Integrated single-cell and metabolomic profiling identifies ovarian metabolic differences and endothelial heterogeneity associated with litter-size variation in Hu sheep.

Background: Hu sheep are highly prolific, but litter size remains variable among individuals. How ovarian metabolism and somatic-cell heterogeneity are associated with this variation remains unclear. Ovarian metabolites from high-litter-size (HLS) and low-litter-size (LLS) Hu ewes were profiled by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Ovarian cell populations and transcriptional states were characterized by single-cell RNA sequencing. Integrated analyses were used to relate metabolic differences to specific cell populations and predicted cell-cell communication. RT-qPCR and multiplex immunofluorescence were used to examine selected metabolic genes and the endothelial localization of ESM1. Separate cell experiments assessed the effects of endothelial cell-conditioned medium (EC-CM) and recombinant human IGFBP3 on primary ovine granulosa cells (GCs), with TMEM219 knockdown used to evaluate receptor involvement.

Results: HLS ovaries showed higher L-carnitine abundance, whereas several representative sphingomyelin species were more abundant in LLS ovaries (nominal P < 0.05). Integrated analysis identified two EC associated genes: DNAJB4 was downregulated, whereas NDRG1 was upregulated in HLS ovaries (P < 0.05). At the EC level, gene set enrichment analysis showed significant enrichment of glutathione metabolism and oxidative phosphorylation in the HLS group. EC subclustering further associated these transcriptional programs with ESM1⁺ angiogenic endothelial cells (A-ECs). Multiplex immunofluorescence localized ESM1 to VWF-positive vascular structures and showed a significantly larger ESM1-positive area and more VWF/ESM1 double-positive cells in HLS ovaries (P < 0.05). Cell-cell communication analysis identified IGFBP3-TMEM219 as a candidate EC-GC interaction. IGFBP3 was detected in EC-conditioned medium, which significantly reduced EdU incorporation and the expression of CYP11A1, FSHR and STAR (P < 0.05) in granulosa cells. Recombinant IGFBP3 produced similar responses, and TMEM219 knockdown partially attenuated several of these effects.

Conclusions: This study identified an association between exploratory ovarian metabolic patterns and EC heterogeneity, while functional experiments showed that EC-derived IGFBP3 affected GC function partly through TMEM219. These findings suggest that endothelial state and signaling may be relevant to follicular development, although whether IGFBP3-TMEM219 signaling contributes to litter-size variation in Hu sheep remains to be established.