Fertilization of mammalian eggs is followed by successive cell divisions and progressive differentiation, first into the early embryo and subsequently into all of the cell types that make up the adult animal. Transfer of a single nucleus at a specific stage of development, to an enucleated unfertilized egg, provided an opportunity to investigate whether cellular differentiation to that stage involved irreversible genetic modification. The first offspring to develop from a differentiated cell were born after nuclear transfer from an embryo-derived cell line that had been induced to become quiescent. Using the same procedure, we now report the birth of live lambs from three new cell populations established from adult mammary gland, fetus and embryo. The fact that a lamb was derived from an adult cell confirms that differentiation of that cell did not involve the irreversible modification of genetic material required for development to term. The birth of lambs from differentiated fetal and adult cells also reinforces previous speculation that by inducing donor cells to become quiescent it will be possible to obtain normal development from a wide variety of differentiated cells.

Viable offspring derived from fetal and adult mammalian cells

La presente revue considere les donnees recentes concernant les produits impliques dans l'activation du genome de l'embryon chez plusieurs especes

https://onlinelibrary.wiley.com/doi/epdf/10.1002/mrd.1080260113

Transition from maternal to embryonic control in early mammalian development: a comparison of several species.

Somatic cell clones often fail at a developmental stage coincident with commencement of differentiation. The transcription factor Oct4 is expressed during cleavage stages and is essential for the differentiation of the blastocyst. Oct4 expression becomes restricted to the inner cell mass and epiblast. After gastrulation Oct4 is active only in germ cells and is silent in somatic cells. Here, Oct4 and an Oct4–GFP transgene were used as markers for which gene reprogramming could be directly related to the developmental potential of somatic cell clones. Cumulus cell clones initiated Oct4 expression at the correct stage but showed an incorrect spatial expression in the majority of blastocysts. The ability of clones to form outgrowths was reduced, and the outgrowths had low or even undetectable levels of Oct4 RNA or GFP. The quality of GFP signals in blastocysts correlated with the ability to generate outgrowths that maintain GFP expression and the frequency of embryonic stem (ES) cell derivation. Abnormal Oct4 expression in clones is either directly or indirectly caused by reprogramming errors and is indicative of a general failure to reset the genetic program. The abnormal Oct4 expression may be associated with aberrant expression of other crucial developmental genes, leading to abnormalities at various embryonic stages. Regardless of other genes, the variations observed in Oct4 levels alone account for the majority of failures currently observed for somatic cell cloning.

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Oct4 distribution and level in mouse clones: consequences for pluripotency

Viable offspring derived from fetal and adult mammalian cells.

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p less than 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p less than 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p greater than 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p less than 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear transplantation in the bovine embryo: assessment of donor nuclei and recipient oocyte.

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.

Nuclear transplantation in bovine embryos.

Enucleated zygotes were compared with enucleated two-cell embryos as recipients for donor nuclei from eight-cell embryos Only one or two cleavage divisions were observed when eight-cell nuclei were transplanted to enucleated zygotes Development of enucleated two-cell embryos containing a transplanted eight-cell nucleus was appreciably better with 51% (45/89) of the embryos forming blastocysts in vitro and 42% (25/60) initiating implantation Of these, eleven implantation sites on Day 10 of gestation were examined histologically and two contained normally developing embryos No development was observed beyond Day 12 of gestation These observations indicate that a major transition occurs between the zygote and two-cell stage that results in the two-cell recipient being more compatible with the eight-cell nucleus than with the zygote

Nuclear transplantation in mouse embryos: assessment of recipient cell stage.

Enucleated mouse zygotes receiving eight-cell nuclei rarely develop beyond the two-cell stage, whereas enucleated two-cell embryos with eight-cell nuclei develop to blastocysts, and a few develop to midgestation. In this study the function of eight-cell nuclei in either the zygote or two-cell recipient was assessed by monitoring several nuclear-dependent events. These included methionine uptake, qualitative changes in protein synthesis, the time of blastocoele formation, and changes in nuclear volume. Although enucleated zygotes with nuclei from eight-cell embryos did not develop past the two-cell stage and had abnormally low levels of methionine uptake, they did show a normal zygote-to-two-cell transition in the types of polypeptides synthesized. Enucleated two-cell embryos with eight-cell nuclei formed blastocysts on schedule with the recipient cell stage and reached levels of methionine uptake equivalent to control embryos. The types of proteins synthesized by two-cell embryos with eight-cell nuclei indicated that the nucleus was developing partly in accord with its own developmental program. Transplanted nuclei did not enlarge to acquire the volume of the recipient cell's nucleus in either the zygote or the two-cell recipient. These results indicate that the difference in development between zygotes and two-cell embryos (each with eight-cell nuclei) may be the result of a greater overlap of function between the two-cell and eight-cell stages, rather than the extent of nuclear reprogramming. Nuclear function was not completely normal in either type of embryo, which may explain their failure to develop to term.

/pdf/nuclear-transplantation-in-mouse-embryos-assessment-of-3osk6ilhik.pdf

Nuclear transplantation in mouse embryos: assessment of nuclear function.

Recent technological advancements in biology including the use of immunological tools have relevance to the livestock industry. These technologies are in support of and expected to contribute most importantly to the cattle artificial breeding and embryo transfer industries. At present nearly 70% of 11 million U.S. dairy cows are mated by artificial insemination. Genetic improvement in dairy cattle through artificial insemination has resulted in a doubling of milk production per cow over the past 30 years. Little progress has been made in genetic improvement by selection of offspring from superior females because a cow produces relatively few calves per year or in a lifetime. To multiply the progeny from an outstanding female or mating, a commercial embryo transfer industry has developed. This industry last year performed approximately 100,000 transfers in the USA and 200,000 in the world of which approximately 25% were with frozen embryos. Its present armament of technologies include the ability to superovulate cows, collect and transfer embryos nonsurgically, store embryos frozen and divide each embryo into two viable embryos. At present 20–30 calves can be produced from one valuable donor cow/year by use of embryo transfer.

J. M. Robl

Papers

Nuclear transplantation in bovine embryos.

Nuclear transplantation in mouse embryos: assessment of recipient cell stage.

Nuclear transplantation in mouse embryos: assessment of nuclear function.

Bovine Embryo: Development, Cloning, Sexing and Transfer of Genes