The Characteristic Function of Blood-Derived Exosomes and Exosomal circRNAs Isolated from Dairy Cattle during the Dry Period and Mid-Lactation.

Exosomes are key mediators of intercellular communication. They are secreted by most cells and contain a cargo of protein-coding genes, long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), which modulate recipient cell behavior. Herein, we collected blood samples from Holstein cows at days 30 (mid-lactation) and 250 (dry period) of pregnancy. Prolactin, follicle-stimulating hormone, luteinizing hormone, estrogen, and progesterone levels showed an obvious increase during D250. We then extracted exosomes from bovine blood samples and found that their sizes generally ranged from 100 to 200 nm. Further, Western blotting validated that they contained CD9, CD63, and TSG101, but not calnexin. Blood-derived exosomes significantly promoted the proliferation of mammary epithelial cells, particularly from D250. This change was accompanied by increased expression levels of proliferation marker proteins PCNA, cyclin D, and cyclin E, as detected by EdU assay, cell counting kit-8 assay, and flow cytometric cell cycle analysis. Moreover, we treated mammary epithelial cells with blood-derived exosomes that were isolated from the D30 and D250 periods. And RNA-seq of two groups of cells led to the identification of 839 differentially expressed genes that were significantly enriched in KEGG signaling pathways associated with apoptosis, cell cycle and proliferation. In bovine blood-derived exosomes, we found 12,747 protein-coding genes, 31,181 lncRNAs, 9374 transcripts of uncertain coding potential (TUCP) candidates, and 460 circRNAs, and 32 protein-coding genes, 806 lncRNAs, 515 TUCP candidates, and 45 circRNAs that were differentially expressed between the D30 and D250 groups. We selected six highly expressed and four differentially expressed circRNAs to verify their head-to-tail splicing using PCR and Sanger sequencing. To summarize, our findings improve our understanding of the key roles of blood-derived exosomes and the characterization of exosomal circRNAs in mammary gland development.