Establishment of in vitro culture of bovine endometrial organoids for the study of maternal-embryonic communication

Author(s)

Thais Sayuri Imura Oshiro

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2024-07-30

Examining board members

Flavio Vieira Meirelles; Mario Binelli; Marcia de Almeida Monteiro Melo Ferraz

• Advisor: Flavio Vieira Meirelles; Tiago Henrique Camara de Bem

Abstract

In cattle, high rates of embryonic losses are significant, with approximately 10% attributed to fertilization failures and 50% to embryonic developmental issues. To better understand these processes, we employed 3D culture to simulate the in vivo endometrial environment. Our study developed an in vitro setting using endometrial gland organoids co-cultured with epithelial and stromal cells (GES), investigating how this communication affects interaction with bovine embryos. We hypothesized that this 3D model mimics conditions closer to the in vivo bovine uterus. The study was divided into two experiments. In Experiment 1, we assessed the morphological effects of glandular organoids and GES under varying progesterone concentrations from D2 to D9. We observed initial variability in the growth of endometrial glands, regardless of hormonal exposure initiated on D2. Effects of hormonal treatments (E2, P4, E2+P4) on organoid height and area on D9 were noted. In both groups (isolated glands and GES), treatments with P4 and E2+P4 resulted in larger spherical areas compared to control. In Experiment 2, we investigated the combined effect of E2+P4 in the Gland versus GES groups, introducing embryos on day 7 of development and evaluating on day 8 of culture. We focused on 382 microRNAs (miRNAs), analyzing their profiles in Gland_CT, Gland_E2+P4, Gland_E2+P4_Emb, GES_CT, GES_E2+P4, and GES_Emb_E2+P4 groups. Unique miRNAs were identified in each group, with bioinformatic analyses predicting modulation of various biological pathways. The Axon Guidance pathway was common across all Gland groups, underscoring its significance. In the GES group, we identified 12 unique miRNAs in GES_CT, 2 in GES_E2+ P4, and 12 in GES_Emb_E2+P4, modulating pathways such as oxytocin signaling and mTOR signaling, among others. The “Pathway in Cancer” was common among subgroups, associated with multiple biological functions. These findings highlight the complexity of regulatory interactions in 3D culture models for studying maternal-embryonic communication in cattle. Co-cultivating glands with epithelial and stromal cells to create an environment mimicking the bovine endometrium in vitro for studying maternal-embryonic communication proved to be a promising model aligned with study expectations. (AU)

• FAPESP's process: 21/13948-9 - Establishment of in vitro cultivation of bovine endometrial organoids for the study of maternal/embryonic communication.
• Grantee: Thais Sayuri Imura Oshiro
• Support Opportunities: Scholarships in Brazil - Master