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.

Zygotes have not been recognized as nuclear recipients since enucleated zygotes receiving nuclei from beyond two-cell stage embryos are not able to form blastocysts. In the present study, a new technique of zygote enucleation is presented, which consists in selectively removing the nuclear membrane with genetic material of pronuclei, but leaving other pronuclear components in the cytoplasm. With selective enucleation it is possible -after transfer of eight-cell stage nuclei -to obtain 70.5 and 7.8% of preimplantation and full-term development respectively. Origin of cloned mice from introduced nuclei was confirmed by the coat colour and glucose phosphate isomerase (GPI) isozyme of the donor. We suggest that some pronuclear factors -taken away from the zygotes in the karyoplasts upon classical enucleation -are needed to reprogram the introduced nuclei.

The objective of the study was to examine preimplantation development of the zygotes derived from bovine oocytes matured in the presence of follicular fluid (bFF) randomly pooled from large (>10 mm in diameter) highly vascularised ovarian follicles originating from slaughter-house ovaries. The maturation medium was supplemented with 20% bFF. The controls comprised 20% bFF+10% FCS, and 10% FCS.The use of three separate batches of bFF have shown that the rates of cleavage (78-87%) and blastocyst formation (about 40%) obtained after bFF-supplemented maturation are similar or higher than those in the FCS control. In another experiment morphological selection of Grade I oocytes was performed after maturation or insemination. Selection of Grade I matured oocytes increased cleavage rate (to 85-91%) and blastocyst yield (to 43%). When Grade I inseminated oocytes were selected, then 92-93% of zygotes divided, and 47% reached blastocyst stage. Blastocyst rates were higher after bFF-supplemented maturation than after FCS-supplemented one, both in Grade I matured (43.0 as opposed to 40.7%) and Grade I inseminated (47.3 against 45.8%) oocytes.The majority of all zygotes underwent the first cleavage division between 27 and 30 hpi. Zygotes originating from oocytes matured in the presence of bFF or bFF+FCS were found dividing until 48 hpi whereas those obtained from control oocytes no longer cleaved after 33 hpi.In conclusion, oocyte maturation in the presence of bovine bFF randomly pooled from large follicles of slaughter-house ovaries supports as high developmental capacity of presumed zygotes as does the FCS-control.KEY WORDS: bovine follicular fluid, in vitro maturation, in vitro insemination, cleavage, blastocyst rate

Universal recipients in the G2 phase of mitotic cell cycle (preactivated oocytes, zygotes, blastomeres) accept embryonic nuclei in all the stages of their cell cycle. To test if recipients in the G2 of meiotic cycle (immature oocytes) are universal recipients, mouse germinal vesicle (GV) oocytes were enucleated and reconstructed with blastomere nuclei in the G1, S, or G2 stages. Analysis of their maturation has shown that about 30% of the G1 nuclei and 60% of G2 nuclei allow for normal metaphase II (MII), both in the oocytes with and without the first polar body (1st PB). Among oocytes reconstructed with the S phase nuclei, only 8% or less have normal MII, although 75% of them extrude 1st PB. No phase of donor cell cycle prevented the abnormal acceleration of 1st PB extrusion, found in reconstructed GV oocytes. In conclusion, enucleated GV oocytes are not universal recipients of embryonic nuclei, because they do not accept the S donors. However, both the G1 and G2 donor nuclei can be reprogrammed in the GV oocyte cytoplasm.

Foetal fibroblasts (FFs) labelled with vital fluorescent dye were microsurgically introduced into eight-cell mouse embryos, three cells to each embryo. FFs were first identified in the inner cell mass (ICM) in about one-third of embryos, whereas in three quarters of embryos FFs were located among trophoblast cells. Some elimination of FFs from trophoblast occurred later on. Eventually, in blastocysts' outgrowths, an equally high contribution from FFs progeny (60%) was found in both ICM and trophoblast. Three days after manipulation, FFs resumed proliferation in vitro. More than three FFs were found in 46.2% of embryos on day 4. On the 7th day in vitro in 70% of embryos more than 12 FFs were found, proving at least three cell divisions.To study postimplantation development, the embryos with FFs were transferred to pseudopregnant recipients a day after manipulation. After implantation, FFs were identified by electrophoresis for isozymes of glucose phosphate isomerase (GPI). A single 11-day embryo delayed to day 8 proved chimeric by expressing both donor isozyme GPI-1B and recipient GPI-1A. Similar chimerism was found in the extraembryonic lineage of 11% of embryos by day 12. Starting from day 11 onwards, in 32% of normal embryos and in 57% of foetal membranes, hybrid GPI-1AB isozyme, as well as recipient isozyme, was present. Hybrid GPI-1AB can only be produced in hybrid cells derived by cell fusion, therefore, we suggest that during postimplantation development, FFs are rescued by fusion with recipient cells. In the mice born, hybrid isozyme was found in several tissues, including brain, lung, gut and kidney.We conclude that somatic cells (FFs) can proliferate in early embryonic environment until early postimplantation stages. Foetuses and the mice born are chimeras between recipient cells and hybrid cells with contributions from the donor FFs. Transdifferentiation as opposed to reprogramming by cell fusion can be considered as underlying cellular processes in these chimeras.

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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.

We have investigated the possibility that mitotic nuclei originating from preimplantation stage embryos and placed in the oocyte cytoplasm can undergo remodelling that allows them to undergo meiosis in the mouse. To address this question, we have used enucleated germinal vesicle (GV) ooplasts as recipients and blastomeres from the 2-, 4-or 8-cell stage as nuclear donors. We employed two methods to obtain ooplasts from GV oocytes: cutting and enucleation. Although efficiency of the reconstruction process was higher after enucleation than after cutting (90% and 70% respectively), the developmental potential of the oocytes was independent of how they had been produced. Nuclei from the 2-, 4-, or 8-cell stage embryos supported maturation in about 35%, 55% and 60% of cases, respectively. The time between nuclear envelope breakdown and the first meiotic division was shortened by up to 5 h in reconstructed oocytes, a period equivalent to the mitotic division of control blastomeres. About onethird of oocytes reconstituted with blastomere nuclei divided symmetrically instead of extruding a polar body; however, in the majority of them metaphase plates were found, suggesting that reconstructed oocytes (cybrids) underwent a meiotic rather than mitotic division. The highest percentage of asymmetric divisions accompanied by metaphase plates was found in cybrids with 8-cell-stage blastomere nuclei, suggesting that the nuclei from this stage appear to conform best to the cytoplasmic environment of GV ooplasts. Our results indicate that the oocyte cytoplasm is capable of remodelling blastomere nuclei, allowing them to follow the path of the meiotic cell cycle.