TMCO5A Antibody

What is TMCO5A and where is it expressed?

TMCO5A belongs to the TMCO (Transmembrane and coiled-coil domains) gene family. This protein is expressed exclusively in the testis of adult male mice, specifically in elongating spermatids of steps 9 to 12 during spermiogenesis . Expression studies using in situ hybridization demonstrate that TMCO5A mRNA is present in round and elongating spermatids in the seminiferous tubules of adult mouse testes .

The protein's expression is developmentally regulated, with detection beginning in mouse testes after 4 weeks of age, coinciding with the first wave of spermatogenesis . Importantly, TMCO5A shows strict tissue specificity, with no detectable expression in other tissues examined (ovary, skeletal muscle, brain, skin, stomach, intestine, colon, spleen) . Even within the reproductive tract, TMCO5A is absent from epididymal extracts, indicating it is not a component of mature sperm .

The specific temporal and spatial expression pattern suggests TMCO5A plays a specialized role during the critical phase of sperm morphogenesis when dramatic remodeling of the sperm head and tail occurs.

What is the domain structure of TMCO5A protein?

TMCO5A possesses a distinctive domain architecture that provides important clues about its function:

• An N-terminal coiled-coil domain: Coiled-coil domains typically mediate protein-protein interactions, oligomerization, and serve as structural components in various cellular processes . This domain may enable TMCO5A to form complexes with other proteins or self-associate into oligomeric structures.

• A C-terminal transmembrane domain: This domain suggests membrane association, potentially allowing TMCO5A to integrate into cellular membranes or interact with membrane-bound organelles .

This domain structure is characteristic of the TMCO gene family members, which have diverse cellular functions. For TMCO5A specifically, this architecture suggests it may function as a linker protein that connects membrane structures or vesicles to the microtubule cytoskeleton during spermatogenesis .

The combination of these domains is particularly relevant considering TMCO5A's localization to the manchette and its effects on Golgi organization when expressed in cell models. The coiled-coil domain likely facilitates interactions with cytoskeletal elements, while the transmembrane domain may enable associations with vesicular compartments being transported along the manchette microtubules.

What is the cellular localization of TMCO5A?

Immunofluorescence studies using monoclonal antibodies have revealed that TMCO5A localizes specifically to the manchette in elongating spermatids . The manchette is a transient microtubule and actin-based structure that emerges during spermiogenesis and contributes to head shaping and tail development in developing sperm cells .

TMCO5A co-localizes with β-tubulin in the manchette, although the localization patterns are not completely identical . TMCO5A appears to be distributed in a slightly thinner region compared to β-tubulin and is not present in the most posterior part of the structure . This suggests TMCO5A associates with specific subdomains of the manchette rather than uniformly binding all microtubules.

Additional immunofluorescent microscopy has clarified that TMCO5A localizes to the opposite side of the acrosome across the nucleus in elongating spermatids . This precise localization pattern, combined with the protein's domain structure, suggests TMCO5A may participate in the transport of Golgi-derived non-acrosomal vesicles along the manchette microtubules during sperm development .

The restricted expression window (steps 9-12 of spermiogenesis) and specific subcellular localization support a specialized function for TMCO5A during the critical period when sperm head shaping and tail formation actively occur.

How are TMCO5A antibodies typically generated?

TMCO5A antibodies can be generated through several methodological approaches, with recombinant protein-based immunization being well-documented in the literature:

• Recombinant protein expression: A partial coding region of TMCO5A (for example, nucleotide positions 162 to 536, NM_026104) is PCR-amplified from adult testis cDNA libraries using primers containing appropriate restriction sites . The amplified fragment is then cloned into an expression vector (such as pRSET A) and transformed into a bacterial expression system (like BL21 (DE3) pLysS) .

• Protein purification: The recombinant protein is purified using metal affinity chromatography (such as TALON Metal Affinity chromatography) following the manufacturer's protocol .

• Immunization: Laboratory animals (rats or rabbits) are immunized with the purified recombinant protein (typically 300 μg) mixed with Freund's adjuvant . Multiple immunizations are performed at 2-week intervals to enhance the immune response .

• For monoclonal antibody production: Following the final immunization, spleen cells from the immunized animals are harvested and fused with myeloma cells (such as P3U1) . Hybridomas are selected using HAT medium and screened for antibody production using conventional ELISA with the recombinant protein and by immunohistochemistry on tissue sections . Positive hybridomas are cloned by limited dilution to establish monoclonal antibody-producing cell lines .

• For polyclonal antibody production: Serum is collected from immunized animals after multiple boosting immunizations, and antibodies are purified using affinity chromatography .

These methodologies have successfully produced specific antibodies against TMCO5A, including the monoclonal antibody RTm01 described in the literature and commercial polyclonal antibodies .

What methods are used to validate TMCO5A antibodies?

Rigorous validation of TMCO5A antibodies requires multiple complementary approaches to ensure specificity and reliability:

• Immunoblotting (Western blot): Antibody specificity is assessed using tissue extracts from multiple organs, particularly including testis as a positive control and various other tissues (ovary, skeletal muscle, brain, skin, digestive organs, spleen) as negative controls . Additionally, developmental series of testes from mice of different ages (3-8 weeks) can confirm the expected developmental expression pattern . Proper validation should show a single band of the appropriate molecular weight exclusively in testis samples from mice 4 weeks and older.

• Immunohistochemistry (IHC): Paraffin sections of various tissues are used to validate antibody specificity through enzymatic immunohistochemistry followed by counterstaining (such as Hematoxylin) . In properly validated antibodies, staining should be restricted to elongating spermatids in seminiferous tubules at specific stages (IX-XII) .

• Immunofluorescence microscopy: This technique determines the subcellular localization of TMCO5A and its co-localization with known structures or proteins (such as β-tubulin) . Validation requires demonstrating the expected manchette localization pattern in elongating spermatids.

• Heterologous expression systems: Inducible expression of TMCO5A in cell lines (like CHO cells using Tet-on systems) followed by immunostaining provides a controlled system for validating antibody specificity . Antibody staining should be absent in non-induced cells and present in induced cells.

• Controls: Appropriate controls include primary antibody omission, isotype controls, and when possible, tissues from knockout or knockdown models.

Multiple validation methods are essential because each technique has different strengths and limitations. A properly validated TMCO5A antibody should show consistent results across these complementary approaches, providing confidence in the specificity and reliability of the reagent for research applications.

What are the common applications for TMCO5A antibodies?

TMCO5A antibodies serve as versatile tools in reproductive biology research with several key applications:

• Immunohistochemistry (IHC): TMCO5A antibodies enable detection and localization of the protein in tissue sections, particularly in testicular tissue . This application helps identify the specific cell types and developmental stages expressing TMCO5A during spermatogenesis. Both enzymatic detection (with chromogenic substrates) and fluorescent detection methods can be employed, with the latter offering better resolution for subcellular localization studies.

• Immunocytochemistry with immunofluorescence (ICC-IF): This technique allows for detailed subcellular localization studies, including co-localization with other proteins or cellular structures . For TMCO5A, this has been particularly valuable in demonstrating its association with manchette microtubules in elongating spermatids and with the microtubule cytoskeleton in heterologous expression systems.

• Western blotting (WB): TMCO5A antibodies can detect and quantify the protein in tissue or cell lysates, enabling comparative expression analysis across tissues, developmental stages, or experimental conditions . Western blotting has revealed the strict testis-specificity of TMCO5A and its developmental regulation during postnatal testis development.

• Developmental studies: These antibodies enable tracking of TMCO5A expression during postnatal testicular development and spermatogenesis . This application has revealed that TMCO5A protein expression begins at 4 weeks of age in mice, coinciding with the appearance of elongating spermatids.

• Functional studies: When combined with protein overexpression or knockdown approaches, TMCO5A antibodies help elucidate the protein's functional roles in cellular processes . The induced expression of TMCO5A in CHO cells, followed by immunostaining, has revealed its effects on Golgi organization and association with microtubules.

These diverse applications make TMCO5A antibodies valuable tools for researchers studying reproductive biology, sperm development, and the cellular mechanisms underlying male fertility.

How can TMCO5A antibodies be used to study spermatogenesis?

TMCO5A antibodies provide sophisticated tools for investigating specific aspects of spermatogenesis, particularly during the critical elongation phase of spermiogenesis:

• Stage-specific expression analysis: Using immunohistochemistry with TMCO5A antibodies on testicular sections allows researchers to precisely identify the stages of seminiferous epithelium and steps of spermatid development where TMCO5A is expressed . Research has demonstrated that TMCO5A protein is specifically expressed in elongating spermatids of steps 9-12, corresponding to stages IX-XII of the seminiferous epithelium cycle . This precise temporal expression pattern suggests TMCO5A functions during critical phases of sperm head shaping and tail formation.

• Manchette structure and function studies: Since TMCO5A localizes to the manchette, antibodies enable detailed investigation of this transient cytoskeletal structure crucial for sperm morphogenesis . Immunofluorescence with TMCO5A antibodies, combined with markers for other manchette components, can reveal the assembly dynamics, protein composition, and potential subdomains within this complex structure.

• Transcription-translation delay investigation: The detection of TMCO5A mRNA in round spermatids contrasted with protein expression only in elongating spermatids highlights the post-transcriptional regulation occurring during spermatogenesis . TMCO5A antibodies, used in conjunction with RNA detection methods, can help study the mechanisms controlling this temporal gap between transcription and translation.

• Functional studies in genetic models: TMCO5A antibodies can assess protein expression and localization in genetic mouse models with defects in spermatogenesis, potentially revealing relationships between TMCO5A and known regulators of sperm development. Changes in TMCO5A expression, localization, or modification in these models could provide insights into the functional pathways involving this protein.

• Vesicular transport investigation: Given TMCO5A's localization to the manchette and its potential role in vesicle transport, antibodies can be used in co-localization studies with markers for vesicular compartments to understand the protein's role in intramanchette transport during sperm development .

These advanced applications allow researchers to gain deeper insights into the molecular mechanisms governing sperm development and the specific contributions of TMCO5A to this complex process.

What is the relationship between TMCO5A and microtubule structures?

Research findings have revealed a significant relationship between TMCO5A and microtubule structures that suggests functional integration:

• Co-localization with β-tubulin: Immunofluorescence studies in elongating spermatids have demonstrated that TMCO5A co-localizes with β-tubulin in the manchette structure . This co-localization indicates a close spatial relationship between TMCO5A and microtubules during spermiogenesis.

• Manchette association: The manchette is a transient microtubule-based structure that forms during sperm head elongation and participates in nuclear shaping and protein transport to the developing tail . TMCO5A's specific localization to this structure suggests involvement in manchette-dependent processes during sperm morphogenesis.

• Specific microtubule subdomain association: While TMCO5A co-localizes with microtubules, its distribution is not identical to that of β-tubulin. The TMCO5A signal appears slightly thinner compared to β-tubulin and is absent from the most posterior part of the manchette structure . This suggests TMCO5A may interact with specific subsets of microtubules or microtubule-associated proteins within the manchette.

• Intrinsic microtubule affinity: When TMCO5A was ectopically expressed in CHO cells, it displayed a fibrous distribution pattern reminiscent of cytoskeletal structures and co-localized with β-tubulin . This observation in a heterologous system indicates that TMCO5A's association with microtubules is an intrinsic property of the protein rather than dependent on testis-specific factors.

• Potential structural role: The coiled-coil domain in TMCO5A could mediate interactions with microtubules or microtubule-associated proteins, potentially allowing TMCO5A to function as a linker between the microtubule cytoskeleton and membrane compartments during sperm development .

These findings collectively suggest TMCO5A functions in close association with microtubules, potentially connecting microtubule structures to membrane compartments during the dramatic cellular remodeling that occurs during spermiogenesis.

How does TMCO5A affect Golgi organization in cellular models?

Experimental evidence demonstrates that TMCO5A exerts significant effects on Golgi organization in cellular models:

These findings highlight a potential role for TMCO5A in organizing cellular architecture through coordinating the relationship between the Golgi apparatus and the microtubule cytoskeleton, which could be particularly important during the dramatic morphological changes that occur during spermiogenesis.

What are optimal sample preparation methods for TMCO5A detection?

Successful detection of TMCO5A requires careful sample preparation tailored to the specific experimental approach:

For immunoblotting (Western blot):

• Tissue extraction: Adult tissue samples should be extracted with 5 times volume (v/w) of SDS sample buffer (5% 2-mercaptoethanol, 10% glycerol, 2% SDS, 0.005% Bromophenol Blue, and 63 mM Tris-HCl pH 6.8) .

• Denaturation: Extracts should be thoroughly denatured by boiling for 5 minutes to ensure complete protein unfolding and exposure of epitopes .

• Clarification: Samples must be centrifuged at high speed (17,400 x g for 10 minutes) to remove insoluble material, and the supernatants collected for analysis .

• Gel electrophoresis: For optimal resolution, 15 μl of each sample should be loaded into wells of a 10% SDS-PAGE gel, and proteins separated according to standard protocols .

• Transfer conditions: Efficient transfer to nitrocellulose membranes is critical for subsequent antibody detection. Transfer efficiency can be verified using reversible protein stains like Ponceau S .

For immunohistochemistry:

• Tissue fixation: Mouse testes should be fixed in Bouin's solution or 4% paraformaldehyde to preserve both cellular morphology and protein antigenicity .

• Processing and embedding: Fixed tissues must be carefully dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin for sectioning .

• Sectioning: Thin sections (5-7 μm) should be mounted on adhesive slides to prevent tissue loss during staining procedures .

• Antigen retrieval: Though not explicitly mentioned in the search results, heat-induced epitope retrieval (using citrate buffer pH 6.0 or EDTA buffer pH 9.0) is often necessary to unmask antigens that may be cross-linked during fixation.

For immunofluorescence:

• Cell fixation: For cultured cells, 4% paraformaldehyde in PBS for 15-30 minutes at room temperature preserves cellular structures while maintaining antigen reactivity .

• Permeabilization: A brief treatment with 0.1-0.5% Triton X-100 in PBS allows antibody access to intracellular antigens without disrupting cellular architecture .

• Blocking: Thorough blocking with appropriate sera (5-10% normal serum from the species in which the secondary antibody was raised) minimizes background staining .

These optimized preparation methods ensure proper preservation of TMCO5A antigenicity while maintaining cellular and tissue morphology for accurate detection and localization studies.

How can specificity of TMCO5A antibody staining be confirmed?

Confirming the specificity of TMCO5A antibody staining is crucial for reliable research outcomes. Multiple validation approaches should be implemented:

• Multi-technique validation: Confirmation of TMCO5A expression should utilize complementary techniques including immunoblotting, immunohistochemistry, and immunofluorescence . Consistent results across these methodologically distinct approaches provides strong evidence of antibody specificity.

• Tissue-specific expression controls: Since TMCO5A is specifically expressed in testis and not in other tissues, a panel of tissues (ovary, skeletal muscle, brain, skin, stomach, intestine, colon, spleen) should be used as negative controls in Western blots and immunostaining . Specific signal should be detected only in testis.

• Developmental timing controls: TMCO5A expression begins in mouse testes after 4 weeks of age . Testing tissues from younger mice (3 weeks) provides a biological negative control where no signal should be detected despite the presence of other testicular proteins .

• Cell-type specificity verification: Within the testis, TMCO5A is exclusively expressed in elongating spermatids of steps 9-12 . The antibody should show this precise pattern rather than generalized staining of all testicular cells. This can be confirmed by counterstaining with Hematoxylin to identify specific seminiferous tubule stages .

• Subcellular localization consistency: TMCO5A specifically localizes to the manchette structure in elongating spermatids . This distinctive pattern should be consistently observed and can be verified by co-localization with known manchette markers like β-tubulin .

• Inducible expression system validation: Using controlled expression systems, such as the Tet-on system in CHO cells, allows definitive validation of antibody specificity . Comparing antibody staining between induced and non-induced cells provides a clear positive/negative control pair.

• Blocking experiments: Pre-incubation of the antibody with the immunizing antigen (recombinant TMCO5A protein) should abolish specific staining in a concentration-dependent manner, providing further evidence of specificity.

These comprehensive validation strategies ensure that the observed staining patterns truly represent TMCO5A expression and localization, rather than non-specific binding or cross-reactivity with other proteins.

What are common challenges in TMCO5A detection and how to overcome them?

Researchers studying TMCO5A may encounter several technical challenges that require specific solutions:

By anticipating these challenges and implementing appropriate strategies, researchers can enhance the reliability and sensitivity of TMCO5A detection in their experimental systems.

How does TMCO5A expression vary during development?

TMCO5A exhibits a tightly regulated developmental expression pattern with important implications for its biological function:

• Postnatal onset: TMCO5A protein is undetectable in mouse testes before 3 weeks of age, becoming detectable at 4 weeks and continuing into adulthood . This timing correlates precisely with the first wave of spermatogenesis in mice, where elongating spermatids (steps 9-12) begin to appear around postnatal day 25 .

• Stage-specific expression: Within the seminiferous epithelium, TMCO5A protein expression is strictly limited to elongating spermatids of steps 9-12, corresponding to stages IX-XII of the seminiferous epithelium cycle . This has been confirmed through careful immunohistochemical analysis with concurrent histological staging of the seminiferous tubules .

• Transcription-translation uncoupling: A notable feature of TMCO5A regulation is the temporal disconnect between mRNA and protein expression. While TMCO5A protein is detected only in elongating spermatids, the mRNA is expressed earlier in round spermatids . This phenomenon reflects the widespread post-transcriptional regulation that occurs during spermatogenesis, where many transcripts are stored in a translationally repressed state until needed .

• Absence in mature sperm: TMCO5A protein is undetectable in epididymal extracts even using highly-sensitive chemiluminescent detection methods . This indicates that TMCO5A is not retained as a structural component of mature sperm but rather functions specifically during the developmental process of spermiogenesis.

• Strict tissue specificity: Beyond developmental regulation, TMCO5A exhibits remarkable tissue specificity, with expression restricted to the testis in adult mice . No detectable expression has been found in other tissues examined (ovary, skeletal muscle, brain, skin, stomach, intestine, colon, spleen) .

This precisely orchestrated developmental expression pattern highlights TMCO5A's specialized role during a critical window of sperm development when dramatic morphological changes are occurring, particularly in nuclear shaping and tail formation.

What functional insights have been gained from TMCO5A localization studies?

Localization studies of TMCO5A have provided several key functional insights:

• Manchette association: TMCO5A specifically localizes to the manchette in elongating spermatids . The manchette is a transient structure composed primarily of microtubules and actin filaments that forms during steps 8-12 of spermiogenesis and plays crucial roles in nuclear shaping and protein transport to the developing sperm tail .

• Microtubule interaction: Immunofluorescence studies have demonstrated that TMCO5A co-localizes with β-tubulin both in elongating spermatids and when ectopically expressed in CHO cells . This consistent association with microtubules across different cellular contexts suggests an intrinsic ability of TMCO5A to interact with the microtubule cytoskeleton.

• Golgi reorganization capacity: When expressed in CHO cells with EGFP-tagged Golgi, TMCO5A induces a dramatic reorganization of the Golgi apparatus . Prior to TMCO5A induction, the Golgi appears scattered around the nucleus, but after TMCO5A expression, it concentrates to a single point at the center of the TMCO5A distribution . This effect suggests TMCO5A may function in organizing membrane compartments along microtubules.

• Subdomain-specific manchette localization: TMCO5A's distribution in the manchette differs slightly from that of β-tubulin, appearing in a thinner region and absent from the most posterior part of the structure . This suggests TMCO5A may associate with specific subdomains of the manchette rather than binding uniformly to all microtubules.

• Potential vesicle transport role: Given that the manchette serves in the transport of Golgi-derived non-acrosomal vesicles during sperm development , and considering TMCO5A's effects on Golgi organization, the protein may participate in coordinating vesicle transport along manchette microtubules during spermiogenesis.

• Functional timing: TMCO5A's expression specifically during steps 9-12 of spermiogenesis coincides precisely with active head shaping and delivery of proteins to the developing tail , supporting a role in these morphogenetic processes.

These localization-based insights provide a foundation for understanding TMCO5A's functional role in spermatogenesis, particularly in coordinating cytoskeletal organization, Golgi dynamics, and potentially vesicle transport during the dramatic cellular remodeling of spermiogenesis.

How does TMCO5A expression in mice compare to other species?

The available research provides limited cross-species comparison data for TMCO5A expression, but some important differences and similarities have been noted:

• Mouse vs. Rat expression patterns: Studies have revealed potential differences in TMCO5A expression patterns between mice and rats. While research with the monoclonal antibody RTm01 found TMCO5A expression restricted to elongating spermatids (steps 9-12) in mice, Kaneko et al. reported that in rats, TMCO5A expression was observed in round and almost developed spermatids in addition to elongating spermatids .

• Potential explanations for species differences:

  • Spermatogenesis cycle variations: The period and stages of the spermatogenesis cycle differ significantly between mice and rats (233.6 hours vs. 310.8 hours for one cycle, and 12 vs. 13 stages, respectively) . These fundamental differences in reproductive physiology might affect the timing and pattern of gene expression.

  • Methodological differences: Studies used different antibody types - a monoclonal antibody against recombinant TMCO5A protein in mouse studies versus a polyclonal antibody against synthetic oligopeptides in rat studies . These differences in antibody specificity might contribute to the observed variations in expression patterns.

• Developmental timing consistency: Despite potential differences in cell type expression, immunoblotting analyses in mouse studies consistently show that TMCO5A expression begins from 4 weeks of age or 28 postnatal days . This suggests that the developmental onset of TMCO5A expression is conserved, at least within rodent species.

• Human TMCO5A: Commercial antibodies against human TMCO5A have been developed , indicating the protein is present in humans, though detailed expression data in human tissues is not provided in the available search results. The conservation of this protein across species suggests important functional roles.

• Evolutionary conservation of function: The existence of TMCO family members across diverse species (including Tango6/TMCO7 in Drosophila) with similar effects on Golgi organization suggests evolutionary conservation of certain functional aspects of these proteins, even if expression patterns may vary .