1 Institute of Animal Production, 2 Center for Cell Therapy and Tissue Repair, Prague, Czech Republic, 3 University of Granada, Department of Biochemistry and Molecular Biology, Campus Fuente Nueva, Granada, Spain, 4 ISCARE IVF, Prague, Czech Republic and 5 MAR & Gen, Molecular Assisted Reproduction and Genetics, Granada, Spain
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Abstract |
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Key words: haploidization/mouse/nuclear transfer/nucleus/oocyte
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Introduction |
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Materials and methods |
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Results |
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When released from dbcAMP block the meiotic cell cycle progression is under the control of the oocyte cytoplasm. This means that the nuclear envelope breakdown and chromosome condensation is typically detected within 1 h of culture in dbcAMP free medium in both intact and enucleated fused cells. The first meiotic spindle could be clearly detected after 6 h of culture. Typically, only one spindle was detected in cells produced by fusion of a somatic cell to an intact oocyte. This spindle contained both the meiotic and mitotic chromosomes resulting from the unification of both groups.
When evaluated after 1416 h of culture in dbcAMP free medium 31% of reconstructed oocytes (somatic cellxintact oocyte) extruded the first polar body (40/127; Table I); however, the evaluation of the second metaphase plates was extremely difficult as they contained both the meiotic and mitotic chromosomes. We may however assume that they were mostly abnormal. This assumption came from the evaluation of those oocytes which did not extrude the first polar bodies. While the organization of meiotic chromosomes was almost exclusively normal, the somatic chromosomes were allocated, in most cases, outside the spindle (Figures 3, 4
). The incompatibility of a meiotic spindle and mitotic chromosomes was even more evident when somatic cells were fused to enucleated oocytes. Here only two oocytes (2/132) extruded the first polar bodies (Table I
). When examined after staining, the chromosomes remaining in the cytoplasm were rather abnormal and formed a cluster of chromatin (Figure 5
). The oocytes without polar bodies were also stained and evaluated. Figure 6
shows the most typical configuration of chromatin where chromosomes are randomly allocated on the spindle.
Next the behaviour of early embryo chromosomes in a meiotic cytoplasm were evaluated. Nuclei were isolated from two-cell stage embryos on the next day after the detection of a vaginal plug because it is known that they are G2-phase staged. This experiment was designed to exclude the possibility that our somatic cell cultures have an adverse effect on cultured cells. Fused cells were cultured with dbcAMP for 1 h and thereafter in an inhibitor free medium for 1416 h. In general, the frequency of oocytes with polar bodies was evidently higher compared with somatic cell fusionin both groups >70% of reconstructed cells exhibited the polar bodyintact oocytexembryonic karyoplast (20/27); enucleated oocytex embryonic karyoplast (23/30; Table I). These polar bodies were only slightly smaller than the oocyte cytoplasm. When these cells were evaluated after staining, again the configuration of mitotic chromosomes showed gross abnormalities which were typically seen as a cluster or patches of chromatin. In conclusion these results show rather the inability of the mitotic cell nucleus (chromosomes) to undergo haploidization in a meiotic cytoplasm. This resulted typically in an abnormal allocation of the chromatin on the meiotic spindle and thus the inability to secure the proper separation of mitotic chromosomes.
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Discussion |
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The abnormal chromosome organization on the first meiotic spindle is not easy to explain; however, some recent results indicate that the absence of chromosome synapsis plays a crucial role. In mouse oocytes from animals homozygous for a targeted disruption of the DNA mismatch repair gene Mlh1, the absence of MLH1 protein dramatically reduces the meiotic recombination. The chromosomes in maturing oocytes are present as univalents and are unable to establish the correct spindle attachment (Woods et al., 1999). Also the absence of Spo 11p results in the defects of chromosome synapsis and a random segregation at meiosis I (Lichten, 2001
). The mouse meiotic mutation mei1 disrupts chromosome synapsis but some oocytes progress to metaphase I; their chromosomes are, however, unpaired and not properly organized on the spindle (Libby et al., 2002
). These results indicate that the meiotic recombination ensures the correct attachment and segregation of chromosomes during meiosis and is essential for its progression, but certainly some other factors may play an important role in the chromosome spindle arrangement and subsequent segregation (Bernard et al., 2001
; Kaplan et al., 2001
). On the other hand, when grasshopper spermatocytes in metaphase I were fused to spermatocytes in metaphase II and a single chromosome was moved from one spindle to the other, chromosomes placed on the spindle of a different meiotic division behaved as they do on their native spindle. Thus metaphase II chromosomes attached to the metaphase I spindle and in anaphase I individual chromatids were separated (Paliulis and Nicklas, 2000
). This phenomenon has been observed also in fused metaphase I to metaphase II mouse oocytes (Fulka et al., 1995a
). However, in both these cases the chromosomes in fused cells still belong to a category of `meiotic chromosomes'. It may be possible that chromosomes in mitotic cells are further modified and thus incompetent to undergo the proper congression and attachment to the spindle.
The frequency of polar bodies extruded was higher when G2 blastomere nuclei were introduced into immature cytoplasts, but the resulting metaphase plates were again abnormal. This higher frequency may be influenced by the absence of cell cycle checkpoint controls (Fulka et al., 2000).
It is not surprising that, after fusion of either somatic or embryonic cells to intact oocytes, polar bodies were frequently observed. It has been shown recently (Fulka et al., 1997; Rieder et al., 1997
) that the cell cycle progression in oocytes with two chromosome groups (spindles) is under the control of the more advanced (or normal) spindle. In the mouse both groups of chromosomes form a single common spindle, on the other hand both spindles are separated in fused pig oocytes (Fulka, 1983
) and also in bovine oocytes. Thus, Salamone et al. postulated the successful haploidization of somatic cells fused to GV stage bovine oocytes (Salamone et al., 2001
, 2002
).
It is interesting to note that the behaviour of meiotic cells injected or fused to immature or maturing oocytes is completely different. Normal metaphase plates are frequently formed and the number of chromosomes is reduced (Ogura et al., 1998; Sasagawa et al., 1998
). When somatic cells were fused to post-metaphase I oocytes, it was shown that the compatibility between this type of cytoplasm and a somatic cell is much better and newly formed metaphase plates seem to be normally organized (unpublished results). This is supported by earlier studies when G2-phase blastomere nuclei were introduced into chemically enucleated oocytes (post-telophase I). Here the metaphase plates were normal and chromosomes segregated properly into their sister chromatids (equatorial division). Other studies claimed the successful haploidization of somatic cells after the injection of their nuclei into mature oocytes which were subsequently activated (Lacham-Kaplan and Daniels, 2001
; Tesarik et al., 2001
). It must be stressed that a meiotic division is not simply a condensation or movement of chromosomes. The first meiotic division requires the pairing and separation of homologous chromosomes; during the second meiotic division the equal distribution of corresponding chromatids must be secured. Our observations suggest that the haploidization of somatic cell by their transition through the `whole' meiotic cell cycle was unsuccessful due to intrinsic characteristics of somatic chromosomes.
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Acknowledgements |
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Notes |
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References |
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Submitted on November 23, 2001; resubmitted on March 15, 2002; accepted on April 10, 2002.