DNAH1 Antibody

What is DNAH1 and why is it important in fertility research?

DNAH1 (Dynein Axonemal Heavy Chain 1) is a protein component critical for sperm flagella structure and function. It plays an essential role in the formation and maintenance of the central pair of microtubules in sperm flagella. Mutations in DNAH1 have been strongly associated with multiple morphological abnormalities of flagella (MMAF) and male infertility across various populations . Unlike many other dynein genes that cause both respiratory ciliary dyskinesia and sperm motility issues when mutated, DNAH1 mutations typically affect sperm motility specifically without manifesting primary ciliary dyskinesia symptoms, making it particularly important for understanding the specialized structural requirements of sperm flagella versus other ciliated structures .

What applications are DNAH1 antibodies commonly used for in research?

DNAH1 antibodies are primarily used for immunohistochemistry, immunofluorescence, and protein detection in research settings. They are valuable tools for:

• Analyzing DNAH1 protein expression in sperm samples from infertile men with MMAF

• Examining the localization of DNAH1 in sperm flagella

• Confirming the effects of DNAH1 mutations on protein expression and localization

• Comparing normal versus abnormal flagellar ultrastructure

The recommended dilution for immunohistochemistry applications is typically 1:200-1:500 . For immunofluorescence studies on sperm samples, protocols typically involve fixation with paraformaldehyde, permeabilization with Triton X-100, and blocking with BSA before antibody application .

How should I prepare sperm samples for DNAH1 immunofluorescence studies?

For effective immunofluorescence analysis of DNAH1 in sperm samples, follow this methodological approach:

• Smear sperm samples on clean slides

• Fix with 4% paraformaldehyde

• Wash three times with phosphate-buffered saline (PBS)

• Permeabilize with 0.5% Triton X-100 for 30 minutes

• Block with 1% BSA

• Incubate with primary DNAH1 antibody (e.g., Abcam, ab122367) overnight at 4°C

• Wash with PBST (PBS containing Triton X-100)

• Incubate with appropriate secondary antibodies (e.g., DAR555) for 1 hour at 37°C

• Wash again and seal with Hoechst and Vectashield

This protocol allows for specific visualization of DNAH1 localization in sperm flagella and can be used for comparative studies between normal controls and patients with suspected DNAH1 mutations.

What controls should I include when using DNAH1 antibodies?

When conducting experiments with DNAH1 antibodies, include the following controls for result validation:

• Positive control: Include sperm samples from fertile males with known normal DNAH1 expression. Normal samples should show DNAH1 localization throughout the sperm flagella .

• Negative control: Include samples where the primary antibody is omitted but all other steps remain identical.

• Comparative control: If investigating potential DNAH1 mutations, include samples from both the affected individuals and unaffected family members when possible.

• Co-staining control: Consider co-staining with α-tubulin (e.g., Sigma, F2168) to mark flagellar structure and SPAG6 (e.g., Proteintech, 12462-1-AP) as a marker for the central pair of microtubules .

These controls ensure specificity of staining and provide critical comparative data for interpreting experimental results.

How do I investigate discrepancies between human and mouse DNAH1 mutation phenotypes?

To investigate this discrepancy:

• Isoform analysis: Examine whether alternative DNAH1 isoforms might compensate in mouse models. Recent research has identified DNAH1 isoform2 in Dnah1Δiso1/Δiso1 mutant mice that may mediate normal ultrastructure formation in the absence of full-length protein .

• Targeted mouse models: Generate specific mouse models that mirror human mutations rather than complete knockouts. CRISPR/Cas9 genome editing can be used to create precise mutations matching those found in human patients .

• Transcriptome comparison: Perform comparative RNA-Seq analysis between human and mouse testicular tissue to identify potential compensatory mechanisms or species-specific differences in flagellar assembly pathways.

• Protein interaction studies: Investigate species-specific differences in DNAH1 protein interactions that might explain the phenotypic differences.

This systematic approach may help elucidate the mechanisms underlying the species-specific differences in DNAH1 mutation consequences.

What methodologies can help determine if DNAH1 mutations affect protein conformation?

Investigating whether DNAH1 mutations affect protein conformation requires multiple complementary approaches:

• In silico structure prediction: Use molecular modeling software such as SWISSMODEL and PyMoL to predict conformational changes induced by specific mutations. This approach revealed that the missense mutation c.4670C>T (p.T1557M) completely altered the spatial structure of DNAH1, affecting random coils, α-helices, and β-sheets .

• Protein stability assays: Employ pulse-chase experiments with tagged DNAH1 constructs (wild-type and mutant) to assess differences in protein half-life and degradation rates.

• Limited proteolysis: Compare trypsin digestion patterns between wild-type and mutant DNAH1 proteins to detect conformational differences that alter protease accessibility.

• Circular dichroism spectroscopy: Analyze secondary structure content and thermal stability of recombinant DNAH1 protein fragments (wild-type versus mutant).

• Co-immunoprecipitation studies: Assess whether mutations alter DNAH1's ability to interact with known binding partners in the axonemal complex.

These methodologies can provide comprehensive insights into how specific mutations might disrupt DNAH1 function through conformational changes rather than simply affecting expression levels.

How can I differentiate between DNAH1-associated MMAF and other genetic causes of flagellar abnormalities?

Differentiating DNAH1-associated MMAF from other genetic causes requires a multi-faceted diagnostic approach:

• Immunofluorescence profiling: Use DNAH1 antibodies to assess protein expression and localization. Patients with DNAH1 mutations typically show absent or severely reduced DNAH1 staining in sperm flagella compared to controls .

• Ultrastructural analysis: Employ transmission electron microscopy to examine flagellar cross-sections. DNAH1 mutations characteristically result in missing central singlet microtubules and disorganized fibrous sheaths .

• SPAG6 co-staining: SPAG6 is a marker for the central pair of microtubules. Its absence or abnormal distribution can confirm disruption of the central pair typically seen in DNAH1-related MMAF .

• Genetic panel testing: Beyond DNAH1, mutations in multiple other genes can cause MMAF, including DNAH10, DNAH17, WDR19, ARMC2, TTC21A, TTC29, FSIP2, AK7, CEP135, SPEF2, QRICH2, DZIP1, BRWD1, DRC1, and STK33 . A comprehensive genetic panel can help distinguish between these causes.

• Clinical correlation: Unlike some other axonemal dynein mutations, DNAH1 mutations typically do not cause primary ciliary dyskinesia (PCD) symptoms, providing an important clinical distinguishing feature .

This integrated approach enables precise identification of DNAH1-associated MMAF cases, facilitating targeted genetic counseling and potential treatment approaches.

What techniques can be used to validate novel DNAH1 mutations as pathogenic?

Validating novel DNAH1 mutations as pathogenic requires a comprehensive approach combining multiple lines of evidence:

• Segregation analysis: Verify that the mutations follow Mendelian inheritance patterns within families. For recessive conditions like DNAH1-associated MMAF, affected individuals should harbor homozygous or compound heterozygous mutations, while parents should be heterozygous carriers .

• Population frequency assessment: Confirm that the variants have minor allele frequencies <0.01 in public databases such as 1000 Genome project, ESP6500, or ExAC database. Exclude variants that are homozygous in fertile control populations .

• In silico pathogenicity prediction: Apply multiple computational tools to predict deleterious effects. Select variants predicted to be deleterious by approximately 10 of 13 commonly employed in silico tools .

• Conservation analysis: Analyze evolutionary conservation of the affected amino acid residues across species. Highly conserved residues (100% conservation) are more likely to be functionally important .

• Functional validation in animal models: Generate mouse models with equivalent mutations using CRISPR/Cas9 genome editing to confirm phenotypic effects .

• Protein expression studies: Perform immunofluorescence with DNAH1 antibodies to confirm reduced or absent protein expression in patient sperm samples .

• Structural modeling: Use protein modeling software to predict the impact of missense mutations on protein conformation and stability .

This multi-faceted approach provides robust evidence for classifying novel DNAH1 variants as pathogenic or likely pathogenic.

How can DNAH1 antibodies be used to study interactions with other axonemal proteins?

DNAH1 antibodies can be instrumental in exploring protein-protein interactions within the complex axonemal structure:

• Co-immunoprecipitation (Co-IP): Use DNAH1 antibodies to pull down protein complexes from testicular or sperm lysates, followed by mass spectrometry analysis to identify interacting partners. This approach can reveal both known and novel interaction partners.

• Proximity ligation assay (PLA): Combine DNAH1 antibodies with antibodies against suspected interaction partners to visualize protein-protein interactions in situ with single-molecule resolution.

• Immunofluorescence co-localization: Perform double immunofluorescence staining with DNAH1 antibodies and antibodies against other axonemal components (such as SPAG6) to assess spatial relationships .

• Comparative studies in mutation models: Compare interaction patterns between wild-type samples and those with specific DNAH1 mutations to determine how mutations might disrupt protein complex formation.

• Super-resolution microscopy: Employ techniques such as STORM or PALM with fluorescently-labeled DNAH1 antibodies to achieve nanoscale resolution of protein localization and interactions within the axonemal structure.

These approaches can provide critical insights into how DNAH1 contributes to axonemal structure and function through its interactions with other flagellar proteins.