Serum and uterine fluid extracellular vesicles and prostaglandin E metabolite concentrations during diestrus and early pregnancy in mares.

Prostaglandins produced by endometrial and conceptus tissues have an established role in maintaining luteal function in early pregnancy and initiating luteolysis during diestrus in mares. The cellular mechanisms controlling the secretion and delivery of prostaglandins have not been fully elucidated. We hypothesized that nanosized lipid bilayer extracellular vesicles (EV) that carry a variety of biological molecules would be involved in these key regulatory processes. Serum and uterine fluid samples were collected from diestrus and pregnant mares on day (D) 10 and D14 post-ovulation, and conceptuses were collected in pregnant mare cycles and cultured for 2-3 h. Extracellular vesicles were isolated using membrane affinity spin columns. Metabolomic EV cargo was assessed using LC-MS/MS, and cytosolic phospholipase A2 (cPLA2) with immunoblotting. Serum and uterine fluid PGEM and conceptus PGE2 concentrations were compared using ELISA. Prostaglandins were below detection limits in all EV serum and uterine fluid eluates. PGEM concentrations in uterine fluid and serum did not differ between pregnant and diestrus mares at these time points. Day 10 and D14 conceptuses produced PGE2. Serum and uterine fluid EV contained AA and cPLA2. While AA abundance did not differ by reproductive status or day at these time points, uterine fluid EV AA abundance was 83 times higher than serum. Serum and uterine fluid EV carry activatable phospholipases and rate-limiting substrates for prostaglandin production rather than the prostaglandins themselves. High levels of AA in uterine fluid EV are likely important in the local regulation of uterine prostaglandin production.