(367) Deciphering the Effect of Ovarian Steroid Stimulation on Bovine Oviductal Organoids Using Single-cell RNA-seq

Brandi Dunn¹, Ahmed Gad², Riley Thompson², Mindy Meyers², Fiona Hollinshead² and Dawit Tesfaye¹

< !1. Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA

< !2.  Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado, USA

Abstract Text:

The oviduct, a crucial yet often overlooked component of the bovine female reproductive tract, plays a vital role in early embryonic development. However, its challenging anatomical location makes it difficult to study directly. Thus, developing an in vitro cell culture system that accurately mimics oviductal physiology and function is essential for modeling maternal-embryo interactions in vitro. In this study, we developed a 3D in vitro model of bovine oviductal organoids overlayed with an organoid culture medium. The culture media was renewed every 2-3 days during organoid growth and development, with organoids passaged every 10 days. During passage 2 (20 days from initial culture),  organoids were grown for 4 days in standard organoid culture media, and then media was completely replaced with organoid culture media supplemented with estradiol (E2) and progesterone (P4) at concentrations reflecting physiological conditions of estrous (300pg/ml E2 & 10ng/ml P4) for 2 days followed by diestrus hormone combination  (75pg/ml E2 & 100ng/ml P4) for 4 days. Vehicle (100% ethanol) treated organoids were used as controls.  To investigate the cellular diversity and cell population-specific response of oviductal organoids to ovarian steroid hormone stimulation, single-cell RNA sequencing (scRNA-seq)  was performed. For this, RNA library preparation and sequencing were conducted using 10X Chromium Single Cell 3' v3 chemistry and Illumina NovaSeq 6000 platform, respectively. Bioinformatic analysis was carried out using Partek Flow, an Illumina web-based bioinformatics software (v12.4.0). Marker gene expression highlighted that the organoids were composed of various key cell types, including secretory and ciliated epithelial, peg, and mesothelial cells.  Cluster analysis of the two treatment groups demonstrated significant alterations in the relative abundance of specific cell populations when subjected to hormonal stimuli, specifically driving cell populations from progenitor-type cells to more differentiated epithelial secretory and ciliate-type cells. Analysis of scRNA-seq data identified 3106 differentially expressed genes (DEGs) between hormone-treated and vehicle-treated organoid groups. Of these DEGs, 3074 genes were downregulated, while 27 were upregulated in the hormone-treated organoids compared to the vehicle-treated control. Pathway enrichment analysis of the DEGs revealed pathways associated with metabolic processes, oxidative phosphorylation, and ribosome-related pathways, as the key affected pathways. These findings demonstrate that translational and metabolic activities were significantly suppressed in hormone-treated organoids compared to vehicle-treated organoids. These results provide valuable insights into the functional biology of these organoids and suggest that bovine oviductal organoids maintain a diverse array of cell types comparable to those found in vivo. Further, cell type-specific DEGs between the two treatment groups yield an extensive list of potential genes and pathways associated with the cell type-specific response to hormones and physiological functions of the different cell types in oviduct-embryo interaction. Our findings demonstrate that bovine organoids serve as a novel and advanced model for studying oviductal-embryo interactions, while also highlighting their promising application in ART systems to improve embryo quality and production.

Funding: BD was supported by the USDA-NIFA National Needs Fellowship (NNF) Grant #2021-38420-34040. Additionally, research funding for this project was partially provided by the Colorado State University College Research Council.