Fusion of large and small karyoplasts produced from metaphase II mouse oocytes with interphase blastomeres from 2-cell and 8-cell embryos (volume ratio of partners, 1:1) results in premature chromosome condensation (PCC) of the interphase nucleus in the majority of the fusion products (hybrids). Fused under the same experimental protocol, oocyte-derived cytoplasts also induce PCC of the blastomere nucleus in the fusion products (cybrids) provided they originate from recently ovulated oocytes (14½ −15h after injection of human chorionic gonadotrophin (HCG)). In cytoplasts derived from older oocytes (16–20 h post-HCG) chromosome condensation activity gradually decreases with time as can be inferred from the increasing proportion of cybrids retaining interphase blastomere nuclei. However, even the oldest cytoplasts (19–20 h post-HCG) can induce PCC if the cytoplast volume significantly exceeds the volume of the interphase partner (7:1)-

We postulate that the condensation activity is predominantly bound to the nuclear apparatus (most probably to the chromosomes), and that in the cytoplasm of metaphase II mouse oocyte it decreases with post-ovulatory age.

Unless they are fertilized or activated (spontaneously or artificially) mammalian oocytes remain arrested in metaphase of the second meiotic division. After fusion with an interphase cell, the metaphase II mouse oocyte promotes premature chromosome condensation (PCC) of the interphase nucleus (Tarkowski & Batakier, 1980). The phenomenon of PCC, commonly observed in mammalian cell hybrids of various origins (meiotic maturing oocytes + interphase blastomeres, mitotic blastomeres + immature oocytes: Balakier, 1978; reviewed by Tarkowski, 1982; somatic mitotic + interphase cells: Johnson & Rao, 1970; reviewed by Rao, 1982) is induced by factors (proteins) that are believed to migrate from a metaphase cell to an interphase nucleus and bind to the condensing chromatin (Rao & Johnson, 1974). Studies by Rao’s group on somatic cell hybrids suggest that a movable factor originates from the cytoplasm of the mitotic partner (Rao & Johnson, 1974; Sunkara et al. 1980; Davis & Rao, 1982).

It has been shown previously that ovarian oocyte-derived cytoplasts separated from the nucleus (germinal vesicle) and cultured in vitro acquire chromosome condensation activity (Balakier & Czolowska, 1977). This activity is attributed to the maturation promoting factor (MPF), which is synthesized, or activated, during oocyte maturation and which in the absence of the nucleus is accumulated in the cytoplasm. In contrast to anucleate oocyte fragments (cytoplasts), in the intact maturing oocyte the chromosome condensation factor is thought - by analogy with somatic cells - to bind preferentially to the condensing meiotic chromosomes. However, no direct evidence has been presented regarding the distribution of the factor between the nuclear and cytoplasmic compartments in the terminal phase of oocyte maturation. With this aim in mind we have assayed the ability of karyoplasts and cytoplasts produced from metaphase II mouse oocytes of different postovulatory age to induce PCC after fusion with interphase blastomeres. We have found that the cytoplasm of recently ovulated oocytes exhibits chromosome condensation activity, and that this activity decreases with post-ovulatory age of oocytes. The bulk of the chromosome condensation activity is bound, however, to the nuclear apparatus.

Animals

Swiss albino (randomly bred colony), Rbl63 and F1 (C57B1/10 ×CBA-H) female mice were used in this study. The animals were kept under a 16/8, day/night, cycle with the middle of the dark period centred at midnight. Oocytes and embryos were obtained from females induced to ovulate with PMSG (pregnant mare’s serum gonadotrophin) and HCG (human chorionic gonadotrophin) (5–10 IU of each given 44—55 h apart) or from spontaneously ovulating animals. Donors of embryos were mated with FdCSTBl/10 ×CBA-H) males.

Cells

Ovulated oocytes

These were collected 16 h after HCG injection. The cumulus cells were dispersed with hyaluronidase (100–300 IU ml−1). In oocytes subjected later to bisection the zona pellucida was removed with 0 ·5% Pronase. Oocytes were stored in culture medium 16 (Whittingham, 1971) or in M2 medium (medium 16 buffered with HEPES: Fulton & Whittingham, 1978).

Blastomeres

Two-cell embryos were recovered from spontaneously ovulating females on the second day of pregnancy (day of vaginal plug is the first day) between 8.30 and 10.30 a.m. Eightcell embryos were obtained on the third day of pregnancy, either from spontaneously ovulating females (autopsy: 9.00 to 11.30a.m.) or from hormonally stimulated females (autopsy: 65 h post-HCG). The majority of the 8-cell embryos were uncompacted and were recovered together with 4-cell to 7-cell embryos. Therefore, we can consider the 8-cell embryos used in our experiments as being in the first half of the cell cycle. After removing the zona pellucida with Pronase, embryos were disaggregated by pipetting in Ca2+/Mg2+-free M2 medium or in Dulbecco’s A medium (Oxoid) supplemented with BSA (bovine serum albumin, fraction V, 1 mgml−1). Disaggregated blastomeres were stored in M2 or in medium 16. Single blastomeres originating from 2-cell and 8-cell embryos are designated in the text as and blastomeres, respectively.

Production of karyoplasts and cytoplasts

Nucleate (karyoplasts) and anucleate fragments (cytoplasts) were produced either by bisection of oocytes by hand, or using microsurgical techniques. Because of the time-consuming procedure, microsurgically produced karyoplasts and cytoplasts could not have been obtained and used for fusion earlier than 16 h post-HCG injection. Younger karyoplasts and cytoplasts were produced by oocyte bisection.

Bisection of oocytes was carried out according to the method of Tarkowski (1977) on an agar-coated Petri dish, in M2 medium, using a glass needle. Only fragments corresponding roughly to half of the oocyte volume were used for further manipulations. The fragments were stored in M2 medium.

Oocytes operated on microsurgically were preincubated for at least 30 min in medium 16 with cytochalasin B (CB, Sigma; 10 μgml−1 of CB dissolved in dimethyl sulphoxide (DMSO)) at 37°C, under liquid paraffin, and in 5% CO2 in air. Operations were performed in M2 medium with CB (10 μgml−1 under the inverted microscope with a Carl Zeiss micromanipulator. Karyoplasts and cytoplasts were produced using the technique of McGrath & Solter (1983). Briefly, the karyoplasts were obtained by aspiration into a sharply pointed pipette (20 μm in diameter) of the region in which the metaphase II spindle was previously localized with a smallest possible amount of the cytoplasm. Karyoplasts thus obtained were about of the oocyte volume, or smaller. Large cytoplasts were produced as a by-product of enucleation (they constituted of the oocyte volume), and small cytoplasts ( of oocyte volume) were produced similarly to karyoplasts. Karyoplasts and cytoplasts (Fig. 1) were transferred to CB-free medium 16’ and stored in standard culture conditions for at least 30 min before further manipulations.

Figs. 1.

1–5. Size relations of fusion partners: karyoplasts and cytoplasts produced from metaphase II mouse oocytes and interphase blastomeres from 2-cell and 8-cell embryos. Fig. 1, ×150; Figs 2 –5, ×1200. Fig. 1. Microsurgically produced cytoplasts (top) and karyoplasts (left) of the size of 18 blastomeres (bottom) placed against enucleated metaphase II oocyte (centre). The smallest fragments (right) are the smallest karyoplasts containing metaphase II apparatus that have been produced by this technique.

Figs. 1.

1–5. Size relations of fusion partners: karyoplasts and cytoplasts produced from metaphase II mouse oocytes and interphase blastomeres from 2-cell and 8-cell embryos. Fig. 1, ×150; Figs 2 –5, ×1200. Fig. 1. Microsurgically produced cytoplasts (top) and karyoplasts (left) of the size of 18 blastomeres (bottom) placed against enucleated metaphase II oocyte (centre). The smallest fragments (right) are the smallest karyoplasts containing metaphase II apparatus that have been produced by this technique.

Fusion

Two methods of fusion were used: polyethylene glycol (PEG)-mediated fusion and fusion in the electric field.

PEG-mediated fusion

Blastomeres and oocyte fragments (karyoplasts, cytoplasts) were incubated for a few minutes in phytohaemagglutinin (PHA, Difco; 100—150 μg ml−1 in BSA-free M2). The fragments were agglutinated with blastomeres into pairs and treated individually for 40—120s with PEG of Mr2000 (Fluka, 50%, w/v, solution) dissolved in BSA-free M2. PEG-treated pairs were thoroughly washed in M2 medium and stored in the same medium until the fusion of partners had occurred. The fusion products were cultured in medium 16 under standard culture conditions; 60—90min later they were processed for haematoxylin-stained whole mounts (Tarkowski & Wrdblewska, 1967).

Electrofusion

Ina few experiments PHA-agglutinated pairs of fusion partners were fused in an electric field according to the method of Zimmermann (1982) adapted to mouse blastomeres by Kubiak & Tarkowski (1985). Pairs of cells were placed between platinum electrodes separated by a distance of 60–100 μm, in 0 ·25M-glucose supplemented with BSA (Imgml−1). Two pulses of 20 μs duration and output voltage of 35 V (fragment + blastomere), or one pulse of 50 μs duration and 7 ·5 V (fragment + blastomere), were applied. Fusion products were cultured and processed as above.

The results obtained in six experimental groups in which different combinations of fusion partners were used are summarized in Table 1.

Table 1.

Behaviour of interphase nuclei in hybrid and cybrid cells produced by fusion of metaphase II mouse oocyte-derived karyoplasts and cytoplasts with interphase blastomeres

Behaviour of interphase nuclei in hybrid and cybrid cells produced by fusion of metaphase II mouse oocyte-derived karyoplasts and cytoplasts with interphase blastomeres
Behaviour of interphase nuclei in hybrid and cybrid cells produced by fusion of metaphase II mouse oocyte-derived karyoplasts and cytoplasts with interphase blastomeres

The reactions of interphase nuclei have been tested in two classes of fusion products: hybrids originating from fusion of karyoplasts with interphase blastomeres, and cybrids resulting from fusion of anucleate cytoplasts with blastomeres (Figs 2 –5). In the majority of experiments the cell volume ratio of oocyte-derived fragment to blastomere was kept constant at 1:1 (Figs 2 –4). Only in one experimental group was the volume of the oocyte-derived fragment substantially larger (ratio 7:1) (Fig. 5).

Fig. 2.

Large cytoplast produced by bisection of an oocyte (left) agglutinated with 12 blastomere (right).

Fig. 2.

Large cytoplast produced by bisection of an oocyte (left) agglutinated with 12 blastomere (right).

In hybrid cells the reaction of the blastomere nucleus was nearly uniform: in the majority of the fusion products the nucleus had undergone PCC (Table 1, lines 1 and 2). Prematurely condensed nuclei either formed metaphase chromosomes or were partially condensed, and frequently accumulated around the metaphase II spindle (Fig. 6). The positive response of the interphase nucleus to the condensing factor from the karyoplast was observed irrespective of the size of karyoplasts, the origin of interphase blastomeres (2-cell and 8-cell embryos), and the post-ovulatory age of karyoplasts at fusion −20h after HCG administration), provided the karyoplast remained arrested in metaphase II (Figs 6, 8, 9). Out of eight fusion products containing the interphase nucleus (Table 1, lines 1 and 2), five cases of spontaneous activation of the oocyte component (release from metaphase block) could be visualized directly on the basis of the presence of anaphase/telophase configuration of chromosomes; in the remaining three hybrid cells it was not possible to ascertain whether or not anaphase lias already begun. The results of experiments shown in line 2 (Table 1) are particularly interesting. The karyoplasts used for fusion were equal in size to blastomere, or even smaller, and were composed mainly of the large metaphase II spindle embedded in a small amount of cytoplasm (Fig. 3). Thus the karyoplast containing a negligible amount of cytoplasm can induce PCC in an interphase cell (Figs 8, 9).

Fig. 3,4

3,4. Small karyoplast (Fig. 3, top) and cytoplast (Fig. 4, top) agglutinated with 18blastomeres (bottom).

Fig. 3,4

3,4. Small karyoplast (Fig. 3, top) and cytoplast (Fig. 4, top) agglutinated with 18blastomeres (bottom).

Fig. 4.

Small karyoplast (Fig. 3, top) and cytoplast (Fig. 4, top) agglutinated with 18blastomeres (bottom).

Fig. 4.

Small karyoplast (Fig. 3, top) and cytoplast (Fig. 4, top) agglutinated with 18blastomeres (bottom).

Fig. 5.

Large cytoplast (left) obtained after enucleation of metaphase II oocyte and its fusion partner, the interphase 18 blastomere.

Fig. 5.

Large cytoplast (left) obtained after enucleation of metaphase II oocyte and its fusion partner, the interphase 18 blastomere.

Figs. 6.

6—12. Fusion products: hybrids (karyoplasts + blastomeres) and cybrids (cytoplasts + blastomeres). ×1200. Fig. 6. After fusion of a large karyoplast obtained by bisection of metaphase II oocyte with 18 blastomere, the interphase nucleus has undergone PCC (arrow) forming the chromosome group beside the metaphase II (arrowhead).

Figs. 6.

6—12. Fusion products: hybrids (karyoplasts + blastomeres) and cybrids (cytoplasts + blastomeres). ×1200. Fig. 6. After fusion of a large karyoplast obtained by bisection of metaphase II oocyte with 18 blastomere, the interphase nucleus has undergone PCC (arrow) forming the chromosome group beside the metaphase II (arrowhead).

Fig. 7.

Cybrid cell obtained after fusion of oocyte-derived large cytoplast and 12 blastomere: PCC of the interphase nucleus.

Fig. 7.

Cybrid cell obtained after fusion of oocyte-derived large cytoplast and 12 blastomere: PCC of the interphase nucleus.

Fig. 8.

9. Two hybrid cells produced by fusion of small 18 karyoplasts with 18 blastomeres. In both hybrids prematurely condensed blastomere chromosomes have been grouped around the metaphase II spindle. Fig. 8, top view of the metaphase; Fig. 9, side view of the metaphase; note the presence of a large meiotic spindle.

Fig. 8.

9. Two hybrid cells produced by fusion of small 18 karyoplasts with 18 blastomeres. In both hybrids prematurely condensed blastomere chromosomes have been grouped around the metaphase II spindle. Fig. 8, top view of the metaphase; Fig. 9, side view of the metaphase; note the presence of a large meiotic spindle.

Fig. 9.

Two hybrid cells produced by fusion of small 18 karyoplasts with 18 blastomeres. In both hybrids prematurely condensed blastomere chromosomes have been grouped around the metaphase II spindle. Fig. 8, top view of the metaphase; Fig. 9, side view of the metaphase; note the presence of a large meiotic spindle.

Fig. 9.

Two hybrid cells produced by fusion of small 18 karyoplasts with 18 blastomeres. In both hybrids prematurely condensed blastomere chromosomes have been grouped around the metaphase II spindle. Fig. 8, top view of the metaphase; Fig. 9, side view of the metaphase; note the presence of a large meiotic spindle.

In cybrid cells (Table 1, lines 3–6) the reaction of the blastomere nucleus was variable. Only cytoplasts originating from recently ovulated oocytes −15h post-HCG injection) were able to induce PCC in all fusion products (line 3, Fig. 7). Cytoplasts at 16− post-HCG induced PCC in 50% of cybrids (line 4), whereas in older cytoplasts −20h post-HCG) condensation was detected in only five out of 29 cybrids studied (line 5) (Figs 10, 11). However, even the oldest (19 ·20h) cytoplasts were able to promote PCC if the volume of PCC-inducing partner was substantially increased (line 6, Fig. 5): the majority of large cytoplasts ( of the oocyte volume) induced PCC in blastomeres (Fig. 12) with the proportion of PCC/interphase nuclei being just the reverse of that obtained after fusion of small cytoplasts with blastomeres (compare lines 5 and 6).

Fig. 10,11

10,11. Two cybrid cells formed by fusion of a small oocyte-derived cytoplast with a 18 blastomere. In one cybrid (age of oocyte cytoplast at fusion 16 h post-HCG injection, Fig. 10). PCC has been induced, whereas in the other one (cytoplast age at fusion 20h after HCG injection, Fig. 11) the interphase nucleus remained intact.

Fig. 10,11

10,11. Two cybrid cells formed by fusion of a small oocyte-derived cytoplast with a 18 blastomere. In one cybrid (age of oocyte cytoplast at fusion 16 h post-HCG injection, Fig. 10). PCC has been induced, whereas in the other one (cytoplast age at fusion 20h after HCG injection, Fig. 11) the interphase nucleus remained intact.

Fig. 11.

Two cybrid cells formed by fusion of a small oocyte-derived cytoplast with a 18 blastomere. In one cybrid (age of oocyte cytoplast at fusion 16 h post-HCG injection, Fig. 10). PCC has been induced, whereas in the other one (cytoplast age at fusion 20h after HCG injection, Fig. 11) the interphase nucleus remained intact.

Fig. 11.

Two cybrid cells formed by fusion of a small oocyte-derived cytoplast with a 18 blastomere. In one cybrid (age of oocyte cytoplast at fusion 16 h post-HCG injection, Fig. 10). PCC has been induced, whereas in the other one (cytoplast age at fusion 20h after HCG injection, Fig. 11) the interphase nucleus remained intact.

Fig. 12.

After fusion of a large cytoplast produced by enucleation of metaphase II oocyte with a small 18 blastomere, PCC of the interphase nucleus has occurred notwithstanding that the cytoplast used for fusion was as old as 20 h after HCG injection.

Fig. 12.

After fusion of a large cytoplast produced by enucleation of metaphase II oocyte with a small 18 blastomere, PCC of the interphase nucleus has occurred notwithstanding that the cytoplast used for fusion was as old as 20 h after HCG injection.

In this study we have demonstrated that fusion of metaphase II mouse oocyte-derived nucleate (karyoplasts) and anucleate fragments (cytoplasts) results in PCC of the interphase blastomere nucleus in the majority of fusion products. PCC frequency is far higher in hybrids (karyoplast + blastomere) than in cybrids (cytoplast 4-blastomere). The post-ovulatory age of a karyoplast at fusion, which varied from to 8h (provided the ovulation occurs about 12 h post-HCG administration; Edwards & Gates, 1959), does not influence the PCC frequency. In contrast, frequency of PCC induced by cytoplasts seems to be inversely correlated with their post-ovulatory age: the older the fragments are at fusion, the smaller the proportion of fusion products showing PCC (frequency of PCC induced by −3h cytoplasts, 100%;4− cytoplasts, 50%; −8h cytoplasts, 17%; see Table 1). Because the volume ratios of oocyte-derived cytoplasts to blastomeres were always the same (1:1) we conclude that with increasing age of cytoplasts chromosome condensation activity gradually decreases until it can be easily diluted or inactivated after fusion with an interphase blastomere. The presence of protein cytoplasmic factors (‘inhibitors of mitotic factors’) acting antagonistically to chromosome condensation factors (‘maturation promoting factor’, ‘mitotic factor’) and operating in interphase cells initiating the cell cycle has been demonstrated in somatic cells and zygotes of mammals (Adlakha et al. 1983; Balakier & Masui, unpublished data, cited by Masui, 1985).

The fact that the chromosome condensation activity of the oocyte cytoplasm was indeed neutralized under the influence of interphase cytoplasm in our oldest, 1:1, cybrids (cf. Table 1, line 5) could be inferred from experiments in which we used large cytoplasts for fusion with small blastomeres (Table 1, line 6). After substantial (sevenfold) increase of the cytoplast volume even ‘old’ cytoplasts were able to induce PCC in the vast majority of cybrids. Thus, although chromosome condensation activity is low in 7–8 h cytoplasts it can still be expressed in fusion products, provided the volume of interphase cytoplasm is small and, consequently, insufficient to dilute or inactivate the condensing factor present in a cytoplast. Fusion of cytoplasts originating from meiotic (or mitotic) cells with interphase cells at different volume ratios may provide a new experimental approach for ‘titration’ of chromosome condensation activity in the cytoplasm of various cells.

Although arrested in karyokinesis the ovulated mouse oocyte is a metabolically active cell (Howlett & Bolton, 1985). Certain post-ovulatory changes are thought to be necessary to render the oocyte competent for sperm penetration or activation (Zamboni, 1970), a concept that finds some support in recent studies on in vitro fertilization of rat oocytes (Shalgi et al. 1985), and in the common experience of embryologists working on experimental parthenogenesis in the mouse, who find it extremely difficult to activate oocytes before 16–17 h post-HCG (i.e. a few hours after ovulation). According to our findings the cytoplasm of the metaphase II oocytes cultured in vitro loses chromatin-condensing activity as the oocytes approach the age 16—17h post-HCG. We postulate that the disappearance of this activity from the oocyte cytoplasm is the manifestation of changes that render the oocyte susceptible to artificial activation and, eventually, may lead to accelerated spontaneous activation of oocytes (more frequent in cumulus-free and zona-free oocytes). The disappearance of the chromosome condensation activity from the cytoplasm that eventually results in the release of the oocyte from metaphase II arrest may indicate that in mammalian oocytes the maturation promoting factor (MPF) responsible for the progression of meiosis and condensation of meiotic chromosomes (Sorensen et al. 1985) is far less stable than MPF in amphibian oocytes, which is thought to be stabilized by the cytostatic factor (Masui & Markert, 1971; Newport & Kirschner, 1984).

Whether or not the changes described occur at the same rate in vivo remains to be investigated. It is quite possible that the gradual release of ovulated oocytes from metaphase II block expresses the process of ageing, and that this process progresses faster in suboptimal in vitro culture conditions. Numerous cytoplasmic changes referred to as ‘ageing’ have been described in ovulated oocytes submitted to prolonged culture in vivo and in vitro (Szollosi, 1971, 1975; Longo, 1974; Webb et al. 1986).

The results of our experiments provide indirect evidence that PCC of the interphase nucleus introduced into metaphase II mouse oocyte is induced by a ‘factor’ that is both cytoplasmically located and associated with the chromosomes and, or, the chromosome-containing region. The condensing activity of the small karyoplasts, which contain mainly the metaphase II spindle enveloped by a narrow cytoplasmic rim, suggests that in this case at least the bulk of the factor responsible for PCC must reside in the meiotic apparatus, probably in the metaphase chromosomes.

Migration and binding of cytoplasmic proteins to prematurely condensed interphase nuclei have been demonstrated in metaphase II amphibian oocytes injected with somatic nuclei (Masui, 1985), and in hybrid (mitotic + interphase) somatic cells (Rao & Johnson, 1974). Whether and how the factor residing in the cytoplasm-deprived karyoplast is mobilized to induce PCC in the recipient interphase cell requires further studies using labelling techniques. At present one can only speculate that PCC is provoked by a factor that either moves freely from the meiotic chromosomes to the interphase nucleus or induces processes that result in activation of MPF in the hybrid cell.

We thank Mrs Alina Szarska and Mrs Marta Modlifiska for their skilful technical assistance. We also acknowledge with thanks the support of the WHO Small Supplies Programme.

This work was partly supported by a research grant (II. 1.6.5) from the Polish Academy of Sciences.

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