Integrated single-cell and metabolomic profiling identifies ovarian metabolic differences and endothelial heterogeneity associated with litter-size variation in Hu sheep.
4 affiliations
- 1Department of Radiology, Tangdu Hospital, Fourth Military Medical University, 569 Xinsi Road, Xi'an, Shaanxi, 710038, People's Republic of China.
- 2Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China.
- 3Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, China. [email protected].
- 4Department of Radiology, Tangdu Hospital, Fourth Military Medical University, 569 Xinsi Road, Xi'an, Shaanxi, 710038, People's Republic of China. [email protected].
Abstract
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.
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BibTeX
@article{ge2026integrated,
title = {{Integrated single-cell and metabolomic profiling identifies ovarian metabolic differences and endothelial heterogeneity associated with litter-size variation in Hu sheep.}},
author = {Ting Ge and Xiaoyu Huang and Kuiming Su and Yifan Wen and Bo Li and Shaohua Jiang and Fan Zhao and Enping Zhang and Wen Wang},
journal = {Journal of animal science and biotechnology},
year = {2026},
month = oct,
doi = {10.1186/s40104-026-01508-6},
url = {https://europepmc.org/article/MED/42850629},
note = {CC BY},
}
RIS
TY - JOUR
TI - Integrated single-cell and metabolomic profiling identifies ovarian metabolic differences and endothelial heterogeneity associated with litter-size variation in Hu sheep.
AU - Ting Ge
AU - Xiaoyu Huang
AU - Kuiming Su
AU - Yifan Wen
AU - Bo Li
AU - Shaohua Jiang
AU - Fan Zhao
AU - Enping Zhang
AU - Wen Wang
PY - 2026
DA - 2026/10/09
JO - Journal of animal science and biotechnology
DO - 10.1186/s40104-026-01508-6
UR - https://europepmc.org/article/MED/42850629
AB - 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.
SN - 1674-9782
SN - 2049-1891
ER -