Single-nucleus RNA sequencing reveals developmental dynamics and cellular heterogeneity in the mammary gland of young fillies and adult Mongolian mares

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.