Single-Nucleus RNA Sequencing and Spatial Transcriptomics Reveal Cellular Heterogeneity and Intercellular Communication Networks in the Hypothalamus–Pituitary–Ovarian Axis of Pregnant Mongolian Cattle

The hypothalamus&ndash:pituitary&ndash:ovarian (HPO) axis orchestrates reproductive functions through intricate neuroendocrine crosstalk. Here, we integrated single-nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics (ST) to decode the cellular heterogeneity and intercellular communication networks in the reproductive systems of pregnant Mongolian cattle. We retained a total of 6161 high-quality nuclei from the hypothalamus, 14,715 nuclei from the pituitary, and 26,072 nuclei from the ovary, providing a comprehensive cellular atlas across the HPO axis. In the hypothalamus, neurons exhibited synaptic and neuroendocrine specialization, with glutamatergic subtype Glut4 serving as a TGF&beta: signaling hub to regulate pituitary feedback, while GABAergic GABA1 dominated PRL signaling, likely adapting maternal behavior. Pituitary stem cells dynamically replenished endocrine populations via TGF&beta:, and lactotrophs formed a PRL&ndash:PRLR paracrine network with stem cells, synergizing mammary development. Ovarian luteal cells exhibited steroidogenic specialization and microenvironmental synergy: endothelial cells coregulated TGF&beta:-driven angiogenesis and immune tolerance, while luteal&ndash:stromal PRL&ndash:PRLR interactions amplified progesterone synthesis and nutrient support. Granulosa cells (GCs) displayed spatial-functional stratification, with steroidogenic GCs persisting across pseudotime as luteinization precursors, while atretic GCs underwent apoptosis. Spatial mapping revealed GCs&rsquo: annular follicular distribution, mediating oocyte&ndash:somatic crosstalk, and luteal&ndash:endothelial colocalization supporting vascularization. This study unveils pregnancy-specific HPO axis regulation, emphasizing multi-organ crosstalk through TGF&beta:/PRL pathways and stem cell-driven plasticity, offering insights into reproductive homeostasis and pathologies.