Transplant Rabbit Embryos1

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1 BIOLOGY OF REPRODUCTION 46, (1992) Effect of Donor Cell Cycle Stage on Chromatin and Spindle Morphology in Nuclear Transplant Rabbit Embryos1 PHILIPPE COLLAS,3 CLARA PINTO-CORREIA, F. ABEL PONCE DE LEON, and JAMES M. ROBL2 Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts ABSTRACT We investigated the influence of the cell cycle stage of the nuclear donor on prematurely condensed chromatin (PCC) and spindle morphology and on chromosome constitution in rabbit nuclear transplant embryos. The configuration of PCC following nuclear transplantation with Gl, early S, and late 5 phase donor nuclei (Gl, early S, and late S transplants, respectively) was characterized in whole mounts and chromosome spreads. In addition, the influence of the donor cell cycle stage on chromosome constitution in cleavage stage-manipulated embryos was determined. Within 2 h after fusion of the donor blastomere, the recipient oocyte cytoplasm was able to induce formation de novo of a metaphase plate associated with a spindle in G 1, early S, and late 5 transplants. Metaphase chromosomes and spindle were intact in most cases of PCC in Gi transplants. However, these structures displayed minor abnormalities in early S transplants and gross abnormalities in late S transplants, such as incomplete or absent spindle formation and incomplete chromatin condensation. Normal chromosomes were present in GI and early S transplants, whereas chromosome abnormalities were detected in late S transplants. The results indicate that morphology of prematurely condensed Gi and early S chromatin has a minor influence on chromosome constitution of manipulated embryos. That of late S chromatin, however, affects chromosome constitution in embryos and may account for reduced development of nuclear transplant embryos when late S phase donor nuclei are used. INTRODUCTION Transplantation of an interphase nucleus into an enucleated, mature oocvte leads to premature condensation of the donor chromatin, followed by formation and swelling of the nucleus [1]. Premature chromosome condensation (PCC) has been observed in mouse [2-6] and rabbit nuclear transplant embryos [1]. PCC and nuclear swelling have been shown to be beneficial for development of reconstituted rabbit embryos [1]. Furthermore, the position of the donor nucleus in the cell cycle influences the extent of development in vitro of nuclear transplant rabbit embryos [7]. Transfer of donor nuclei in the Gi phase of the cell cycle yields high rates of development to blastocysts; however, development is reduced with donor nuclei in early S phase and is dramatically impaired with those in late S phase. Therefore, as donor nuclei progress in the cell cycle, the extent of development of reconstituted embryos is reduced [7]. Fusion of a somatic cell in interphase with a mitotic cell also results in PCC of the interphase nucleus [8-10]. In somatic cells, PCC is manifested by breakdown of the nuclear envelope and condensation of the interphase chromatin without formation of a spindle [8]. In addition, the morphological changes that occur in the interphase chromatin have been shown to depend on the position of the interphase cell in the division cycle. In particular, PCC in Gi and G2 phases results in elongated chromosomes with single- and double-stranded chromatids, respectively [8-il]. Chromatin in S phase, however, condenses less completely Accepted November 4, Received July 17, This work was supported in part by USDA grant No to J.M.R. Correspondence. tcurrent address: GeivMark. Inc Wakara Way. Salt Lake Cirs-, UT and displays numerous fragments, separated by condensed regions [8-ui]. These studies indicate, therefore, that the cell cycle stage of the interphase nucleus greatly affects the configuration of PCC in mitotic somatic cells. The stage of the cycle of the interphase donor nucleus not only influences the morphology of PCC, it also affects the extent of the genetic contribution of the interphase nucleus to the genome of the hybrid cell [12-15]. Several studies have demonstrated that chromosome fragments, hut not intact chromosomes, resulting from prematurely condensed chromatin, are able to survive in the descendents of heterokaryons [12, 13]. It was suggested that such fragments survive either independently or by integration into chromosomes of the partner nucleus. However, results demonstrating the stability of these integrations have been inconsistent [12, 14, 15]. In addition, it has been reported that the likelihood of contributing genetic markers to the partner nucleus was higher with prematurely condensed Gi or G2 chromosomes than with S phase chromatin [13]. These results support the conclusion that the developmental rate of nuclear transplant embryos is higher with GI than with S phase donor nuclei [7]. The importance of cell cycle stage of the donor nucleus on development of nuclear transplant embryos has already been documented. The present study investigated the effect of donor cell cycle stage on the morphology of PCC and spindle, and on chromosome constitution in nuclear transplant embryos. We characterized the morphology of chromatin that had been prematurely condensed in the Gi, early 5, and late S phases of the cell cycle. The influence of donor cell cycle stage on chromosome constitution in embryos reconstituted in the cleavage stage was also determined. 501

2 502 COLLAS ET AL. MATERIALS AND METHODS Embryo Micromanipulation Recipient Oocyte and Donor Embrpo Collection Mature mixed-breed female rabbits were superovulated with six injections of 0.3 mg FSH (Burnes Biotech, Omaha, NE) [16] followed by a single administration of 75 IU hcg (Sigma, St. Louis, MO). Ovulated oocytes were recovered from the oviducts h after hcg injection by flushing with 37#{176}C gassed Earles balanced salt solution and 10% fetal calf serum (EBSS/FCS; ph 7.2), and the cumulus was removed with hyaluronidase (Sigma) as described elsewhere [1, 17, 18]. Nonsuperovulated females served as embryo donors and were mated to an intact male. Late 16-cellstage embryos were recovered from the oviducts with 37#{176}C PBS 54 h after mating and were cultured until blastomere synchronization. Embryo Synchronization Donor blastomeres were used in the Gi phase (Gi transplants), early S phase (early S transplants), and late S phase (late S transplants) of the cell cycle. A procedure for synchronizing donor blastomeres in Gi in a reversible manner was previously determined [7]. Briefly, 16-cell-stage embryos were cultured for 10 h in the presence of 0.5 p.g/ ml of the microtuhule inhibitor, colcemid, to arrest them in M phase between the 16- and 32-cell stages. They were then extensively washed in PBS and immediately incubated in 0.1 g/ml of the DNA synthesis inhibitor aphidicolin. Cleavage to the 32-cell stage in aphidicolin was allowed, and embryos remained arrested at the GuS transition until used for nuclear transfer [7]. Blastomeres were isolated in the presence of aphidicolin as described previously [1, 17, 18]. Synchronization of 32-cell-stage embryos in early S entailed culturing 16-cell-stage embryos in 0.5 g/ml colcemid for 10 h. After a thorough wash in PBS, they were allowed to cleave in culture without aphidicolin, and blastomeres were used within 1.5 h after mitosis. Thirty-two-cell-stage embryos used in late S were cultured without any inhibitor for 16 h after recovery from the oviducts at the 16-cell stage. Early and late S donor blastomeres were isolated. Colcemid and aphidicolin (Sigma) were reconstituted at 2 mg/mi in PBS and dimethylsulfoxide (DMSO), respectively, aliquoted, and stored at -10#{176}C, Embryo Culture Embryos were cultured in vitro in vitreous humor (VH) from the eyes of female rabbits as reported elsewhere [1, 7; P. Collas, R.T. Duby, and J.M. Robl, unpublished results]. Briefly, VH was aspirated aseptically from the eyes of killed rabbits and centrifuged at X g for 2 mm to pellet cellular debris. The supernatant was filtered (0.2 pan) in microdrops overlaid with equilibrated paraffin oil. Culture dishes (Falcon No. 1007, \WR Scientific, Boston, MA) were incubated at 39#{176}C in an atmosphere of 5% CO2 in air for 12 h prior to use [1]. Nuclear transfers were performed in gassed EBSS/FCS with 7.5 pg/ml cytochalasin B (Sigma) as previously described [1, 18]. With Gi donor nuclei, however, micromanipulation was carried out in the presence of 0.1 g/ml aphidicolin, and embryos were cultured with aphidicolin until fusion and activation as described previously [7]. Blastomere Fusion and Oocyte Activation Fusion and activation were performed electrically [1, 17, 18]. Manipulated embryos were placed between two electrodes 0.5 mm apart, overlaid with 0.30 M mannitol containing 100 p.m CaCl2 and MgC12 [16] at 30#{176}C. Six direct current pulses of 2.2 ky/cm for 60 p.sec, each pulse 30 mm apart, starting 18 h post-hcg, were given using a BTX Electro Cell Manipulator 200 (BTX, Inc., San Diego, CA); and embryos were placed in culture between each pulse. For Gi transplants, fused embryos were cultured with 0.1 p.g/ ml aphidicolin for 30 mm after the first pulse, to prevent DNA synthesis prior to chromosome condensation [1]. Embryos allowed to develop in vitro were incubated in 7.5 p.g/ml cytochalasin B for 1 h after the last stimulation [16]. In all cases, time zero was considered to be the time of cell fusion, which occurs about 15 mm after the first pulse. Spnchronization Controls A control was established to determine whether colcemid or aphidicolin treatments had an effect on the morphology of PCC in Gi transplants. Donor embryos were synchronized in Gi without colcemid, using two different procedures. (1) Isolated late 16-cell-stage blastomeres were cultured in vitro, and cleavage to the 32-cell-stage was monitored every 15 mm starting 60 h after mating. Elongated (cleaving) blastomeres were manipulated in the presence of 0.1 p.g/ml aphidicolin until complete fusion to the recipient oocyte. (2) To test the effect of aphidicolin, a portion of cleaving 16-cell-stage blastomeres were slowed in development by cooling at 4#{176}C in PBS for 2-3 h, until micromanipulation and fusion as previously described [7]. The maximum interval between cold treatment and complete blastomere fusion was 30 mm in this experiment. Chromatin and microtubule immunolocalization and chromosome analyses were performed as described below. The two synchronization procedures were directly compared, and two replicates were carried out. Localization of Chromatin and Microtubules Chromatin and microtubules were detected by immunofluorescence in whole mounts of nuclear transplant embryos. Immunofluorescence was performed with mature oocytes 18 h post-hcg and with Gi, early 5, and late S transplants 2 h after nuclear transplantation, once PCC had occurred [1]. Oocytes and embryos were permeabilized for 1 h at 37#{176}C in microtubule-stabilization buffer [19] with 2.5%

3 CHROMOSOME CONDENSATION IN NUCLEAR TRANSPLANT EMBRYOS 503 Triton X-100 and fixed for 30 mm in 2% paraformaldehyde. After six washes in PBS and a 10-mm treatment with 0.1 M glycmne, embryos were incubated overnight at 4#{176}C with antia tubulin monoclonal antibody (Sigma) diluted 1:100 in PBS containing 0.1% Tween-20 (PBS-T). As a control for specificity of the anti-a tubulin, monoclonal antibody oocytes and embryos were treated with the same concentration of affinity-purified mouse IgG (Sigma). After several washes in PBS-T, oocytes and embryos were again incubated overnight at 4#{176}C in fluorescein isothiocyanate (FITC)- conjugated affinity-purified goat anti-mouse IgG (Sigma) diluted 1:20 in PBS-T. After an overnight wash at 4#{176}C with PBS-T, specimens were mounted in glycerol containing 0.5 p.g/ml Hoechst (Sigma). Coverslips were sealed to glass slides with nail polish, and slides were stored in the dark at 4#{176}C. Three replicates were performed with each twe of donor nucleus. In all cases, statistical comparisons were made by means of chi-square analysis [20]. Chromosome Spreads Chromosome spreads were prepared at the same time as whole mounts in each replicate. Eighteen-hour post-hcg mature oocytes and G 1, early 5, and late S transplants 2 h after nuclear transplantation were used; and spreads were prepared on acid-washed coverslips according to Matsuda and Tobari [21]. After alcohol dehydration, coverslips were allowed to air-dry and were mounted in anti-fade solution containing 2.3% of 1,4-diazabicyclo(2,2,2)octane (DAPCO, Sigma) [22; unpublished results] and 0.2 p.g/ml propidium iodide (Sigma). As for whole mounts, coverslips were sealed to a glass slide and stored at 4#{176}C in the dark. Three replicates were performed with each type of nuclear donor. Chromosome spreads were also prepared from nonmanipulated in vivo-fertilized 16-cell-stage embryos and Gi (8-16-cell stage), early S (8-16-cell stage), and late S (8- cell stage) transplants (n = 3, 2, and 4, respectively). Embryos were arrested in metaphase (M phase) with 0.5 p.g/ ml colcemid and 10 p.g/ml ethidium bromide (Sigma) for 2-5 h. The zona pellucida and the mucin coat, when present, were removed [18], and embryos were processed for chromosome spreads as described. In all cases, comparisons were made by means of chi-square analysis. Microscopy Whole mounts and chromosome spreads were examined at 100 x under a Zeiss Axioskop microscope equipped with epifluorescence. Hoechst, propidium iodide, and FITC signals were detected using 365/400, 546/590, and 450/ 520 nm excitation/barrier filter combinations, respectively. An SIT 66 video camera (DAGE MTI, Michigan City, IN) fixed to the microscope was used to capture images that were digitized using the Image 1 AT software (Universal Imaging, Media, PA). Images were stored in 44-mb disk cartridges, and photomicrographs were prepared with a video printer (Sony, model UP 5000, Park Ridge, NJ). RESULTS Chromatin and Microtubule Organization in Whole Mounts Two Hours after Nuclear Transfer Unfertilized oocytes. Mature oocytes 18 h post-hcg showed, as expected, an M phase plate (12/12 oocytes, 100%; Fig. Ia) at the equator of the meiotic spindle (Fig. ub). The morphology of this spindle differed from that of a typical meiotic barrel-shaped spindle, such as that in oocytes 14 h post-hcg. This difference in spindle morphology may be due to aging of the oocyte [C. Pinto-Correia, P. Collas, and J.M. RobI, unpublished results]. The spindle was consistently located in the cortex of the oocyte, parallel to the plasma membrane. Gi transplants. PCC of donor nuclei in the Gi phase was manifested by the formation of an M phase plate in 16/19 embryos (84%; Fig. 2, a and c). In some instances (44%, 7/16), the plate was perfectly regular (Fig. 2a); however, in most cases (9/16, 56%), it showed a few isolated chromosomes or, possibly, chromosome fragments in its vicinity (Fig. 2c). Chromosomes were consistently aligned at the equator of spindles. These spindles were morphologically similar to those of 18-h post-hcg oocytes (Fig. ib) and to mitotic spindles (Fig. le). The spindles were located in the oocyte cortex, either parallel or perpendicular to the plasma membrane. In 3/19 embryos (i6%), PCC were scattered in the cytoplasm with no visible microtubules (Fig. 2e, f). Early S transplants. PCC in the early S phase was manifested by the formation of M phase plates in 10/11 embryos (91%; Fig. 2, i, k, and m). This proportion did not differ from that in Gi transplants (p > 0.1). One or two M phase plates were formed (7/ui embryos, 64%, and 3/11 embryos, 36%, respectively), and each was associated with a spindle (Fig. 2, j, 1, and n). Interestingly, when two spindles were formed, their width and length were half those of single spindles. Single metaphase plates were less organized than those of Gi transplants, and all single plates were disrupted. In addition, spindles surrounding these plates showed microtubules radiating from the poles and from the sides of the spindle into the cytoplasm (Fig. 2, and 1). One embryo had no spindle and contained only dispersed chromatin (not shown). Late S transplants. PCC in the late S phase was characterized in 7/13 embryos (54%) by the formation of a very irregular, large, and thick M phase plate, and 85% of these plates (6/7) were surrounded by isolated chromosomes and/ or chromosome fragments. The M phase plates were located at the equator of a spindle with microtubules radiating from the poles and/or from the sides. This configuration was very similar to that found in early S transplants. The morphology of these spindles was similar to that of early S transplants. In 3/i3 embryos (23%), two distinct masses of chromatmn were located outside a small spindle,

4 504 COLLAS ET AL. FIG. 1. Meiotic apparatus in metaphase II rabbit oocyte 18 h post-hcg (a-c) and mitotic apparatus in pronuclear embryo id-f) (whole mounts). Oocytes and embryos were double-stained with the DNA-specific Hoechst (a, d) and by indirect immunofluorescence with anti-tubulin antibody (b, e). The M phase plate occupies the equator the meiotic spindle (arrows). (c, f) lgg control. Bar, 10 gm. adjacent to the poles (Fig. 2, q and r). In addition, 2/13 embryos (15%) showed unorganized chromatin with corresponding half spindles (Fig. 2, s and t) and, in one embryo, three M phase plates were formed at the equators of three small spindles (Fig. 2, u and v). Despite the discrepancies in configuration, the overall frequency of spindle and plate formation in late S transplants was not different from that of Gi and early S transplants (8/13 embryos,p > 0.1). Controls. Omission of colcemid and aphidicolin for blastomere synchronization did not alter the morphology of PCC in G 1 transplants. Synchronization without colcemid but with aphidicolmn resulted in embryos with PCC organized in an M phase plate at the equator of an intact spindle (7/7 embryos; Fig. 3, a and b). Similarly, when blastomeres were synchronized in Gi without colcemid or aphidicolin (cold treatment), M phase plates were formed at the equators of spindles (5/6 embryos). One embryo was already in anaphase (Fig. 3, d and e). Therefore, morphology of PCC in Gi transplants was not caused by colcemid or aphidicolin treatments themselves. Rather, it reflected the stage of the cell cycle of the donor blastomere at the time of fusion. In addition, we examined whether the structures observed in manipulated embryos were due to the introduction of the exogenous nucleus and not to fusion or activation stimuli themselves. An anucleate cytoplasmic bleb was fused to an enucleated oocyte. As expected, no DNA label was detected (4/4 embryos; Fig. 3g). Furthermore, there was no detectable microtubule organization, and the pool of unpolymerized tubulin was scattered in the entire oocyte cytoplasm (Fig. 3h). This confirms, therefore, that M phase structures and spindles form only in the presence of the exogenous nucleus. Chromosome Spreads from Embryos Tu o Hours after Nuclear Transplantation. Unfertilized oocvtes. Chromosome spreads of nonmanipulated 18-h post-hcg oocytes showed haploid sets of 22 chromosomes, each with 2 distinct chromatids (17/17 oocytes, 100%; Fig. 4a). Gi transplants. PCC in GI revealed two types of configuration. In 8/12 transplants (67%), chromosomes were formed but were not fully individualized (Fig. 4b). The remaining embryos (33%) displayed individualized chromosomes (Fig. 4c) together with unorganized chromatmn (Fig. 4c). In both cases, some chromosomes seemed paired (Fig. 4, b and c). In addition, these chromosomes were shorter and less defined than typical mitotic chromosomes (see Fig. 5a) and displayed a meiotic configuration, similar to that of oocyte chromosomes (Fig. 4a). Early S transplants. PCC in the early S phase resulted in the formation of chromosomes that were not individualized (3/5 embryos, 60%; Fig. 4d). Alternatively, no chromosomes were formed, and only patches of unorganized chromatmn were present (Fig. 4e). Late S transplants. PCC in late S phase was unorganized and never formed chromosomes. Rather, the chromatin remained in 1, 2 or 4 patches (5/1 0, 4/10, and 1/10 transplants, respectively; Fig. 4, f and g).

5 CHROMOSOME CONDENSATION IN NUCLEAR TRANSPLANT EMBRYOS 505 FIG. 2. PCC and newly formed spindle after transfer of nuclei in Gi, early S and late S phases (whole mounts). DNA was stained with Hoechst (Gi: a, c, e, g; early S: i, k, m, o; late S: q, s, u, w). Microtubules were stained with anti-tubulin antibody (Gi: b, d, f; early S: i. I, n; late S: r, t, v). Control embryos (h, p. x). Gi transplants: (a, b) metaphase plate formed at the equator of a spindle (arrows); (c, dl metaphase plate with isolated chromosomes or, possibly, chromosome fragments (arrowheads); (e, f) chromosomes dispersed in the oocyte without a spindle; (g, h) lgg controls. Early S transplants: (i, i) metaphase plate and spindle with radiating microtubules (arrows); (k, I) disrupted metaphase plate, and spindle with radiating microtubules (arrows); (m, n) two small metaphase plates formed at the equator of two distinct spindles; (0, p) IgG controls. Late S transplants: (q, r) two sets of chromatin at the poles of a small spindle (arrows); is, t) unorganized chromatin within an uneven number of half spindles; (u, v) three small metaphase plates formed at the equator of three spindles; (w, x) lgg controls. Bar, 10 p.m.

6 506 COLIAS ET AL. FIG. 3. PCC and spindle in synchronization and anucleate control manipulated embryos. Prior to nuclear transfer, non-colcemid-treated donor biastomeres were synchronized in Gi with aphidicolin (a-c) or cold treatment id-f). (a, b) Whole mounts showing a metaphase plate at the equator of an intact spindle; (c) smear with individualized chromosomes. (d, e) Whole mounts with chromosomes already in anaphase along a normal spindle; if) smear with not fully individualized chromosomes. (g, h) When an anucleate cytoplasmic fragment was transferred into a nenucleated oocyte, no chromatin was present (g), and only the unpolymerized pool of tubulin was detected without any signs of spindle formation (h). Bar, 10 p.m. Synchronization controls. Synchronization in the Gi phase without colcemid but with aphidicolin resulted in spreads with individualized chromosomes (5/5 embryos; Fig. 4c). Synchronization in the Gi phase by cold treatment resulted in spreads with chromosomes not fully individualized (5/5 embryos; Fig. 4f). These results indicate that use of colcemid and aphidicolin for blastomere synchronization did not affect chromosome configuration in Gi transplants. Early S transplants. Two 8-16-cell stage embryos were processed. Fourteen M phase spreads were obtained; 12 (86%) revealed normal chromosomes (Fig. 5c), and 2 blastomeres contained a total of 7 chromosomes and chromosome fragments (2/14, 14%, Fig. 5d). Chromosome morphology did not differ from that of G1 transplants or nonmanipulated embryos. Chromatmn fragments were structures irregular in shape and size and smaller than in- Chromosome Spreads of Cleavage Stage Nuclear Transplant Embryos GI transplants. Three manipulated embryos were successfully prepared at the 8-16-cell stage, and 7 M phase spreads were obtained. Chromosome counts (n = 44) and morphological comparisons with chromosome spreads from nonmanipulated control embryos (Fig. Sa) indicated that chromosomes were normal (7/7 M phase spreads; 100%; Fig. Sb). FIG. 4. Chromosome spreads from a mature oocyte, and PCC in Gi, early S, and late S transplants. (a) Haploid set of 22 bivalent chromosomes in a metaphase II rabbit oocyte. Note the short and condensed morphology, characteristic of meiotic chromosomes (compare with mitotic chromosomes in Fig. 5a). In Gi transplants, the donor chromatin (b) forms plates with chromosomes not fully individualized or (c) condenses into chromosomes with two chromatids (arrowheads), chromosomes with one chromatid (short arrow), and random clusters (long arrow). (dl In early S transplants, the chromatin is either organized into aggregated chromosomes, or (e) displays an unorganized conformation. (f, g) In late S transplants, chromosomes are never formed, and the chromatin condenses into one or two clusters. Bar, 10 p.m.

7 CHROMOSOME CONDENSATION IN NUCLEAR TRANSPLANT EMBRYOS 507

8 508 COLLAS ET AL. tact chromosomes. In addition, one blastomere contained 2 interphase micronuclei (Fig. Sd). Micronuclei consisted of round entities smaller than an intact nucleus. They probably resulted from the formation of a nuclear envelope around a single chromosome or small cluster of chromosomes [23]. Therefore, although each embryo contained cells with normal chromosomes, some defects also occurred in other blastomeres. Late S transplants. Four 8-cell-stage embryos were examined, and 20 M phase spreads were obtained. In 15 of these spreads (75%), chromosomes tended to be larger than those of GI transplants, early S transplants, and nonmanipulated embryos (Fig. Se). Width and length of chromosomes 7 and 18 were measured in one blastomere of nonmanipulated 8-cell- and 64-cell-stage embryos, as well as of Gi (8-16-cell), early S (8-16-cell), and late S (8-cell) transplants (Table 1). Chromosomes 7 and 18 were chosen because they were easily identifiable owing to their morphology. Since only 2 chromosomes were measured in each type of embryo, the data indicate only a trend, and statistical comparisons were not made. However, this phenomenon was consistently observed in these 15 chromosome spreads. The fact that chromosome size was similar in 8- and 64-cell-stage controls indicates that the increase in chromosome size in late S transplants was not due to a difference in embryo cell stage at the time chromosome spreads were made; rather, it reveals a peculiarity of late S transplants. The increase in width/length (W/L) ratio in late S transplants indicates that an increase in size is due more to an increase in chromosome width than in length. In addition, 4 chromosome spreads revealed extremely damaged chromatin (4/20; 20%; Fig. 5h). It is likely that these cells were already degenerated when embryos were fixed. Furthermore, one spread displayed paired chromosomes, as in diplo-chromosomes (1/20, 5%; Fig. 5g). In addition to the 20 M phase spreads, over 300 interphase micronuclei were detected in a total of 8-10 cells (Fig. Sf). In contrast, micronuclei were not detected in nonmanipulated embryos cultured in vitro. A characteristic of late S transplants, therefore, was that each embryo contained blastomeres with large chromosomes, and a high proportion of cells with abnormalities. DISCUSSION In this study, we investigated the effect of donor cell cycle stage on morphology of PCC and spindle and on chromosome structure in nuclear transplant embryos. The results indicate that after nuclear transfer, the recipient oocyte cytoplasm was typically able to induce the formation de novo of an M phase plate associated with a spindle. M phase plates and spindles were intact in most cases of PCC in GI transplants. However, their integrity was lost in most early S transplants, and gross abnormalities were detected in late S transplants. Furthermore, normal chromosomes were obtained in Gi and most of early S transplants, whereas a large proportion of chromosome abnormalities was present in late S transplants. M Phase Plate and Spindle in Nuclear Transplant Embryos Two Hours after Nuclear Transfer Interactions between donor chromatin and recipient oocyte microtubules may determine spindle and M phase plate conformation. Formation of a spindle in Gi, early 5, and, to some extent, late S transplants suggests that initiation of microtubule nucleation in the oocyte is the result of a signal common to donor blastomeres in G1 and S phases. Nevertheless, a normal spindle can be associated with an intact or an abnormal M phase plate (Gi transplants). Alternatively, an abnormal spindle is always associated with an abnormal M phase plate (early and late S transplants). These observations suggest that morphology of the new spindle influences integrity of the M phase plate. It is not excluded, however, that the condensed chromatin also sends specific instructions to oocyte microtubules; these instructions may differ with the cell cycle stage at which the chromatmn is condensed. Thus, it is possible that only Gi donor chromatin sends correct instructions for spindle assembly, whereas altered information from chromatin condensed in the S phase may result in abnormal or incomplete spindle formation. Alternatively, final spindle assembly may depend simply on the physical conformation of the condensed chromatin. It is suggested, therefore, that a first set of instructions, nonspecific for the stage of the donor blastomere in the cell cycle, triggers the nucleation of microtubules. Then, physical configuration of the condensed chromatin or a signal specific for the cell cycle stage of the condensed chromatmn may determine the extent of final spindle assembly. The comparison between M phase and spindle morphology in whole mounts and chromatin configuration in chromosome spreads 2 h after nuclear transfer supports this hypothesis. It is likely that M phase spreads showing chromosomes in Gi and early S transplants (Fig. 4, b and d) may correspond to the M phase plates found in whole mounts (Fig. 2, a, c, i, and k). Furthermore, in both Gi and early S transplants, isolated chromosomes or chromosome fragments present in whole mounts (Fig. 2, FIG. 5. Chromosome spreads in cleavage stage G1, early S, and late S transplants. DNA was double-stained with Hoechst and propidium iodide. (a) M phase chromosomes from a nonmanipulated embryo. (b) Chromosomes from a Gi transplant; no morphological differences with (a) were detected. (c) Within early S transplants, some blastomeres show normal chromosomes, and (d) others reveal abnormalities, such as a micronucleus (arrow) coexisting with chromosomes (arrowheads), or, possibly, chromosome fragments. (e-h) Late S phase transplants containing (e) M phase chromosomes, larger than in controls, Gi transplants, or early S transplants; if) a micronucleus (arrow) next to a normal interphase nucleus; (g) paired chromosomes; and (h) abnormal chromatin. The proportion of abnormalities within embryos was increased between early S and late S transplants. Bar, 10 p.m.

9 CHROMOSOME CONDENSATION IN NUCLEAR TRANSPLANT EMBRYOS 509

10 510 COLIAS ET AL. TABLE 1. Chromosome size in cleavage stage Gi, early S (ES), and late S (LS) transplants. Embryo Typeb Big submetacentric (Chrom. 71 Big acroc (Chrom. entric 18) Width Length W/L WxL Width Length W/L WxL Ctrl 8c Ctrl 64c G1 trans ES trans LS trans Chromosomes 7 and 18 were chosen because they were easily identifiable due to their morphology. Width and length, p.m; W x L, width x length product, p.m2; W/L, width/length ratio. 6Ctrl 8c and Mc: nonmanipulated 8-cell- and 64-cell-stage embryos, respectively; one embryo was examined in each group. c, i, and k) were not found in chromosome spreads (Fig. 4, h, and d). It is possible that the were lost during chromosome spread preparation. In addition, absence of M phase plate formation revealed in whole mounts ma\ he manifested in chromosome spreads with non-uniformly condensed chromatin (Fig. 4, c, e, f, and g). Also, the multiple M phase plates in earl and late S transplants (Fig. 2, m and u) ma have been manifested h multiple chromatin patches in chromosome spreads (Fig. 4, e and g). These obseryations suggest that chromosomes, as opposed to non-uniform chroniatin, may he present in the M phase plates of GI and early S transplants. However, defects in spindle formation may he related to the non-chromosome structures observed in M phase spreads. l remature Condensation of Gi and S Chronzatin in Nuclear Transplant Embryos Gi chromatin condenses into chromosomes in nuclear transplants; however, the pattern of PCC differs between somatic cell h brids and nuclear transplants. First, PCC was found not to he accompanied by formation of a spindle in somatic cell hybrids [8]. This may reflect a difference in recipient cell type, as discussed below. Second, in contrast to findings in somatic cell hybrids [8, 11], not all chromosomes were fully individualized in nuclear transplants. Because presumably the nuclear envelope breaks down after blastomere fusion, remnants of nuclear matrix not completelv extracted at permeahilization ma have held the chromosomes together. Third, chromosomes in GI transplants were extremely short and condensed and showed a meiotic configuration. Ln contrast, prematurely condensed Gi chromosomes in somatic hybrids have been found to he abnormally elongated when compared to control meiotic chromosomes [8, 11]. Again, a difference in recipient cell wpe may he responsible for this phenomenon (see below). Fourth, a proportion of chromosomes in Gi transplants revealed a hivalent structure, similar to that of metaphase II chromosomes. The fact that Gi chromosomes are normally nionoyalent suggests that some chromosome pairing occurred. Whether pairing occurred between homologous chromosomes, however, was not investigated. PCC in S phase also displays morphological differences in somatic cell hybrids and nuclear transplants. As with Gi cells, no evidence of spindle formation has been reported after FCC in S phase in somatic hybrids [8]. Furthermore, PCC in S phase results in extensive chromatmn fragmentation in somatic hybrids [8, 1 1 ]. Fragmentation was not observed in rabbit nuclear transplants; however, as in somatic cell hybrids [8, 1 1 ], chromosomes were never formed with late S chromatin. The reason for condensation into a nonchromosome structure may be similar in both studies. Replication forks in S phase DNA create a discontinuity and, presumably, weak points in the chromatin due to absence of base pairing. This condition may be responsihle for nonuniform condensation, s ielding the structures observed. As with PCC in somatic cells, our findings support the conclusion that the ability of the donor nucleus to participate fulls iii nietaphase plate formation in the oocvte decreases as donor blastomeres progress in the cell cycle. Several factors may account for differences in the morphology of FCC in somatic cells and nuclear transplants. Differentiated cells have previously been used in somatic cell fusion experiments [6-9], whereas embryonic blastomeres were used in our study. Therefore, the differentiated state of the interphase nucleus may have affected FCC. In a separate experiment, however, differentiated granulosa cell nuclei fused to enucleated oocvtes also condensed into a metaphase plate within a spindle [unpublished observation]. This finding, therefore, rules out the above hpothesis. Another factor that may influence the spindle and the chromatin in nuclear transplants is the absence of the meiotic spindle in recipient oocytes. Since the metaphase plate no longer has to he maintained, the tubulin pool is entirely available for the establishment of a new spindle. Further studies indicate, however, that metaphase plate and spindle form de novo even in non-enucleated oocvtes [unpublished observation]. Thus, it is unlikely that absence of the meiotic spindle per se allowed spindle formation in nuclear transplants. The most significant factor affecting FCC is probably the pe of M phase recipient cell used. Mitotic cells were employed in previous cell fusions [8-11], whereas a meiotic cell (oocyte) was used for nuclear transfer. It is possible that meiotic-specific factors present in the oocte ma\ be responsible for PCC into a metaphase plate, rneioticlike configuration of chromosomes, and chromosome pairing in nuclear transplants. These factors max or may not be associated with a maturation-promoting factor. Mature oocytes have also been used as recipient cells in mouse thymocvte nuclear transplants [2-6]; however, absence of control of the cell cle stage of donor thvmocvtes makes comparisons difficult. The data suggest, therefore, that the mature oocvte is a privileged environment for condensanon of the chromatin into a metaphase plate and a spindle.

11 CHROMOSOME CONDENSATION IN NUCLEAR TRANSPLANT EMBRYOS 511 Chromosome Constitution in Cleavage Stage Nuclear Transplant Embryos The morphology of the metaphase plate upon PCC in nuclear transplants does not, to some extent, affect chromosome structure and number and early embryo development In particular, formation of an intact metaphase plate upon FCC is compatible with normal chromosome complement (Gi transplants). Similarly, a less defined or disrupted metaphase plate inside an abnormal spindle does not alter subsequent chromosome structure in Gi and early S transplants. Furthermore, Gi and early S transplants developed to blastocysts at high and similar rates in a separate study (71% and 59%, respectively; p > 0.1) [7]. These results suggest that formation of a metaphase plate and a spindle upon PCC is compatible with normal preimplantation development of manipulated embryos. These indications do not imply, however, that all embryos with intact chromosomes develop to blastocysts. In addition, a small proportion of Gi and early S transplants do not activate or develop beyond 2 or 3 cleavages [7]. The data suggest that these embryos may be those in which the chromatin condenses into a non-uniform mesh with no spindle. Furthermore, FCC of late S chromatin is also compatible with subsequent formation of chromosomes in cleavage stage embryos. These chromosomes, however, are larger than those of Gi and early S transplants. Also, a low percentage of these embryos develop to blastocysts (15% [7]). FCC in late 5, therefore, is detrimental, and blastomeres containing abnormally thick chromosomes are likely to be nonviable. Reasons for this defect remain to be determined; they may entail reduced chromosome compaction at M phase. These observations indicate that the ability of the donor nucleus to direct development in vitro of nuclear transplant embryos does not depend solely on the morphology of FCC and, therefore, on the cell cycle stage of the donor nucleus. Other defects that may not be related to PCC morphology may arise during embryo development. Absence of cell division immediately after FCC in nuclear transplants may account for the normal chromosome structure in cleavage stage embryos. Completion of mitosis after FCC in somatic cell hybrids has been shown to be responsible for the deleterious effect of PCC in such systems [12-15]. In nuclear transplant embryos, however, the condensed chromatin decondenses into a pronucleus upon activation of the oocyte [1]. It is possible, therefore, that the chromatin may be reorganized in the interphase pronucleus. However, this does not exclude the possibility that chromosomes or chromosome fragments may be lost during this process. Alternatively, chromosome abnormalities that would not be detected in chromosome spreads may arise. These defects may, however, remain compatible with development of nuclear transplant embryos in vitro. ratorv. ACKNOWLEDGMENT The authors are grateful to John Balise for his technical expertise in the laho. REFERENCES 1. Collas P. RobI JM. Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos. Biol Reprod 1991; 45: Czolowska R, Modlinski JA, Tarkowski AK. Behaviour of thymoevte nuclei in nonactivated and activated mouse oocvtes. J Cell Sci ]984; 69: Balakier H, Masui Y. Chromosome condensation activity in the cytoplasm of anucleate and nucleate fragments of mouse oocvte. Dev Biol 1986; 113: Czolowska R, Waksmundzka M, Kubiak JZ, Tarkowski AK. Chromosome conden. sation activity in ovulated metaphase II mouse oocvtes assayed by fusion with interphase blastomeres. J Cell Sci 1986; 84: Szollosi D, Czolowska R, Soltynska M5, Tarkowski AK. 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