Extracellular vesicles as a platform for diagnostic and manipulation of the in vitro embryo production system: the next generation in animal reproduction

• Grant number: 21/06645-0
• Support Opportunities: Research Grants - Young Investigators Grants - Phase 2
• Start date: March 01, 2022
• End date: February 28, 2027
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Juliano Coelho da Silveira
• Grantee: Juliano Coelho da Silveira
• Host Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil
• Associated researchers: Felipe Perecin ; Flávio Vieira Meirelles ; Guilherme Pugliesi ; Lawrence Charles Smith ; Lucimara Gaziola de la Torre ; Marcelo Bispo de Jesus ; Marcia de Almeida Monteiro Melo Ferraz ; Samantha Cristina de Pinho
• Associated research grant: 14/22887-0 - Cell-secreted vesicles containing miRNAs modulate epigenetic changes during in vitro culture of bovine gametes and embryos, AP.JP
• Associated research grant(s): 23/01511-0 - Analysis of the mechanisms involved in miRNA delivery mediated by small extracellular vesicles to COCs and bovine embryos, AP.R; 22/01433-7 - Multi-user equipment approved in the grant FAPESP 21/06645-0: Inverted Microscope Yhunder Imager 3D Assay, AP.EMU; 22/01505-8 - Multi-user equipment approved in grant 21/06645-0: CytoFLEX System B3-R0-V3, AP.EMU
• Associated scholarship(s): 24/03052-6 - Extracellular Vesicles Containing NPR2 and EGFr Supplemented During Oocyte Maturation: Effects on Transzonal Projection Communication, Meiotic Resumption, and Oocyte Quality, BP.MS; 23/02746-1 - Influence of body energy reserves on the environment of the uterotubal junction and the anterior uterine horn portion of Nellore cows, BP.MS; 23/16444-7 - Influence of palmitic acid on the content of Extracellular Vesicles in uterine epithelial cells and its consequences in the in vitro culture of bovine embryos, BP.IC; + associated scholarships 24/01143-4 - Characterization of extracellular vesicles isolated from the oviductal and follicular fluid from the reproductive tract of bovine females., BP.TT; 23/08030-8 - ANALYSIS OF THE EXTRACELLULAR VESICLES ENDOCYTOSIS PROCESS IN OVARIAN FOLLICLE CELLS, BP.IC; 23/05684-7 - Characterization of extracellular vesicles isolated from the oviductal and follicular fluid from the reproductive tract of bovine females., BP.TT; 22/09461-0 - Transfection of extracellular vesicles from follicular fluid with specific microRNAs: a form of miRNA delivery to bovine cumulus-oocyte complex cells, BP.MS; 22/01235-0 - Extracellular vesicles as an amplifier of embryonic signals: implications to maternal immune response, BP.PD; 21/12560-7 - Extracellular vesicles from bovine follicular fluid isolated by different methods have distinct characteristics and microRNA profile, BP.MS; 22/01236-7 - Hybrid and synthetic vesicles formed by extracellular vesicles and liposomes for the supplementation of embryo cells during bovine in vitro production, BP.PD; 22/02701-5 - Generation of microfluidic devices with different geometries for in vitro fertilization, BP.PD; 20/13075-2 - Dairy cows metabolic status effects on epigenetic changes in the embryo and endometrium: the role of extracellular vesicles, BP.PD - associated scholarships

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)

Scientific publications

(11)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

GIULIANA DE AVILA FERRONATO; FRANCIELE FLORES VIT; JULIANO COELHO DA SILVEIRA. 3D culture applied to reproduction in females: possibilities and perspectives. Animal Reproduction, v. 21, n. 1, 2024-03-08. (21/06645-0, 22/02701-5, 19/25675-7)

DLAMINI, NOTSILE H.; BRIDI, ALESSANDRA; DA SILVEIRA, JULIANO COELHO; FEUGANG, JEAN M.. Unlocking Gamete Quality Through Extracellular Vesicles: Emerging Perspectives. BIOLOGY-BASEL, v. 14, n. 2, p. 23-pg., 2025-02-01. (21/06645-0)

BARCELOS, STEFHANI MARTINS; ROSA, PAOLA MARIA DA SILVA; MOURA, ANA BEATRIZ BOSSOIS; VILLARROEL, CARLA LUJAN PEREIRA; BRIDI, ALESSANDRA; BISPO, ELIZABETE CRISTINA ISEKE; GARCEZ, EMANUELLA MELGACO; OLIVEIRA, GABRIELA DE SOUZA; ALMEIDA, MARIA ALICE; MALARD, PATRICIA FURTADO; et al. Extracellular vesicles derived from bovine adipose-derived mesenchymal stromal cells enhance in vitro embryo production from lesioned ovaries. CYTOTHERAPY, v. 26, n. 10, p. 11-pg., 2024-09-24. (22/01505-8, 21/06645-0, 19/23840-0)

FIORENZA, MARIANI FARIAS; BRIDI, ALESSANDRA; DOS SANTOS, GISLAINE; ROSA, PAOLA MARIA; ALVES, LUANA; FERST, JULIANA GERMANO; FERRAZ, PRISCILA ASSIS; PUGLIESI, GUILHERME; POHLER, KY; PERECIN, FELIPE; et al. Labeled extracellular vesicles can be found in the blood plasma shortly after intrauterine infusion in bovine. ANIMAL REPRODUCTION, v. 21, n. 3, p. 6-pg., 2024-01-01. (22/01505-8, 19/06696-3, 22/01235-0, 22/01433-7, 21/06645-0)

DA SILVA ROSA, PAOLA MARIA; BRIDI, ALESSANDRA; DE AVILA FERRONATO, GIULIANA; PRADO, CIBELE MARIA; BASTOS, NATALIA MARINS; SANGALLI, JULIANO RODRIGUES; MEIRELLES, FLAVIO VIEIRA; PERECIN, FELIPE; DA SILVEIRA, JULIANO COELHO. Corpus luteum presence in the bovine ovary increase intrafollicular progesterone concentration: consequences in follicular cells gene expression and follicular fluid small extracellular vesicles miRNA contents. JOURNAL OF OVARIAN RESEARCH, v. 17, n. 1, p. 14-pg., 2024-03-18. (21/06645-0, 20/08478-0, 21/09886-8)

FERRONATO, GIULIANA DE AVILA; DOS SANTOS, CAROLINA MONICA; ROSA, PAOLA MARIA DA S.; BRIDI, ALESSANDRA; PERECIN, FELIPE; MEIRELLES, FLAVIO VIEIRA; SANGALLI, JULIANO RODRIGUES; DA SILVEIRA, JULIANO COELHO. Bovine in vitro oocyte maturation and embryo culture in liquid marbles 3D culture system. PLoS One, v. 18, n. 4, p. 17-pg., 2023-04-21. (19/25675-7, 14/22887-0, 21/06645-0)

MOUGENOT, MARCEL FRANCO; PEREIRA, VANESSA SOUSA; RODRIGUES COSTA, ANA LETICIA; LANCELLOTTI, MARCELO; PORCIONATTO, MARIMELIA APARECIDA; DA SILVEIRA, JULIANO COELHO; DE LA TORRE, LUCIMARA GAZIOLA. Biomimetic Nanovesicles-Sources, Design, Production Methods, and Applications. PHARMACEUTICS, v. 14, n. 10, p. 25-pg., 2022-10-01. (21/06645-0, 21/11564-9)

SARAIVA, HELENA FABIANA REIS DE ALMEIDA; SANGALLI, JULIANO RODRIGUES; ALVES, LUANA; DA SILVEIRA, JULIANO COELHO; MEIRELLES, FLAVIO VIEIRA; PERECIN, FELIPE. NPPC and AREG supplementation in IVM systems alter mRNA translation and decay programs-related gene expression in bovine COC. ANIMAL REPRODUCTION, v. 21, n. 2, p. 19-pg., 2024-01-01. (21/08759-2, 13/08135-2, 22/01433-7, 21/09886-8, 21/06645-0)

ROSA, PAOLA MARIA DA SILVA; BRIDI, ALESSANDRA; FERRONATO, GIULIANA DE AVILA; NOCITI, RICARDO PERECIN; DOS SANTOS, ANGELICA CAMARGO; CATALDI, THAIS REGIANI; DOS SANTOS, GISLAINE; CHIARATTI, MARCOS ROBERTO; SILVA, LUCIANO ANDRADE; PUGLIESI, GUILHERME; et al. Corpus luteum proximity alters molecular signature of the small extracellular vesicles and cumulus cells in the bovine ovarian follicle environment. Molecular and Cellular Endocrinology, v. 592, p. 12-pg., 2024-08-28. (21/06645-0, 20/08478-0, 21/09886-8)

FERRONATO, GIULIANA DE AVILA; VIT, FRANCIELE FLORES; DA SILVEIRA, JULIANO COELHO. 3D culture applied to reproduction in females: possibilities and perspectives. ANIMAL REPRODUCTION, v. 21, n. 1, p. 18-pg., 2024-01-01. (21/06645-0, 22/02701-5, 19/25675-7)

ANGULO, DANIEL LOPEZ; LOURENCO, RODRIGO VINICIUS; BRIDI, ALESSANDRA; CHAVES, MATHEUS ANDRADE; DA SILVEIRA, JULIANO COELHO; SOBRAL, PAULO JOSE DO AMARAL. Enhancing Bovine Embryo Development In Vitro Using Oil-in-Water Nanoemulsions as Specific Carriers for Essential Lipids. BIOTECH, v. 13, n. 2, p. 16-pg., 2024-06-01. (13/07914-8, 21/06645-0)