/ . Embryol. exp. Morph. Vol. 55, pp. 319-330, 1980
Printed in Great Britain © Company of Biologists Limited 1980
3J9
Nucleo-cytoplasmic
interactions in cell hybrids between mouse oocytes,
blastomeres and somatic cells
By ANDRZEJ K. TARKOWSKI 1 AND HANNA BALAKIER 1
From the Department of Embryology, Institute of Zoology,
University of Warsaw
SUMMARY
With the help of the technique of Sendai virus-mediated cell fusion, hybrid cells were
produced between two maturing oocytes, between maturing oocytes or mature secondary
oocytes and interphase blastomeres from 2-cell embryos, and between secondary oocytes and
follicle cells (FC). In the first case giant oocytes form and in these the two groups of condensing bivalents join on a common spindle, undergo a first meiotic division, and become
arrested in metaphase II; these hybrids are heterozygous diploids. When blastomeres are
fused with oocytes undergoing maturation, then the blastomere nucleus undergoes premature
chromosome condensation (PCC), and two sets of chromosomes (meiotic and mitotic)
come close to each other but the mitotic chromosomes are not fully incorporated into the
meiotic spindle. The behaviour of the nuclei from blastomeres or from follicle cells fused with
secondary ovulated oocytes, depends on whether or not the oocyte undergoes activation.
When the oocyte is not activated, then the introduced nuclei undergo PCC but the chromosomes remain separate from the spindle of metaphase II. When the oocyte is activated, then
the introduced nuclei remain in interphase; the FC nuclei may increase their volume eight
times and undergo some other structural changes but during the 6 h period after fusion their
appearance remains distinct from that of the female pronucleus. Since some oocytes are not
activated after fusion has occurred, it follows that the fusion process itself is not sufficient to
trigger egg activation.
INTRODUCTION
The application of the cell hybridization technique to mammalian eggs and
early embryonic cells offers the opportunity to study various problems concerned
with the cytoplasmic control of nuclear activity in oogenesis and development,
whose solution would otherwise require the microsurgical transfer of nuclei or
the injection of cytoplasm. So far this technique has been used to introduce
somatic cell nuclei into the early embryo (Graham, 1969; Barariska &
Koprowski, 1970; Bernstein & Mukherjee, 1972, 1973), and to make tetraploid
embryos (Graham, 1971). It has also been particularly useful in the analysis of
the role of cytoplasmic factors in the resumption of meiosis in mouse oocytes
(Balakier & Czolowska, 1977; Balakier, 1978).
In the present study we have used this technique to produce various types of
1
Authors' address: Department of Embryology, Institute of Zoology, University of
Warsaw, 00-927 Warszawa 64, Poland.
320
A. K. TARKOWSKI AND H. BALAKIER
hybrid cells between maturing oocytes, mature secondary oocytes, blastomeres,
and differentiated cells. In particular we have made heterozygous diploid oocytes,
investigated the phenomenon of premature condensation of mitotic chromosomes under the influence of the cytoplasm of maturing oocytes, and followed
the fate of nuclei from blastomeres and from follicle cells when they were fused
into the cytoplasm of activated and non-activated secondary oocytes.
MATERIALS AND METHODS
Animals. Eight- to twelve-week-old Swiss albino (outbred colony), A,
129/terSv, CBA-T6T6, C57BL/10, and F x (CBA/H x C57BL/10) females were
used in this study. The animals were kept under a 16 h light/8 h dark cycle with
the middle of the dark period at midnight.
Cells. Four types of cells were used for fusion: fully grown ovarian oocytes
with germinal vesicles which resumed meiosis in vitro (maturing oocytes),
ovulated oocytes in metaphase II, single blastomeres from 2-cell embryos, and
follicle cells.
Oocytes with germinal vesicles were obtained by teasing apart the ovaries
from untreated 6- to 8-week-old females. The follicle cells were dispersed with
vigorous pipetting, and the oocytes cultured for 1-3 h before hybridization.
During this period, meiosis resumed and the germinal vesicle broke down and
bivalents condensed.
Ovulated oocytes were obtained from females which had been induced to
ovulate (5 i.u. each of PMSG and HCG given 45-53 h apart) and which were
autopsied 13-18 h after the HCG injection. The follicle cells were dispersed
with hyaluronidase (100 i.u. per ml in PBS). Occasionally, oocytes from
spontaneous ovulations were also used.
Follicle cells (FC) were obtained from the cumuli oophori surrounding the
ovulated oocytes. They were dispersed with the same concentration of hyaluronidase which was subsequently replaced with Ca- and Mg-free phosphatebuffered saline (solution A of Dulbecco & Vogt, 1954).
Two-cell embryos were recovered on the second day of development between
9.00 and 12.00 a.m. from females which had ovulated spontaneously (day of
vaginal plug = first day). After removal of the zona pellucida the embryos were
dissociated into single blastomeres by gentle pipetting.
From both types of oocytes and from 2-cell embryos the zona pellucida was
removed with pronase (method of Mintz, 1962).
The zona-free oocytes and blastomeres, and FC were stored in Ca- and Mgfree PBS with the addition of 0-1 % (w/v) bovine serum albumin for 14-45 min
at room temperature before being used for fusion. The lack of Ca 2+ and Mg 2+
ions in the medium is supposed to facilitate fusion (Poste & Allison, 1971).
Fusion procedure. The cells were next transferred to drops of Sendai virus
(virus-inactivated with beta-propiolactone; Neff & Enders, 1968, and, depending
Nucleo-cytoplasmic interactions
321
on the fusion efficiency of a particular batch, diluted to 125, 500 or 1000 HAU)
in a siliconized glass Petri dish at 4 °C. Oocytes and blastomeres were exposed
to the virus for 3-10 min and FC for 20-30 min. FC exposed to the virus for a
shorter time did not fuse efficiently. Close contact between oocytes and between
oocytes and blastomeres was achieved by sucking the adhering cells into a
mouth-controlled pipette with a narrow lumen so that the cells were squeezed
together. Contact between FC and oocytes was achieved by rolling the oocytes
across the follicle cells which were on the bottom of the dish.
Cells were fused in the following combinations: (1) two maturing oocytes,
(2) maturing oocyte + blastomere, (3) ovulated oocyte + blastomere, (4) ovulated
oocyte + FC. Sometimes three, rather than two cells, were put in contact in
order to increase the chance of fusion (e.g. one oocyte + two blastomeres or
vica versa). Since no difference between strains in respect to the incidence of
successful fusions was observed in any cell combination, the data were pooled.
Culture and examination of cells. Virus-agglutinated cells were cultured in
separate drops of Whitten's medium (Whitten, 1971) under liquid paraffin at
37 °C with a gas mixture of 5 % CO2 in air. They were examined for signs of
fusion and lysis every 30 min for up to 8 h. At various times after the start
of the experiment, the cells were fixed and either air-dried (Tarkowski, 1966)
or made into haematoxylin-stained whole mounts (Tarkowski, 1971).
RESULTS
(1) Fusion of two maturing oocytes
In all, 1148 oocytes, which had begun maturation in vitro, were used for these
experiments but only 27 pairs fused to form 'giant' oocytes (Fig. 1). The viruscoated oocytes only weakly adhered to each other and it was much more difficult
to obtain close contact between these cells when compared to ovulated oocytes
and blastomeres.
Twelve giant oocytes and 145 single unfused oocytes lysed within 1-24 h of
culture. At 1-4 h after fusion, the giant oocytes contained two groups of
chromosomes (seven cells examined, see Fig. 2), but at 5-6 h after fusion only a
single group of 40 typical bivalents was observed (three cells examined). Three
out of the five hybrid oocytes which were alive after 24 h culture had extruded
the first polar body (Fig. 1) and contained 40 chromosomes whose appearance
was characteristic of metaphase II (Fig. 3). In the two other cases the first polar
body was not extruded but the homologous chromosomes had separated from
each other and the oocytes contained a single group of 80 metaphase II chromosomes.
(2) Fusion of maturing oocytes with blastomeres
Thirty hybrid cells were produced (Table 1, Fig. 4) but only 21 were available
for analysis (one lysed, eight damaged). In all the hybrid cells, the chromosomes
of the blastomere condensed and this process could begin as early as during
322
A. K. T A R K O W S K I AND H. BALAKIER
Figs. 1-3. Giant oocytes produced, by fusion of two maturing oocytes.
Fig. 1. Giant oocyte which has extruded first polar body (1PB) and three control
oocytes (all three oocytes have extruded 1PB but in two of them it was detached
during manipulations), x 200.
Fig. 2. Two h post fusion the oocyte still contains two separate groups of bivalents.
Whole mount preparation, x 450.
Fig. 3. Forty chromosomes of metaphase II from a giant oocyte shown in Fig. 1
which has extruded 1PB in culture. Air-dried preparation, x 1000.
Table 1. Fusion of maturing oocytes with interphase blastomeres
Hybrid cells
No. of lysed cells
No. of
oocytes
473
Oocyte Blastomere ,
No. of
+
+
Hybrid
blastomeres blastomere blastomere
cells
686
30*
8
1
Oocytes
Blastomeres
42
47
* In three cases one oocyte fused with two blastomeres.
fusion when the two cells were only connected by a cytoplasmic bridge (Fig. 5).
During the first 1-2 h after fusion the chromosomes of the two original cells
remained separate (Fig. 6), but in all cells examined at 3-7 h after fusion the
two chromosome sets had joined together into one group composed of 20
bivalents and 40 mitotic chromosomes (Fig. 7, 8).
Two hybrid cells were examined at 20 h after fusion. They had not dividedand
contained 80 chromosomes: 40mitotic chromosomes and40chromosomes with
an appearance characteristic of the metaphase of the second meiotic division.
Nucleo-cytoplasmic interactions
323
In contrast to their behaviour in hybrid cells, the nuclei of blastomeres which
had not fused with the oocytes and those from blastomeres which had fused
with each other remained in interphase. The oocytes which had not fused had
proceeded to metaphase I.
(3) Fusion of ovulated oocytes with blastomeres
Thirty-five oocytes were fused with blastomeres and in 29 cases the fusion
was with a single blastomere while in six cases the fusion was with two blastomeres (Table 2). Fourteen of these hybrid cells lysed during the first 6 h of
culture. The behaviour of the interphase nucleus from the blastomere depended
on whether or not the oocyte underwent activation (it is not known whether
activation of the oocyte occurred before or at the time of fusion). In the situation in which the oocyte was not activated, then the nucleus of the blastomere
reacted by premature chromosome condensation (12 hybrid cells examined).
Six hybrids were examined in whole mount preparations and it was found that
there were two or three separate groups of chromosomes each arranged on its
own spindle (Fig. 9); those with three sets of chromosomes originated from cases
in which one oocyte fused with two blastomeres. The morphology of these two
kinds of chromosomes could be easily distinguished in air-dried preparations
where it was possible to observe the rod-like morphology of the blastomere
mitotic chromosomes and the metaphase-II appearance of the 20 meiotic
bivalents from the oocyte (two cells fixed at 3 h after fusion). In four other
hybrid cells examined in whole mounts the chromosomes from both partners
had formed a single group. In the two cases in which it could be seen, the
spindle was abnormally long and the chromosomes were scattered along its
length.
In some cases the oocytes became activated and the behaviour of the nucleus
from the blastomere was quite different from the situation described above.
Nine hybrids were made with activated oocytes and of these five underwent
immediate cleavage (Fig. 10), two extruded the second polar body and formed a
single pronucleus, and in two cases second polar body formation was suppressed
and three or four oocyte subnuclei were formed (for routes of normal egg activation see Tarkowski, 1975). Irrespective of the type of development of the
activated oocyte, the nucleus from the blastomere always remained in interphase
as did all the blastomeres which had not fused and which were in culture for
6 h. Thirty out of the 160 unfused oocytes underwent activation.
(4) Fusion of ovulated oocytes with follicle cells (FC)
It was not possible to directly observe the fusion of small FCs with oocytes
and so there is no information about the actual fusion frequency: some oocytes
may have fused with FCs and subsequently lysed. Eighteen hybrid cells were
examined and, as in the previous experiment, it was found that the behaviour of
the nucleus from the follicle cell depended on whether or not the recipient
324
A. K. T A R K O W S K I AND H. BALAKIER
12
Figs. 4-8. Cells obtained by fusion of a maturing oocyte with a blastomere in
interphase.
Fig. 4. A cell shortly after fusion: the dark cytoplasm originating from the blastomere is clearly delineated from the oocyte cytoplasm. Adhering to this cell are the
sister unfused blastomere and 2PB. x 400.
Fig. 5. A cell in the process of fusion with the two contributing cells still connected
by a cytoplasmic bridge. The blastomere chromosomes have already begun to
Nucleo-cytoplasmic
interactions
325
oocyte was activated: in inactivated oocyte cytoplasm the nuclei from the FC
underwent premature chromosome condensation while in activated oocytes they
remained in interphase. Despite the fact that many FC adhered to the oocyte
only one or rarely two FC actually fused with it: it may be that when many FC
fuse with the oocyte then it tends to lyse, and it is noticeable that lysis of oocytes
was very common in this series of experiments (Table 2).
The nuclei from the FC never formed one group with the metaphase-JI
chromosomes from the oocyte. The FC chromosomes were either arranged in a
group resembling a metaphase plate (Fig. 11), or they were scattered over a
large area beneath the egg membrane.
In seven cases the hybrid cells underwent activation and a second polar body
was formed. These cells contained a typical female pronucleus and one or two
other nuclei with clearly different basophilic-staining properties and a distinct
structure (Figs. 12-15). The female pronucleus contained a single nucleolus in
clear nucleoplasm, while the additional nuclei had many chromatin granules
condense (on the right); on the left - chromosomes of the oocyte. Whole mount
preparation. x450.
Fig. 6. A cell fixed 1 h post fusion. Chromosomes of the oocyte in the center and
chromosomes of the blastomere near the surface of thecell. Whole mount preparation.
x55O.
Fig. 7. A common group of chromosomes from the oocyte and the blastomere.
Fixed 8 h post fusion. Whole mount preparation, x 450.
Fig. 8. Bivalents of metaphase I and blastomere chromosomes from a hybrid cell
fixed 2 h post fusion (fragment of an air-dried preparation), x 1000.
Figs. 9-10. Hybrid cells formed by fusion of an ovulated oocyte with an interphase
blastomere. Whole mount preparations.
Fig. 9. A cell fixed 2 h post fusion. A spindle with blastomere chromosomes is in the
center of the cell; a spindle of metaphase II is out of focus (arrow). Note that despite
fusion the oocyte has not undergone activation, x 450.
Fig. 10. A hybrid cell which underwent immediate cleavage as a consequence of
oocyte activation during fusion. Very early pronuclei are visible in each cell and
one cell contains also an interphase nucleus of the blastomere. Fixed 2 h post
fusion, x 300.
Figs. 11-15. Hybrid cells formed by fusion of ovulated oocyte with one follicular
cell. Whole mount preparations.
Fig. 11. Metaphase II (bottom) and a group of chromosomes originating from the
nucleus of FC. Note that fusion did not result in activation of the oocyte. x 600.
Figs. 12,13. Activated hybrid cell which extruded 2PB and formed one pronucleus.
Fixed 6 h post fusion. Fig. 12 focused on the female pronucleus and Fig. 13 on the
nucleus of FC and 2PB. The FC nucleus has a more deeply stained nucleoplasm than
the pronucleus and contains several chromatin granules and/or nucleolus-like
spheres. The FC nucleus is slightly smaller from the pronucleus. x 650.
Figs. 14, 15. The nuclei of a hybrid cell which has extruded 2PB. The FC nucleus
(Fig. 15) has enlarged in the egg cytoplasm and is much larger than the pronucleus
(Fig. 14). The two nuclei clearly differ in structure. Fixed 5 h post fusion, x 1500.
191
215
237
Oocyte + blastomere
Oocyte -f follicle cells
35*
18
Total
no.
A.
12
lit
9
71
Nonactivated Activated
oocytes
oocytes
14
Hybrid
cells
14
51
27
—
Oocytes Blastomeres
A
No. of lysed cells
* In six cases one oocyte fused with two blastomeres; four such hybrid cells lysed during 2 h of culture.
t Three oocytes contained two groups of chromosomes beside metaphase II.
% One oocyte contained two somatic nuclei beside a pronucleus and a 2PB.
No. of
blastomeres
No. of
oocytes
Combination
of
cells
Hybrid cells
Table 2. Fusion of ovulated oocytes with blastomeres and with follicle cells
O
CO
w
E
^
>•
'SKI
Nucleo-cytoplasmic interactions
327
Table 3. Volume of nuclei {in /tra3) in activated oocytes fused with
follicle cells§ (cells fixed after 5-6 h of culture)
Egg
no.
Nucleus
of 2PB
Female
pronucleus
FC
nucleus
Nuclei of
unfused FC
1
2
3
4
5
6
7
151
626
437
1000
dl*
113:214
n = 14
139
231
1151
x = 1361
dl
1100
462
S.D. = 54-4
dl
164
1000
Range = 55-220
dl
310
111
269
739
183
* 2PB detached and lost during processing.
§ The diameter of nuclei was measured twice at right angles with the eye-piece micrometer
and the average diameter was used to calculate the volume. AH measurements were made in
whole mounts.
and/or nucleolus-like bodies and they still bore some resemblance to the nuclei
of unfused FC. It is shown in Table 3 that in two hybrid cells the FC nuclei
remained small and within the size range of the nuclei in unfused FC, while in
the other five hybrid cells the FC nuclei had definitely enlarged reaching a
maximum volume eight times that of the nuclei from unfused FC.
One important feature of these hybrid cells is that the volume of the FC
nucleus may be smaller or larger than that of the female pronucleus within the
same cell. This variation may be due to the fact that the FC nucleus could
enter the oocyte either at the time of activation or later. The relationship between
the time of activation and the time of fusion is not clear because it is impossible
to directly observe either event in these experiments.
In this series of experiments the activation frequency of unfused oocytes was
high (64 out of 168) and the most common form of activation was the extrusion
of the second polar body and the formation of a single pronucleus.
DISCUSSION
The formation of heterozygous diploid oocytes
Heterozygous diploid oocytes were made by fusing together two oocytes
which had resumed meiosis in vitro. In these giant oocytes the two groups of
condensing chromosomes join together to form a common tetraploid plate of
metaphase I; in some cases they undergo the first meiotic division with extrusion
of the first polar body and then become arrested in metaphase II, as do normal
oocytes maturing in vivo or in vitro.
These giant oocytes could give rise to diploid heterozygous parthenogenetic
embryos. However, it is unlikely that they would develop to advanced embryonic
stages because eggs fertilized or activated after maturation in vitro generally
328
A. K. TARKOWSKI AND H. BALAKIER
develop rather poorly (Cross & Brinster, 1970; Mukherjee, 1974; Eppig, 1978).
For this reason the fusion of activated ovulated oocytes can prove a more
efficient method for making such diploid heterozygotes (Soupart, Anderson &
Repp, 1977; Soupart, 1978).
One problem in working with these giant oocytes is that they are difficult to
make because the cells fuse at low frequency even when treated in exactly the
same way as the other cell combinations used in these experiments. It is likely
that this problem is caused by the smooth or slightly ruffled cell membrane of
these cells after germinal vesicle breakdown (see Nicosia, Wolf & Mastroianni,
1978, for their recent SEM studies and for references to previous TEM work).
On the contrary, in the other cell combinations at least one partner is known
to have a cell surface with many microvilli and according to Poste & Allison
(1971) microvilli facilitate cell fusion.
Control of nuclear activity in the hybrid cells
{a) Premature chromosome condensation (PCC)
When blastomeres with interphase nuclei were fused with maturing oocytes,
then the blastomere nucleus was invariably induced into premature chromosome
condensation; this effect must be rapid because it can take place when the two
cells are only connected by a cytoplasmic bridge, and this observation shows that
the maturation promoting factor in the oocyte cytoplasm can either diffuse to or
initiate a rapid change in the surroundings of the blastomere nucleus. Premature
chromosome condensation also occurred when blastomeres or follicle cells
were fused with ovulated oocytes which had remained in metaphase II. These
findings confirm our previous observations on the induction of PCC by oocyte
cytoplasm; a phenomenon which occurs even in the absence of the germinal
vesicle (Balakier & Czolowska, 1977), which is not species specific (Balakier,
(1979), and which can act between mitotic and meiotic cells (Balakier,
1978). Our results are similar to those obtained with amphibians (Gurdon,
1968; Masui & Markert, 1971; Wasserman & Masui, 1976; Wasserman & Smith,
1978).
After the induction of PCC the mitotic chromosomes of the blastomere and
the meiotic chromosomes of the oocyte appeared by light microscopy to be
arranged on a common spindle. However, electron microscopy shows that the
mitotic chromosomes are often scattered along the spindle or are situated in the
plane of the metaphase plate outside the spindle (Szollosi, Czolowska, Balakier
& Tarkowski, 1976, and in preparation). This arrangement is in contrast to the
situation when blastomere nuclei are induced into PCC in ovulated oocytes; in
this case the two groups of chromosomes usually remained separate. In the same
situation, the prematurely condensed chromosomes of follicle cells could either
be clumped or widely dispersed beneath the egg surface.
Nucleo-cytoplasmic interactions
329
(b) Nuclear swelling
In contrast to the previous behaviour, the nuclei from blastomeres and from
follicle cells remained in interphase when they were fused with ovulated eggs
which were activated and which were themselves developing to interphase.
Once again there is concordance between the behaviour of nuclei in a common
cytoplasm.
The behaviour of the follicle cell nucleus is particularly interesting because
this is a nucleus from a differentiated cell and because only a small amount of
'differentiated' cytoplasm enters the oocyte during fusion; the situation is
equivalent to 'nuclear transfer' in amphibian experiments. In amphibian
nuclear transfer experiments one of the first morphological signs of donor
nucleus reactivation is nuclear swelling (Graham, Arms & Gurdon, 1966). In
our experiments it has been shown that the nuclei from follicle cells can swell to
a maximum volume which is eight times that of control follicle cell nuclei; this
enlargement is greater than that previously observed when spleen nuclei were
fused with fertilized eggs (Graham, 1969). However, the degree of swelling of
the follicle cell nuclei was variable and the structure of the swollen nuclei was
not identical to that of a normal pronucleus. We conclude that it will only be
possible to show that the differentiated cell nucleus is completely reactivated
when it has been shown to code for normal development.
Cell fusion and egg activation
It has been previously suggested from similar fusion experiments that cell
fusion itself and by implication the fusion of sperm with egg in normal fertilization is alone sufficient to trigger normal egg activation and embryonic development (Soupart et al. 1977; Soupart, 1978). However our observation that the
fusion of blastomeres and follicle cells with metaphase II oocytes did not
inevitably lead to egg activation shows that this conclusion is not correct and
prompts us to suggest that the activating stimulus provided by the sperm in
normal fertilization is more subtle than it might first appear.
This work was financed in part by the Committee of Cell Pathophysiology of the Medical
Section of the Polish Academy of Sciences. The authors wish to thank Mrs Alina Szarska
for excellent technical assistance.
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