Developmental potential of selectively enucleated immature mouse oocytes upon nuclear transfer.

Selective enucleation (SE) was applied to germinal vesicle (GV) oocytes by removing the chromatin attached to nuclear envelope, and leaving the liquid contents of GV in the cytoplast. However, after reconstruction with 1/8 blastomeres or fetal fibroblasts (FFs) neither the maturation efficiency nor the frequency of normal (asymmetric) division was improved as compared with completely enucleated (CE) oocytes. Chromosomal aberrations introduced with somatic nuclei were not rescued in SE oocytes either. On the other hand, timing of maturation division in SE GV oocytes, but not in CE GV oocytes, reconstructed with GV-karyoplasts was like in the control. After maturation and fertilization in vitro, SE oocytes reconstructed with 1/8 blastomeres developed nucleolated donor pronuclei, contrary to CE oocytes. The latter could be rescued with nucleoli-containing nucleus, but not anucleolate nucleus, from a 1/2 blastomere. SE oocytes reconstructed with FFs contained nucleolated pronuclei upon activation, unlike CE GV oocytes. These experiments show that the ooplast nucleolar material and/or embryonic nucleolus are indispensable for pronuclei formation. SE oocytes reconstructed with 1/8 blastomeres or FFs failed to cleave after activation or in vitro fertilization. Control GV oocytes enucleolated before fertilization seized cleavage at the 6-cell stage, as oppose to intact GV oocytes, which in 50.9% yielded morulae/blastocysts. These results suggest that ooplast nucleolar material is essential for the cleavage divisions. Activation of cumulus-enclosed SE GV oocytes matured in hormone-supplemented medium and fused to 1/2 blastomere-karyoplasts, yielded morulae, and blastocysts in 45.5% and 23.4% of reconstructed oocytes, respectively.

[Somatic cloning in mammals]. / Klonowanie somatyczne ssaków.

Somatic cloning in mammals became possible due to refinement of the nuclear transfer technique consisting of using nuclear donor cells in GO or activating reconstituted oocytes few hours after nuclear transfer (post-activation). Sheep, goats, pigs, cattle and mice have been cloned this way; the latter two species - of both sexes. Cloning of transgenic mammals producing human therapeutic proteins in milk (or urine) can find application in pharmacology and medicine. Somatic cloning of a goat producing human antithrombine III in its milk has already been achieved.

The possible role of cell cycle stage in mammalian cloning.

Progress in mammalian cloning started from cloning embryos (of mice, rats, rabbits, sheep, goats, pigs, cattle and rhesus monkeys) and culminated in obtaining clones of sheep, cattle, pigs and mice from adult somatic cells. Knowing the relationship between the cell cycles of the recipient and the donor of cell nucleus in embryonic cloning by nuclear transfer one can adjust the phases of the cell cycle properly. Metaphase II recipients accept G1 (in most species) or G2 donors (in the mouse). Interphase recipients can harbour nuclei in all stages of cell cycle. Relatively little is known about somatic cloning. Two attitudes are applied: either the donor is in the G0 phase or the recipient is in a prolonged MII phase.

Effects of preactivation of ooplasts or synchronization of blastomere nuclei in G1 on preimplantation development of rabbit serial nuclear transfer embryos.

Blastomeres from eight-cell-stage rabbit embryos have been fused with enucleated metaphase II oocytes (ooplasts) or with ooplasts that were preactivated before fusion. Preactivation of ooplasts before nuclear transfer (NT) raises the rate of preimplantation development from 15% to 56%, which remains elevated in the next series of NT (48.6% and 47.2% in the second and third rounds, respectively). Transfer of eight-cell embryos from the third round to the recipient resulted in the birth of normal young. Synchronization of blastomere nuclei in the G1 phase with nocodazole before fusion results in 42% morula/blastocyst formation. However, in the second generation of NT embryos, the yield drops to as low as 17%, indicating deleterious effects of the second nocodazole treatment on blastomeres. The calculated number of clones per one round of cloning was 4.5, 3.9, and 3.8 in subsequent series; the highest number of morulae and blastocysts that developed from individual donor embryos after three rounds were 26 and 27, respectively.

Development of bovine embryos on Vero/BRL cell monolayers (mixed co-culture).

The objective of this study was to test a new co-culture system of bovine embryos, which we call "mixed co-culture." This system consists of culturing embryos on cell monolayers composed of both Vero and BRL cells (Vero/BRL). Cumulus-oocyte complexes from ovaries of slaughtered cows were matured and fertilized in vitro. The presumptive zygotes were cultured with Vero/BRL (Group 1), BRL (Group 2) or Vero (Group 3) cell monolayers, in 40 microL drops of Menezo B2 medium supplemented with 10% FBS and antibiotics. The development of the presumptive zygotes was compared on Day 2 [48 h post insemination (pi)] and Day 7 (168 h pi). On Day 2, there was no difference between the groups. On Day 7, the highest percentage of compacted morulae/blastocysts was observed in mixed co-culture of Vero/BRL cells: 40% versus 36% on BRL versus 27% on Vero cell monolayers. The differences were statistically significant (P < or = 0.05). Among compacted morulae/blastocysts, blastocysts prevailed in mixed co-culture: 67% on Vero/BRL as compared with 55% on BRL and 27% on Vero cell monolayers. The differences were highly statistically significant (P < or = 0.01). The results suggest that Vero/BRL cells improve the development of bovine embryos.

Development of isolated sheep inner cell masses/embryonic discs in vitro.

Ovine inner cell masses (ICMs)/embryonic discs cultured in vitro, in conditions copying those in which mouse embryonic stem cells (ESCs) arise from mouse blastocysts, give rise to ectodermal colonies. Day 10-11 ICMs/epiblasts produce ectodermal colonies sufficiently often (55-60%) for it to be considered worthwhile trying to generate presumed ESCs from them. Younger ICMs can only be taken into account if culture conditions can be improved so that ICM/ectodermal cells are more numerous. Older embryonic discs (12-13 day) are inconvenient because of the problem of endoderm overgrowing ectoderm. Secondary cultures of ectodermal colonies form epithelial or mesenchymal cells, which can be passaged at least seven times (50 days).

Autonomous cortical activity in mouse eggs controlled by a cytoplasmic clock.

Mouse eggs of Swiss albino origin, both parthenogenetic and fertilized, were bisected into nucleate (NHs) and anucleate halves (AHs) and observed in vitro (semicontinuous observations) for up to 40 h for possible manifestations of cortical activity. Three experimental groups were studied: (1) Non-fertilized eggs activated 17 h after administration of hCG with a heat-shock and bisected 4 h later. (2) Non-fertilized eggs first bisected, and the resulting sister halves activated 17 h after administration of hCG with ethyl alcohol. (3) In vivo fertilized eggs bisected 27 h after administration of hCG into an AH and a binucleate half. Parthenogenetic eggs (intact, zona-free, and incompletely bisected), and fertilized eggs collected 17, 20, and 27 h after administration of hCG were also studied. In the middle of the first cell cycle the cell surface in all types of cells studied changed from smooth to slightly undulate. In nucleate cells the surface deformations lasted for several hours and disappeared shortly before the first mitosis. In contrast, in AHs the indentations of the cell surface deepened, and developed into manifold furrows, thus leading to fragmentation. However, in 20% of AHs fragmentation was partially or completely reversed. The incidence and the intensity of fragmentation were lower, and its reversibility was more common in AHs carrying the 2nd polar body. We suggest that the interphase nucleus, i.e. the pronucleus in whole eggs and NHs, and the 2nd polar body nucleus (if 2nd polary body is attached to an AH) exerts a moderating effect on cortical activity. However, the initiation of cortical activity is nucleus-independent, as shown by the behaviour of AHs separated before activation. We believe that the observed phenomena reflect autonomous cortical activity which is regulated by a cytoplasmic clock.