In vitro maternal-embryonic interaction mediated by extracelular vesicles in cattle

Author(s)

Amanda Nespolo Silva

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2023-04-13

Examining board members

Flavio Vieira Meirelles; Mario Binelli; Juliano Coelho da Silveira

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

Abstract

In ruminants, maternal recognition of pregnancy (MRP) involves the production of interferon tau (IFNT) by the trophectoderm (TE) cells of the embryo to prevent luteolysis. Although IFNT signaling is well characterized, the production and release of extracellular vesicles (Evs) has emerged as a potential mechanism of cellular communication between mother and embryo during MRP. Thus, the hypothesis of this project is that endometrial and TE cells cultivated in vitro can produce and release Evs, which can be internalized and modulate the transcriptome in the target cells. To test this hypothesis, we generated cultures of endometrial cells (epithelial and stromal) and TE cells from IVF blastocysts. After, Evs were isolated from the culture media. The epithelial and stromal cell lineages (n = 5) were maintained until the 4th passage and were characterized by immunofluorescence (anti-cytokeratin, epithelial cells and anti-vimentin, stromal cells). TE cells from IVF embryos were cultured until the 2nd passage and characterized by immunofluorescence (anti-CDX2). Evs were evaluated for particle size and concentration using Nanoparticle Tracking Analysis (NTA). Regarding the characterization of endometrial cells, only epithelial cells were positive for cytokeratin and stromal cells were positive for vimentin, as expected. TE cells were positive for CDX2. The Evs showed average size of 131.92 ± 5.52, 153.46 ± 7 and 143.66nm ± 4.60 and concentration 6.54E+08 ± 4.57E+07, 8.15E+08 ± 4, 59E+07 and 2.56E+11 ± 1.61E+10 particles/mL, for epithelial, stromal and TE cells, respectively. There was no significant difference (P>0.05) between the groups. The characterization of the Evs by Western blotting confirmed the presence of ALIX and the absence of the GRP78 protein in the Evs. Furthermore, transmission electron microscopy (TEM) showed that Evs have the expected morphology and size (<150nm). To simulate maternal- embryonic crosstalk in vitro and investigate how Evs modulate transcripts in target cells, we performed the treatment of endometrial cells with Evs from TE and vice versa, the treatment of TE cells with Evs from endometrial cells. After 6 hours of treatment, RNAseq revealed that Evs from the TE lineage more broadly altered the transcriptional profile of stromal cells compared to epithelial cells. Some of the significantly altered genes (P<0.05) are related with important signaling pathways such as PI3K/Akt pathway, fundamental during the pre- implantation development period, ECM-receptor interaction pathway, directly related with the early contact of the embryo with endometrium, Cytokine-cytokine signaling pathway, crucial during immunological and inflammatory responses, among others. Herein, we generate for the first time, an in vitro model that mimics the microenvironment of early embryonic maternal interaction by the exchange of Evs between endometrial and embryonic cells. The knowledge generated here will clarify specific effects of Evs, providing the IVF environment more similar to the in vivo. (AU)

• FAPESP's process: 21/02458-0 - In vitro maternal-embryary interaction mediated by extracellular vesicles in cattle
• Grantee: Amanda Nespolo Silva
• Support Opportunities: Scholarships in Brazil - Master