Recombinant Mouse Actin-like protein 9 (Actl9)

What is the tissue expression pattern of Actl9 in mice?

Actl9 is predominantly expressed in the testis, as confirmed through both database analysis (GTEx) and real-time PCR validation. Expression analysis shows negligible levels in other adult tissues, making it a testis-specific actin-related protein . When designing experiments involving Actl9, researchers should consider this tissue-specific expression pattern, particularly when selecting appropriate control tissues.

What is the subcellular localization of Actl9 in mouse sperm?

In normally capacitated mouse sperm, Actl9 is primarily localized in two distinct regions: the equatorial segment of the sperm head (observed in approximately 84.4% ± 11.1% of sperm) and the neck region (observed in 100% of sperm). A smaller proportion (11.6% ± 10.7%) exhibits Actl9 in the acrosomal segment of the head . This localization pattern is critical for proper sperm function, particularly in fertilization processes.

How do Actl9 mutations affect sperm morphology?

Mutations in Actl9 lead to structural abnormalities in the perinuclear theca (PT) of sperm. Transmission electron microscopy (TEM) analysis reveals that Actl9-mutated sperm exhibit a loosened PT structure where the acrosome becomes detached from the nuclear envelope. Additionally, during spermiogenesis, proacrosomal vesicles appear atypical and separated in the Golgi phase, with failed fusion of additional proacrosomal vesicles in the cap phase .

What methodology can be used to generate Actl9-mutated mouse models?

Actl9-mutated mouse models can be effectively generated using the CRISPR/Cas9 system. The process involves designing an sgRNA targeting the desired change, injecting the constructs into C57BL/6 embryonic stem cells, and selecting offspring expressing the mutation through deep sequencing. To eliminate potential CRISPR off-target effects, selected mice should be back-crossed to wild-type C57BL/6 mice for at least three generations . Genotyping can be performed using allelic discrimination RT-PCR with appropriate SNP-PCR probes.

How can Actl9 expression be verified in mouse models?

Verification of Actl9 expression requires a multi-faceted approach:

• Real-time PCR to quantify mRNA expression levels in testicular tissue

• Immunostaining to visualize protein localization in sperm

• Western blot analysis to confirm protein expression

• Deep sequencing to verify genetic modifications in mutant models

For immunostaining, researchers should focus on the equatorial segment of the sperm head and the neck region, as these are the primary sites of Actl9 localization in normal sperm.

How does Actl9 interact with Actl7A to maintain proper perinuclear theca structure?

Actl9 and Actl7A co-localize in the subacrosomal layer of the PT that links the acrosome to the nuclear envelope. Intensity profile analysis of immunostaining shows that Actl9 and Actl7A signals overlap almost completely in the acrosomal or equatorial segments of normal sperm. In the equatorial region, they co-localize in the outer peri-acrosomal layer of the PT, exhibiting strong signals in the outer layer of the nuclear envelope .

The interaction between these proteins can be studied using co-immunoprecipitation (co-IP) assays. The methodology involves:

• Co-transfection of wild-type and mutant Actl9 with Actl7A into HEK293T cells

• Protein isolation using appropriate lysis buffer with protease inhibitors

• Incubation with protein A/G magnetic beads cross-linked with tag-specific antibodies

• Elution and detection via immunoblotting analysis

Research indicates that wild-type Actl9 forms complexes with Actl7A, while mutant forms show weakened or complete loss of interaction, suggesting these complexes are critical for PT formation and acrosomal anchoring.

What mechanisms explain the link between Actl9 mutations and PLCζ mislocalization?

PLCζ (phospholipase C zeta) is a key oocyte activation factor normally localized in the PT. In wild-type sperm, the PLCζ signal overlaps with Actl9 in the equatorial segment of the sperm head. In Actl9-mutated sperm, two abnormal patterns emerge:

• Complete absence of PLCζ signal (observed in 32.9%-52.6% of mutant sperm compared to 18.0% in normal sperm)

• Abnormal localization of PLCζ in the neck region (observed in 47.4%-67.1% of mutant sperm but not in normal sperm)

The current hypothesis suggests that Actl9 mutations disrupt the PT structure, which serves as the scaffold for proper PLCζ localization. This disruption likely occurs through the weakened interaction between Actl9 and Actl7A, affecting the integrity of the PT structure that anchors PLCζ in the correct position.

What experimental approaches can assess the functional consequences of Actl9 mutations on fertilization?

To assess fertilization capacity in Actl9-mutated models, intracytoplasmic sperm injection (ICSI) represents the gold standard methodology. The procedure involves:

• Collection of mature oocytes from superovulated female mice

• Collection and preparation of sperm from wild-type and Actl9-mutated male mice

• Microinjection of individual sperm into oocytes

• Culture of injected oocytes and assessment of fertilization markers (pronuclei formation)

• Monitoring of embryonic development to blastocyst stage

Research shows that sperm from Actl9-mutated mice result in total fertilization failure (TFF) following ICSI, mirroring the phenotype observed in humans with ACTL9 mutations. This experimental approach enables direct assessment of the fertilization capacity independent of sperm motility or zona penetration ability.

How can the developmental defects in proacrosomal vesicle formation be quantitatively analyzed in Actl9-mutated mice?

Quantitative analysis of proacrosomal vesicle formation defects requires sophisticated imaging and analytical techniques:

• Transmission Electron Microscopy (TEM): Prepare testicular tissue using standard fixation protocols for TEM. Analyze sections from different stages of spermatogenesis with particular attention to the Golgi phase and cap phase.

• Quantification Parameters:

  • Number and size of proacrosomal vesicles

  • Distance between vesicles

  • Degree of fusion between vesicles

  • Completeness of acrosome formation

• Statistical Analysis:

  • Compare metrics between wild-type and Actl9-mutated samples

  • Establish developmental timeline of defect manifestation

  • Correlate vesicle abnormalities with mature sperm PT structure

These analyses can help establish the precise developmental stage at which Actl9 dysfunction impacts acrosome biogenesis and PT formation.

What are the optimal conditions for expressing recombinant Actl9 in cell culture systems?

For successful expression of recombinant Actl9 in cell culture systems, researchers should consider the following methodology:

• Plasmid Construction:

  • Create expression plasmids encoding wild-type Actl9 with appropriate tags (e.g., His-tag)

  • Include the complete coding sequence with optimal Kozak consensus sequence

  • Consider using mammalian expression vectors with strong promoters (CMV)

• Cell Culture and Transfection:

  • Maintain HEK293T cells in DMEM supplemented with 10% fetal bovine serum

  • Grow cells to 70-80% confluence before transfection

  • Use Lipofectamine 3000 for transient transfections following manufacturer's protocol

  • Harvest cells 48 hours post-transfection for optimal protein expression

• Protein Extraction and Purification:

  • Lyse cells in appropriate buffer (e.g., NP-40 Lysis Buffer with 1mM PMSF)

  • Purify using affinity chromatography based on the chosen tag

  • Verify expression and purity using Western blot with Actl9-specific antibodies

This approach allows for production of functional recombinant Actl9 for subsequent biochemical and functional studies.

What immunostaining protocols are most effective for visualizing Actl9 in sperm samples?

For optimal visualization of Actl9 in sperm samples, the following immunostaining protocol is recommended:

• Sample Preparation:

  • Collect and wash sperm in PBS

  • Fix sperm with 4% paraformaldehyde

  • Permeabilize with 0.2% Triton X-100

• Immunostaining:

  • Block with 5% BSA

  • Incubate with primary antibodies against Actl9

  • For co-localization studies, include antibodies against Actl7A

  • Use peanut agglutinin (PNA) as an acrosomal marker

  • Counterstain nuclei with DAPI

  • Employ fluorescently-labeled secondary antibodies specific to the host species of primary antibodies

• Imaging and Analysis:

  • Use confocal microscopy for high-resolution imaging

  • Perform line-intensity profile analysis to precisely determine subcellular localization

  • Compare signal distribution between the acrosomal segment, equatorial segment, and neck region

  • Quantify the percentage of sperm showing Actl9 in each compartment

This approach enables precise determination of Actl9 localization and potential co-localization with interacting partners.

How can the interaction between Actl9 and other actin-related proteins be systematically investigated?

A comprehensive approach to study Actl9 interactions with other actin-related proteins involves:

• Yeast Two-Hybrid Screening:

  • Use Actl9 as bait to screen for potential interacting partners

  • Focus on testis-specific cDNA libraries

• Co-Immunoprecipitation (Co-IP):

  • Co-transfect wild-type and mutant Actl9 with potential partners (e.g., Actl7A)

  • Isolate proteins using appropriate lysis buffers

  • Incubate with protein A/G magnetic beads cross-linked with tag-specific antibodies

  • Detect complexes via immunoblotting with specific antibodies

• Proximity Ligation Assay (PLA):

  • Perform on fixed sperm or testicular sections

  • Use antibodies against Actl9 and potential interacting partners

  • Quantify interaction signals in different sperm regions

• FRET or BiFC Analysis:

  • Create fusion constructs of Actl9 and potential partners with appropriate fluorescent proteins

  • Express in suitable cell lines

  • Analyze protein-protein interactions in living cells

This multi-methodological approach provides robust evidence for protein interactions and can identify the specific domains involved in these interactions.

How is Actl9 evolutionarily related to other actin-like proteins in mammals?

Actl9 belongs to a family of actin-related proteins that includes several testis-specific members. Evolutionary analysis should consider:

• Sequence Homology Analysis:

  • Compare Actl9 with other actin-related proteins, particularly Actl7A, Actl7B, Actrt1, Actrt2, and Actrt3

  • Construct phylogenetic trees to establish evolutionary relationships

  • Identify conserved domains and motifs across family members

• Functional Domain Comparison:

  • Analyze the conservation of functional domains across species

  • Identify species-specific adaptations in Actl9 structure

• Expression Pattern Analysis:

  • Compare tissue-specific expression patterns of Actl9 and related proteins across mammalian species

  • Correlate expression patterns with reproductive strategies

Current understanding indicates that actin-related proteins often function in pairs or multimeric complexes. Actl7A is recognized as an important paralog of Actl9, and both proteins appear to form complexes involved in PT formation and acrosomal anchoring .

What functional differences exist between mouse Actl9 and human ACTL9?

Understanding the functional differences between mouse Actl9 and human ACTL9 is crucial for translational research. Key comparisons should include:

• Expression Pattern Comparison:

  • Both mouse Actl9 and human ACTL9 show testis-specific expression

  • Quantitative comparison of expression levels during spermatogenesis

• Localization Pattern:

  • Human ACTL9 is primarily localized in the equatorial segment of the head and neck regions of sperm

  • Similar localization pattern is observed in mouse Actl9

• Mutation Effects:

  • In both species, ACTL9/Actl9 mutations lead to loosened PT structure

  • Both result in total fertilization failure following ICSI

  • Proacrosomal vesicle formation defects are observed in both species

• Protein Interactions:

  • Both mouse Actl9 and human ACTL9 interact with ACTL7A

  • The interaction domain appears to be conserved between species

Research indicates high functional conservation between mouse and human proteins, making the mouse model valuable for understanding human fertility disorders associated with ACTL9 mutations.

How can Actl9 research inform clinical approaches to male infertility treatment?

Research on Actl9 has significant implications for clinical management of specific types of male infertility:

• Diagnostic Applications:

  • Genetic screening for ACTL9 mutations in cases of unexplained total fertilization failure (TFF)

  • Immunostaining of sperm samples to assess ACTL9 localization and PT structure

• Therapeutic Approaches:

  • Assisted oocyte activation using calcium ionophores has been successful in overcoming TFF in couples with ACTL9 mutations

  • This approach addresses the downstream effects of PLCζ mislocalization

• Prognostic Value:

  • ACTL9 mutation status can serve as a genetic marker for predicting ICSI outcomes

  • Enables personalized treatment approaches for affected individuals

Research has demonstrated that assisted oocyte activation by calcium ionophore exposure successfully overcame TFF and achieved live births in couples with ACTL9 variants . This translational application directly addresses the molecular mechanism of fertilization failure in these cases.

What experimental design is optimal for testing rescue strategies in Actl9-mutated mouse models?

When designing experiments to test rescue strategies in Actl9-mutated mouse models, researchers should consider the following approach:

• Experimental Groups:

  • Wild-type control

  • Homozygous Actl9-mutated

  • Actl9-mutated with rescue intervention

• Rescue Interventions to Test:

  • Calcium ionophore treatment during ICSI

  • Recombinant PLCζ injection during ICSI

  • Viral vector-mediated expression of wild-type Actl9

• Outcome Measures:

  • Fertilization rate (pronuclear formation)

  • Embryo development to blastocyst stage

  • Implantation rate following embryo transfer

  • Live birth rate

• Molecular and Cellular Analyses:

  • Calcium oscillation patterns in oocytes post-ICSI

  • Ultrastructural analysis of sperm PT in rescued models

  • PLCζ localization in treated sperm

This comprehensive experimental design enables assessment of both the efficacy of rescue interventions and their mechanistic basis.

How should researchers quantitatively analyze PT structural abnormalities in Actl9-mutated sperm?

Quantitative analysis of PT structural abnormalities requires:

• Sample Preparation Protocol:

  • Collect sperm from wild-type and Actl9-mutated mice

  • Process for transmission electron microscopy (TEM) using standard fixation protocols

  • Obtain multiple sections per sample to ensure representative sampling

• Measurement Parameters:

  • Distance between acrosomal membrane and nuclear envelope

  • Thickness of PT layer

  • Continuity of PT structure (% intact vs. disrupted)

  • Degree of acrosomal detachment from nuclear envelope

• Imaging and Quantification:

  • Capture high-resolution TEM images at standardized magnification

  • Use image analysis software to measure defined parameters

  • Analyze at least 50-100 sperm per sample

  • Have multiple blinded observers score structural abnormalities

• Statistical Analysis:

  • Compare measurements between wild-type and mutant samples

  • Apply appropriate statistical tests (t-test or ANOVA)

  • Calculate effect sizes to determine the magnitude of differences

This approach provides objective quantification of PT structural abnormalities and enables correlation with functional defects.

What statistical approaches are most appropriate for analyzing fertilization outcomes in Actl9 research?

When analyzing fertilization outcomes in Actl9 research, the following statistical approach is recommended:

• Experimental Design Considerations:

  • Ensure adequate sample sizes based on power calculations

  • Include appropriate controls (wild-type, heterozygous mutants)

  • Replicate experiments across multiple cohorts

• Primary Outcome Measures:

  • Fertilization rate (number of fertilized oocytes/total oocytes)

  • Developmental progression (rates of 2-cell, 4-cell, morula, blastocyst formation)

  • Live birth rate (if embryo transfer is performed)

• Statistical Tests:

  • For comparison of fertilization rates: Chi-square test or Fisher's exact test

  • For developmental progression: Kaplan-Meier survival analysis

  • For multiple group comparisons: ANOVA with post-hoc tests

• Data Presentation:

  • Present data as both percentages and absolute numbers

  • Include 95% confidence intervals

  • Create graphical representations to highlight key differences

What are the common pitfalls in generating and maintaining Actl9-mutated mouse lines?

Researchers should be aware of several technical challenges when working with Actl9-mutated mouse lines:

• Generation Challenges:

  • Off-target effects from CRISPR/Cas9: Minimize by selecting sequences with minimal off-target potential and back-crossing for at least three generations

  • Mosaicism in founder animals: Screen multiple offspring to identify those with germline transmission

  • Reduced fertility: May require superovulation protocols or IVF/ICSI for line maintenance

• Genotyping Challenges:

  • Design allele-specific primers for accurate discrimination between wild-type and mutant alleles

  • Optimize PCR conditions for reliable results

  • Consider using SNP-PCR probes for allelic discrimination RT-PCR

• Phenotypic Variability:

  • Monitor for potential genetic drift over generations

  • Maintain consistent background strain by periodic back-crossing

  • Document housing conditions, as environmental factors may influence phenotype expression

• Controls Selection:

  • Use littermate controls whenever possible

  • Match for sex and age in all experiments

  • Consider including heterozygous animals to assess gene dosage effects

Addressing these challenges ensures robust and reproducible results in Actl9 research.

How can researchers troubleshoot failed co-immunoprecipitation experiments when studying Actl9 interactions?

When troubleshooting failed co-immunoprecipitation experiments for Actl9 interactions, consider the following methodological adjustments:

• Expression Level Issues:

  • Verify protein expression by Western blot before attempting co-IP

  • Optimize transfection efficiency to ensure adequate expression

  • Consider using stronger promoters or codon-optimized constructs

• Lysis Conditions:

  • Test different lysis buffers (NP-40, RIPA, etc.)

  • Adjust salt concentration to optimize interaction conditions

  • Add protease inhibitors to prevent protein degradation

• Antibody Selection:

  • Validate antibody specificity before co-IP experiments

  • Test different tag positions (N-terminal vs. C-terminal)

  • Consider using different tags (His, FLAG, cMYC) if one approach fails

• Binding and Washing Conditions:

  • Optimize incubation time with antibody-conjugated beads

  • Adjust washing stringency (number of washes, buffer composition)

  • Test different elution methods

By systematically addressing these potential issues, researchers can successfully optimize co-IP protocols for studying Actl9 interactions.

What are the key unanswered questions about Actl9's role in spermatogenesis?

Several critical questions remain unanswered regarding Actl9's function:

• Temporal Expression Pattern:

  • At which stage of spermatogenesis is Actl9 first expressed?

  • How is Actl9 expression regulated during spermiogenesis?

  • What transcription factors control Actl9 expression?

• Protein Modification:

  • Does Actl9 undergo post-translational modifications?

  • How do these modifications affect its function and interactions?

  • What enzymes are responsible for these modifications?

• Additional Binding Partners:

  • Beyond Actl7A, what other proteins interact with Actl9?

  • Are there stage-specific interactions during sperm development?

  • How do these interactions contribute to PT formation?

• Cytoskeletal Organization:

  • How does Actl9 contribute to actin cytoskeleton organization in developing spermatids?

  • What is the relationship between Actl9 and other cytoskeletal elements?

Addressing these questions will provide a more comprehensive understanding of Actl9's role in spermatogenesis and may reveal additional therapeutic targets for male infertility.

How might novel genome editing approaches improve Actl9 research?

Emerging genome editing technologies offer new opportunities for Actl9 research:

• Base Editing Applications:

  • Enables introduction of specific point mutations without double-strand breaks

  • Reduces off-target effects compared to traditional CRISPR/Cas9

  • Allows creation of specific Actl9 variants to study structure-function relationships

• Conditional Knockout Strategies:

  • Develop testis-specific or stage-specific Actl9 knockout models

  • Use inducible Cre-lox systems to control timing of gene deletion

  • Enables study of Actl9 function at specific developmental stages

• Tagging Endogenous Actl9:

  • Insert fluorescent protein tags at the endogenous locus

  • Enables live imaging of Actl9 during spermatogenesis

  • Maintains physiological expression levels and regulation

• Humanized Mouse Models:

  • Replace mouse Actl9 with human ACTL9

  • Introduce specific human ACTL9 variants found in infertile patients

  • Provides clinically relevant model for testing therapeutic interventions

These approaches will enable more precise manipulation of Actl9 and provide new insights into its function in normal and pathological conditions.