(242) Hormonal modulation of bovine endometrial epithelial organoids

Hormonal modulation of bovine endometrial epithelial organoids

Iebu Devkota¹; Zachary L. Bonomo¹; Dailin M. Fuego¹; Shavahn C. Loux¹; Constantine A. Simintiras¹

1. School of Animal Sciences, Agricultural Center, Louisiana State University, Baton Rouge, LA 70803, USA.


Abstract Text:

The bovine endometrium plays a crucial role in key reproductive processes, including conceptus (embryo and extraembryonic membrane) elongation and implantation, which are critical to successful pregnancy. These events, however, coincide with periods of high pregnancy loss. Studying bovine endometrial physiology in vivo presents numerous challenges, including ethical, technical, and financial limitations. Bovine endometrial epithelial organoids (EEO) offer a promising alternative, providing a versatile platform to investigate endometrial biology within a controlled and manipulable environment. Organoids are three-dimensional in vitro systems closely resembling the in vivo architecture and function of their tissue of origin. Notably, the intra-organoid fluid (IOF) composition reflects the proteomic and metabolomic profile of the corresponding tissue lumen. Therefore, our primary objective was to assess the extent to which bovine EEO replicate in vivo endometrial physiology. EEO were derived from synchronized crossbred beef cattle (n=3) post-mortem on Day 5 of the estrous cycle. After undergoing two subcultures and a 6-day growth period, EEO were treated with 10 nM 17β-estradiol (E2) for 48 hours. Subsequent treatments included: (a) vehicle control, (b) 10 nM E2, (c) 1 µM medroxyprogesterone acetate (MPA), (d) E2+MPA (to mimic the diestrus phase), and (e) E2+MPA+5 nM recombinant interferon tau (IFN𝛕) to simulate pregnancy. EEO were imaged every 24 hours following media replenishment. After 6 days of treatment, EEO morphology, immunohistochemistry (IHC), gene expression (RT-PCR), and IOF metabolomics were analyzed. Bovine EEO expressed epithelial markers such as cytokeratin and FOXA2, a marker of uterine glandular epithelium. Hormonal responsiveness was validated through nuclear estrogen and progesterone receptor IHC. While EEO count, area, and circularity did not vary across treatments, EEO area increased (P< 0.05) over time in all treatment groups. Interestingly, treatment influenced IOF composition, with the most significant shift observed following MPA supplementation. Ongoing research aims to examine the gene expression of interferon-stimulated genes in bovine EEO and compare all results with in vivo data. However, these findings demonstrate that bovine EEO represent a valuable model for studying key reproductive processes in agriculturally relevant species. This study was approved by the Louisiana State University (LSU) Agricultural Center Animal Care and Use Committee. Funding was provided by the State of Louisiana Board of Regents [LEQSF(2023‐26)‐RD‐A‐03], Audubon Nature Institute Center for Research of Endangered Species (GR-00013800), the Joe W. and Dorothy Dorsett Brown Foundation (BG008584), and USDA Research Capacity (Hatch) funds (LAB-94578).