Aggregating Embryonic but Not Somatic Nuclear Transfer Embryos Increases Cloning Efficiency in Cattle1

Abstract

Our objectives were to compare the cellular and molecular effects of aggregating bovine embryonic vs. somatic cell nuclear transfer (ECNT vs. SCNT) embryos and to determine whether aggregation can improve cattle cloning efficiency. We reconstructed cloned embryos from: 1) morula-derived blastomeres, 2) six adult male ear skin fibroblast lines, 3) one fetal female lung fibroblast line (BFF), and 4) two transgenic clonal strains derived from BFF. Embryos were cultured either singularly (1X) or as aggregates of three (3X). In vitro-fertilized (IVF) 1X and 3X embryos served as controls. After aggregation, the in vitro development of ECNT but not that of SCNT or IVF embryos was strongly compromised. The inner cell mass (ICM), total cell (TC) numbers, and ICM:TC ratios significantly increased for all the aggregates. The relative concentration of the key embryonic transcript POU5F1 (or OCT4) did not correlate with these increases, remaining unchanged in the ECNT and IVF aggregates and decreasing significantly in the SCNT aggregates. Overall, the IVF and 3X ECNT but not the 1X ECNT embryos had significantly higher relative POU5F1 levels than the SCNT embryos. High POU5F1 levels correlated with high in vivo survival, while no such correlation was noted for the ICM:TC ratios. Development to weaning was more than doubled in the ECNT aggregates (10/51 or 20% vs. 7/85 or 8% for 3X vs. 1X, respectively; P < 0.05). In contrast, the SCNT and IVF controls showed no improvement in survival. These data reveal striking biological differences between embryonic and somatic clones in response to aggregation.

Exposure to DNA is insufficient for in vitro transgenesis of live bovine sperm and embryos

“…For zona-free IVM, oocytes were matured individually in 5 ml droplets of IVM medium. Mature zona-free oocytes were cultured individually in 5 ml droplets of IVF medium and inseminated with 2!10 5 spermatozoa/ml as described (Misica-Turner et al 2007). Following insemination for 18-24 h, embryos were cultured singularly in 5 ml droplets of biphasic ESOF/LSOF as described (Oback & Wells 2003).…”

Developmental Ability of Somatic Cell Nuclear Transferred Embryos Aggregated at the 8-cell Stage or 16- to 32-cell Stage in Cattle

“…In a study of cattle, however, aggregation of three embryonic NT embryos improved the calving and survival rates, but no improvement was observed when SCNT embryos were used for aggregation [8]. In this report, however, the timing of aggregation was different between embryonic NT and SCNT, and aggregation was performed at the 1-cell stage for SCNT and at the 2-cell stage for embryonic NT [8]. Furthermore, cloning efficiency is improved by aggregating two mouse SCNT embryos at the 4-cell stage [7].…”

“…In fact, aggregation of two to three somatic cell NT (SCNT) embryos leads to an increase in cell number, blastocysts with normal Oct4 gene expression and full-term development by eightfold in mice [7]. In a study of cattle, however, aggregation of three embryonic NT embryos improved the calving and survival rates, but no improvement was observed when SCNT embryos were used for aggregation [8]. In this report, however, the timing of aggregation was different between embryonic NT and SCNT, and aggregation was performed at the 1-cell stage for SCNT and at the 2-cell stage for embryonic NT [8].…”

18 Embryo Aggregation in Pig Improves Cloning Efficiency and Embryo Quality

“…This situation could be due to a) an epigenetic compensation since some epigenetic defects during cell reprogramming could be overcome by the combination of three embryos derived from the same cell line but with epigenetic differences; b) an increase in embryo cell number at day 0 of their development and a larger embryonic volume in the microwell system that may help embryos compact more easily, or c) a microenvironment generated by the interaction of beneficial and paracrine factors liberated by the aggregate embryo could increase rates of embryonic development; or d) a combination of all those situations. Additionally, embryo aggregation has been proposed as an alternative to avoid the increased level of heteroplasmy caused by the fusion of enucleated oocytes during the handmade cloning procedure [12,16,18]. In general, the term "heteroplasmy" refers to different mtDNA genomes in a single cell.…”

“…Embryo aggregation consists of placing more than one zona free embryos in the same well; some of the reported benefits in mammals are: improvement in embryo development in pig [6], equine [8,9], feline [10] and mouse [11], increasing the density of cells in the inner cell mass on cattle [12], blastocyst diameter in equine and in bovine [8,13], reduction in apoptosis in bovine and pigs [13,14,15], normalization of gene expression in bovine [15]and in some cases, it has also improved in vivo embryo development and early pregnancy rates in mouse [11], equine [8] and in cattle [13,16,17]. As a single clone may have epigenetic defects [15], embryo aggregation could compensate the reprogramming deficiencies through the interactions between blastomeres [11] or by paracrine pathways [11].…”