Recombinant Human T-complex protein 11 homolog (TCP11)

What is the expression pattern of human TCP11 compared to its mouse ortholog?

Human TCP11 shows a testis-enriched expression pattern with detectable expression in brain and epididymis, suggesting broader functionality compared to mouse Tcp11. In contrast to the mouse, RT-PCR detects human TCP11 in the brain and epididymis in addition to strong expression in the testis . This tissue distribution pattern indicates that human TCP11 likely serves functions beyond those identified in mouse models, particularly in neural and reproductive tissues outside the testes.

How is TCP11 genetically conserved across species?

TCP11 is highly evolutionarily conserved, suggesting fundamental biological importance. The TCP11 domain is specific to TCP11 homologs and has been identified across multiple species. Interestingly, this domain shares approximately 25% identity and 49% similarity with the yeast protein SOK1, indicating ancient evolutionary origins . The conservation of this protein across diverse species points to its essential biological function that has been maintained throughout evolutionary history.

What protein isoforms of TCP11 exist and how can they be detected?

Multiple TCP11 isoforms have been documented with varying molecular weights:

These isoforms can be detected using specific antibodies targeting different regions of TCP11. Research has utilized antibodies raised against amino acids 15-32 of mouse TCP11 as well as full-length TCP11 antibodies . When developing detection methods for human TCP11 isoforms, researchers should consider the possible presence of multiple variants with distinct functional properties.

How does TCP11 influence sperm motility at the molecular level?

TCP11 appears to function through the cAMP/PKA signaling pathway, which is crucial for regulating sperm motility. Sperm from Tcp11-null mice showed decreased PKA activity compared to wild-type controls, suggesting that TCP11 positively regulates this pathway . Western blot analysis using an antibody recognizing the phosphorylated target motif of PKA (R-R-X-S/T-X) revealed decreased signal in Tcp11 knockout samples, confirming reduced phosphorylation of PKA targets .

The molecular mechanism likely involves:

• TCP11 expression during spermiogenesis

• Influence on PKA signaling components

• Resulting impact on flagellar function and energy metabolism

This suggests that human recombinant TCP11 could potentially serve as a research tool for investigating PKA pathway modulation in human sperm motility disorders.

What are the experimental challenges in determining TCP11 localization in cells?

Contradictory findings regarding TCP11 localization highlight important methodological considerations:

SignalP and transmembrane prediction algorithms do not detect a signal sequence or transmembrane region in TCP11, challenging earlier claims of surface localization . When studying human TCP11 localization, researchers should employ multiple detection methods, include appropriate controls (ideally knockout validations), and use fractionation approaches to distinguish between true localization and artifacts.

How do TCP11 paralogs differ in their expression and function?

The TCP11 gene family exhibits diverse expression patterns and potentially distinct functions:

This diversity suggests that while TCP11 itself may have specialized reproductive functions, the gene family as a whole might have broader physiological roles . When studying human TCP11, researchers should consider potential functional redundancy or complementation by its paralogs.

What approaches are effective for studying TCP11 function in fertility research?

Multiple complementary methods can assess TCP11's role in fertility:

• In vivo fertility assessment: Breeding studies tracking pregnancy rates and litter sizes provide functional endpoints. Tcp11-null male mice showed severe subfertility, siring significantly fewer pups compared to control males over a three-month period .

• In vitro fertilization (IVF) with varying conditions:

  • Tcp11-null sperm failed to fertilize cumulus-intact and cumulus-free oocytes

  • The same sperm successfully fertilized zona pellucida-free oocytes

  • Partial rescue was achieved by treating eggs with glutathione

• Computer-Assisted Sperm Analysis (CASA): Provides objective measurements of sperm motility parameters, revealing decreased motility in Tcp11-null sperm .

These approaches collectively provide a comprehensive assessment of TCP11's functional impact on fertility.

How can researchers effectively generate and validate TCP11 antibodies?

Development of specific TCP11 antibodies requires careful validation:

• Target selection: Choose unique epitopes based on sequence analysis across species

  • N-terminal antibodies (amino acids 15-32) successfully detected 62kD and 54kD isoforms

  • Full-length protein antibodies may detect additional isoforms

• Validation methods:

  • Western blot with wild-type and knockout tissue (essential control)

  • Testing multiple extraction conditions (RIPA buffer with 5mM DTT)

  • Immunofluorescence with co-localization markers

• Cross-reactivity assessment: Commercial antibodies showed several non-specific bands, highlighting the importance of knockout controls .

When developing antibodies against human TCP11, researchers should prioritize validation with appropriate negative controls and multiple detection methods.

What experimental design is most effective for studying recombinant TCP11 in PKA pathway research?

Based on TCP11's involvement in the PKA pathway, researchers should consider:

• PKA activity assays:

  • Measure phosphorylation of PKA target proteins using phospho-specific antibodies

  • Compare activity in the presence/absence of recombinant TCP11

  • Assess dose-dependent effects

• Interaction analyses:

  • Co-immunoprecipitation to identify binding partners

  • Proximity ligation assays to confirm in situ interactions

  • Pull-down assays with recombinant proteins

• Rescue experiments:

  • Add recombinant TCP11 to Tcp11-null sperm

  • Measure restoration of PKA signaling and motility

  • Evaluate structure-function relationships with mutated forms

This multi-faceted approach would provide mechanistic insight into how TCP11 influences PKA signaling in reproductive physiology.

How should researchers approach functional studies of human TCP11 given the limitations of animal models?

Mouse Tcp11 studies provide important insights, but researchers must consider species differences:

• Expression pattern differences:

  • Human: Testis-enriched with brain and epididymis expression

  • Mouse: Predominantly testis-specific

• Methodological considerations:

  • Generate humanized mouse models expressing human TCP11

  • Utilize human tissue/cell samples when available

  • Perform comparative studies across species

  • Consider the implications of different paralog expression patterns

• Translational approaches:

  • Correlate TCP11 variants in human male infertility cases

  • Study TCP11 expression in testicular biopsies from infertile men

  • Investigate potential roles in brain tissue based on expression pattern

These approaches would help bridge the gap between mouse model findings and human applications while accounting for species-specific differences in TCP11 biology.

What are the implications of TCP11 variants in human male infertility?

Databases list approximately 8,000 variants in human TCP11, though their clinical significance remains largely undetermined . Given the severe motility defects in Tcp11-null mice, human TCP11 variants may contribute to asthenozoospermia (reduced sperm motility). Potential research approaches include:

• Screening infertile men for TCP11 variants

• Functional characterization of identified variants using recombinant proteins

• Development of in vitro assays to assess variant TCP11 effects on PKA signaling

• Correlation of variant types with specific motility parameter defects

This line of investigation could yield valuable diagnostic and potentially therapeutic insights for male infertility cases with unexplained motility defects.

How might tissue-specific expression of human TCP11 in brain and epididymis relate to its function?

Unlike mouse Tcp11, human TCP11 exhibits expression in brain and epididymis, suggesting expanded functionality . Investigation into these non-testicular roles could involve:

• Region-specific expression analysis in brain tissue

• Evaluation of TCP11 in epididymal secretions and their impact on sperm maturation

• Assessment of potential neurological phenotypes in TCP11-deficient models

• Exploration of PKA pathway involvement in brain regions expressing TCP11