20 IMMUNOCYTOCHEMICAL CHARACTERIZATION OF DAYS 17 AND 19 OVINE IN VIVO AND SOMATIC CELL NUCLEAR TRANSFER EMBRYOS
N. I. Alexopoulos A B , K. Schauser B , M. K. Holland A , T. T. Peura C , K. M. Hartwich D , S. K. Walker D , N. T. D'Cruz A , A. J. French E F and P. Maddox-Hyttel AA Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia;
B University of Copenhagen, Copenhagen, Denmark;
C Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, Queensland, Australia;
D South Australian Research and Development Institute, Rosedale, South Australia;
E Stemagen Corporation, La Jolla, California, Australia;
F Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia
Reproduction, Fertility and Development 21(1) 110-110 https://doi.org/10.1071/RDv21n1Ab20
Published: 9 December 2008
Abstract
The present study aimed to characterize development of in vivo and somatic cell nuclear transfer (SCNT) Merino ovine embryos on Days 17 and 19 using the mesoderm marker vimentin, the neuroectoderm marker β-tubulin III, and the pluripotency marker OCT4 for primordial germ cells. In vivo embryos were obtained by transferring 10 to 20 zygotes to each of 20 intermediate recipient ewes. On Day 6, 4 final recipients were used to obtain embryos for recovery at Day 17 (n = 2) and at Day 19 (n = 2). SCNT embryos were constructed from in vitro-matured oocytes and adult granulosa cells. On Day 6, 9 embryos (with intact inner cell masses) were transferred to each recipient for collection at Day 17 (n = 2) and Day 19 (n = 2). Ewes were euthanazed with phenobarbitone and excised reproductive tracts flushed with saline. A total of 24 embryos at Day 17 (14 in vivo and 10 SCNT) and 23 embryos at Day 19 (11 in vivo and 12 SCNT) were collected and processed for immunohistochemistry. On Day 17, length of embryo proper for in vivo and SCNT embryos was 6.2 and 5.6 mm, width of allantois was 9.3 and 3.8 mm, and number of somites was 19 and 13, respectively. On Day 19, length of embryo proper for in vivo and SCNT embryos was 11.5 and 7.3 mm, width of allantois was 70.6 and 13.7 mm, and number of somites was 28 and 22, respectively. On both Day 17 and Day 19, in vivo embryos had a much larger allantois, more somites, and were longer in length. Vimentin staining was observed in all in vivo embryos at Day 17 and Day 19; however, in 75% of Day 17 SCNT embryos, differentiation of the somites into dermatome with underlying sclerotome was either delayed or absent. Similarly, at both Day 17 and Day 19, a larger proportion of embryos did not stain for β-tubulin III, and of those that did, only a small amount was found in the neural tube. The intensity of both stains was much weaker in the SCNT embryos compared to in vivo embryos. Oct-4 was initially found in the splanchnic mesoderm lining the endoderm in the wall of the yolk sac and later in the dorsal mesentery and medial aspect of the mesonephros with in vivo embryos having double the number of positively stained cells than SCNT embryos. The delay in mesoderm compartments of SCNT embryos could imply future problems in the musculoskeletal system. However, lack of any neuroectoderm staining in more than half SCNT embryos could point to neurological problems being more of an issue in terms of survival and well-being. Equally, the smaller allantois could induce later placental abnormalities.
The current project was funded by the Co-operative Research Centre for Innovative Dairy Products (CRC-IDP), Australia.
