/. Embryol. exp. Morph. Vol. 36, 2, pp. 283-290, 1976
Printed in Great Britain
283
Postimplantation development of blastomeres
isolated from 4- and 8-cell mouse eggs
By J. ROSSANT 1
From the Department of Zoology, Oxford
SUMMARY
Blastomeres isolated from 4- and 8-cell mouse eggs were inserted into empty zonae and
transferred to the oviduct. The products of both types of blastomere were capable of inducing decidual formation. One implant produced by an isolated blastomere from a 4-cell egg
contained a small, retarded embryo at 5£ days but most decidua from blastomeres of either
4- or 8-cell eggs contained only a few trophoblast giant cells. It is suggested that this lack
of totipotency is due to insufficient cells being present at cavitation rather than restriction in
developmental potential.
INTRODUCTION
The prospective potency of isolated cleavage-stage blastomeres has been
studied in several mammalian species in order to discover whether the
morphological similarity between early blastomeres is reflected in their developmental potential (see review, Wilson & Stern, 1975). In the mouse, adult animals have been produced from 1/2 blastomeres (Tarkowski, 1959a, b: Hoppe
& Whitten, 1972) but the development of 1/4 and 1/8 blastomeres has only been
studied as far as the blastocyst stage (Tarkowski, 1959a, b; Mulnard, 1965;
Tarkowski & Wroblewska, 1967; Sherman, 1975). (The notation used is that of
Tarkowski & Wroblewska (1967), where 1/4 blastomere is one blastomere from
a 4-cell egg, 1/8 blastomere is one blastomere from an 8-cell egg, and so on.)
The present experiments were designed to assess the postimplantation potential
of isolated 1/4 and 1/8 mouse blastomeres after development in vivo. If isolated
blastomeres are obtained by destroying all other blastomeres within the zona,
incompletely destroyed cells and cell debris remaining inside the zone might
interfere with development, and so blastomeres were isolated by disaggregating zona-free cleavage stages. The blastomeres were then inserted into empty
zonae prior to transfer to the oviduct, since naked blastomeres and cleavage
stages do not survive in the oviduct in vivo (Bronson & McLaren, 1970; Modlinski, 1970).
1
Author's address: Department of Zoology, South Parks Rd, Oxford, 0X1 3PS, U.K.
284
J. ROSSANT
MATERIALS AND METHODS
Recovery of embryos from donor females
CFLP mice (Anglia Laboratory Animals Ltd) were used throughout this
study. Cleaving eggs for preparation of isolated blastomeres were obtained following natural mating. Four-cell eggs were flushed from the oviducts between
8.00 and 9.30 and 8-cell eggs between 12.00 and 15.00 on the 3rd day of pregnancy. Fertilized or unfertilized one-cell eggs for the preparation of empty zonae
were obtained from the ampullary region of the oviduct on the morning after
mating with fertile or vasectomized males. The cumulus cell mass was removed
by incubation in hyaluronidase solution (100 i.u./ml phosphate buffered saline).
PB1 +10 % foetal calf serum (Whittingham & Wales, 1969) was used for recovery,
storage, microsurgery and transfer of embryos.
Preparation of isolated blastomeres and insertion into zonae
The zonae were removed from 4- and 8-cell eggs by incubation in pronase
(Mintz, 1962). Blastomeres were separated by incubation in a 0-25 % solution of
trypsin for 20min at 37 °C. A single 1/4 or 1/8 blastomere was inserted into
each empty zona prepared by sucking out the contents of a fertilized or unfertilized egg (Rossant, 1975; Tarkowski & Rossant, 1976). Where possible all
blastomeres from each egg were inserted into zonae. A control experiment was
also performed to test whether this procedure damaged the blastomeres in any
way. Blastomeres from single 4-cell eggs were separated into pairs and one such
pair injected into a zona from a fertilized egg and the other into the zona from
an unfertilized egg. For brevity, the zonae themselves will be termed fertilized
and unfertilized from now on.
Transfer to pseudopregnant recipients
Isolated 1/4 and 1/8 blastomeres in zonae were transferred to the oviducts of
recipient females on the first day of pseudopregnancy. Where possible in the
control series, 2/4 blastomeres in fertilized zonae were transferred to one oviduct while 2/4 blastomeres in unfertilized zonae were transferred to the opposite
oviduct in the same mouse.
Examination of preimplantation development
The oviducts of some recipient females were flushed 2 days after transfer and
the flushings examined for zonae containing blastomere derivatives. The oviducts of other recipients were fixed 2 days after transfer, processed and embedded in wax. Serial sections were cut at 5-6 jum and stained with haemalum and
eosin. The structures derived from isolated blastomeres were identified in the
sections and, where possible, cell counts were made by serial reconstruction
from camera lucida drawings or photographs,
Postimplantation development of blastomeres
285
Table 1. Postimplantation development of 2/4 blastomeres in fertilized
and unfertilized zonae
Recipients killed 5\ days p.c, only pregnant females considered
Unfertilized
Type of zona
Fertilized
No. transferred
No. of recipients
No. of decidua
No. of egg cylinders
No. of disorganized egg
cylinders
Total no. of decidua with
embryonic derivatives
35
6
39
14(40%)
8 (23 %)
21(54%)
8 (21 %)
3 (9%)
4(10%)
11 (31%)
12(31%)
7
Table 2. Preimplantation development of isolated blastomeres in zonae after
2 days in oviduct
Combined data from flushings and sections
Type of blastomere
...
No. of recipients
No. recovered or identified
No. transferred
Type of structure recovered
Blastocyst
Disorganized ' trophectoderm'
'False blastocyst'
1/4
1/8
4
4
9/21
8/19
6
3
0
0
6
2
Examination of postimplantation development
Recipient females were killed on the 6th day of pregnancy and their uteri
fixed and cleared (Orsini, 1962), so that even very small decidual swellings could
be detected. All such swellings were embedded in wax and sectioned at 6 ju,m.
The sections were stained with haemalum and eosin and examined for the
presence of embryonic derivatives in the decidua.
RESULTS
Development of 2/4 blastomeres in fertilized and unfertilized zonae
Histological analysis at 5^- days established that 2/4 blastomeres in both fertilized and unfertilized zonae could induce decidual formation and give rise to
normal egg cylinders (Table 1). Some abnormal egg cylinder-like structures
were also found. The percentages of normal and abnormal egg cylinders formed
are very similar for 2/4 blastomeres in both types of zona (Table 1).
The low percentage for embryo formation and the occasional formation of
19
EMB 36
286
J. ROSSANT
Fig. 1. Blastocyst developed from 1/4 blastomere in zona after 2 days in the oviduct.
Table 3. Postimplantation development of isolated blastomeres in zonae
Recipients killed 5% days p. c, only pregnant females considered
Type of blastomere
...
No. transferred
No. of recipients
No. of implants
Contents of decidual chamber
Embryo
Trophoblast giant cells
Lumps of cells
Nothing identified
Isolated 1/4
25
5
13(52%)
1
7
0
5
Isolated 1/8
26
5
9 (35 %)
0
4
2
3
abnormal, badly organized structures is probably due to use of half-size
embryos. Such embryos are known to develop less well than normal embryos,
although development to term has been achieved (Tarkowski, 1959 a, b;
Gardner, 1974). However, production of some morphologically normal egg
cylinders showed that the procedure adopted for the study of isolated blastomeres is compatible with continued development. The results also showed that
the type of zona used had no effect on blastomere development.
Development of isolated 1J4 blastomeres
The proportion of transferred blastomeres identified after 2 days in the oviduct was rather low. Nevertheless, six morphologically normal '1/4' blastocysts
were recovered (Table 2, Fig. 1). Cell counts were made on four of the '1/4'
Postimplantation development of blastomeres
287
Fig. 2. 5^-day conceptus developed from 1/4 blast omere in zona.
19-2
288
J. ROSSANT
^^^tf ^
50 fan
Fig. 3. Section of group of cells developed from 1/8 blastomere
in zona after 2 days in the oviduct.
blastocysts recovered. The total number of cells was 13-14, of which 2-3 were
inner cell mass (ICM) cells. Three groups of disorganized trophectoderm-like
cells were also recovered (Table 2). The number of cells in these was lower than
in the blastocysts (ca. 4-10 cells by visual estimation).
Isolated 1/4 blastomeres in zonae were capable of initiating a decidual reaction in recipient females (Table 3). However, in only 1 out of 13 decidua was a
recognizable embryo detected (Fig. 2). This embryo was apparently retarded by
approximately 12-15 h since it lacked extra-embryonic ectoderm. It was smaller
than normal embryos of similar recipient age or embryonic stage. However, it
was not abnormal and contained proliferating polar trophoblast, mural giant
cells, proximal endoderm, as well as embyonic ectoderm. Little distal endoderm was present. It is unlikely that it would have survived to term.
Of the remaining 12 decidua, five had no detectable embryonic cells. In the
other seven implants, no ICM derivatives were found, but groups of trophoblast giant cells were detected. The number of giant cells varied widely (range
5-16, average 11) but did not exceed the maximum number of cells that could
be derived by four cell divisions from one isolated 1/4 blastomere (24 cells).
Development of isolated 1/8 blastomeres
Developing isolated 1/8 blastomeres were identified 2 days after transfer
(Table 2). No true blastocysts containing enclosed cells were found. Six out of
eight recovered structures consisted of disorganized trophectoderm-like cells
Postimplantation development of blastomeres
289
(Fig. 3) while the other two were 'false blastocysts' (Tarkowski & Wroblewska,
1967), in which no cells were actually enclosed.
Isolated 1/8 blastomeres were capable of inducing decidua, despite their small
size (Table 3). No organized embryo was found in any of the decidua, but four
contained trophoblast giant cells. The number of giant cells found was lower
than for 1 /4 blastomeres (range 2-9, average 5) and did not exceed (except by
one cell in one instance) the maximum number of cells that could be derived by
three cell divisions from an isolated 1/8 blastomere (23 cells). Two further decidua
contained small lumps of cells whose nature could not be accurately determined
due to poor fixation, but visual observation suggested that they were of embryonic rather than maternal derivation. The remaining three decidua were empty.
Unfortunately, the low implantation rates of both 1/4 and 1/8 blastomeres
made it impractical to attempt to assess the developmental potential of all
blastomeres from one egg.
DISCUSSION
Tn the present study, isolated 1/4 mouse blastomeres usually formed small
blastocysts in vivo as reported by previous workers (Tarkowski, 1959a, b). However, when the blastomeres were allowed to proceed to implantation, only one
egg cylinder was found in 13 implants analysed at 5^ days p.c. This embryo was
small and retarded (Fig. 2). Most other implants contained trophoblast giant
cells only. Isolated 1/8 blastomeres never formed true blastocysts in the present
experiments (c.f. Tarkowski & Wroblewska, 1967) and, although capable of
implanting and forming trophoblast giant cells, never produced egg-cylinder
structures. Thus, isolated 1/4 and 1/8 blastomeres seem unable to form complete
foetuses (c.f. rabbit blastomeres, Moore, Adams & Rowson, 1968). However,
Kelly (1975) has shown that 1/4 and 1/8 blastomeres can contribute to both
ICM and trophectoderm derivatives in postimplantation conceptuses, when
combined with enough 'carrier' blastomeres to restore the normal cell number.
Thus, the apparent lack of totipotency in the present experiments is probably
not due to restriction in developmental potential of the cells. Since blastocyst
formation begins at the same time in isolated blastomeres as in intact eggs
(Tarkowski & Wroblewska, 1967), insufficient cells may be present at the time of
cavitation of 1/4 and 1/8 blastomeres to produce a functional ICM. Isolated 1/4
blastomeres normally form blastocysts containing 2-3 ICM cells but only one
egg cylinder was produced from such a structure. This suggests that a certain
minimum number of ICM cells (probably greater than 3) is required for postimplantation embryonic development (Snow, 1976). On the other hand, the
formation of functional trophoblast cells does not seem to be greatly affected
by cell number during cleavage since structures containing 1/8 of the normal preimplantation cell number can implant and transform into trophoblast giant cells.
290
J. ROSSANT
I should like to thank Dr R. L. Gardner for valuable discussion and Mrs L. Ofer for
technical assistance. The author was supported by a Medical Research Council Research
Studentship and a Beit Memorial Junior Research Fellowship.
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(Received 25 February 1976)