Recombinant Pongo pygmaeus Sperm protein associated with the nucleus on the X chromosome N2 (SPANXN2)

What is SPANXN2 and how is it characterized in primates?

SPANXN2 (Sperm protein associated with the nucleus on the X chromosome N2) belongs to the SPANX gene family, which is primarily expressed in the testis. The SPANX family in humans includes SPANXA/D genes that encode proteins with distinctive characteristics including high percentages of charged amino acids (>30%) and glutamic acid-rich composition . The Pongo pygmaeus (orangutan) SPANXN genes show evolutionary conservation with human SPANX-N genes, though with some distinctive features.

In terms of biochemical characterization, SPANX proteins typically show variable isoelectric points (pI). Human SPANX-N proteins have pIs ranging from 3.9 to 9.2, while other primate SPANX proteins, including those from orangutans, demonstrate pIs ranging from 4.1 to 9.5 . These properties reflect their charged nature and potential functional roles in nuclear processes during spermatogenesis.

How does SPANXN2 expression differ between normal and pathological tissues?

In normal tissues, SPANXN2 expression is predominantly limited to the testis. Bioinformatics and northern blot analyses reveal that SPANXA/D genes are expressed among normal tissues only in testes . In pathological contexts, particularly in testicular germ cell tumors (TGCTs), SPANXN2 expression is significantly downregulated compared to adjacent normal tissues .

This differential expression pattern has been validated through multiple approaches. The Gene Expression Profiling Interactive Analysis (GEPIA) database demonstrates significant downregulation of SPANXN2 in TGCT tissues compared to normal counterparts. Quantitative real-time PCR (qRT-PCR) analysis of TGCT samples and normal samples confirmed this differential expression pattern . This expression profile suggests SPANXN2 may function as a tumor suppressor in testicular tissues, with its decreased expression potentially contributing to tumor progression.

What are the structural characteristics of recombinant SPANXN2 protein?

Recombinant SPANXN2 protein, like other SPANX family members, is characterized by high glutamic acid content and a significant proportion of charged amino acids (>30% of the total amino acid composition) . SPANX proteins contain predicted PEST sequences, which are regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T) that often serve as signals for protein degradation.

In terms of specific structural features, SPANXN2 shares the common SPANX family trait of having two exons separated by a small intron. While human SPANXA, SPANXC, and SPANXD encode proteins of 97 amino acids, and SPANXB encodes a 103-amino acid protein, the precise length of Pongo pygmaeus SPANXN2 would need to be determined through sequence analysis and comparison . This structural information is crucial for producing functional recombinant protein for experimental applications.

What role does SPANXN2 play in normal cellular processes?

SPANXN2, like other SPANX family proteins, appears to play specialized roles in spermatogenesis. The SPANX proteins demonstrate unique morphogenetic movements during spermatid nuclear condensation, being restricted to non-acrosomal regions of the spermatid nuclear envelope . This localization pattern suggests SPANXN2 may be involved in nuclear remodeling during spermiogenesis.

In the context of primate evolution, SPANX gene family replication was initiated 6–11 million years ago when primates were becoming more human-like. This evolutionary timing suggests that SPANXN2 and related proteins may contribute to species-specific aspects of post-meiotic spermatid nuclear differentiation . Comparative studies between human and Pongo pygmaeus SPANXN2 could provide insights into evolutionary adaptations in sperm development across primate species.

How does SPANXN2 function as a migration inhibitor in cancer cells?

Research has demonstrated that SPANXN2 functions as a cell migration inhibitor in testicular germ cell tumors. When SPANXN2 is overexpressed in TGCT cell lines (TCAM-2 and NCCIT), it significantly reduces cell migration capability as measured by both wound healing and transwell migration assays .

The mechanism behind this inhibitory effect involves modulation of epithelial-mesenchymal transition (EMT) and AKT signaling pathways. Western blot analysis revealed that SPANXN2 overexpression downregulates the expression of EMT-related proteins including Vimentin and Snail. Additionally, SPANXN2 overexpression reduced the levels of AKT and phosphorylated AKT (p-AKT) . These molecular changes collectively contribute to decreased cell migration, suggesting that loss of SPANXN2 expression in tumors may promote metastatic potential.

Does SPANXN2 affect cell proliferation and apoptosis?

Interestingly, while SPANXN2 exhibits clear effects on cell migration, research indicates that it does not significantly impact cell proliferation or apoptosis. In TGCT cell lines transfected with SPANXN2 expression plasmids, MTT assays revealed no significant difference in cell proliferation rates compared to control groups .

Similarly, flow cytometric analysis performed 48 hours after transfection showed no significant differences in cell cycle distribution between SPANXN2-overexpressing cells and control cells. No marked cell apoptosis was observed at 24 and 48 hours after transfection with the SPANXN2 plasmid . These findings suggest that SPANXN2's tumor suppressive function operates primarily through inhibition of migration rather than through effects on proliferation or apoptosis, pointing to a specialized role in regulating cellular motility.

What are the optimal conditions for expressing recombinant SPANXN2 in experimental systems?

For effective expression of recombinant SPANXN2, researchers should consider the following methodological approach:

• Vector Selection: Based on successful expression studies, plasmid vectors with strong promoters (such as CMV) are recommended for mammalian cell expression. For TGCT cell transfection studies, plasmid transfection has achieved >75% efficiency as confirmed by fluorescence markers .

• Expression System: For functional studies, mammalian expression systems are preferable to maintain proper post-translational modifications. TGCT cell lines such as TCAM-2 and NCCIT have been successfully used for SPANXN2 overexpression .

• Transfection Protocol: Transient transfection protocols using lipid-based reagents have proven effective, with confirmation of expression by quantitative RT-PCR using β-actin as a reference gene .

• Validation Approaches: Expression should be validated through multiple methods, including fluorescence microscopy for tagged proteins and qRT-PCR for transcript quantification. Western blotting with specific antibodies provides confirmation of protein expression levels .

When adapting these protocols specifically for Pongo pygmaeus SPANXN2, codon optimization may be necessary to ensure efficient expression in commonly used experimental systems.

What techniques are most effective for studying SPANXN2 localization in cells?

For studying SPANXN2 localization, immunolocalization techniques have proven most effective. Based on studies of human SPANX proteins, the following methodology is recommended:

• Antibody Selection: Using antibodies that specifically recognize SPANXN2 is crucial. For cross-species studies, antibodies targeting conserved regions of SPANX proteins should be selected to enable comparison between human and Pongo pygmaeus SPANXN2 .

• Fixation Protocol: Appropriate fixation is critical for maintaining nuclear architecture. Paraformaldehyde fixation (typically 4%) followed by permeabilization with a detergent such as Triton X-100 allows for preservation of nuclear structures while enabling antibody access .

• Imaging Techniques: Confocal microscopy provides the necessary resolution to observe nuclear localization patterns. Co-staining with nuclear envelope markers and chromatin stains helps establish the precise subnuclear localization .

• Developmental Staging: For studies in testicular tissue, careful staging of spermatogenesis is essential to track the dynamic localization of SPANXN2 during spermiogenesis, as SPANX proteins show stage-specific localization patterns .

These approaches enable detailed characterization of SPANXN2's subcellular distribution and potential interactions with other nuclear components.

How can researchers effectively measure SPANXN2's effects on cell migration?

To evaluate SPANXN2's effects on cell migration, researchers should employ multiple complementary assays as demonstrated in previous studies:

• Transwell Migration Assay: This quantitative method involves seeding cells in the upper chamber of a transwell insert and counting cells that migrate through a membrane toward a chemoattractant in the lower chamber. For SPANXN2 studies, significant differences have been observed 48 hours post-transfection .

• Wound Healing Assay: This method involves creating a "wound" in a cell monolayer and measuring the rate at which cells migrate to close the gap. Measurements at multiple time points (e.g., 24h and 48h) provide dynamic information about migration rates .

• Controls and Validation: Proper controls include empty vector transfection and untransfected cells. Validation of SPANXN2 expression levels by qRT-PCR and Western blot is essential to correlate expression with observed phenotypic effects .

• Molecular Pathway Analysis: To understand mechanisms, Western blot analysis of EMT markers (Vimentin, Snail) and signaling proteins (AKT, p-AKT) should be performed in parallel with migration assays to correlate behavioral changes with molecular alterations .

This multi-assay approach provides robust assessment of SPANXN2's effects on cellular motility and the underlying molecular mechanisms.

How does Pongo pygmaeus SPANXN2 differ from human SPANXN2 in structure and function?

Comparative analysis of Pongo pygmaeus and human SPANXN2 reveals both conservation and divergence:

The SPANX gene family shows evidence of rapid evolution under positive selective pressure, with coding regions evolving faster than non-coding regions . While specific data on Pongo pygmaeus SPANXN2 is limited in the available literature, analysis of related SPANX-N proteins shows that orangutan SPANX proteins maintain the characteristic high percentage of charged amino acids seen in human SPANX proteins .

Functional differences may exist due to evolutionary adaptation, though these would need to be experimentally determined. Of note, orangutan SPANX-N3 contains two predicted PEST regions, similar to human SPANXB, SPANX-N3, and SPANX-N4 proteins . This suggests some conservation of regulatory elements that may affect protein stability and turnover.

For definitive comparison, recombinant expression of both human and Pongo pygmaeus SPANXN2, followed by detailed biochemical characterization and functional assays, would be necessary to identify species-specific properties that might reflect evolutionary adaptations in sperm development or other functions.

What is the relationship between SPANXN2 expression and tumor progression in clinical samples?

The relationship between SPANXN2 expression and tumor progression appears significant based on available research. SPANXN2 expression is downregulated in testicular germ cell tumors compared to adjacent normal tissues, suggesting its potential role as a tumor suppressor .

This expression pattern has important clinical implications. Given that SPANXN2 inhibits cell migration in vitro, its reduced expression in tumors may contribute to increased metastatic potential. While SPANXN2 does not affect cell proliferation, it significantly inhibits colony formation ability, indicating its influence on long-term cellular survival and establishment .

For definitive clinical correlations, larger scale studies examining SPANXN2 expression across tumor stages and correlating with patient outcomes would be valuable. The gene's location on chromosome Xq27, a region associated with susceptibility to TGCT and prostate malignancy, further suggests its potential importance in cancer progression . Comprehensive analysis of SPANXN2 expression in patient cohorts, correlated with clinical parameters such as metastasis, recurrence, and survival, would provide crucial insights into its prognostic value.

How can SPANXN2 research contribute to understanding the evolution of reproductive proteins across primates?

SPANXN2 research provides a valuable window into the evolution of reproductive proteins across primates for several reasons:

The SPANX gene family originated relatively recently in evolution, with replication initiated 6–11 million years ago during primate evolution toward human-like characteristics . This timing makes SPANX genes, including SPANXN2, particularly interesting for studying recent adaptive evolution in reproductive proteins.

Comparative analysis of SPANX genes across primates reveals significant differences: human SPANXA, SPANXC, and SPANXD encode 97-amino acid proteins, while SPANXB contains an 18-nucleotide insertion and encodes a 103-amino acid protein that is absent in Gorilla and Pan . These molecular differences may reflect species-specific adaptations in sperm development.

Furthermore, the expression patterns of SPANX genes show individual variation even within species. In humans, not all SPANX-A/D genes are expressed in all individuals, with SPANXC expressed in only a subset of individuals . This polymorphic expression adds another layer of complexity to understanding the evolution and function of these genes.

Studying Pongo pygmaeus SPANXN2 in comparison with human and other primate SPANX proteins can provide insights into how reproductive proteins evolve under sexual selection and what functional consequences these changes may have for sperm development and function across different primate lineages.

Through which signaling pathways does SPANXN2 inhibit cell migration?

SPANXN2 inhibits cell migration through at least two interconnected signaling pathways:

• EMT-Related Pathway: SPANXN2 overexpression downregulates key EMT-related proteins, including Vimentin and Snail . Epithelial-mesenchymal transition is a process whereby epithelial cells gain migratory and invasive properties, becoming mesenchymal-like. By suppressing these EMT markers, SPANXN2 helps maintain a less migratory cellular phenotype.

• AKT Signaling Pathway: SPANXN2 overexpression reduces both total AKT and phosphorylated AKT (p-AKT) protein levels . The AKT pathway is a central regulator of various cellular processes including migration, and its inhibition by SPANXN2 likely contributes to the reduced migratory capacity of TGCT cells.

These pathways are likely interconnected, as AKT signaling can promote EMT through various mechanisms including activation of transcription factors that regulate EMT markers. The dual suppression of both pathways by SPANXN2 suggests a coordinated mechanism for inhibiting cell migration, potentially explaining its tumor suppressive function in testicular tissues.

What protein interactions does SPANXN2 form during spermatogenesis?

While specific protein interactions of SPANXN2 during spermatogenesis have not been fully characterized in the provided search results, studies of SPANX family proteins provide some insights:

SPANX proteins undergo unique morphogenetic movements during spermatid nuclear condensation, being restricted to non-acrosomal regions of the spermatid nuclear envelope . This specific localization suggests potential interactions with nuclear envelope proteins and chromatin components during sperm development.

The presence of PEST sequences in many SPANX proteins, including SPANX-N proteins from primates, suggests that these proteins may undergo regulated degradation . This implies possible interactions with components of the ubiquitin-proteasome system that regulate protein turnover during spermatogenesis.

For comprehensive identification of SPANXN2 protein interactions, techniques such as co-immunoprecipitation followed by mass spectrometry, proximity labeling approaches (BioID or APEX), or yeast two-hybrid screening would be necessary. These approaches could reveal binding partners that mediate SPANXN2's functional roles in both normal spermatogenesis and potential tumor suppressive functions.

How does post-translational modification affect SPANXN2 function?

While the search results do not provide specific information about post-translational modifications (PTMs) of SPANXN2, several aspects of SPANX protein structure suggest important roles for PTMs:

The high content of serine and threonine residues in SPANX proteins, particularly within PEST sequences, suggests potential sites for phosphorylation . Phosphorylation could regulate protein stability, localization, or interactions with other proteins.

The dynamic localization of SPANX proteins during spermiogenesis implies regulated transport mechanisms that may involve PTMs . Nuclear envelope association may be regulated by modifications that affect protein-protein or protein-membrane interactions.

For experimental investigation of SPANXN2 PTMs, methodologies such as mass spectrometry-based phosphoproteomics, site-directed mutagenesis of potential modification sites, and the use of modification-specific antibodies would be valuable approaches. Understanding how PTMs regulate SPANXN2 function could provide insights into both its normal role in spermatogenesis and its potential tumor suppressive functions in testicular tissues.

How has the SPANX gene family evolved across primate species?

The SPANX gene family demonstrates fascinating evolutionary dynamics across primate species:

The SPANX gene family originated relatively recently, with replication initiated 6–11 million years ago when primates were evolving toward more human-like characteristics . This recent origin makes it particularly valuable for studying gene family evolution.

The SPANX genes show evidence of rapid evolution under positive selective pressure. The SPANXA/D coding regions exhibited a 2-fold acceleration in the rate of synonymous and non-synonymous substitutions compared with non-coding regions, suggesting that selective pressures are acting specifically on the protein-coding sequences .

Structural differences exist between species: human SPANXB contains an 18-nucleotide insertion encoding a 103-amino acid protein that is absent in Gorilla and Pan species . This indicates species-specific adaptations within the gene family.

The Pongo pygmaeus (orangutan) SPANX genes maintain the characteristic features of the family, including high glutamic acid content and a high percentage of charged amino acids, but may have species-specific features that reflect evolutionary adaptations in reproductive biology .

This evolutionary pattern suggests that SPANX genes, including SPANXN2, may be involved in species-specific aspects of reproduction, potentially contributing to reproductive isolation and speciation processes.

What are the key differences in protein sequence and expression patterns between human and Pongo pygmaeus SPANXN2?

While comprehensive comparative data specifically on SPANXN2 between humans and orangutans is not fully detailed in the search results, analysis of the SPANX family provides some insights:

PEST sequences, which are regions associated with protein degradation, are present in various SPANX proteins across species. Specifically mentioned is that orangutan SPANX-N3 contains two predicted PEST regions, similar to human SPANXB, SPANX-N3, and SPANX-N4 proteins . This suggests some conservation of regulatory elements affecting protein stability.

For definitive characterization of differences, direct sequence comparison and expression analysis of SPANXN2 from both species would be necessary, along with functional studies to determine if these differences result in altered biological activities.

What role might SPANXN2 play in reproductive isolation between primate species?

The potential role of SPANXN2 in reproductive isolation between primate species is a fascinating evolutionary question:

The rapid evolution of SPANX genes under positive selection is consistent with patterns observed in reproductive proteins that often evolve rapidly due to sexual selection or sexual conflict. Such rapid evolution can contribute to reproductive barriers between species.

The SPANX gene family originated 6–11 million years ago, coinciding with significant divergence in the primate lineage . This timing suggests these genes may have played a role in the reproductive differentiation of emerging primate species.

Individual variation in SPANX gene expression even within species (as shown by the variable expression of SPANXC in individual human testes) adds another layer of complexity that could influence reproductive compatibility.

The localization of SPANX proteins to specific regions of developing sperm nuclei suggests they may influence sperm morphology or function in species-specific ways, potentially affecting cross-species fertilization success.

While direct evidence of SPANXN2's role in reproductive isolation is not presented in the search results, its characteristics as a rapidly evolving, testis-specific protein make it a candidate for contributing to reproductive barriers between primate species. Comparative functional studies examining the effects of human versus Pongo pygmaeus SPANXN2 on sperm development and function could provide insights into this evolutionary question.

What are the most promising therapeutic applications of SPANXN2 research?

Given SPANXN2's role as a migration inhibitor in testicular germ cell tumors, several promising therapeutic directions emerge:

• Diagnostic Biomarker Development: The downregulation of SPANXN2 in TGCTs compared to normal testicular tissue suggests its potential as a diagnostic biomarker. Further validation in larger patient cohorts could establish SPANXN2 expression as a clinically useful indicator of tumor development or progression.

• Prognostic Indicator: Since SPANXN2 inhibits cell migration, its expression levels might correlate with metastatic potential and patient outcomes. Research examining correlations between SPANXN2 expression and clinical progression could establish its value as a prognostic marker .

• Therapeutic Target: Restoring SPANXN2 expression or function in tumors represents a potential therapeutic strategy. Gene therapy approaches delivering SPANXN2 to tumor cells or small molecules that mimic its effects on EMT and AKT signaling pathways could inhibit tumor cell migration and potentially reduce metastatic spread .

• Combination Therapies: Given that SPANXN2 affects migration but not proliferation , combining SPANXN2-targeted therapies with conventional cytotoxic approaches might provide complementary anti-tumor effects, addressing both growth and metastatic potential simultaneously.

These applications would require further research to validate SPANXN2's clinical utility and develop effective intervention strategies based on its biological functions.

What key questions remain unanswered about SPANXN2 function across primate species?

Despite the progress in understanding SPANXN2, several critical questions remain unanswered regarding its function across primate species:

• Functional Conservation: To what extent is SPANXN2 function conserved between humans and other primates, including Pongo pygmaeus? Does it maintain similar roles in sperm development and tumor suppression across species?

• Regulatory Mechanisms: What mechanisms control the species-specific and individual-specific expression patterns of SPANX genes? The variable expression of SPANXC among individual humans raises questions about regulatory mechanisms that might also apply to SPANXN2.

• Evolutionary Pressure: What specific selective pressures drove the rapid evolution of SPANX genes in primates? Does this reflect sexual selection, pathogen resistance, or other evolutionary forces?

• Reproductive Implications: Does variation in SPANXN2 structure or expression between species contribute to sperm function differences or reproductive barriers?

• Comprehensive Interaction Networks: What are the complete protein interaction networks of SPANXN2 in different contexts (normal spermatogenesis versus cancer) and how do these compare across primate species?

Addressing these questions would require comparative studies across primate species, combining genomic, proteomic, and functional approaches to build a comprehensive understanding of SPANXN2's evolutionary and functional significance.

What novel methodologies could advance SPANXN2 research in comparative primatology?

Advancing SPANXN2 research in comparative primatology would benefit from several innovative methodological approaches:

• CRISPR-Based Genome Editing: Using CRISPR/Cas9 to create species-specific mutations or to "humanize" SPANXN2 in model organisms could help evaluate functional differences between primate variants. This approach could reveal how specific sequence differences translate to functional divergence.

• Single-Cell Transcriptomics: Applying single-cell RNA sequencing to testicular tissue from different primate species would provide unprecedented resolution of SPANXN2 expression patterns during spermatogenesis, revealing species-specific regulation and potential co-expression networks.

• Cryo-Electron Microscopy: Structural determination of SPANXN2 proteins from different primates using cryo-EM could reveal subtle structural differences that might influence function, particularly in nuclear localization or protein-protein interactions.

• Organoid Models: Developing testicular organoids from different primate species would provide valuable in vitro systems for studying SPANXN2 function in a species-specific cellular context while reducing the need for primate specimens.

• Comparative Proteomics: Applying techniques such as BioID or APEX proximity labeling followed by mass spectrometry across species would identify species-specific protein interaction networks for SPANXN2, potentially revealing functional divergence.