(60) Characterizing Extracellular Vesicles and their Metabolic Cargo from the Bovine Uterine Epithelium 

Kassandra Sandoval¹; Malia D. Berg²; Bruce Southey²; Matthew Dean^1,2 

1. Division of Nutritional Sciences, University of Illinois at Urbana Champaign, Urbana, IL, United States of America

2. Department of Animal Sciences, University of Illinois at Urbana Champaign, Urbana, IL, United States of America


Abstract Text:

Extracellular vesicles (EVs) can transfer material from the uterine epithelium to the embryo. Many factors regulate EV secretion, uptake, and cargo. The role of estradiol and progesterone on EVs has not yet been fully elucidated. Inappropriate levels of nutrients like glucose can be lethal to an embryo. We aimed to explore the in vivo secretion of EVs and the localization of proteins related to EV secretion in the bovine uterine epithelium during the estrus cycle. We confirmed the secretion of EVs by immortalized bovine uterine epithelial (BUTE) cells in vitro and explored the effect of estradiol and progesterone treatment on morphology, secretion, and metabolic contents of uterine EVs. Transmission electron microscopy (TEM) analysis of uterine biopsies from abattoir collected bovine samples in the luteal phase showed EVs in the glandular lumen. Immunohistochemical localization of proteins involved in vesicular trafficking/recycling (RAB11) and exocytosis (RAB3A, RAB27A) was observed in uterine sections on day 1 and day 11 of the reproductive cycle. On day 1, RAB11 was localized in the glandular epithelium and throughout the stroma. On day 11, RAB11 was mainly localized to the glandular epithelium. On day 1 and 11, Rab3A was expressed diffusely throughout the stroma and glandular epithelium with more intense staining in the glandular epithelium on day 11. RAB27A was similarly localized to the glandular and stromal epithelia with no evident differences across the cycle. RNA sequencing performed on progesterone-treated BUTE cells corroborates these observations; RAB3A expression increased and RAB27A expression remained unchanged. These results suggest there are cyclic differences in vesicular trafficking and EV secretion during the reproductive cycle. To determine if we could use BUTE cells to study factors regulating EV secretion, ultra-centrifugation was used to isolate EVs from the conditioned media of BUTE cells. TEM showed nanoparticles that matched the morphology of EVs observed in vivo. NanoSight NS300 showed an abundance of particles ranging from 50-300 nm with two distinct peaks at 188 nm and 208 nm. Western blot of isolated EVs expressed known extracellular vesicle markers, CD9, CD63, HSP70, and β-tubulin. This confirms our ability to isolate and study EVs from BUTE cells. Treatment with estradiol or progesterone led to a significant increase in EV concentration, with progesterone treatment having the greatest effect. Neither hormone significantly altered the particle size of the EVs. This confirms our ability to isolate EVs from BUTE cells for further study and establishes a hormonal effect from hormone treatment on in vitro collected EVs from BUTE cells. Gas chromatography-mass spectrometry (GC-MS) was performed on EVs isolated from hormone treated BUTE cells to identify the metabolites contained within EVs. Notably, lactic acid and glucose were identified within the EVs. Estradiol or progesterone treated EVs yielded different metabolic cargo. Estradiol treatment changed levels of hexadecanoic acid, pyrophosphate, ribose, dodecanoic acid, hexadecanol, adenine, and erythronic acid (P< 0.1). Progesterone treatment altered levels of octadecanoic acid, tetradecanol, hexadecanoic acid, lactic acid, and heptadecanol (P< 0.1). GC-MS identified 1-cyanohexadecane, 1-oleyglycerol, aminomalonic acid solely in the progesterone treated EVs. Overall, this supports our working theory that the uterus secretes EVs with nutrients to nourish the early embryo in response to reproductive hormones.

Financial support for this project was provided by USDA National Institute of Food and Agriculture, Hatch Project ILLU-538-949 to M.D. and the Kraft Heinz Company Human Nutrition Fellowship to K.S.