77 LOSS OF SAND CAT CLONED EMBRYOS DURING EARLY PREGNANCY MAY BE ASSOCIATED WITH ABERRANT Oct-4 EXPRESSION

Abstract

Genetic incompatibilities between the donor nucleus and the recipient cytoplast of inter-species cloned embryos may be associated with a low frequency of development to the blastocyst stage and/or in vivo developmental competence. We have established pregnancies after the transfer of inter-species Black-footed cat (BFC; Felis nigripes) cloned embryos and live offspring after the transfer of inter-species African Wildcat (AWC; Felis silvestris lybica) cloned embryos into domestic cat recipients (G�mez et al. 2006 Theriogenology 66, 72; 2004 Cloning Stem Cells 6, 217). However, many of the pregnancies were re-absorbing before Day 30 of gestation. According to Arat et al. (2003 Mol. Reprod. Dev. 66, 334), inter-generic mouse–bovine cloned embryos do not express the POU transcription factor Octamer-4 (Oct-4), and this abnormal gene expression may compromise pluripotency and contribute to the failure of cloned embryos to undergo post-implantation development (Boiani et al. 2002 Genes Dev. 16, 1209). The purpose of the present study was to evaluate (1) the in vivo developmental competence of nuclear transfer embryos derived by fusion of Sand cat (SC; Felis margarita) fibroblasts with domestic cat (DSH; Felis catus) cytoplasts after transfer into domestic cat recipients, and (2) protein distribution of Oct-4 in derived cloned blastocysts. Fibroblast cell lines were established from skin biopsies of 2 adult SC (both males). Cloned embryos were produced as described by G�mez et al. (2004 Cloning Stem Cells 6, 217). Reconstructed SC (n = 1113) Day 1 NT embryos were transferred by laparoscopy into the oviducts of 29 gonadotropin-treated DSH recipients on Day 1 after induced ovulation. Pregnancy was assessed by ultrasonography on Day 22. Seven (24%) cats receiving somatic cell nuclear transfer (SCNT) embryos became pregnant. Two SC cloned embryos developed to term; one kitten died in utero on Day 61 of gestation and the second kitten died 2 h after being delivered by caesarean section on Day 62 of gestation. The clonal status of the SC kittens was assessed by multiplex PCR amplification of 20 microsatellite markers. Immunocytochemical localization of Oct-4 was determined on Day 8 SCNT (n = 16) and DSH/IVF (n = 10) blastocysts. Embryos were incubated in 1 : 100 anti-human Oct-4 polyclonal antibody for 1 h, followed by incubation in 1 : 128 FITC-labeled anti-goat IgG for 1 h. Embryos were then counter-stained with propidium iodide, and examined with epifluorescence microscopy. Most cloned blastocysts had a detectable signal of Oct-4 (69%), but some exhibited aberrant expression in the trophectoderm (TPD; 44%). In contrast, the signal in most DSH/IVF blastocysts was restricted to the inner cell mass (ICM; 80%). Oct-4 expression was not detected in 2-cell DSH/IVF embryos. These observations suggest that aberrant Oct-4 expression may contribute to the embryonic loss observed in the present study.