CATSPERE Antibody

What is CATSPERE and what is its significance in reproductive biology?

CATSPERE is a key component of the CatSper ion channel complex that plays an essential role in sperm hyperactivation and the acrosome reaction, processes critical for successful fertilization. The CatSper complex functions as a voltage-sensitive calcium channel specifically expressed in the testis, and dysfunction of CATSPERE has been linked to male infertility . Understanding CATSPERE is valuable for researchers investigating sperm physiology, infertility mechanisms, and potential therapeutic strategies for male reproductive disorders . Recent research has revealed that CATSPERE's extracellular domains are particularly important for proper channel assembly and activity modulation .

What validated applications are available for CATSPERE antibodies?

CATSPERE antibodies have been validated for several research applications:

• Western Blotting (WB): Used to detect CATSPERE protein in tissue lysates (1:500-1:5000 dilution)

• Immunohistochemistry (IHC): For localization in tissue sections (1:20-1:200 dilution)

• Immunocytochemistry/Immunofluorescence (ICC/IF): To visualize cellular localization (1:50-1:200 dilution)

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection (1:2000-1:10000 dilution)

Commercial antibodies are typically tested against human and mouse samples, though reactivity may vary between products .

What is the typical subcellular localization pattern of CATSPERE in sperm?

CATSPERE exhibits a characteristic quadrilinear localization pattern in the principal piece of mature sperm tails . Research has shown that only properly assembled CatSper channel complexes are trafficked to the flagellar membrane . In contrast to some other CatSper subunits that have been detected in various sperm regions, CATSPERE is primarily found along the sperm flagellum. This specific localization is critical for its role in sperm motility and hyperactivation .

How do the extracellular domains of CATSPERE contribute to CatSper channel assembly and function?

Recent research indicates that CATSPERE's extracellular domains (ECDs) are essential for proper CatSper channel assembly. Studies have demonstrated that truncated CATSPERE lacking these domains fails to incorporate into functional CatSper complexes . Co-immunoprecipitation experiments reveal that while native full-length CATSPERE associates with other CatSper subunits, truncated versions do not form stable complexes with other channel components . This suggests that CATSPERE's ECDs play a crucial structural role, potentially forming part of the "canopy" structure of the channel complex. The failure of truncated CATSPERE to integrate into the channel complex indicates that the extracellular domains are required not just for modulating channel activity but for fundamental assembly processes necessary for trafficking to the sperm flagellar membrane .

What are the molecular mechanisms by which anti-CatSper antibodies could affect male fertility?

Anti-CatSper antibodies have shown potential as non-hormonal contraceptives through several mechanisms:

• Channel blockade: Antibodies targeting the extracellular loop of CatSper can physically block ion conduction through the channel, preventing calcium influx required for sperm hyperactivation .

• Interference with sperm motility: By binding to extracellular domains, antibodies can impair the conformational changes necessary for proper channel function, thereby reducing sperm motility .

• Prevention of hyperactivation: Even when basic motility remains, antibodies may specifically prevent the hyperactivated motility pattern required for penetration of the zona pellucida .

Experimental studies have validated this approach by immunizing mice with extracellular fragments of CatSper1, which produced antibodies that significantly reduced sperm motility (p<0.05) . These antibodies specifically recognized the antigen and demonstrated potential contraceptive effects without affecting other physiological processes since CatSper expression is highly sperm-specific .

How do researchers distinguish between functional effects of different CatSper subunits?

Distinguishing the functional contributions of individual CatSper subunits requires a multi-faceted experimental approach:

• Subunit-specific antibodies: Using highly specific antibodies against each subunit (including CATSPERE) allows researchers to track localization patterns and potential differences in expression timing or subcellular distribution .

• Genetic models: Knockout or knockdown of specific subunits helps identify their individual contributions to channel assembly, trafficking, and function .

• Co-immunoprecipitation studies: These reveal physical interactions between subunits and help determine which components directly interact with CATSPERE versus other auxiliary subunits .

• Functional assays: Calcium imaging, patch-clamp electrophysiology, and sperm motility analyses with subunit-specific interventions help correlate molecular changes with functional outcomes .

Research has revealed distinct but complementary roles for different CatSper subunits. For example, while CatSper1-4 form the core channel pore, CATSPERE appears critical for proper assembly and trafficking, with its extracellular domains playing a particularly important role in these processes .

What are the optimal conditions for Western blot detection of CATSPERE?

For optimal Western blot detection of CATSPERE:

• Sample preparation: Use testis or sperm lysates prepared with protease inhibitors to prevent degradation of the target protein. For cell lines, confirm expression as CATSPERE has been detected in U87, HepG2, A549, and PC-3 cell lines .

• Protein loading: Load 20-40 μg of total protein per lane.

• Recommended dilution: Use antibody at 1:500-1:5000 dilution depending on the specific antibody and sample type .

• Detection system: Use a secondary antibody such as goat polyclonal to rabbit IgG conjugated with HRP or fluorescent dye .

• Expected band size: Look for bands corresponding to the full-length CATSPERE protein. The exact molecular weight may vary depending on post-translational modifications.

• Controls: Include positive controls such as testis lysate and negative controls from tissues not expected to express CATSPERE .

What validation methods should be used to confirm CATSPERE antibody specificity?

To ensure CATSPERE antibody specificity:

• Blocking peptide competition: Pre-incubate the antibody with the immunogen peptide before application to demonstrate signal reduction when the specific epitope is blocked .

• Multiple antibody comparison: Use antibodies raised against different epitopes of CATSPERE to confirm consistent localization patterns .

• Genetic models: Test antibody on samples from CATSPERE knockout or knockdown models to confirm absence of signal .

• Cross-reactivity testing: Examine reactivity with recombinant CATSPERE protein versus other CatSper family members .

• Multiple application validation: Confirm consistent results across different techniques (WB, IHC, IF) to build confidence in specificity .

• Mass spectrometry verification: Use immunoprecipitation followed by mass spectrometry to confirm the identity of the protein being detected by the antibody .

What immunofluorescence protocol is recommended for CATSPERE detection in sperm?

For optimal immunofluorescence detection of CATSPERE in sperm:

• Fixation: Use 4% paraformaldehyde for 15-30 minutes at room temperature to preserve protein structure while allowing antibody access .

• Permeabilization: Treat with 0.1-0.2% Triton X-100 for 10 minutes to allow antibody access to intracellular epitopes while preserving membrane structures .

• Blocking: Use 5-10% normal serum (from the species in which the secondary antibody was raised) with 1% BSA for 1 hour to reduce non-specific binding .

• Primary antibody: Apply CATSPERE antibody at 1:50-1:200 dilution and incubate overnight at 4°C .

• Secondary antibody: Use fluorophore-conjugated secondary antibodies specific to the primary antibody species (typically anti-rabbit) at manufacturer-recommended dilutions .

• Counterstaining: Include DAPI for nuclear staining and consider phalloidin for actin filament visualization to provide structural context .

• Mounting: Use anti-fade mounting medium to preserve fluorescence signal during imaging and storage .

This protocol has successfully revealed the characteristic quadrilinear localization pattern of CATSPERE in the principal piece of mature sperm tails .

How does CATSPERE expression and function compare across different species?

Research has revealed both conservation and divergence in CATSPERE across species:

Interestingly, while mammals show primarily testis-specific expression, studies in C. intestinalis (ascidians) detected CatSper expression in multiple tissues including heart, siphon, and gill, suggesting potentially broader functions in some species . This comparative analysis highlights the importance of species-specific validation when using CATSPERE antibodies for evolutionary or comparative studies.

What are common technical issues when working with CATSPERE antibodies and how can they be addressed?

Researchers may encounter several challenges when working with CATSPERE antibodies:

• Background signal: High background may occur due to non-specific antibody binding. Increase blocking time/concentration, optimize antibody dilution, and include additional washing steps .

• Inconsistent detection: Variability between experiments may result from sample preparation differences. Standardize lysate preparation protocols and include positive controls in each experiment .

• Cross-reactivity: Some antibodies may detect other CatSper family members due to sequence similarity. Validate specificity using knockout controls or peptide competition assays .

• Epitope accessibility: Some epitopes may be masked in native conformation. Try different fixation and permeabilization methods or use antibodies targeting different regions of CATSPERE .

• Signal degradation: CATSPERE may be sensitive to proteolysis. Always include protease inhibitors during sample preparation and avoid repeated freeze-thaw cycles of antibodies .

• Species cross-reactivity limitations: Commercial antibodies may have limited cross-reactivity across species. Verify reactivity for your species of interest or consider generating custom antibodies for non-model organisms .

What are promising applications of CATSPERE antibodies in fertility research?

CATSPERE antibodies offer several promising avenues for fertility research:

• Diagnostic tools: Developing immunoassays to detect abnormal CATSPERE expression or localization in sperm from infertile men could provide new diagnostic markers .

• Contraceptive development: Building on research showing antibodies against CatSper extracellular domains can inhibit sperm function, CATSPERE-specific antibodies could lead to novel non-hormonal contraceptives .

• Structure-function analysis: Using domain-specific antibodies to probe the relationship between CATSPERE structure and channel function could reveal new therapeutic targets .

• Evolutionary conservation studies: Comparing CATSPERE localization and function across species using cross-reactive antibodies may reveal evolutionary adaptations in fertilization mechanisms .

• Sperm selection: Antibodies against CATSPERE could potentially be used to select functional sperm for assisted reproductive technologies, improving success rates in clinical settings .

Research demonstrating that antibodies targeting CatSper extracellular domains can significantly reduce sperm motility suggests particularly promising applications in contraceptive development, offering a highly specific approach with minimal side effects due to the testis-specific expression pattern .

How might CATSPERE antibodies be used to study the interplay between ion channels and other sperm signaling pathways?

CATSPERE antibodies can serve as valuable tools for investigating complex signaling networks in sperm:

• Co-localization studies: Dual immunofluorescence using CATSPERE antibodies alongside markers for other signaling molecules can reveal spatial relationships and potential interaction sites .

• Signaling pathway modulation: Combining CATSPERE antibodies that block channel function with pharmacological modulators of other pathways can help delineate pathway interactions and hierarchies .

• Protein complex analysis: Using CATSPERE antibodies for co-immunoprecipitation followed by mass spectrometry can identify novel interaction partners and regulatory proteins .

• Calcium dynamics: Pairing CATSPERE antibody treatments with calcium imaging can directly link channel modulation to downstream calcium signaling events .

• Phosphorylation studies: Combining phospho-specific antibodies with CATSPERE detection can reveal how phosphorylation states affect channel assembly, trafficking, and function .

These approaches could address key questions about how CatSper channels integrate with other essential sperm signaling pathways involved in capacitation, hyperactivation, and the acrosome reaction, potentially revealing new therapeutic targets for both fertility enhancement and contraception .