Identification of Regulatory Mechanisms Involved in Sexual Maturity and Fertility of Female Goats Based on Regulatory Network Analysis and Functional Pathway Approaches

Document Type : Research Paper

Authors

Department of Animal Science, Faculty of Agriculture and Natural Resources, University of Tehran, Karaj, Iran

Abstract

Sexual maturity and fertility in female animals are key components for enhancing reproductive efficiency and breeding strategies in livestock production; however, the gene regulatory mechanisms underlying these processes remain incompletely characterized. In the present study, to investigate the molecular regulations associated with fertility in goats, RNA-Seq data from the ovarian tissue of female Jining Black Grey goats at four developmental stages (birth, two months, four months, and six months old) were analyzed. Genes exhibiting upregulation and downregulation were separately classified. For each group, miRNA–mRNA regulatory networks and related functional modules were reconstructed. Hub genes were identified based on two approaches: multi-partite regulatory network analysis, which led to the identification of hub genes CCDC39, CCDC40, CCDC65, DNAH1, DNAI2, LRGUK, and LOC102177295. Additionally, by assessing gene participation levels in biological pathways, DNAH2, DNAH6, DNAH7, DNAH5, ENSCHIP00000030934, and DNAI2 emerged as hub genes. The gene DNAI2 was identified as a hub gene by both methods. Moreover, two hub miRNAs, mir-187-5p and mir-147a, were identified within the upregulated gene network, demonstrated prominent interactions with their target genes. These genes are involved in various biological processes including ciliary motility, dynein arm assembly, organelle organization, microtubule-based transport, germ cell differentiation, and acrosome activation. The findings of this study provide deeper insights into the genetic regulation affecting sexual maturity and fertility in female goats and may serve as a foundation for developing improved strategies for reproductive management in female goat populations.

Keywords

Main Subjects

Extended Abstract

Introduction

    The global increase in protein demand has made livestock a crucial component of food security. Among livestock, goats have the fastest population growth due to their high reproductive efficiency and resilience to environmental challenges, with over 73% of the world's goat meat production coming from Asia. The Jining Black Grey (JBG) breed is notable for its early puberty and high fertility, making it a valuable candidate for genetic improvement programs. Female puberty is regulated by the hypothalamic-pituitary-gonadal (HPG) axis, initiating reproductive capacity through the body’s hormonal regulation of folliculogenesis and ovulation. While optimizing the age at first estrus can boost reproductive output, the molecular mechanisms involved are still not fully understood. RNA sequencing (RNA-Seq) allows for high-throughput, transcriptome-wide profiling of reproductive tissues, uncovering regulatory elements such as mRNAs and miRNAs. However, systematic analyses across different stages of puberty are limited. This study aims to analyze transcriptome profiles from ovarian samples of female goats at four crucial developmental stages (D1, M2, M4, and M6) to characterize gene expression dynamics, reconstruct miRNA-mRNA interaction networks, and identify functional modules that support sexual maturation.

 

Materials and Methods

This study utilized an ovarian RNA-Seq dataset from JBG female goats, collected at four developmental stages: D1, M2, M4, and M6. The dataset is available in the GEO database under accession number GSE262297, with each age group comprising five biological replicates. Raw reads were assessed for quality using FastQC and subsequently trimmed with Trimmomatic software. The processed sequences were aligned to the genome reference using HISAT2, quantified with FeatureCounts, and analyzed for differential expression with DESeq2. Functional enrichment analyses for detecting biological processes, molecular functions, and cellular components were performed utilizing the DAVID and STRING databases, with KEGG pathway analysis also integrated. For the reconstruction of regulatory networks, protein-protein interaction (PPI) and predicted miRNA-mRNA interactions were sourced from STRING and TargetScan. Functional modules, hub genes and hub miRNAs were identified using the MCODE plugin in Cytoscape, which facilitated the characterization of the molecular frameworks associated with ovarian maturation.

 

Results and Discussion

Transcriptomic analysis of ovarian tissue at four developmental stages identified a total of 27,215 genes. Among these, 712 genes were significantly upregulated, and 214 were downregulated (log₂ fold change ≥ 2, FDR ≤ 0.05). Comparative analysis revealed that 27 transcripts were commonly upregulated across all groups, while no overlap was found among the downregulated transcripts. Reconstruction of the protein-protein interaction (PPI) network showed four modules in the upregulated group and five modules in the downregulated group. Key hub genes such as CCDC39, CCDC40, CCDC65, DNAH1, DNAI2, LRGUK, and LOC102177295 demonstrated extensive connectivity. Further analysis identified additional hub genes based on their participation in biological pathways, which included DNAH2, DNAH6, DNAH7, DNAH5, ENSCHIP00000030934. The gene DNAI2 was identified as a hub gene by both methods. Additionally, two hub miRNAs, mir-187-5p and mir-147a, were identified in the upregulated gene network, showing significant interactions with their target genes. These genes were enriched in pathways associated with dynein arm assembly, ciliary motility, cytoskeletal organization, oocyte maturation, and organelle regulation. Upregulated modules were linked to reproductive processes including folliculogenesis, acrosome activation, oocyte development, and gamete transport. In contrast, downregulated modules indicated a suppression of ciliary assembly, axonemal transport, epithelial organization, and nucleoside metabolism. These findings suggest that coordinated regulation of cilia-related genes and cytoskeletal components is fundamental to ovarian maturation, with implications for oocyte competence and fertilization potential..

 

Conclusion

The findings indicate that combining ovarian transcriptomic profiling with miRNA–mRNA network reconstruction and pathway-based enrichment analysis provides a strong method for investigating key molecular regulators of sexual maturation in female goats. By concentrating on different developmental stages and utilizing bioinformatic tools, this study identified several key genes and interaction modules that play roles in folliculogenesis, ciliary motion, and oocyte maturation. This integrative approach offers valuable insights into the gene regulatory mechanisms that influence fertility and supports the creation of targeted breeding strategies in goat reproductive systems.

 

 

 

 

Author Contributions

Conceptualization, Zeynab Aslzare Razlighi, Seyed Reza Miraei Ashtiani, Mostafa Sadeghi and Farzad Ghafouri; methodology, Zeynab Aslzare Razlighi; formal analysis, Zeynab Aslzare Razlighi and Farzad Ghafouri; writing—original draft preparation, Zeynab Aslzare Razlighi; supervision, Mostafa Sadeghi, Seyed Reza Miraei Ashtiani, and Farzad Ghafouri. All authors have read and agreed to the published version of the manuscript.

 

Data Availability Statement

The datasets analyzed during the current study are publicly available in the NCBI Gene Expression Omnibus (GEO) repository under accession number GSE262297. Additional details are available upon request from the corresponding author.

 

Acknowledgements

The authors would like to thank all participants of the present study.

 

Ethical considerations

The study did not involve human or animal subjects and therefore did not require ethical approval. The authors confirm that no data fabrication, falsification, plagiarism, or misconduct occurred.

 

Conflict of interest

   The author declares no conflict of interest.

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Iranian Journal of animal Science
Volume 57, Issue 2
July 2026
Pages 277-301

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History

  • Receive Date: 03 August 2025
  • Revise Date: 30 September 2025
  • Accept Date: 01 October 2025

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