Multi-omics profiling of the testes in Kazakh horses across sexual maturation.

Overview

• This research investigates the molecular changes in the testes of Kazakh horses during sexual maturation by analyzing multiple biological layers including RNA, proteins, and metabolites.
• The study offers a comprehensive regulatory network that elucidates how various molecules interact to regulate testicular development and sexual maturity in horses.

Research Objective and Background

• Equus caballus (the domestic horse) holds significant economic and cultural value.
• Understanding the molecular mechanisms behind sexual maturation in horses is essential to improve reproductive success and breeding strategies.
• Prior to this study, the regulatory networks controlling equine sexual maturation were not well understood.
• The focus was on Kazakh horses at two key developmental stages: 2 years old (pre-maturation) and 3 years old (post-maturation).

Methodological Approach

• Sample Collection:
  • Testicular tissues were collected from eight Kazakh horses (four at 2 years and four at 3 years).
• Multi-omics Profiling Techniques:
  • Whole transcriptome sequencing to profile coding and non-coding RNAs, including circRNAs and LncRNAs.
  • Data-independent acquisition (DIA) proteomics to quantify protein expression changes.
  • Untargeted metabolomics to identify and quantify metabolites present in testicular tissues.
• Integration:
  • Constructed a regulatory network combining circular RNA (circRNA), long non-coding RNA (LncRNA), microRNA (miRNA), mRNA, proteins, and metabolites.

Key Findings in Transcriptomics

• Significant alterations were found in the expression of several types of RNAs during sexual maturation:
  • Circular RNAs (circRNAs) and long non-coding RNAs (LncRNAs) changed significantly.
  • Key microRNAs identified included eca-miR-34-5p, eca-miR-34c, eca-miR-449a, and eca-miR-486-3p.
  • Target genes regulated by these miRNAs included TRAF1, TRAF3, ALPK3, SLC29A4, and CPNE3.
• A total of 277 genes showed differential expression at both mRNA and protein levels, underscoring their importance in testicular development.

Functional Pathways and Biological Processes

• The genes and proteins identified were mainly involved in pathways such as:
  • PI3K-AKT signaling pathway – critical for cell growth and survival.
  • Focal adhesion – important for cell adhesion and communication with the extracellular matrix.
  • Extracellular matrix (ECM) receptor interactions – essential for tissue structure and signaling.
  • Phospholipase D signaling – associated with lipid signaling and membrane trafficking.
  • Ovarian steroidogenesis – hormone biosynthesis fundamental for reproductive function.

Regulatory RNA Networks

• LncRNAs and circRNAs were found to influence gene expression indirectly by interacting with miRNAs:
  • COL1A1 gene expression was regulated by competitive binding of miR-7844-x to LncRNAs and circRNAs.
  • SPARC gene regulation was modulated through interactions involving eca-miR-146-5p and eca-miR-328 with LncRNAs or circRNAs.

Proteomic and Metabolomic Insights

• Several central proteins were identified that may play key roles during testicular maturation:
  • TAP1, COL14A1, PRDX3, and SEBENP1 were highlighted as core proteins.
• Key metabolites associated with testicular development included:
  • Guanosine
  • Austocystin B
  • Methanone
  • Glycerophosphocholine
• These molecules were predominantly involved in metabolic and biosynthesis pathways such as:
  • Ovarian steroidogenesis
  • Steroid hormone biosynthesis
  • Propionate metabolism

Implications and Conclusions

• The study provides a detailed molecular map of the regulatory networks driving testicular development and sexual maturation in Kazakh horses.
• It highlights the interplay among non-coding RNAs, mRNAs, proteins, and metabolites linking transcriptional and post-translational regulatory processes.
• The findings may help improve reproductive strategies and breeding efficiency by identifying molecular markers and pathways critical for sexual maturation.
• This integrative multi-omics approach could be applied to other species for advancing reproductive biology research.