Extracellular Vesicles from BOEC in In Vitro Embryo Development and Quality.

Ricaurte Lopera-Vásquez,Miguel Ángel Ramirez,Beatriz Fernandez-Fuertes,Paula Beltrán-Breña,Meriem Hamdi,Verónica Maillo,Alfonso Gutierrez-Adán,Alexandra Calle,Soraya López-Martín,María Yañez-Mó,Alberto Redruello,Dimitrios Rizos

doi:10.1371/journal.pone.0148083

Abstract

To evaluate the effect of conditioned media (CM) and Extracellular Vesicles (EVs) derived from bovine oviduct epithelial cell (BOEC) lines on the developmental capacity of bovine zygotes and the quality of embryos produced in vitro, presumptive zygotes were cultured under specific conditions. In experiment 1, zygotes were cultured either on monolayers from BOEC extended culture (E), together with fresh BOEC suspension cells, or with BOEC-CM from fresh or E-monolayers. In experiment 2, EVs were isolated from BOEC-CM and characterized (150–200 nm) by Nanosight® and electron microscopy. Zygotes were cultured in the presence of 3x105EVs/mL, 1.5x105EVs/mL or 7.5x104EVs/mL of fresh or frozen BOEC-EVs. In experiment 3, zygotes were cultured in absence of FCS but with EVs from BOEC-E that had been cultured in different culture media. In experiment 4, zygotes were cultured in SOF+5% normal-FCS, or EV-depleted-FCS. In all cases, cleavage rate (Day 2) and blastocyst development (Day 7–9) was assessed. Blastocysts on Days 7/8 were used for quality evaluation through differential cell count, cryotolerance and gene expression patterns. No differences were found among all FCS-containing groups in cleavage rate or blastocyst yield. However, embryos derived from BOEC-CM had more trophectoderm cells, while embryos derived from BOEC-EVs, both fresh and frozen, has more trophectoderm and total cells. More embryos survived vitrification in the BOEC-CM and BOEC-EV groups. In contrast, more embryos survived in the EV-depleted-FCS than in normal-FCS group. Gene expression patterns were modified for PAG1 for embryos cultured with EVs in the presence of FCS and for IFN-T, PLAC8, PAG1, CX43, and GAPDH in the absence of FCS. In conclusion, EVs from FCS have a deleterious effect on embryo quality. BOEC-CM and EVs during in vitro culture had a positive effect on the quality of in vitro produced bovine embryos, suggesting that EVs have functional communication between the oviduct and the embryo in the early stages of development.

Highlights

In vitro embryo production is a useful tool to study early embryonic development in mammals, to solve reproductive issues in humans and to conserve gametes from animals with high genetic merit or endangered species
The use of conditioned media from a Bovine oviduct epithelial cells (BOEC) extended culture monolayer has a positive effect on the quality of bovine embryos
It is well known that culture environment during embryo development has an impact on the quality of the produced embryos in terms of cryotolerance [1,14], ultrastructure morphology [48]; embryo cell number [49] and gene expression [50,51]

Summary

Introduction

In vitro embryo production is a useful tool to study early embryonic development in mammals, to solve reproductive issues in humans and to conserve gametes from animals with high genetic merit or endangered species. The first stages of bovine embryo development occur in the oviduct, where the embryo spends around 4 days [5]. The oviduct is an active organ that maintains and modulates the milieu for sperm capacitation, transport and fertilization of the mature oocyte and early embryonic development [6,7,8]. Embryonic Genome Activation (EGA), the time at which the embryo starts to synthetize and use its own mRNA, is the most important step and occurs at the 8–16 cell stage [10], ensuring normal preimplantation and early fetal development [11]

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