TMEM95 Antibody

What is TMEM95 and why is it significant in reproductive research?

TMEM95 is a sperm acrosomal membrane protein that plays a critical role in mammalian fertilization, specifically in the membrane fusion of sperm and eggs. Research has demonstrated that TMEM95 ablation in mice causes complete male-specific infertility, with sperm unable to fuse with the egg membrane despite normal morphology, motility, and ability to penetrate the zona pellucida . TMEM95 joins IZUMO1 and SPACA6 as the only sperm proteins currently known to be indispensable for sperm-egg membrane fusion, making it a significant target for reproductive research . The protein appears to bind to egg membranes through a putative receptor-binding surface, suggesting a receptor-mediated interaction that facilitates membrane fusion .

How do TMEM95 antibodies affect sperm-egg interaction?

TMEM95 antibodies have been shown to significantly impair sperm-egg fusion without blocking sperm-egg binding. In sperm penetration assays, monoclonal antibodies against TMEM95 reduced the number of human sperm fused with hamster eggs while allowing normal binding to occur . Specifically, treatment with TMEM95 antibody Fab fragments (3A01 and 6B08) decreased the average numbers of fused sperm per egg from 9.1 ± 0.7 in untreated controls to 4.1 ± 0.9 and 3.4 ± 0.6, respectively . This distinct pattern—inhibition of fusion without affecting binding—suggests that TMEM95 functions specifically in the membrane fusion process rather than in the initial attachment of sperm to eggs .

What types of TMEM95 antibodies are available for research?

Current research has utilized several types of TMEM95 antibodies:

• Monoclonal antibodies (e.g., 3A01 and 6B08) generated against the TMEM95 ectodomain

• Fab fragments of these monoclonal antibodies, which bind antigens at nanomolar affinities and may have less steric hindrance in fusion experiments

• Commercial antibodies from suppliers such as MyBioSource used for immunocytochemistry applications

These antibodies have been employed in various experimental contexts, including sperm penetration assays, immunolocalization studies, and protein-protein interaction analyses .

How can researchers use structural insights about TMEM95 to develop more specific antibodies?

X-ray crystallography has revealed an evolutionarily conserved, positively charged region of TMEM95 as a putative receptor-binding surface . Researchers can utilize this structural information to design antibodies targeting specific epitopes within this region. The crystal structure indicates that amino acid residues R70 and R73 are particularly important for egg-binding activity, as substitutions in these positions significantly reduce TMEM95's ability to bind to egg membranes . By generating antibodies that specifically recognize these functional domains, researchers can develop more precise tools for studying TMEM95-receptor interactions. This structure-based approach could lead to antibodies that differentially affect various aspects of TMEM95 function, enabling more nuanced experimental manipulation of sperm-egg fusion .

What is the relationship between TMEM95 antibodies and other fertilization-essential protein antibodies?

While TMEM95, IZUMO1, and SPACA6 antibodies all target sperm proteins essential for fertilization, they exhibit distinct effects that reflect the different roles of their target proteins. IZUMO1 antibodies completely block sperm-egg interaction, whereas TMEM95 antibodies specifically inhibit membrane fusion without affecting binding . This difference suggests these proteins function at different stages of fertilization or through different mechanisms. When designing experiments using multiple antibodies, researchers should consider potential synergistic or antagonistic effects. For instance, combining TMEM95 and IZUMO1 antibodies might completely abolish fertilization or provide insights into the sequential nature of their functions. Understanding these relationships is crucial for dissecting the molecular mechanisms of fertilization and developing targeted reproductive interventions .

What controls should be included when using TMEM95 antibodies in sperm penetration assays?

When using TMEM95 antibodies in sperm penetration assays, researchers should implement several critical controls:

Additionally, researchers should quantify both bound and fused sperm per egg to distinguish between binding and fusion effects . This comprehensive approach enables accurate interpretation of how TMEM95 antibodies specifically affect the fertilization process .

How should TMEM95 antibodies be validated for research applications?

Thorough validation of TMEM95 antibodies is essential for reliable research outcomes. A comprehensive validation protocol should include:

• Specificity testing: Western blot analysis comparing wild-type and TMEM95-knockout samples to confirm antibody specificity

• Binding affinity measurement: Biolayer interferometry or similar techniques to determine antibody-antigen binding kinetics

• Epitope mapping: Identifying the specific regions of TMEM95 recognized by the antibody

• Cross-reactivity assessment: Testing against related proteins or TMEM95 from different species

• Functional validation: Confirming that antibodies produce expected effects in sperm penetration assays

• Immunolocalization verification: Comparing antibody staining patterns in permeabilized versus non-permeabilized conditions to confirm proper localization

Researchers should also validate antibodies in the specific experimental conditions they will be used in, as fixation methods can affect antibody performance .

What are the optimal conditions for immunolocalization of TMEM95 using antibodies?

For optimal TMEM95 immunolocalization in sperm cells, researchers should consider several protocol variables:

• Fixation: Both standard fixation (4% PFA for 5 minutes) and alternative protocols (4% PFA at 4°C for 30 minutes) have been successfully used for TMEM95 detection

• Permeabilization: TMEM95 can be detected with or without permeabilization (0.1% Triton X-100 for 10 minutes), with the choice depending on whether researchers want to visualize total TMEM95 or only surface-exposed protein

• Blocking: 5% FCS in PBS for 45 minutes at 4°C effectively reduces non-specific binding

• Antibody incubation: Overnight incubation at 4°C with anti-TMEM95 antibody (1:100 dilution) yields optimal results

• Secondary antibody: For TMEM95 detection, Alexa Fluor 596 goat anti-rabbit IgG (1:500) with 2-hour incubation at room temperature works effectively

• Co-staining: For acrosome visualization, FITC-PNA conjugate (1 μg/ml) can be used alongside TMEM95 antibodies

For dual labeling of TMEM95 and IZUMO1, sequential overnight incubations with respective primary antibodies followed by appropriate secondary antibodies have proven successful .

How can TMEM95 antibodies be used to investigate sperm-egg receptor interactions?

TMEM95 antibodies provide powerful tools for investigating the hypothesized receptor-mediated interaction between sperm TMEM95 and eggs. Researchers can:

• Use competition assays with TMEM95 antibodies and recombinant TMEM95 proteins to identify potential binding partners on egg membranes

• Employ antibody-based pull-down assays to isolate TMEM95-receptor complexes for mass spectrometry analysis

• Utilize antibodies in proximity labeling experiments to identify proteins near TMEM95 during fertilization

• Develop antibody-based blocking strategies that target specific TMEM95 domains to determine which regions are critical for receptor binding

Additionally, TMEM95-Fc fusion proteins can be used alongside antibodies in avidity-based extracellular interaction screening (AVEXIS) to detect direct interactions between TMEM95 and potential egg receptors . These approaches can help identify the elusive TMEM95 receptor on eggs, which would be a significant advancement in reproductive biology .

What are the best approaches for using TMEM95 antibodies to study acrosome reaction dynamics?

TMEM95 undergoes relocalization during the acrosome reaction, making antibodies valuable tools for studying this critical fertilization event. Researchers should:

• Compare TMEM95 localization before and after inducing the acrosome reaction with calcium ionophore (20 μM for 20 minutes)

• Use dual labeling with TMEM95 antibodies and acrosomal markers (such as FITC-PNA conjugate) to track protein redistribution

• Employ time-course experiments with fixed samples at various stages of the acrosome reaction to capture dynamic changes

• Utilize live-cell imaging with fluorescently-labeled TMEM95 antibody fragments to observe real-time protein movement

• Compare TMEM95 and IZUMO1 relocalization patterns using appropriate antibodies to understand their functional relationship

This approach provides insights into how TMEM95 positioning changes during capacitation and the acrosome reaction, helping to elucidate its precise role in the fertilization process .

How can TMEM95 antibodies contribute to developing fertility diagnostics or contraceptives?

The emerging understanding of TMEM95's essential role in fertilization, facilitated by antibody studies, opens several translational research avenues:

• Diagnostic applications: TMEM95 antibodies could be used to develop assays evaluating TMEM95 expression, localization, or function in sperm samples from infertile males, potentially identifying a previously undiagnosed cause of male infertility

• Contraceptive development: The ability of TMEM95 antibodies to significantly reduce sperm-egg fusion without affecting other sperm functions suggests they could serve as models for targeted contraceptives

• Therapeutic strategies: For TMEM95-related infertility, antibody studies might guide the development of assisted reproductive technologies that bypass fusion defects

• Species-specific approaches: Comparative studies using antibodies against TMEM95 from different species could identify conserved functional domains important for contraceptive development

These applications underscore the significant translational potential of TMEM95 antibody research beyond basic scientific discovery .

What new TMEM95 antibody tools are needed to advance the field?

Several new antibody-based tools would significantly enhance TMEM95 research:

• Domain-specific antibodies: Antibodies targeting distinct functional domains of TMEM95, particularly the evolutionarily conserved, positively charged region

• Species-comparative antibodies: Tools to compare TMEM95 function across species, helping identify conserved mechanisms

• Conformation-specific antibodies: Antibodies that recognize TMEM95 in specific conformational states, potentially revealing structural changes during fertilization

• Non-blocking fluorescent tags: Minimally disruptive fluorescent antibody tools for live imaging of TMEM95 during fertilization

• Intrabodies: Cell-permeable antibody fragments for manipulating TMEM95 function within living sperm

Development of these specialized antibody tools would enable more sophisticated investigations into TMEM95's structure-function relationships and its interactions with other fertilization-essential proteins .

How might combinatorial approaches using multiple antibodies advance our understanding of fertilization?

Combinatorial antibody approaches could reveal complex interactions between fertilization proteins:

• Sequential blocking experiments: Using TMEM95 antibodies alongside antibodies against other fertilization proteins (IZUMO1, SPACA6) in specific sequences to determine the order of protein action

• Protein complex identification: Employing proximity-dependent biotinylation with antibody-based targeting to identify TMEM95-associated protein complexes

• Synergistic effects assessment: Testing combinations of antibodies at sub-inhibitory concentrations to identify synergistic relationships between fertilization proteins

• Conditional manipulation: Using antibodies against various proteins under different physiological conditions to understand context-dependent functions

Such approaches could help construct a comprehensive model of the fertilization cascade, defining the precise temporal and spatial relationships between key players like TMEM95, IZUMO1, and their partners .