Multi-user equipment approved in the grant FAPESP 21/06645-0: Inverted Microscope Yhunder Imager 3D Assay

Abstract

In vitro production of embryos (IVP) is widely used in humans and animals as alternatives to infertility problems and a tool to increase the production efficiency of livestock animals, especially in cattle. Since its beginning, IVP relied on the constant evolution of cell culture media composition based on the observed composition of reproductive biofluids. Extracellular vesicles (EVs) are nano-sized shuttles present in different body fluids, including ovarian follicular fluid, oviductal and uterine fluid, and are believed to be involved in cell-to-cell communication. EVs play an important role in mediating the exchange of bioactive materials such as miRNAs, mRNAs, and proteins. In addition, EVs are used for diagnostics and therapeutics purposes, enabling for the detection of cellular changes and modifying target cells, respectively. Previously from our laboratory focused on investigated miRNAs, proteins and mRNAs contents of EVs in ovarian follicular fluid, oviductal fluid, uterine fluid, bull seminal plasma, and blood serum. These experiments uncovered a list of many miRNAs, protein and mRNA targets in EVs, which were demonstrated to be associated with oocyte and sperm competence as well as with embryo development. However, considerable gaps still need to be addressed, including identifying the mechanisms involved in EVs cargo (miRNA and mRNA) delivery to cumulus and oocytes, the development of non-invasive and reliable platforms to perform IVP diagnostics, and the development of new-generation non-animal-based culture media, to treat gametes and embryos in vitro. This project aims to answer these gaps and test the hypothesis that small extracellular vesicles are a diagnostic and therapeutic tool to improve in vitro embryo production. To test this hypothesis, we propose three main objectives: 1) Determine the mechanisms involved in extracellular vesicles delivery of miRNAs and mRNAs to cumulus cells and early embryos using EVs from follicular fluid and oviductal fluid, as well as their impact on cumulus-oocyte and embryos upon small EVs supplementation. 2) Develop a microfluid system to allow for embryo-conditioned media sampling and EVs analysis. 3) Determine the role of ovarian granulosa cell cultures as bioreactors for the generation of extracellular vesicles compared to liposomes and hybrid vesicles as delivery agents. Upon completion, this project will identify new tools to allow the use of EVs for specific cargo delivery and the use of these vesicles for small volume cell-free based diagnostics. This project will improve our understanding of EVs cargo delivery, therapeutic and diagnostic, which will accelerate the development of a next generation platform for animal reproduction, impacting domestic animal IVP industry and human health. Thus, at the end of this grant we aim to have miRNAs and mRNAs suitable to be used as supplement for IVP and as tools to determine gamete and embryo quality increasing the IVP efficiency. (AU)