Nuclear Transplantation in Bovine Embryos

Abstract

This study was conducted to develop a method for transplanting nuclei in bovine embryos and to test the development of several stages of donor nuclei transplanted to enucleated pronuclear recipient embryos. Pronuclear embryos were centrifuged to reveal nuclei. Nuclei were removed without penetrating the plasma membrane as membrane-bound karyoplasts, and were inserted into enucleated zygotes by electrically induced cell fusion. The highest rate of fusion (79%) occurred in Zimmerman Cell Fusion medium at 100 V for 20 to 40 microseconds with the fusion membranes oriented parallel to the electrodes. The effect of nuclear transplantation on development was tested in pronuclear embryos in which nuclei were removed and reinserted and the embryos were then transferred to sheep oviducts for 5 d. Of the intact nuclear transplant embryos recovered, 5/29 (17%) developed to morulae or blastocysts compared with 11/30 (37%) of the non-manipulated embryos. Two nuclear transplant embryos were transferred to a recipient cow, and both developed to normal offspring. When nuclei from two-, four-, or eight-cell embryos were transplanted to pronuclear recipient embryos, no development was observed.

Improved Development to Blastocyst of Ovine Nuclear Transfer Embryos Reconstructed during the Presumptive S-Phase of Enucleated Activated Oocytes

“…Similarly, when pronuclear exchange is carried out in both bovine or porcine zygotes, the reconstructed embryos are capable of development [3,5]. However, experiments in cows using donor blastomeres from 2-, 4-, and 8-cell embryos resulted in no development [3]. The lack of development observed in ungulate embryos reconstructed from enucleated zygotes suggests that other as yet unidentified factors are required for the development of reconstructed embryos.…”

“…Experiments in both cattle [29] and pigs [30] have shown that visualization of the pronuclei by centrifugation does not affect the developmental potential of one-cell zygotes. Similarly, when pronuclear exchange is carried out in both bovine or porcine zygotes, the reconstructed embryos are capable of development [3,5]. However, experiments in cows using donor blastomeres from 2-, 4-, and 8-cell embryos resulted in no development [3].…”

“…The reconstruction of early embryos by the transfer of a nucleus from a donor cell (or karyoplast) to an enucleated oocyte or pronuclear zygote (cytoplast) has been accomplished in a variety of mammalian and amphibian species, including mice [1], cattle [2,3], sheep [4], pig [5], rabbit [6], and amphibians (i.e., Rana [7,8]; Xenopus [9]). Although the methods used for embryo reconstruction across these species vary (for review see [10]) they fall broadly into two groups dependent upon the cell cycle stage of the recipient cytoplast.…”

Performance of Holstein Clones in the United States

“…The cloning technologies of embryo splitting (Willadsen, 1979) and nuclear transfer (Robl et al, 1987) were introduced to dairy cattle breeding in the 1980s. Holstein Association USA first registered calves from embryo splitting in 1982 and from nuclear transfer in 1989.…”

Cloning: Eight Years After Dolly

“…The use of enucleated metaphase II oocytes as recipient cytoplasts proved more successful and in 1986 resulted in the production of live lambs using blastomeres from 8 to 16-cell stage embryos as nuclear donors (Willadsen 1986). Although this success in sheep was repeated in other species including cattle (Robl et al 1987) and pigs (Prather et al 1989), there were major limitations to the utility of the technology; first the frequency of development was very low limiting the number of identical animals which could be produced; secondly this number was further limited by the number of cells in the embryonic stage from which successful development could be obtained. To counteract these problems, the search for a suitable nuclear donor cell type which could be maintained in culture became the aim of many groups.…”