The success of development of invitro embryo production needs to mimic culture conditions in the maternal environment. Recently, it has been seen that extracellular vesicles (EVs) secreted by oviducal or endometrial cells may improve development and quality of embryos produced invitro. Extracellular vesicles are a mechanism of cellular communication; they carry molecules that are delivered into the target cells changing gene expression and function. Due to the size range and characteristics of EVs, they require specific methods for purification and characterisation. However, the possible contamination with other nanoparticles and their effect on embryo development have not been considered. Based on that, the goal of this work was to evaluate the effect on invitro bovine embryo development, of the addition to culture medium EVs secreted by oviducal and endometrial cells and isolated by centrifugation and concentrates with Amicon filters. For this purpose, cells were isolated from bovine oviduct and endometrium collected in local abattoir and primary cultures of epithelial and stromal cells were derived. The primary cultures from both sources were exposed or not to progesterone (P4; 15ngmL−1) for 4 days and then cultured for 24h in EV depleted media. The supernatant was harvested and EVs were isolated by serial centrifugations and subsequently concentrated by a 100 kDa Amicon filter system. The isolated EVs were characterised by transmission electron microscopy, nanoparticle tracking analysis, and flow cytometry. Oocytes were obtained from ovaries collected in the abattoir. The cumulus-oocyte complexes were matured invitro for 22h and subsequently fertilised for 18h. Presumptive zygotes were invitro cultured in synthetic oviducal fluid with EVs (1000ngmL−1 of total proteins) or not according to experimental group (1: EVs− (control); 2: EVs−OP4+; 3: EVs−OP4−; 4: EVs−EP4+ and 4: EVs−EP4−). Embryos were cultured for 7 days in 5% CO2, 5% O2, and 90% N2 (25 embryos/well in 4-well plates). At Day 7, embryo development was evaluated considering the blastocyst yield. Transmission electron microscopy showed typical structures and morphology of EVs and they were positive for CD9, CD63, and CD81 markers, and negative for CD40. According to nanoparticle tracking analysis, the mean size of EVs was 160±62nm and concentration of 3.29×1011 particlesmL−1 for oviducal and endometrial cells, respectively. A significant reduction of blastocyst rate was observed when embryos were cultured with cell-derived EVs; control: 152/44 (28.9%) vs. treatments with EVs; OP4+: 74/3 (4.1%), OP4−:76/2 (2.6%), EP4+: 74/6 (8.1%), and EP4−: 73/2 (2.7%) (P ≤ 0.01). Our results indicate that the use of nanoparticles, including EVs, isolated from cells of oviduct or endometrium, has a blocking effect on embryonic development and compromises the performance of blastocysts on Day 7 when used at concentrations of 1000ngmL−1 total protein, independent of the use or not of P4 and the source. These data provide insights regarding the use and protocols of acquiring exosomes for embryo supplementation.
This research was supported by FONDECYT, Chile-1170310.
Evidence for soluble factors regulating cell death and cell proliferation in primary cultures of rabbit endometrial cells grown on collagen.