Embryo aggregation and adipose-derived mesenchymal donor cells in bovine somatic cell nuclear transfer

Abstract

Somatic cell nuclear transfer (SCNT) is a powerful tool, but its efficiency remains low. The use of less differentiated donor cells or the embryo aggregation (EA) strategy improves the SCNT rates in several species. It remains unexplored whether the combined use of both strategies results in a synergistic effect that improves SCNT efficiency in bovine. To evaluate that, we assessed the optimal time of EA using IVF embryos (aim 1) and we evaluated whether the use of adipose-derived mesenchymal stem cells (ASC) as donor for SCNT together with EA improves the blastocyst rates and quality (aim 2). For aim 1, cumulus–oocyte complexes (COCs) were collected from slaughterhouse ovaries, in vitro matured (TCM-199), fertilized (16 × 106 spermatozoa mL−1 for 5 h) and cultured (synthetic oviductal fluid media in a humidified gas mixture at 39°C). After IVF, the zona pellucida was enzymatically removed and zona-free (ZF) embryos were cultured individually (1X) or 2 embryos placed together within a microwell (2X) (Day 0, n = 70). This procedure was performed at Days 3, 4, 5, 6, or 7 (n = 76, 78, 94, 96, 90, respectively) and blastocyst rate was assessed at Day 8. Contribution of both embryos to the 2X blastocyst was confirmed by staining Day 0 IVF embryos either with green or red Mitotracker (ThermoFisher Scientific) before EA. For aim 2, fibroblast (FB) and ASC cells were isolated from the skin and subcutaneous adipose tissue of the same adult animal, respectively. Cloned embryos were produced by ZF enucleation, fusion of one ASC or FB cell, and activation with 5 μM ionomycin/6-(dimethylamino)purine (6DMAP). After activation, cloned embryos were aggregated (FB2X or ASC2X) or individually cultured (FB1X or ASC1X). Blastocyst rates were recorded at Day 7 of in vitro culture. Three biological replicates were evaluated for each aim. Embryo developmental differences were determined using Fisher’s exact test. Relative expression of OCT4, SOX2, and KRT18 was measured by RTqPCR at the blastocyst stage and analysed by Kruskal–Wallis statistical test. Regarding aim 1, no differences for developmental rates were found for Day 0, 3, 4, and 5 groups (57%, 60%, 61.5%, 61%), but the blastocyst rate was only improved in Day 0 and Day 3 relative to their respective 1X controls (Day 0 2X 54.2% vs. Day 0 1X 25.5% and Day 3 2X 52.6% vs. Day 3 1X 25.3%). No aggregation occurred in Day 6 and Day 7 groups. All blastomeres were homogeneously distributed in the 2X blastocyst. Regarding aim 2, no effect of the donor cell was observed on the blastocyst rate (FB1X 26.8%, n = 82; ASC1X 21.7%, n = 198; FB2X 39.7%, n = 126; ASC2X 33%, n = 204), whereas EA improved the blastocyst rate of ASC-derived embryos (ASC1X 21.7% vs. ASC2X 33%). Overall, no synergistic effect of the use of both strategies was observed. Relative expression of KRT18 was significantly different between ASC1X and ASC2X embryos. Although OCT4 and SOX2 expression did not differ between groups, EA tended to bring the values closer to that of an IVF embryo. No effect of the donor cell was observed on the embryo relative expression. Our results suggest that EA at Day 0 improves the blastocyst rate in bovine SCNT and IVF embryos. EA of 2 ASC-derived embryos seemed to normalise the embryo quality and may improve post-implantation development.