Differential expression of bovine milk-derived exosomal miRNAs and their role in modulating endometrial receptivity during early pregnancy

Endometrial receptivity is critical for successful implantation of bovine embryos. MicroRNA (miRNA), as a key regulator of uterine receptivity, is involved in physiological processes such as cell differentiation, proliferation, and apoptosis. The aim of this study was to identify pregnancy-specific miRNAs derived from milk exosomes of non-pregnant and early pregnant cows. In addition, bioinformatics analysis was used to assess the differential expression, target genes, and functions of these miRNAs in order to examine their significance in endometrial cell regulation. Exosomes were isolated from milk using an exosome extraction kit and then identified by Western blotting and transmission electron microscopy. We used Illumina high-throughput sequencing to profile miRNAs and identify differentially expressed miRNAs in bovine milk-derived exosomes at different stages of pregnancy (days 15, 25 and 30) and in non-pregnant cows (day 0). The sequencing data revealed a significant upregulation of bta-miR-125b in pregnant cows at days 15 and 25 compared to non-pregnant cows. Bta-miR-125b targets the Leukemia inhibitory factor (LIF), which is thought to play a critical role in the development of endometrial receptivity by regulating gene expression. KEGG pathway enrichment and Gene Ontology analysis indicated that the target genes of the differential miRNAs were significantly enriched in the key signaling pathways, including the MAPK, phosphatidylinositol signaling system and PI3K-Akt signaling pathways, as well as physiological activities such as RNA polymerase II transcriptional regulation, protein phosphorylation, apoptosis control and cell proliferation regulation. These signaling pathways and physiological activities are all indispensable parts during the process of pregnancy. These findings emphasize bta-miR-125b critical function in regulating endometrial receptivity via important signaling pathways, providing potential indicators for early pregnancy detection and insights into enhancing reproductive efficiency in dairy cows.