Nuclear transplantation by microinjection of inner cell mass and granulosa cell nuclei

The developmental potential of bovine inner cell mass (ICM) and somatic differentiated (granulosa cell) nuclei was investigated using nuclear transplantation. ICM blastomeres were isolated after immunosurgery of day 7 in vitro produced blastocysts and cumulus granulosa cells recovered from in vitro matured oocytes. Nuclear transplantation was carried out by microinjection of the lysed donor cells into enucleated mature oocytes. Oocytes were activated by three 0.2 kVcm−1/20 μs pulses in mannitol containing 100 μM Ca2+, with each pulse 22 min apart. Embryos were cultured in vitro for 7 days and blastocysts were transferred into recipients. ICM and granulosa cell donor nuclei directed 7% (20/304) and 9% (19/213) development to blastocysts, respectively. Fifteen blastocysts from ICM donors resulted in four pregnancies (27%) and two births. No pregnancy was detected with granulosa cell donors. The results illustrate the totipotency of ICM nuclei and indicate that granulosa cell nuclei promote preimplantation development of nuclear transplant embryos. © 1994 Wiley‐Liss, Inc.

[1]  J. Gurdon,et al.  Adult frogs derived from the nuclei of single somatic cells. , 1962, Developmental biology.

[2]  P. Collas,et al.  Development of rabbit nuclear transplant embryos from morula and blastocyst stage donor nuclei , 1991 .

[3]  R. Briggs,et al.  Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs' Eggs. , 1952, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. D. Di Berardino,et al.  Development and chromosomal constitution of nuclear-transplants derived from male germ cells. , 1971, The Journal of experimental zoology.

[5]  J. Modliński.  The fate of inner cell mass and trophectoderm nuclei transplanted to fertilized mouse eggs , 1981, Nature.

[6]  F. Barnes,et al.  The kinetics of oocyte activation and polar body formation in bovine embryo clones , 1992, Molecular reproduction and development.

[7]  R. Briggs,et al.  Nuclear transplantation studies on the early gastrula (Rana pipiens). I. Nuclei of presumptive endoderm. , 1960, Developmental biology.

[8]  R. Briggs,et al.  Changes in the nuclei of differentiating endoderm cells as revealed by nuclear transplantation , 1957 .

[9]  M. Diberardino.  Genomic potential of differentiated cells analyzed by nuclear transplantation , 1987 .

[10]  P. Collas,et al.  Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution, and development in nuclear transplant bovine embryos , 1993, Molecular reproduction and development.

[11]  K. Illmensee,et al.  Nuclear transplantation in mus musculus: Developmental potential of nuclei from preimplantation embryos , 1981, Cell.

[12]  T. Kono,et al.  Development of mouse oocytes receiving embryonic nuclei and thymocytes , 1991 .

[13]  K. Bondioli,et al.  Freeze survival of bovine embryos produced by maturation, fertilization and culture of oocytes , 1992 .

[14]  T. Tokunaga,et al.  Nuclear transplantation of male primordial germ cells in the mouse. , 1989, Development.

[15]  P. Collas,et al.  Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos. , 1992, Biology of reproduction.

[16]  N. H. Orr,et al.  Feeding tadpoles cloned from Rana erythrocyte nuclei. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[17]  P. Collas,et al.  Electrically induced calcium elevation, activation, and parthenogenetic development of bovine oocytes , 1993, Molecular reproduction and development.

[18]  I. Wilmut,et al.  Influence of nuclear and cytoplasmic activity on the development in vivo of sheep embryos after nuclear transplantation. , 1989, Biology of reproduction.