Validation of lncRNA expression in a 3D culture model for pregnancy recognition

Abstract

Early embryonic mortality is responsible for a large proportion of reproductive losses and usually occurs before maternal recognition of pregnancy (around day 16) in cattle. The IFNT secreted by the embryo signals its presence in the uterus to the mother, preventing luteolysis and embryonic loss. For the pregnancy to be maintained and the offspring to be healthy, proper communication between the conceptus and the uterine environment is essential. On day 20, the bovine conceptus elongates and, during this process, it depends on secretions from the uterus (histotroph) to survive and grow. The endometrium undergoes many changes to become receptive to the embryo, including transcriptomic changes, which provide an ideal environment for embryonic development. In this context, lncRNAs have been shown to be fundamental transcripts for the regulation of many physiological and pathological processes at the transcriptional, post-transcriptional and translational levels. They are part of gene co-expression networks that enable a more in-depth assessment of the biological processes involved in fertility, embryonic development and pregnancy maintenance, allowing us to identify who is involved in hormonal regulation, development of embryonic structures and tissues, interaction and transfer of molecules between the conceptus and the mother, etc. Therefore, its study in the context of the uterine environment is essential for a better understanding of the establishment and maintenance of pregnancy. This is why it is important to associate endometrial cells with embryos through co-culture, but for this to happen, an in vitro model that is more similar to the in vivo environment is indispensable, and this is where three-dimensional cell culture models come in, which make it possible to mimic the physiological environment in a much more appropriate way for cell growth and differentiation, by mimicking the extracellular matrix in vivo, since they show better results in terms of cell morphology, proliferation, response to stimuli, differentiation, etc. Three-dimensional cell culture systems make it possible to maintain embryonic architecture and enable physical interaction with the surrounding environment, improving development. Thus, in this project, bovine embryos will be produced in vitro and cultured together with a layer of epithelial and stromal cells in a 3D model made from endoECM. Subsequently, on D8 of the culture, the epithelial and stromal cells of the endometrium will be collected for PCR and then the previously identified and selected lncRNAs will be compared and validated based on the preliminary results of the student's scientific initiation scholarship in Brazil.