Bovine endometrial organoids: A new tool to study conceptus-maternal interactions in mammals

In cattle, communication between endometrium and conceptus during the peri-implantation period is crucial for successful pregnancy. Understanding these interactions is vital as most early pregnancy loss occurs during this time. A major challenge in understanding uterine function and early pregnancy is lack of appropriate in-vitro models. Two-dimensional models are available, but do not recapitulate the endometrium's complex multicellular structure. Here, we describe a hormonally responsive organoid model of the bovine endometrium, developed as a tool for studying endometrial function and early pregnancy. Bovine glandular epithelial cells were isolated from reproductive tracts and cultured in an extracellular matrix hydrogel (Cultrex 2) at 38.5°C, 5% CO (n=3). RNA was extracted and qPCR confirmed the presence of gland markers: leukemia inhibitory factor, mucin-1, insulin-like growth factor binding protein-1, kruppel-like factor-5 and forkhead box protein-A2. Organoids were imaged at specific time-points to monitor growth and passaged 3 times in 1:2 or 1:3 ratios after growing for a minimum of 10 days per passage. Morphologically, organoids were spherical and fast-growing at passages 0 and 1, but this declines following passage 2. Bovine endometrial organoids (n=3, passage 0) were treated with 1000 ng/ml recombinant ovine Interferon Tau (IFNT) or 10 µg/ml progesterone (P4) for 24 hours and analysed by RNASeq to assess hormone responsiveness. Differential expression analysis by DeSeq2 negative binomial distribution model followed by Wald test and Benjamini-Hochberg correction identified 373 transcripts significantly upregulated (padj<0.05 or log2fold change >0.05) in response to P4 treatment, with downstream analysis showing significant overrepresentation (FDR<0.05) of genes associated with positive regulation of protein localisation to plasma membrane and cell periphery. Of the 240 genes significantly downregulated by P4 these were significantly overrepresented (FDR<0.05) in biological processes of cilium and cytoskeleton organisation. IFNT treatment resulted in significant upregulation of 414 genes and downregulation of 119 genes. The largest cluster associated with differentially expressed genes in response to IFNT is defence to virus and interferon signalling. There were 30 genes altered by both P4 treatment and IFNT treatment. Organoids were also shown to express conserved microRNAs, and it was possible to culture them in a microfluidics device - making them a useful model for a multitude of potential investigations. This model provides a tool to investigate bovine endometrial function and peri-implantation communication, subsequently allowing species comparison to understand diversity in reproductive strategies. Overall design: Bovine endometrial organoid cultures were treated with control (media only), vehicle control (EtOH), Progesterone, PBS, PDI, CAPG, and Interferon tau for 24 hours and RNASeq carried out to assess hormone/protein responsiveness