Two-staged nuclear transfer can enhance the developmental ability of goat–sheep interspecies nuclear transfer embryos in vitro

Abstract

The technique of interspecies somatic cell nuclear transfer, in which interspecies cloned embryos can be reconstructed by using domestic animal oocytes as nuclear recipients and endangered animal or human somatic cells as nuclear donors, can afford more opportunities in endangered animal rescue and human tissue transplantation, but the application of this technique is limited by extremely low efficiency which may be attributed to donor nucleus not fully reprogrammed by xenogenic cytoplasm. In this study, goat fetal fibroblasts (GFFs) were used as nuclear donors, in vitro-matured sheep oocytes were used as nuclear recipients, and a two-stage nuclear transfer procedure was performed to improve the developmental ability of goat-sheep interspecies clone embryos. In the first stage nuclear transfer (FSNT), GFFs were injected into the ooplasm of enucleated sheep metaphase-II oocytes, then non-activated reconstructed embryos were cultured in vitro, so that the donor nucleus could be exposed to the ooplasm for a period of time. Subsequently, in the second stage nuclear transfer, FSNT-derived non-activated reconstructed embryo was centrifuged, and the donor nucleus was then transferred into another freshly enucleated sheep oocyte. Compared with the one-stage nuclear transfer, two-stage nuclear transfer could significantly enhance the blastocyst rate of goat-sheep interspecies clone embryos, and this result indicated that longtime exposure to xenogenic ooplasm benefits the donor nucleus to be reprogrammed. The two-stage nuclear transfer procedure has two advantages, one is that the donor nucleus can be exposed to the ooplasm for a long time, the other is that the problem of oocyte aging can be solved.

In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors

“…Some reports have focused on this step, which has demonstrated to be beneficial to epigenetic reprogramming and normal embryo development in different mammalian species [ 13 , 14 , 15 ]. However, when this interval was prolonged embryo development was compromised [ 16 , 17 , 18 ], probably due to oocyte aging [ 19 ].…”

Timing Factors Affecting Blastocyst Development in Equine Somatic Cell Nuclear Transfer

“…The optimal R-A delay varies among species (Choi et al, 2004;Liu et al, 2013;Wakayama et al, 1998). It is likely that the optimum period of exposure of donor nuclei to the nonactivated ooplast is related to the duration of maintenance of oocyte developmental competence after the oocyte reaches MII in each species (Ma et al, 2011). Oocytes matured in vitro for 20 h had a significantly higher rate of blastocyst formation after NT than did oocytes matured for 24 h (experiment 3); however, at 20 h over half the membrane-intact oocytes were still in MI.…”

“…However, reduced embryo development has resulted when intervals from donor nucleus transfer to activation were prolonged (Choi et al, 2004;Liu et al, 2013;You et al, 2010). In a notable report on this subject, Ma et al (2011), working on goatsheep nuclear transfer, separated the effects of prolonged exposure of donor chromatin to the oocyte cytoplasm from the effects of prolonged post-metaphase II (MII) ''aging'' of the oocyte by a two-stage transfer procedure. They found that blastocyst development decreased after 6 h if the nucleus remained in the primary host ooplast, but continued to increase up to 18 h exposure if the nucleus was then transferred to a second, ''young'' MII ooplast for activation.…”

Improved development of somatic cell cloned bovine embryos by a mammary gland epithelia cellsin vitromodel

“…Nuclear transfer was performed according to the method reported by Ma et al [ 20 ]. Briefly, in vitro –matured eggs with the first polar body were cultured in hepes-synthetic oviductal fluid (H-SOF) medium supplemented with 10% (v/v) FBS, 7.5 g/mL cytochalasin B (CB; Sigma) and 10 g/mL Hoechst 33342 (Sigma) at 38.5℃ for 15 min.…”