolutionarily conserved in mammals .

ACTL7A's importance lies in its critical role in male fertility. Research has established that ACTL7A is indispensable for proper acrosomal biogenesis, attachment, and sperm-egg fusion processes. Mutations or antibodies targeting this protein can lead to male infertility, making it a significant target for reproductive biology research .

How is the structure of ACTL7A characterized and what domains are targeted by antibodies?

ACTL7A consists of two major domains:

• A conserved actin domain that shares structural similarity with conventional actins

• A unique N-terminal domain (~70 amino acids) that is intrinsically disordered and mammalian-specific

Many commercially available antibodies target the N-terminal region (approximately amino acids 41-67) of human ACTL7A . This region is particularly useful for antibody generation because it represents a unique sequence that distinguishes ACTL7A from other actin-related proteins, ensuring specificity.

What are the primary subcellular localizations of ACTL7A during spermatogenesis?

ACTL7A exhibits dynamic localization patterns during spermatid development:

• Initially present within the nucleus of developing germ cells

• Located in the subacrosomal space during acrosome formation

• Associated with postacrosomal regions in elongating spermatids

• Present in the acrosome and tail of mature spermatozoa

This spatiotemporal pattern suggests multiple roles for ACTL7A throughout spermatogenesis, particularly in acrosomal biogenesis and attachment. Recent research also indicates potential nuclear functions related to chromatin remodeling .

What experimental techniques can ACTL7A antibodies be used for?

ACTL7A antibodies are versatile research tools applicable across multiple methodologies:

Most commercially available antibodies are polyclonal, rabbit-derived, and purified through protein A columns followed by peptide affinity purification .

How can researchers validate the specificity of ACTL7A antibodies?

Validating specificity is crucial for antibody-based experiments. For ACTL7A, consider these approaches:

• Knockout validation: Use tissues from Actl7a knockout mice as negative controls in immunoassays

• Peptide competition: Pre-incubate antibody with the immunizing peptide to block specific binding

• Tissue specificity testing: Confirm testis-specific reactivity and absence of signal in other tissues

• Western blot verification: Confirm detection of a band at the expected molecular weight (~47 kDa)

• Recombinant protein testing: Use purified ACTL7A as a positive control

This multi-modal validation approach ensures the antibody specifically recognizes ACTL7A without cross-reactivity to other actin-related proteins.

What considerations should be made when selecting host species for ACTL7A antibodies?

Most commercial ACTL7A antibodies are developed in rabbits, though some mouse-derived antibodies are available . When selecting:

• Consider experimental design: Avoid host species that match other antibodies in multiplexed experiments

• Evaluate sensitivity needs: Rabbit polyclonals often offer higher sensitivity for low-abundance proteins

• Consider cross-reactivity with sample species: Check reactivity data for human, mouse, or other species

• Review purification method: Affinity-purified antibodies generally offer higher specificity

• Assess conjugation needs: Determine if direct conjugates (FITC, HRP, Biotin) are required or if secondary detection is preferred

How does ACTL7A contribute to acrosomal biogenesis and what methods best demonstrate this?

ACTL7A plays a critical role in acrosomal biogenesis through several mechanisms:

• Regulation of subacrosomal filamentous actin (F-actin) formation

• Mediation of acrosomal attachment to the nuclear surface via the acroplaxome

• Facilitation of proper acrosomal granule migration during spermatid elongation

To investigate these roles, researchers have employed:

• Actl7a knockout mouse models that demonstrate disrupted acrosomal biogenesis

• Immunofluorescence microscopy to track ACTL7A localization during spermatogenesis

• F-actin staining to visualize cytoskeletal arrangements in wild-type versus knockout models

• Electron microscopy to observe ultrastructural abnormalities in acrosomal attachment

Notably, knockout studies revealed complete loss of subacrosomal F-actin structures, establishing ACTL7A as essential for the formation or maintenance of this cytoskeletal element .

What protein interactions has ACTL7A been shown to participate in during spermatogenesis?

ACTL7A interacts with several proteins critical for cytoskeletal dynamics and motor function:

• Actin-related protein 2 (ARP2): Component of the ARP2/3 complex involved in actin nucleation

• Dynactin subunit 1 (DCTN1): Mediates dynein-cargo binding for intracellular transport

• Myosin 6 (MYO6): Unconventional myosin involved in vesicular trafficking

• Profilin 4 (PFN4): Actin-binding protein that regulates actin polymerization

• Testin (via LIM1-2 domains): Scaffold protein that connects actin filaments

These interactions suggest ACTL7A functions as part of a larger cytoskeletal regulatory complex involved in acrosomal development and anchoring. Techniques used to identify these interactions include co-immunoprecipitation, proximity ligation assays, and mass spectrometry .

How does mutation of ACTL7A affect male fertility and what experimental models demonstrate this?

Mutations in ACTL7A have significant consequences for male fertility:

• Homozygous frameshift mutations (e.g., c.1101dupC; p.S368Qfs*5) cause complete fertilization failure

• Other mutations lead to defects in acrosomal structure and early embryonic arrest

• Knockout mouse models show sterility due to impaired acrosomal attachment and function

Researchers have demonstrated these effects through:

• Generation of Actl7a knockout mouse models showing consistent disruption of acrosomal biogenesis

• Whole-exome sequencing of infertile patients to identify causative mutations

• In vitro fertilization experiments showing reduced fertilization capacity

• Ultrastructural analysis revealing abnormal acrosomal morphology and "peeling acrosomes" during spermatid elongation

These findings establish ACTL7A as an essential fertility factor with potential clinical significance for cases of unexplained male infertility.

How can ACTL7A antibodies be used to investigate immunological infertility?

Anti-ACTL7A antibodies have been implicated in immunological infertility. Researchers investigating this connection utilize ACTL7A antibodies in several sophisticated approaches:

• ELISA detection of anti-ACTL7A antibodies in patient sera:

  • Developed specifically for detecting antibody concentrations in fertile (n=267) and infertile sera (n=193)

  • Shows significantly higher antibody concentrations in infertile sera (p<0.0001)

  • Particularly effective for male sera detection (AUC=0.9899)

• In vitro fertilization impact studies:

  • Treatment of mouse spermatozoa with ACTL7A antibody-containing serum

  • Measurement of reduced fertilizing capacity

  • Assessment of sperm agglutination patterns

• Active immunization models:

  • Immunization of mice with ACTL7A protein to induce antibody production

  • Monitoring of fertility outcomes

  • Correlation of antibody titers with fertility reduction

These approaches provide mechanistic insights into how anti-ACTL7A antibodies contribute to immunological infertility and offer potential diagnostic applications.

What are the emerging nuclear roles of ACTL7A and how can researchers investigate them?

Recent research has uncovered potential nuclear functions for ACTL7A beyond its established cytoskeletal roles:

• Nuclear localization during early spermatogenesis stages

• Possible involvement in epigenetic regulation during spermiogenesis

• Predicted interactions with chromatin remodeling complexes

To investigate these emerging functions, researchers employ:

• Fluorescence microscopy to document intranuclear localization

• RNA-seq analysis in ACTL7A knockout models to identify transcriptional changes

• AI-driven approaches to predict interactions with chromatin remodeling complexes

• Analysis of lysine acetylation and HDAC levels in developing spermatids

These approaches suggest ACTL7A may have dual functionality in both cytoskeletal regulation and nuclear processes during spermatogenesis.

How can researchers use ACTL7A antibodies to detect abnormalities in clinical fertility samples?

For clinical research applications, ACTL7A antibodies offer valuable diagnostic potential:

• Detection of anti-ACTL7A antibodies in patient sera:

  • ELISA-based detection shows high sensitivity and specificity

  • When set at strongly positive values, positive predictive value reaches 100% with zero false positives

  • More accurate than traditional tray agglutination tests (TAT) for detecting immunological fertility issues

• Analysis of ACTL7A expression in patient sperm samples:

  • Immunofluorescence localization to assess proper distribution

  • Correlation with acrosomal morphology and function

  • Potential biomarker for specific forms of male infertility

• Genetic screening correlation:

  • Linking ACTL7A mutations identified through genome sequencing with protein expression patterns

  • Functional validation of novel mutations

  • Understanding genotype-phenotype relationships in male infertility

These applications provide researchers with tools to investigate clinical cases of unexplained infertility and develop more precise diagnostic approaches.

What optimization strategies improve Western blot detection of ACTL7A?

For optimal Western blot detection of ACTL7A, researchers should consider:

• Sample preparation:

  • Fresh testicular tissue yields best results

  • Protect from degradation with protease inhibitors

  • Use RIPA buffer with sodium deoxycholate for membrane protein extraction

• Gel electrophoresis and transfer:

  • 10-12% polyacrylamide gels recommended

  • Expected molecular weight: ~47 kDa

  • Standard transfer conditions suitable (100V for 1-2 hours)

• Blocking and antibody incubation:

  • 5% non-fat milk in TBST recommended for blocking

  • Primary antibody dilution typically 1:1000

  • Overnight incubation at 4°C for highest sensitivity

• Detection considerations:

  • HRP-conjugated secondary antibodies work well

  • ECL substrate provides sufficient sensitivity

  • Exposure time optimization may be required due to variable expression levels

How should researchers design experiments to study ACTL7A-cytoskeletal interactions?

To effectively investigate ACTL7A's interactions with the cytoskeleton:

• Co-localization studies:

  • Double immunofluorescence with markers for F-actin (phalloidin) and other interacting proteins

  • Super-resolution microscopy for detailed structural relationships

  • Z-stack acquisition to capture 3D relationships

• Biochemical interaction analysis:

  • Co-immunoprecipitation using ACTL7A antibodies

  • Proximity ligation assays for in situ interaction detection

  • Pull-down assays with recombinant ACTL7A

• Functional perturbation approaches:

  • CRISPR/Cas9 knockout or knockdown models

  • Domain-specific mutations to disrupt specific interactions

  • Cytoskeletal drug treatments to assess dependency relationships

• Dynamic analysis:

  • Live-cell imaging with fluorescently tagged ACTL7A

  • Tracking of acrosomal development in conjunction with cytoskeletal elements

  • FRAP (Fluorescence Recovery After Photobleaching) for mobility assessment

These complementary approaches provide comprehensive insights into ACTL7A's role in cytoskeletal organization during spermatogenesis.

What controls are essential when using ACTL7A antibodies in immunofluorescence studies?

Proper controls are critical for reliable immunofluorescence with ACTL7A antibodies:

• Negative controls:

  • Primary antibody omission

  • Isotype control antibodies

  • Tissue from Actl7a knockout models

  • Non-reproductive tissues (ACTL7A is testis-specific)

• Specificity controls:

  • Peptide competition (pre-incubation with immunizing peptide)

  • Multiple antibodies targeting different epitopes

  • Parallel Western blot verification of specificity

• Positive controls:

  • Wild-type testis sections with established staining patterns

  • Developmental staging validation (showing expected dynamic localization)

  • Co-staining with established markers of relevant structures (acrosome, manchette)

• Technical controls:

  • Autofluorescence assessment

  • Secondary antibody-only controls

  • Counterstaining with DAPI or other nuclear markers