Comparative analysis of mRNA and circRNA expression profiles and interaction networks in testicular tissues of Mongolian horses before and after sexual maturity.

Researchers compared RNA molecules in the testes of Mongolian horses before and after sexual maturity and found widespread changes in gene and circular RNA activity. They mapped how specific circular RNAs may control gene expression through microRNAs, spotlighting TGF-β signaling and a newly validated regulatory axis tied to spermatogenesis.

Study goal and rationale

• Determine how gene (mRNA) and circular RNA (circRNA) expression changes as stallion testes transition from immature to sexually mature states.
• Reveal regulatory networks—especially circRNA–miRNA–mRNA interactions—that may coordinate testicular development and spermatogenesis in Mongolian horses.

Experimental overview

• Whole-transcriptome RNA sequencing of testicular tissue from pre- and post-sexual maturity animals to profile mRNAs and circRNAs.
• Histological assessment using H.E. and DAPI staining to confirm morphological differences between developmental stages.
• Differential expression analysis with DESeq2 to identify stage-associated changes in mRNAs and circRNAs.
• Functional interpretation via Gene Ontology (GO) and KEGG pathway enrichment.
• Integration of circRNA “host-gene” annotations to relate circRNAs to their originating genes.
• Construction of circRNA–miRNA–mRNA regulatory (ceRNA) networks and targeted experimental validation by real-time PCR; functional validation of a specific ceRNA axis.

Key histological findings

• H.E. and DAPI staining showed clear, stage-specific testicular morphology, consistent with immature versus mature tissue architecture and cell composition.
• These structural differences support the biological relevance of the transcriptome shifts observed.

Differential expression highlights

• Genes: 10,317 differentially expressed genes between stages (5,280 upregulated and 5,033 downregulated).
• Circular RNAs: 356 differentially expressed circRNAs (263 upregulated and 93 downregulated).
• Real-time PCR validation confirmed the reliability of RNA-seq trends for selected transcripts.

Functional enrichment (GO/KEGG) and biological themes

• Enriched GO terms point to reproductive development and gamete generation, aligning with expected maturation processes.
• KEGG pathways enriched among differentially expressed genes include signaling cascades central to testis development and cell fate regulation.
• Together, functional annotations suggest coordinated activation/suppression of pathways governing spermatogenesis, germ cell differentiation, and testicular microenvironment remodeling.

circRNAs and their host genes

• Annotation of differentially expressed circRNAs to their host genes revealed congruent expression trends in selected pairs, indicating shared regulatory control or co-regulation.
• Identification of eca_circ_0005223 and its host gene TGFBRAP1 as highly expressed in immature testes implicates TGF-β pathway involvement early in testicular development.
• This points to a potential mechanism where circRNAs derived from pathway components reinforce stage-specific signaling states.

Regulatory network: circRNA–miRNA–mRNA (ceRNA) model

• Authors constructed interaction networks showing circRNAs potentially “sponging” miRNAs, thereby relieving repression on target mRNAs.
• A validated axis—eca_circ_0008614 → eca-miR-432 → AASDH—demonstrates a post-transcriptional control route relevant to spermatogenesis.
• In this model, eca_circ_0008614 likely binds eca-miR-432, reducing its availability to suppress AASDH, ultimately modulating gene output important for germ cell processes.

Biological interpretation and mechanistic insights

• TGF-β signaling emphasis: Elevated eca_circ_0005223/TGFBRAP1 in immature testes suggests TGF-β pathway components help establish early testicular architecture and cell-state programs (e.g., Sertoli/germ cell communication, proliferation cues).
• ceRNA control of spermatogenesis: The eca_circ_0008614–eca-miR-432–AASDH axis provides a concrete example of how circRNAs can tune gene expression during germ cell development.
• System-level view: Thousands of mRNA and hundreds of circRNA changes indicate a broad transcriptional and post-transcriptional reconfiguration accompanying sexual maturation.

Strengths of the study

• Multi-layered design integrates morphology, transcriptomics, bioinformatics, and experimental validation.
• Large set of differentially expressed transcripts offers a comprehensive catalog of maturation-associated changes.
• Both pathway-level (GO/KEGG) and specific network-level (ceRNA axis) evidence strengthen biological interpretability.

Limitations to consider

• Abstract does not specify sample size, biological replicates, or statistical thresholds, which are important for assessing robustness.
• Most findings are correlative; functional causality beyond the validated ceRNA axis remains to be established.
• Testicular tissue heterogeneity (cell-type composition shifts) may confound bulk expression changes without cell-type–resolved analyses.
• Species- and breed-specific regulatory features may limit generalization to other horses or mammals.

Implications and applications

• Foundational resource for understanding molecular programs driving stallion sexual maturation.
• Candidate biomarkers (mRNAs, circRNAs, and ceRNA relationships) for monitoring testicular development and reproductive health.
• Potential targets for improving breeding management, fertility assessment, and conservation strategies in equine populations.
• Comparative value for reproductive biology across mammals, informing mechanisms of spermatogenesis and testis maturation.

Future directions

• Functional perturbation of key nodes (e.g., eca_circ_0005223, eca_circ_0008614, TGFBRAP1, AASDH, eca-miR-432) via knockdown/overexpression in relevant testicular cell types.
• Single-cell or spatial transcriptomics to resolve cell-type–specific dynamics and microenvironmental signaling.
• Proteomic and phospho-signaling assays to link transcript changes to pathway activity, especially for TGF-β signaling.
• Longitudinal sampling across developmental milestones to refine timing and sequence of regulatory shifts.
• Cross-breed and cross-species comparisons to identify conserved versus lineage-specific regulatory circuits.

Key terms and concepts

• circRNA: Covalently closed RNA molecules often acting as molecular sponges for miRNAs, thereby modulating target mRNA expression.
• miRNA: Small non-coding RNAs that typically repress target mRNAs by promoting degradation or inhibiting translation.
• ceRNA network: Competing endogenous RNA interactions where RNAs (e.g., circRNAs) sequester miRNAs, influencing the expression of other mRNAs.
• Host gene: The gene locus from which a circRNA originates; expression of host gene and circRNA can be co-regulated.
• DESeq2: A statistical framework for identifying differentially expressed genes/RNAs from count-based RNA-seq data.
• GO/KEGG: Databases for functional annotation and pathway mapping used to interpret large gene lists in biological context.