Ex utero embryonic development: the effect of endometrial spheroids on conceptus elongation in cattle.

• Grant number: 24/05039-7
• Support Opportunities: Research Grants - Young Investigators Grants
• Start date: July 01, 2025
• End date: June 30, 2030
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Tiago Henrique Camara de Bem
• Grantee: Tiago Henrique Camara de Bem
• Host Institution: Instituto de Zootecnia. Agência Paulista de Tecnologia dos Agronegócios (APTA). Secretaria de Agricultura e Abastecimento (São Paulo - Estado). Nova Odessa , SP, Brazil
• Associated researchers: Fabio Morato Monteiro ; Felipe Perecin ; Flávio Vieira Meirelles ; Juliano Coelho da Silveira ; Lawrence Charles Smith ; Lígia Garcia Mesquita ; Lindsay Unno Gimenes ; Maria Eugênia Zerlotti Mercadante ; William Allan King ; yeda fumie watanabe

Abstract

The establishment of pregnancy in ruminants involves complex morphological and molecular changes including the elongation of the blastocyst into a filamentous conceptus and maternal recognition of pregnancy. At this period, the conceptus does not attach to the endometrium and depends on nutrients available in the uterine luminal fluid, formed and secreted by the endometrium. Therefore, the endometrium is the main player in forming a receptive microenvironment favorable to the development of the embryo by secreting molecules and components called as histotroph. Therefore, the histotroph promotes nutrition and supports the elongation of the conceptus. In parallel to this, embryos cultured in vitro for long periods fail to achieve elongation. A critical point in conventional in vitro culture is that it does not mimic the adequate physiology that the uterine environment imposes on conceptuses. Recently, endometrial spheroids (ES) have been suggested as an option for studying uterine physiology in vitro. Spheroids are self-organized three-dimensional (3D) cellular aggregates collected from in vivo tissues that mimic the functional, structural, and biological complexity of the organ. Therefore, ES can be used to investigate the mechanisms involved in embryo elongation. Other factors that can also affect the elongation of conceptuses are the pressure exerted by the uterus, as physical confinement is crucial for the embryo to develop into an ovoid/cylindrical shape and the composition of culture media, with the supplementation of extracellular vesicles (EVs) of endometrial origin, for example. Interestingly, both factors can be promoted by ES. In this way, the in vitro culture of embryos with ES represents a promising tool to allow elongation to occur in vitro as they are capable of secreting molecules similar to the histotroph (histotrophe-like), providing an in vitro 3D environment closer to the physiological and imposing forces physics to concepts. Therefore, considering that: 1) the in vitro culture of bovine embryos is currently restricted up to blastocysts hatch around day 9 of development, compromising the understanding of embryonic development in advanced stages; 2) the establishment of in vitro culture of ES provides a good model to understand uterine physiology; and 3) providing the post-hatching embryo with in vitro culture conditions with modifications in the medium, pressure and physical contact with the ES could promote stimuli for in vitro elongation and study of cross-talk between the conceptus and the mother, the goal of this project is to establish a study model to allow the elongation of bovine embryos in vitro and investigate possible factors involved in this mechanism. To this end, ES will be produced in vitro from bovine uteruses. Blastocysts will also be produced by in vitro fertilization and will be co-cultured with ES, with extracellular vesicles from different origins and subjected to different atmospheric pressures to investigate in vitro elongation competence. The endometrial and embryonic lineages will be characterized, and gene expression and mRNA-sequencing analysis will be performed to validate the proposed study. The results obtained will certainly provide a valuable model for investigating the establishment of ES, the development and differentiation of embryonic lineages, as well as the mechanism of elongation in vitro. Additionally, ES will represent a highly valuable tool to open new avenues in the research of bovine reproductive physiology, study of reproductive pathologies, assessment of fertility and fertilization, in addition to enabling testing of drugs and therapies using less animals in the experiments. Finally, the results achieved in this project will also contribute to the understanding of embryonic development in other species, including humans. (AU)