Bovine endometrial organoids show selective transcript-level responses to a sequential hormone, cAMP and interferon tau regimen

The bovine endometrium responds to ovarian and conceptus-derived signals that are essential for early pregnancy establishment, yet available in vitro systems remain limited in their ability to combine controlled stimulation with sustained three-dimensional epithelial organisation. Here, we generated bovine endometrial organoids (EGOs) from endometrial cell fractions enriched in glandular fragments and evaluated their transcript-level response to a sequential differentiation and stimulation regimen including oestradiol, progesterone, cyclic adenosine monophosphate and interferon tau. EGOs were maintained in Matrigel under defined expansion and differentiation conditions and formed cystic structures lined by epithelial-like cells around a central lumen. Histology confirmed this epithelial-like cystic morphology, whereas representative confocal immunofluorescence performed with organoids from one uterus documented marker-associated signals. Quantitative PCR identified a selective transcriptional response, most consistently reflected by increased expression of canonical interferon tau-responsive genes, including ISG15, MX1, MX2, and OAS1Y. Additional transcripts showed more heterogeneous responses, whereas several expected hormone-associated genes did not differ clearly under the conditions evaluated. Because molecular readouts were assessed primarily at the transcript level and immunofluorescence comparisons were descriptive, these findings should be interpreted accordingly. Overall, organoids generated from gland-enriched bovine endometrial fractions formed epithelial-like cystic structures and showed selective transcript-level responses to the complete sequential differentiation and stimulation regimen.