Abstract

Background

Normal testicular development is essential for maintaining male fertility and reproductive performance in livestock. Leydig cells (LCs) play a central role in testicular physiology; however, the epigenetic mechanisms regulating their development remain largely unclear. Methyltransferase-like 3 (METTL3), a key m6A methylation enzyme, and microRNAs are increasingly recognised as critical regulators of this process.

Results

METTL3 expression in goat LCs markedly decreased during testicular development. This downregulation reduced m6A modification on pri-miR-145, impairing DiGeorge syndrome critical region 8-mediated processing and resulting in decreased levels of mature miR-145-3p. This reduction in miR-145-3p increased the expression of phosphoenolpyruvate carboxykinase 1 (PCK1), which activated gluconeogenesis, increased intracellular glucose levels, and increased mitochondrial membrane potential. Consequently, this metabolic shift upregulated cell cycle-related genes (cyclin B1 and cyclin E2), promoting LC proliferation and testicular growth.

Conclusions

Our findings demonstrate that the METTL3/miR-145-3p/PCK1 axis is a key regulatory pathway linking epigenetic modification to the metabolic activity and proliferation of LCs. This mechanism provides novel insights into the molecular control of testicular development in male goats and may offer new targets for improving male reproductive capacity in livestock.

Data Availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

  • 3-MPA:: 3-Mercaptopicolinic acid
  • AKT:: Protein kinase B
  • ALKBH5 :: AlkB homolog 5
  • AMPK:: AMP-activated protein kinase
  • ATP:: Adenosine triphosphate
  • CCK8:: Cell Counting Kit-8
  • CCNB1 :: Cyclin B1
  • CCNE2 :: Cyclin E2
  • DEPC:: Diethylpyrocarbonate
  • DGCR8:: DiGeorge syndrome critical region 8
  • DNMT3b:: DNA methyltransferase 3 beta
  • EdU:: 5-Ethynyl-2'-deoxyuridine
  • FTO:: Fat mass and obesity-associated protein
  • GATA4:: GATA binding protein 4
  • HE:: Hematoxylin and eosin
  • ID4:: Inhibitor of DNA binding 4
  • IGF1 :: Insulin-like growth factor 1
  • INSL3 :: Insulin-like 3
  • KEGG:: Kyoto Encyclopedia of Genes and Genomes
  • LCs:: Leydig cells
  • lncRNAs:: Long noncoding RNAs
  • m6A:: N6-methyladenosine
  • MeRIP:: Methylated RNA immunoprecipitation
  • METTL3 :: Methyltransferase like 3
  • METTL14 :: Methyltransferase like 14
  • mRNAs:: Messenger RNAs
  • NF-κB:: Nuclear factor kappa-light-chain-enhancer of activated B cells
  • PCK1 :: Phosphoenolpyruvate carboxykinase 1
  • PDGF:: Platelet-derived growth factor
  • PI3k:: Phosphoinositide 3-kinase
  • PLZF :: Promyelocytic leukemia zinc finger protein
  • TLR4 :: Toll-like receptor 4
  • WTAP :: Wilms tumor 1-associating protein

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Acknowledgements

We sincerely thank Xishui Fuxing Husbandry Co., Ltd. for providing experimental animals and the members of the College of Animal Science, Guizhou University, for their technical support and valuable discussions throughout the study.

Funding

This work was supported by the National Natural Science Foundation of China (32260835), Guizhou High- level Innovative Talents Project (Qiankehe Platform Talents [2022] 021–1), and Guizhou Province Mutton Sheep Genetic Improvement and Innovative Utilization Science and Technology Innovation Talent Team Project (Qian Kehe Platform Talent-CXTD[2023]025), and the Natural Science Special Research Fund of Guizhou University (Gui Da Te Gang He Zi [2025] 12), and Project on the Development of a Technical Support System for the Meat Sheep Industry in Guizhou Province (GZRYCYJSTX-02).

Ethics Declaration

Ethics approval and consent to participate

All animal experiments were conducted in accordance with the guidelines approved by the Ethics Committee of Guizhou University (Approval No. EAE-62 U-2022-T055).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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