SPATC1L Antibody

What is SPATC1L and why are antibodies against it important in research?

SPATC1L (spermatogenesis and centriole associated 1 like) is a germ cell-specific protein that plays a crucial role in maintaining the integrity of the sperm head-tail junction. It is expressed as a 38-kDa protein in spermatogenic cells and testicular sperm, with expression beginning in the postmeiotic phase of spermatogenesis . SPATC1L has been found to associate with the regulatory subunit of protein kinase A (PKA), specifically with PRKAR1A (RIα), PRKACA isoform 2 (Cα2), and AKAP11 .

Antibodies against SPATC1L are particularly important in reproductive biology research because:

• They enable localization studies of SPATC1L in spermatogenic cells and sperm

• They facilitate investigation of protein-protein interactions between SPATC1L and PKA components

• They allow detection of SPATC1L expression patterns during different stages of spermatogenesis

• They support research into male infertility conditions, particularly acephalic spermatozoa syndrome

Recent studies have established that SPATC1L deficiency leads to male sterility in mice due to the separation of sperm heads from tails, and biallelic mutations in human SPATC1L have been linked to acephalic spermatozoa syndrome .

What techniques are validated for SPATC1L antibody applications?

SPATC1L antibodies have been validated for multiple research applications through rigorous testing protocols. Based on available data, the following techniques have been successfully employed with SPATC1L antibodies:

For optimal results, validation protocols typically include:

• Verification of antibody specificity using competitive immunoblotting

• Use of appropriate positive and negative controls

• Confirmation of expected molecular weight and localization patterns

• Cross-validation using multiple antibodies or detection methods

How should SPATC1L expression be analyzed during spermatogenesis?

Analysis of SPATC1L expression during spermatogenesis requires a methodical approach that accounts for the dynamic nature of this process. Based on published research methodologies, the following protocol is recommended:

• Developmental staging: Collect mouse testis samples at different days after birth (particularly important are days 20-21 when SPATC1L expression begins) .

• Cell isolation: Separate different spermatogenic cell populations:

  • Testicular spermatogenic cells (including spermatogonia, spermatocytes, and round spermatids)

  • Testicular sperm (including elongating and condensing spermatids, and fully developed sperm)

  • Epididymal sperm (mature sperm)

• Protein detection: Perform immunoblot analyses using validated SPATC1L antibodies. Western blotting has demonstrated that SPATC1L is expressed in testicular spermatogenic cells and testicular sperm but not in epididymal sperm .

• Immunofluorescence localization:

  • In round spermatids, SPATC1L co-localizes with γ-tubulin in centrosomal regions

  • During spermiogenesis, SPATC1L signals separate from γ-tubulin-enriched regions

  • In testicular sperm, SPATC1L localizes to the neck/connecting piece in close proximity to the centriole

• Co-localization studies: Perform double immunofluorescence with antibodies against SPATC1L and other components of interest, such as PRKAR1A (RIα), to track their relative distributions during spermatogenesis .

This methodical approach allows precise tracking of SPATC1L expression patterns throughout spermatogenesis and reveals its stage-specific localization.

What controls should be used when employing SPATC1L antibodies?

Proper controls are essential for reliable results when using SPATC1L antibodies. Based on established research protocols, the following controls should be implemented:

For antibody specificity validation:

• Competitive immunoblotting using the recombinant antigen (such as GST-fusion protein of mouse SPATC1L fragment corresponding to amino acids 101-200)

• Comparison of results using different antibodies against SPATC1L

• Use of SPATC1L knockout tissue/cells as negative control

For immunoblotting:

• Positive control: Testicular tissue lysates from adult mice or humans

• Negative control: Tissue lysates where SPATC1L is not expressed (e.g., epididymal sperm, non-reproductive tissues)

• Loading control: Standard housekeeping protein appropriate for the cell type being studied

For immunohistochemistry/immunofluorescence:

• Positive tissue control: Adult testis sections

• Negative tissue control: Testis sections from prepubertal animals (before day 20 postpartum in mice)

• Technical negative controls: Primary antibody omission and isotype control

• Co-localization markers: γ-tubulin for centrosome identification

For developmental studies:

• Temporal controls: Testis samples from different postnatal days (particularly days before and after day 20, which marks SPATC1L expression onset)

For studies involving SPATC1L-deficient models:

• Wild-type controls should be age-matched and of identical genetic background

Implementation of these controls ensures reliable interpretation of results and strengthens the validity of experimental findings.

How can SPATC1L antibodies be utilized to investigate male infertility conditions?

SPATC1L antibodies represent powerful tools for investigating specific forms of male infertility, particularly acephalic spermatozoa syndrome. A comprehensive research approach should include:

Diagnostic applications:

• Immunofluorescence analysis of patient sperm samples using anti-SPATC1L antibodies to assess:

  • Presence/absence of SPATC1L protein

  • Subcellular localization patterns

  • Co-localization with other structural proteins at the head-tail junction

• Western blot analysis of testicular biopsy samples to quantify SPATC1L protein levels and detect potential truncated forms resulting from mutations

Genotype-phenotype correlation studies:

• Parallel analysis of SPATC1L mutations identified through whole-exome sequencing and SPATC1L protein expression/localization using antibodies

• Investigation of the impact of specific mutations (e.g., c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X) on protein expression and function

Functional studies:

• Immunoprecipitation of SPATC1L from patient samples followed by analysis of interaction partners, particularly PKA regulatory subunits

• Assessment of PKA activity in relation to SPATC1L expression levels through phosphorylation assays

Therapeutic development assessment:

• In vitro studies utilizing SPATC1L antibodies to monitor expression and localization of wild-type or mutant SPATC1L proteins in cellular models

• Evaluation of potential therapeutic interventions on SPATC1L expression or localization

This multifaceted approach can provide valuable insights into the molecular mechanisms underlying acephalic spermatozoa syndrome and potentially inform the development of diagnostic and prognostic tools for patients with SPATC1L-related infertility.

What methods are most effective for co-localization studies of SPATC1L and PKA regulatory subunits?

Advanced co-localization studies of SPATC1L and PKA regulatory subunits require sophisticated imaging and biochemical techniques. Based on published methodologies, the following approaches are recommended:

Immunofluorescence confocal microscopy:

• Double immunostaining of testicular cells or tissue sections using:

  • Anti-SPATC1L antibody

  • Anti-PRKAR1A (RIα) antibody

  • Nuclear counterstain (e.g., DAPI)

  • Additional markers (e.g., acetylated tubulin for flagella structures)

• High-resolution image acquisition using:

  • Confocal microscopy with appropriate wavelength settings

  • Z-stack imaging to capture the three-dimensional relationship

  • Super-resolution techniques (STED, STORM, or PALM) for nanoscale co-localization

• Quantitative co-localization analysis:

  • Pearson's correlation coefficient

  • Manders' overlap coefficient

  • Object-based analysis methods

Proximity ligation assay (PLA):
A more sensitive approach for detecting protein interactions within a 40 nm distance:

• Primary antibodies against SPATC1L and PRKAR1A from different species

• Species-specific PLA probes

• Rolling circle amplification and fluorescent detection

• Quantification of interaction points

Biochemical fractionation and co-immunoprecipitation:

• Subcellular fractionation of testicular cells

• Immunoprecipitation from each fraction using anti-SPATC1L or anti-PRKAR1A antibodies

• Western blot analysis of precipitates to detect co-precipitated proteins

FRET (Förster Resonance Energy Transfer):
For live-cell studies using tagged proteins:

• Expression of fluorescently-tagged SPATC1L and PRKAR1A

• FRET measurements to detect direct molecular interactions

• Analysis of interaction dynamics in response to cAMP or other stimuli

Research has demonstrated that SPATC1L and RIα co-localize in round spermatids and at the connecting piece in testicular sperm, providing key insights into the functional relationship between these proteins in spermatogenesis .

How can researchers analyze SPATC1L's role in PKA signaling pathways?

Analysis of SPATC1L's role in PKA signaling requires a multifaceted approach combining biochemical, cellular, and genetic techniques:

In vitro PKA activity assays:

• Prepare lysates from cells/tissues expressing or lacking SPATC1L

• Measure phosphorylation levels of PKA-specific substrates:

  • In wild-type vs. SPATC1L-knockout models

  • In cells with different SPATC1L expression levels

  • With/without cAMP analog stimulation (e.g., dibutyryl cAMP)

Quantification of C-subunit association with R-subunit:

• Immunoprecipitate RIα from samples with/without SPATC1L

• Analyze co-precipitated Cα by immunoblotting

• Calculate the relative amount of Cα associated with RIα in different conditions

SPATC1L and PKA substrate identification:

• Phosphoproteomic analysis:

  • Compare phosphorylation profiles in wild-type vs. SPATC1L-deficient samples

  • Identify differentially phosphorylated proteins containing PKA consensus sites

• Targeted analysis of potential substrates:

  • Example: Capping protein muscle Z-line beta has been identified as a candidate target of phosphorylation by PKA in testis

Structure-function analysis:

• Generate SPATC1L deletion mutants (e.g., lacking the N-terminal coiled-coil domain)

• Assess their effect on:

  • Interaction with RIα

  • PKA activity

  • Subcellular localization

Experimental findings supporting SPATC1L's role in PKA signaling:

These findings suggest that SPATC1L competitively inhibits the association between RIα and Cα, thereby increasing PKA activity .

What approaches can be used to study SPATC1L protein interactions?

Understanding SPATC1L's protein interactions is crucial for elucidating its function in sperm development and PKA signaling. Multiple complementary approaches can be employed:

Mass spectrometry-based interactome analysis:

• Immunoprecipitate SPATC1L from testicular lysates using anti-SPATC1L antibodies

• Subject precipitates to tryptic digestion followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)

• Identify co-precipitated proteins through database searching

• Validate hits by reciprocal co-immunoprecipitation

This approach has successfully identified key SPATC1L interactors including:

• Protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A/RIα)

• Protein kinase cAMP-activated catalytic subunit alpha isoform 2 (PRKACA isoform 2/Cα2)

• A kinase anchor protein 11 (AKAP11)

Yeast two-hybrid screening:

• Create SPATC1L bait constructs (full-length and domain-specific)

• Screen against testis-expressed prey libraries

• Validate positive interactions through secondary assays

Protein domain mapping:

• Generate deletion constructs of SPATC1L lacking specific domains (e.g., SPATC1LΔC lacking the N-terminal coiled-coil domain)

• Assess interaction with partners like RIα through co-immunoprecipitation

• Determine functional consequences on protein localization and PKA activity

Proximity-dependent biotinylation (BioID or TurboID):

• Express SPATC1L fused to a biotin ligase in spermatogenic cells

• Allow proximity-dependent biotinylation of interacting proteins

• Purify biotinylated proteins and identify by mass spectrometry

• This approach can identify transient or weak interactions that might be missed by co-immunoprecipitation

In situ analysis of protein complexes:

• Proximity ligation assay to visualize SPATC1L-containing complexes in situ

• Immunofluorescence co-localization with candidate interactors at different stages of spermatogenesis

These approaches provide complementary information about SPATC1L's interaction network, helping to elucidate its molecular function in sperm development and fertility.

What are the challenges in detecting SPATC1L in different stages of spermatogenesis?

Detecting SPATC1L across different stages of spermatogenesis presents several technical challenges that researchers must address:

Developmental timing challenges:

• SPATC1L expression is temporally regulated, appearing first at postnatal day 20 in mice, corresponding to the beginning of the postmeiotic phase

• Expression patterns change dynamically during spermatogenesis, requiring precise staging and timing of sample collection

Localization pattern shifts:

• SPATC1L localization changes during spermatogenesis:

  • Initially co-localizes with γ-tubulin in round spermatids

  • Separates from γ-tubulin-enriched regions during spermiogenesis

  • Finally localizes to the neck/connecting piece in testicular sperm

• This dynamic relocalization necessitates multiple markers and careful image analysis

Cell type heterogeneity in testicular samples:

• Testicular tissue contains mixed cell populations at various developmental stages

• Solutions include:

  • Separation of specific cell populations through density gradient centrifugation or FACS

  • Use of staged animal models (e.g., first wave of spermatogenesis)

  • Single-cell approaches for greater resolution

Protein detection sensitivity:

• SPATC1L is expressed at different levels across developmental stages

• Western blot detection may require:

  • Optimization of extraction buffers for different spermatogenic cells

  • Enhanced chemiluminescence or fluorescent detection systems

  • Concentration of samples from specific cell populations

Antibody specificity concerns:

• Ensuring antibody specificity is critical due to:

  • Potential cross-reactivity with related proteins

  • Background in complex testicular tissues

• Validation strategies include:

  • Competitive immunoblotting with recombinant SPATC1L

  • Use of SPATC1L knockout tissues as negative controls

  • Multiple antibodies targeting different epitopes

Absence in mature sperm:

• SPATC1L is not detected in epididymal (mature) sperm, limiting analysis to earlier developmental stages

• This necessitates working with testicular samples rather than easily collected ejaculated sperm

Addressing these challenges requires careful experimental design and validation strategies to ensure accurate detection and interpretation of SPATC1L expression patterns throughout spermatogenesis.

How should researchers design experiments to study SPATC1L function in male fertility?

Designing robust experiments to study SPATC1L function in male fertility requires a multidisciplinary approach spanning genetic, cellular, and physiological analyses:

Genetic approaches:

• CRISPR/Cas9-mediated genome editing to:

  • Generate complete SPATC1L knockout models

  • Create specific mutations mirroring human variants (e.g., c.910C>T:p.Arg304Cys and c.994G>T:p.Glu332X)

  • Develop conditional knockout models for temporal control

• Patient cohort analysis:

  • Whole-exome sequencing of patients with acephalic spermatozoa syndrome

  • Filtering and in silico analysis of candidate variants

  • Comparison with control cohorts of fertile subjects

Cellular and biochemical analyses:

• Protein localization studies:

  • Immunofluorescence analysis using SPATC1L antibodies

  • Co-localization with structural markers (e.g., acetylated tubulin) and functional partners (e.g., PRKAR1A)

• Protein-protein interaction studies:

  • Co-immunoprecipitation followed by Western blotting

  • Mass spectrometry analysis of immunoprecipitates

  • Proximity ligation assays in testicular cells

• PKA activity assays:

  • Measurement of PKA substrate phosphorylation in wild-type vs. SPATC1L-deficient samples

  • Analysis of RIα-Cα association in the presence/absence of SPATC1L

Morphological and functional assessments:

• Sperm analysis:

  • Light microscopy with specific staining (e.g., Papanicolaou staining)

  • Electron microscopy of the head-tail junction

  • Assessment of acephalic sperm percentage

• Fertility assessment:

  • Natural mating trials

  • Assisted reproductive technology outcomes

  • Early embryonic development monitoring

Expression systems for mechanistic studies:

• Overexpression systems:

  • Transfection of cell lines (e.g., TM4 Sertoli cells) with wild-type or mutant SPATC1L

  • Assessment of protein localization, stability, and function

• Structure-function analysis:

  • Creation of domain deletion constructs (e.g., SPATC1LΔC)

  • Evaluation of effects on localization and interaction partners

A well-designed experimental approach should combine these methods to establish a comprehensive understanding of SPATC1L's role in maintaining sperm head-tail integrity and male fertility.

What are the optimal conditions for using SPATC1L antibodies in immunohistochemistry?

Optimizing immunohistochemistry (IHC) protocols for SPATC1L detection requires careful consideration of tissue preparation, antigen retrieval, and detection methods:

Tissue preparation:

• Fixation:

  • Freshly collected testicular tissue should be fixed in 4% paraformaldehyde for 12-24 hours

  • Bouin's fixative may provide better morphological preservation of testicular architecture

  • Overfixation should be avoided as it can mask epitopes

• Processing and sectioning:

  • Paraffin embedding following standard protocols

  • Section thickness of 4-6 μm is optimal for testicular tissue

  • Mount sections on positively charged slides to prevent tissue loss

Antigen retrieval methods:

• Heat-induced epitope retrieval:

  • Citrate buffer (pH 6.0) at 95-98°C for 20 minutes

  • EDTA buffer (pH 9.0) may provide better results for some SPATC1L epitopes

  • Pressure cooker methods can enhance retrieval efficiency

• Enzymatic retrieval:

  • Proteinase K digestion (10 μg/ml) for 10-15 minutes at room temperature

  • This may be necessary if heat-induced methods prove insufficient

Blocking and antibody incubation:

• Blocking:

  • 5-10% normal serum (from the species in which the secondary antibody was raised)

  • 1% BSA in PBS

  • Add 0.1-0.3% Triton X-100 for permeabilization

• Primary antibody:

  • Optimal dilution typically ranges from 1:100 to 1:500 (specific to each antibody)

  • Incubation overnight at 4°C produces best results

  • PBS with 1% BSA as diluent

• Secondary antibody:

  • Species-appropriate HRP-conjugated or fluorescently-labeled antibodies

  • Incubation for 1-2 hours at room temperature

  • Thorough washing between steps (3-5 washes with PBS)

Detection systems:

• For chromogenic detection:

  • DAB (3,3'-diaminobenzidine) substrate for 5-10 minutes

  • Hematoxylin counterstaining for nuclear visualization

• For fluorescent detection:

  • Alexa Fluor or similar conjugated secondary antibodies

  • DAPI or other nuclear counterstain

  • Mounting medium containing anti-fade agent

Controls and validation:

• Positive control: Adult testis sections (post-day 20 in mice)

• Negative control: Pre-pubertal testis or non-testicular tissue

• Technical negative: Primary antibody omission

• Verification of specificity: Competitive blocking with immunizing peptide

Following these optimized conditions will maximize the specificity and sensitivity of SPATC1L detection in testicular tissues.

How can researchers troubleshoot issues with SPATC1L antibody specificity?

Troubleshooting SPATC1L antibody specificity issues requires systematic investigation and optimization of experimental conditions:

Common specificity issues and solutions:

Validation approaches for confirming specificity:

• Competitive immunoblotting/immunostaining:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare results with and without competition

  • Specific signals should be abolished or significantly reduced

• Genetic validation:

  • Use SPATC1L knockout tissues as negative controls

  • Compare wild-type vs. knockout signal patterns

  • True signals should be absent in knockout samples

• Multiple antibody validation:

  • Use antibodies targeting different SPATC1L epitopes

  • Compare localization patterns and Western blot results

  • Consistent results across antibodies support specificity

• Recombinant protein controls:

  • Express tagged SPATC1L in heterologous systems

  • Use as positive controls for Western blotting

  • Compare migration patterns with endogenous protein

• Correlation with mRNA expression:

  • Perform parallel analysis of SPATC1L mRNA expression by RT-PCR or in situ hybridization

  • Protein expression should correlate with mRNA presence

• Mass spectrometry verification:

  • Excise Western blot bands of interest

  • Perform mass spectrometry analysis to confirm identity

  • Verify that peptides match SPATC1L sequence

• Orthogonal detection methods:

  • Compare results from different detection techniques (e.g., immunofluorescence vs. Western blot)

  • Consistent patterns across methods increase confidence in specificity

Implementing these troubleshooting and validation strategies will help ensure reliable and reproducible results when using SPATC1L antibodies for research.