Single-nucleus RNA sequencing reveals developmental dynamics and cellular heterogeneity in the mammary gland of young fillies and adult Mongolian mares.
Abstract: Mongolian horses are famous for their lactation traits. Their milk contains a high protein content and low levels of fatty acids. Given their superior milk composition and historical use in dairy production across Inner Mongolia and Central Asia, Mongolian horses serve as a valuable model for understanding lactational biology. Multiple factors regulate the lactation process; however, a detailed study of this biological process has not been performed with single-nucleus RNA sequencing (snRNA-seq) technology. Insights gained from snRNA-seq of their mammary glands can inform molecular strategies to enhance milk production both in Mongolian horses and in other less productive equine breeds. These findings may aid in selective breeding, nutritional interventions, and health management practices aimed at improving lactational efficiency, as snRNA-seq of frozen mammary gland tissue samples from young fillies and adult Mongolian mares provides high-resolution insights into the transcriptional dynamics and cellular heterogeneity associated with mammary gland development. In this study, we employed snRNA-seq and histological analyses to map the cellular landscape of the mammary gland in Mongolian mares across 4 physiological stages: 6-mo-old weanlings (6M), 2-yr-old yearlings (2Y), 4-yr-old lactating adults (LT), and 4-yr-old nonlactating adults (NL). Frozen parenchymal mammary gland tissues were surgically collected and processed for snRNA-seq via iodixanol gradient-based nuclei isolation, enabling high-resolution transcriptomic profiling, and complementary tissues were processed for histology. This study employed integrated analysis to reveal stage-specific shifts in epithelial, stromal, and immune cell populations, to highlight dynamic changes in mammary gland development and function. A total of 28,287 nuclei were profiled via transcriptome sequencing and categorized into 8 major cell types: basal myoepithelial, luminal secretory, luminal hormone-sensing, endothelial, fibroblasts, macrophages, T cells, and B cells. The LT-group samples (11,136) exhibited the greatest nuclei diversity and expansion, particularly in the luminal compartments, compared with the other stages, 6M (3,067 nuclei), 2Y (5,654 nuclei), and adult NL (8,430 nuclei), which shows the structure and maturation of the mammary gland. Hematoxylin and eosin staining confirmed structural remodeling during lactation, including increased epithelial thickness and ductal complexity. Pseudotime analysis revealed a dynamic transition from basal progenitors to differentiated luminal cells, identifying 3 major epithelial branches. Expression analysis of key genes and functional enrichment (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]) was performed using the entire dataset across all physiological stages to capture shared transcriptional programs. To further investigate stage-specific responses, pathway enrichment and intercellular signaling analyses were conducted separately for each stage, enabling identification of both common and unique regulatory events. Key genes such as TP63 (basal identity), ERBB4 and NRG1 (epithelial signaling), and SORBS1, SLPI, SLC12A2, KCNMA1, and TAGLN (cytoskeletal and immune remodeling) were differentially expressed across stages, reflecting their roles in epithelial maintenance, lactational function, and structural adaptation. The GO and KEGG analyses identified differentially expressed genes in each cluster, mainly enriched in the TGF-β and VEGF signaling pathways, suggesting coordinated regulation of tissue remodeling, vascular development, and immune modulation. These findings deepen our understanding of mammary gland maturation and offer insights into the cellular and molecular architecture of equine mammary biology and its relevance to reproductive health and lactation studies.
The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Publication Date: 2025-08-28 PubMed ID: 40885302DOI: 10.3168/jds.2025-26480Google Scholar: Lookup
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Summary
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Overview
- This research used single-nucleus RNA sequencing (snRNA-seq) to study the developmental changes and cellular diversity in the mammary glands of young fillies and adult Mongolian mares.
- The study identified how various cell types and gene expressions change across different physiological stages, providing new insights into mammary gland development and lactation in horses.
Background and Significance
- Mongolian horses are notable for their high-quality milk, characterized by high protein and low fatty acid content.
- These horses have historically been important in dairy production in Inner Mongolia and Central Asia, making them an ideal model for studying lactational biology.
- Lactation is regulated by multiple factors, but prior to this study, snRNA-seq had not been used to analyze the mammary gland’s cellular and molecular landscape in horses.
- Understanding these processes can help in selective breeding, nutritional support, and improving lactational efficiency in horses and possibly other species.
Research Objectives and Methods
- The study aimed to analyze the transcriptional dynamics and cellular heterogeneity of the mammary gland at various developmental stages using snRNA-seq.
- Mammary gland tissue samples were collected from four physiological stages: 6-month-old weanlings (6M), 2-year-old yearlings (2Y), 4-year-old lactating mares (LT), and 4-year-old non-lactating mares (NL).
- Frozen parenchymal tissues underwent iodixanol gradient-based nuclei isolation for high-resolution snRNA-seq profiling.
- Complementary histological assays, such as hematoxylin and eosin staining, were performed to confirm structural changes in the tissue.
- Integrated bioinformatics analysis included clustering nuclei by cell types, pseudotime trajectory analysis, gene expression profiling, and functional enrichment (Gene Ontology and KEGG pathways).
Key Findings: Cellular Composition and Dynamics
- In total, 28,287 nuclei were profiled and classified into 8 major cell types:
- Basal myoepithelial cells
- Luminal secretory cells
- Luminal hormone-sensing cells
- Endothelial cells
- Fibroblasts
- Macrophages
- T cells
- B cells
- The lactating group (LT) showed the greatest cellular diversity and expansion, especially among luminal epithelial cells, indicating active gland maturation and function during lactation.
- The younger groups (6M and 2Y) and the non-lactating adults (NL) showed less cellular diversity and complexity, reflecting less developed or inactive mammary tissue.
- Histological analysis confirmed structural changes such as increased epithelial thickness and ductal complexity during lactation.
Developmental Trajectory and Gene Expression
- Pseudotime analysis mapped the differentiation trajectory from basal progenitor cells to mature luminal cells, revealing three major epithelial branches in the developmental process.
- Gene expression analysis identified key genes with stage-specific roles:
- TP63: Associated with basal cell identity and maintenance.
- ERBB4 and NRG1: Involved in epithelial cell signaling important for gland function.
- SORBS1, SLPI, SLC12A2, KCNMA1, TAGLN: Related to cytoskeletal reorganization and immune system remodeling during lactation.
Functional Enrichment and Pathway Insights
- Gene Ontology (GO) and KEGG pathway analyses revealed enrichment in pathways involved in:
- TGF-β signaling: Related to tissue remodeling and development.
- VEGF signaling: Important for vascular development and adaptation in the mammary gland.
- Immune modulation pathways: Reflecting the involvement of immune cells in gland function and remodeling.
- Pathway and intercellular signaling analyses were conducted both globally and by individual physiological stages to identify common and unique regulatory mechanisms at each stage.
Conclusions and Implications
- This study provides high-resolution insights into the complex cellular and molecular landscape of the equine mammary gland across development and lactation.
- The data enhances understanding of mammary gland maturation, identifies critical pathways and gene networks involved, and highlights the role of different cell populations during lactation.
- Findings have practical implications for improving dairy traits in Mongolian horses and possibly other breeds through targeted breeding and management strategies.
- The research also opens pathways for future studies aimed at reproductive health, lactation biology, and comparative mammary gland physiology in mammals.
Cite This Article
APA
Jafari H, Wang Z, Li C, Yang G, Yang Q, Han J, Hu Q, Muhatai G, Lei C, Dang R.
(2025).
Single-nucleus RNA sequencing reveals developmental dynamics and cellular heterogeneity in the mammary gland of young fillies and adult Mongolian mares.
J Dairy Sci, 108(11), 12792-12813.
https://doi.org/10.3168/jds.2025-26480 Publication
Researcher Affiliations
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
- College of Animal Science and Technology, Tarim University, Alar 843300, China.
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
- Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China. Electronic address: dangruihua@nwsuaf.edu.cn.
MeSH Terms
- Animals
- Horses
- Female
- Mammary Glands, Animal / growth & development
- Mammary Glands, Animal / metabolism
- Lactation
- Sequence Analysis, RNA / veterinary
- Milk / chemistry
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