Chromosomal Aneuploidy in African Wildcat Somatic Cells and Cloned Embryos

Abstract

In the present study, we compared the incidence of aneuploidy in in vitro fertilized domestic cat embryos (DSH-IVF) with that of African Wildcat (AWC) cloned embryos reconstructed with AWC fibroblast donor cells from different passages (AWC-NT). Fibroblast cells were cultured to passages 1 (P1), 3 (P3), 4 (P4), and 9 (P9), after which cells at each passage were karyotyped and serum-starved before being frozen for nuclear transfer. AWC-NT embryos were produced by fusion of a single AWC somatic cell at P1, P3, P4, or P9 to enucleated domestic cat cytoplast derived from in vitro matured (IVU) oocytes. DSH-IVF embryos were produced after IVU oocytes were fertilized in vitro with domestic cat spermatozoa. To determine chromosome numbers, embryos (2-4-cell) or fibroblast cells were cultured in medium containing 0.28 microg/mL of Colcemid for 22-24 h or 15-24 h, respectively. Subsequently, embryos and cells were placed in hypotonic solution, fixed, and stained for analysis of chromosome spreads by bright field microscopy. Chromosomal abnormalities in AWC fibroblast cells increased progressively during culture in vitro: P1 (43%), P3 (46%), P4 (62%), and P9 (59%). In fibroblast cells, hypoploidy (94/202, 46%) was the major chromosomal abnormality, and it occurred more frequently than hyperploidy (14/202, 7%; p < 0.05). While the percentage of hyperploid cells remained stable during all passages, the proportion of hypoploidy in fibroblast cells increased significantly after P4. The overall incidence of chromosomal abnormalities in AWC-NT embryos at P1 (45%), P3 (60%), and P4 (50%) was similar to that of the fibroblast cells from which they were derived; however, the incidence was higher for embryos reconstructed with donor fibroblasts at P9 (89%). Hypoploidy was the most common chromosomal abnormality observed in either AWC-NT or DSH-IVF embryos. AWCNT embryos reconstructed with donor cells at early passages (P1, P3, and P4) had similar frequencies of chromosomal diploidy, as did DSH-IVF embryos. Accordingly, based on the present results, for NT we are currently using cat donor cells at early passages, when the percentage of cells with chromosomal abnormalities is low. It is recommended that the chromosomal stability of each cell line be analyzed before use as NT donor cells to reduce the incidence of chromosomal anomalies in reconstructed embryos and to possibly produce a subsequent increase in cloning efficiency.

Cloning in companion animal, non-domestic and endangered species: can the technology become a practical reality?

“…Bovine donor cell lines with a high percentage of abnormal cells produced embryos with high rates of chromosomal abnormalities compared with donor cell lines with a low percentage of abnormal cells (Slimane Bureau et al 2003). Similar results were obtained by Gomez et al (2006b) using African wild cat fibroblasts, which were 98% abnormal after only nine passages in vitro. Lack of embryo development beyond the 16-32 cell stage in vitro was observed following Argali sheep SCNT (Loi et al 2001).…”

Hypodiploidy as a prominent attributor to chromosomal aneuploidy in transgenic rabbit embryos

“…However, hypodiploid cells occurred in both transgenic (100%) and non-transgenic (14.3% in microinjected and 73.33% in intact, respectively) rabbit blastocysts at different frequencies. This report is consistent with the works of Gómez et al (2006), who compared the incidence of aneuploidy in in vitro fertilized domestic cat embryos (DSH-IVF) with that of African wildcat (AWC) cloned embryos reconstructed with AWC fibroblast donor cells from different passages (AWC-NT). They reported that hypoploidy was the most common chromosomal abnormality in either AWC-NT or DSH-IVF embryos, and it occurred more frequently than hyperploidy.…”

Investigating Markers of Reprogramming Potential in Somatic Cell Lines Derived from Matched Donors

“…In Experiment 1, cell lines were assessed for variability on a cellular level to ensure uniformity in response to in vitro culture by measuring known factors, including morphology, growth kinetics, genomic stability, apoptosis, and cell type contribution (Gómez et al, 2006;Hayflick and Moorhead, 1961;Mastromonaco et al, 2006;Rhinn et al, 2019;Slimane Bureau et al, 2003). Alterations in many of these metrics can lead to genomic instability (i.e., DNA and telomere damage/dysfunction), which has been shown to affect SCNT embryo development (Davoli et al, 2010;Jagannathan et al, 2016;Slimane Bureau et al, 2003).…”