Endometrial stromal cell (ESC) plasticity is critical to embryo survival and conceptus elongation; however, their genetic regulation remains unclear. To elucidate the molecular mechanisms by which ESR1 regulates sheep fecundity through endometrial function, we investigated the effects of ESR1 on ovine ESCs in vitro. Tissue distribution analysis revealed elevated ESR1 expression in the reproductive organs, particularly the endometrium, compared with that in non-reproductive tissues. Both ESR1 mRNA and its encoded ERα protein were significantly upregulated in the endometrium of high-fecundity Hu sheep compared to those in the endometrium of low-fecundity Hu sheep. Given the specific localisation of ERα in ESCs, we examined the functional roles of ESR1 in these cells in vitro. ESR1 knockdown in ESCs inhibited cell proliferation and induced apoptosis, concomitant with increased Caspase3 and Caspase9 mRNA expression. Furthermore, ESR1 interference triggered G0/G1-phase cell cycle arrest via the upregulation of P53 and P21 and significantly impaired cell migration capacity. Collectively, these results demonstrate that ESR1 critically regulates ESC proliferation, apoptosis, cell cycle progression, and migration. Our findings establish ESR1 as a key determinant of sheep fecundity and provide mechanistic insights into the high reproductive efficiency of Hu sheep.

Background: Endometrial stromal cells (ESCs) plasticity is critical for embryo survival and conceptus elongation; however, their genetic regulation remains unclear. Method: To elucidate the molecular mechanisms by which ESR1 regulates sheep fecundity through endometrial function, we investigated the effects of ESR1 on ovine ESCs in vitro. Results: Tissue distribution analysis revealed elevated ESR1 expression in the reproductive organs, particularly the endometrium, compared with that in non-reproductive tissues. Both ESR1 mRNA and its encoded ERα protein were significantly upregulated in the endometrium of high-fecundity Hu sheep compared to that in low-fecundity Hu sheep endometrium. Given the specific localisation of ERα in ESCs, we examined the functional roles of ESR1 in these cells in vitro. ESR1 knockdown in ESCs inhibited cell proliferation and induced apoptosis, concomitant with increased Caspase3 and Caspase9 mRNA expression. Furthermore, ESR1 interference triggered G0/G1 phase cell cycle arrest via upregulation of P53 and P21 and significantly impaired cell migration capacity. Collectively, these results demonstrate that ESR1 critically regulates ESCs proliferation, apoptosis, cell cycle progression, and migration. Conclusions: Our findings establish ESR1 as a key determinant of sheep fecundity and provide mechanistic insights into the high reproductive efficiency of Hu sheep.

Chronic cold stress disrupts hepatic metabolism and fetal development of Mongolian sheep in northern pastoral zones, while systematic nutritional mitigation strategies remain poorly characterized. N-carbamylglutamate (NCG) promotes endogenous arginine synthesis; however, its hepatic regulatory roles against cold stress in pregnant ewes and fetuses remain unclear. This study explored the potential protective effects of NCG via integrated hepatic transcriptome and metabolome analysis. Twenty-four late-gestation ewes were assigned to warm control, cold stress, and cold stress + NCG supplementation groups. Hepatic mRNA, lncRNA, circRNA, and miRNA sequencing, untargeted LC-MS metabolomics, multi-omics conjoint analysis, and qRT-PCR verification were performed using ewe and fetal liver tissues. The results indicated that cold stress potentially was associated with mTOR, PI3K/AKT, amino acid, and energy metabolic pathways, inducing extensive transcriptomic and metabolic alterations. NCG supplementation might reverse cold-induced transcriptional and metabolic disorders and restore glutathione and cysteine–methionine metabolism. qRT-PCR validated the differential expression of core hub genes AK4, AHCY, and RRM2, linking transcriptomic and metabolic reprogramming. Divergent hepatic molecular responses to NCG were observed between ewes and fetuses. Multi-omics correlation analysis suggested that maternal NCG may modulate hepatic energy and amino acid metabolism through mTOR and redox-related pathways under cold stress and potentially exert transgenerational hepatic effects on fetuses. These findings provide preliminary omics-based mechanistic hypotheses and a theoretical basis for nutritional intervention against cold stress in sheep production.

Chronic cold stress impairs hepatic metabolism, antioxidant balance and fetal development of Mongolian sheep in northern pastoral zones, yet systematic research on nutritional mitigation remains limited. N-carbamylglutamate (NCG) boosts endogenous arginine synthesis, but its hepatic regulatory mechanisms against cold stress in pregnant ewes and fetuses remain unclear. This study explored NCG’s protective effects via integrated liver transcriptome and metabolome analysis. Twenty-four late-gestation ewes were divided into warm control, cold stress, and cold stress + daily NCG supplementation groups. Hepatic mRNA, lncRNA, circRNA, miRNA sequencing, untargeted LC-MS metabolomics, multi-omics conjoint analysis and qRT-PCR verification were conducted on ewe and fetuses liver samples. The results showed that, cold stress disturbed mTOR, PI3K/AKT, amino acid and energy metabolic pathways, triggering massive differential RNA and metabolite changes. NCG supplementation reversed cold-induced transcriptional and metabolic disorders, restoring glutathione and cysteine-methionine metabolism. Core hub genes AK4, AHCY and RRM2 linking transcriptomic and metabolic shifts were validated by qPCR, with divergent hepatic NCG responses observed between ewes and fetuses. Maternal NCG alleviates cold-caused liver oxidative damage, remodels energy and amino acid metabolism via mTOR and redox pathways, and confers transgenerational protection to fetuses through the maternal-placental-fetal axis. These findings elucidate NCG’s anti-cold-stress molecular mechanism and provide a basis for nutritional intervention for cold-region sheep farming.