Characterizing extracellular vesicles and their metabolites from bovine uterine epithelial cells

Pregnancy loss under normal and healthy conditions still occurs at an inherently high rate of ~50% in humans and cattle alike. The dairy industry and human medicine would benefit from understanding more about reproductive mechanisms influencing early embryonic development and the uterine environment. Estradiol and progesterone are important hormones for cyclicity and pregnancy. Secretions produced by the endometrium (i.e., histotroph) is the only source of pre-implantation embryonic nutrition. Luminal and glandular epithelial cells are important cells that line the uterine lumen and secrete histotroph. One component of histotroph are extracellular vesicles (EVs), which can carry a wide variety of cargo. Our objective was to explore the secretion of EVs by immortalized bovine uterine epithelial (BUTE) cells in vitro and investigate the effect of estradiol and progesterone treatment on morphology, secretion, and metabolic contents of EVs from BUTE cells. Transmission electron microscopy (TEM) analysis of uterine biopsies from abattoir collected bovine samples in the luteal phase showed EVs in the glandular lumen. These nanoparticles were consistent with TEM images of BUTE EVs isolated through ultracentrifugation. Western blot of isolated EVs from BUTE cells 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. Neither hormone significantly altered the 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 decreased levels of guanine, adenine, erythronic acid, hexadecanol, and increased dodecanoic acid (FDR<0.1). Ribose, pyrophosphate and hexadecenoic acid were decreased with estradiol treatment (FDR<0.1). Progesterone treatment altered levels of four metabolites (FDR<0.1). Lactic acid, hexadecanol, and hexadecenoic acid increased in abundance with progesterone treatment. The fourth metabolite tetradecanol decreased in abundance. KEGG analysis determined that the gene sets implicated by these metabolites sets are related to fatty acid metabolism, the pentose phosphate pathway and purine metabolism. Overall, this supports our working theory that the uterus secretes EVs with metabolites to nourish the early embryo and support embryonic development in response to ovarian hormones.

Name thesis:degree_name

M.S.

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Nutritional Sciences

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Sandoval, Kassandra

Contributors dc:contributor

• Dean, Matthew J.
• Chen, Hong
• Nowak, Romana
• Wheeler, Matthew

• Extracellular Vesicles
• Bovine

Statement dc:rights

• Copyright 2025 Kassandra Sandoval

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/130066

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F130066