J. Embryol. exp. Morpli. Vol. 52, pp. 209-225, 1979
Printed in Great Britain © Company of Biologists Limited 1979
209
Cytoplasmic and nuclear protein synthesis in
preimplantation mouse embryos
By C. C. HOWE 1 AND D. SOLTER 1
From the Wistar Institute of Anatomy and Biology, Philadelphia
SUMMARY
Cytoplasmic and nuclear proteins synthesized by mouse embryos at different stages of
preimplantation development were analyzed by two-dimensional polyacrylamide gel electrophoresis. Several nuclear-specific proteins (i.e. proteins more abundant in the nucleus than
in the cytoplasm) and numerous cytoplasmic-specific proteins were observed. The trends of
changes in the nuclear and cytoplasmic protein synthesis are similar. Moderate changes
occur between the unfertilized egg and the zygote. Striking changes characterized by the
disappearance of numerous major oocyte-specific proteins and the appearance of a large
number of new, stage-specific proteins occur between the zygote and the 4- to 8-cell stages. In
contrast, between the 4- to 8-cell and early blastocyst periods, only a few new proteins appear,
and a small number of oocyte-specific or other stage-specific proteins disappear. Minor
differences in protein synthesis were observed between the trophoblast and inner cell mass.
INTRODUCTION
During the early development of the mouse embryo, a series of specific
changes accompany the transition of a single-cell zygote to a multicellular
blastocyst. Optical and scanning electron microscopic studies (Mulnard, 1967;
Ducibella, 1977) demonstrated that in the developing mouse embryo the blastomeres undergo a dramatic change in shape, called compaction, during the 8-cell
stage. Subsequently, during early blastulation, the first grossly observable
segregation of the embryonic cells into two phenotypically distinct populations
trophectoderm and inner cell mass (ICM), occurs.
Changes in metabolic pathways, enzymic activities and macromolecular
synthesis during this period have been described (Epstein, 1975; Manes, 1975;
McLaren, 1976; Johnson, Handyside & Braude, 1977). In particular, changes in
the pattern of protein synthesis have been the subject of several investigations.
One- and more recently two-dimensional polyacrylamide gel electrophoresis
has shown that major changes in protein synthesis occur between the 2-cell
and the 4- to 8-cell stages of development (Epstein & Smith, 1974; Van
Blerkom & Brockway, 1975; Levinson, Goodfellow, Vadeboncoeur & McDevitt,
1
Authors' address: The Wistar Institute of Anatomy & Biology, 36th Street at Spruce,
Philadelphia, Pennsylvania 19104, U.S.A.
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210
C. C. HOWE AND D. SOLTER
1978; Martin, Smith & Epstein, 1978). In addition, two-dimensional electrophoresis has revealed that the ICM and the trophectoderm synthesize qualitatively distinct sets of proteins (Van Blerkom, Barton & Johnson, 1976;
Handyside & Barton, 1977; Dewey, Filler & Mintz, 1978) and that the
inside cells of the morula synthesize ICM-speciflc proteins but not trophectoderm-specific proteins (Handyside & Johnson, 1978).
The present paper reports additional details of the changes in protein synthesis
during preimplantation development of mouse embryos from parallel studies of
the cytoplasmic as well as nuclear protein fractions.
MATERIALS AND METHODS
Preparation of embryos
Unfertilized eggs were isolated from oviducts of superovulated ICR female
mice 14 h after injection of human chorionic gonadotropin (Pregnyl, Organon),
and cumulus oophorous cells were removed with hyaluronidase (Solter &
Schachner, 1976). Zygotes, 2-cell and 4- to 8-cell stage embryos were flushed
from the oviducts and blastocysts from the uteri of ICR female mice and collected
as described (Solter & Schachner, 1976). Zonae pellucidae were removed by
pronase (Mintz, 1962). ICM were isolated by immunosurgery (Solter & Knowles,
1975).
Radioactive isotope labeling ofproteins andpreparation of nuclear and cytoplasmic
proteins
Embryos were labeled at 37 °C for 5 h in Whitten's medium (Whitten, 1971)
containing 0-4% of dialyzed bovine serum albumin and 500/^Ci/ml of [35S-]
methionine (New England Nuclear, specific activity > 400 Ci/m-mole) in moist
air containing 5 % CO2. After the radiolabeling, embryos were washed extensively in protein-free Whitten's medium. The embryos were then separated with
minimal manipulation into nuclear and cytoplasmic fractions by the modified
procedures of Berkowitz, Kakefuda & Sporn (1969). The washed embryos were
transferred to 50/d of 0-01 M-NaCl, 0-01 M Tris HC1, pH 7-4, 1 % NP-40
(Shell Chemical), and 2 mM phenylmethylsulfonylfluoride (PMSF) as a protease
inhibitor. The suspension was kept on ice for 1 h with occasional vortexing, and
then underlayed with 30 [A of 0-32 M sucrose, 3 mM-MgCl2, and 0 01 M TrisHC1, pH 7-5 and centrifuged at 2000 rev./min for 20 min in an International
centrifuge. As a preliminary experiment, we used this same fractionation procedure for pluripotent teratocarcinoma stem cells. The resulting nuclei were
virtually free of unbroken cells and cytoplasmic components as examined by
phase-contrast or electron microscopy. The supernatant, which contained the
cytoplasmic proteins, was lyophilized. The lyophilized cytoplasmic proteins
and the nuclear proteins (the pellet) were prepared for two-dimensional gel
electrophoresis according to the procedures described by Peterson & McConkey
Protein synthesis in mouse embryos
211
(1976a). The samples were then centrifuged to remove any insoluble proteins,
stored at — 70 °C and used for electrophoresis as soon as possible to avoid
possible degradation of the proteins.
Two-dimensional gel electrophoresis of proteins
Separation of proteins with two-dimensional polyacrylamide gel electrophoresis was carried out according to the method of O'Farrell (1975). The
ampholine compositions used in the first-dimension isoelectrofocusing were
1-6% of pH 5-8, 0-1 % of pH 2-5-4 and 0-1 % of pH 4-6. This ampholine
composition gave a pH range of 4-5-6-7. This narrow range was chosen to
achieve clear resolution of proteins. The pH gradient was determined as described
by OTarrell (1975). The second-dimension electrophoresis was carried out in
an SDS gel with a 9-14-4% exponential acrylamide gradient. The resulting gels
were dried and exposed to Kodak NS2T No-screen X-ray film (Eastman Kodak
Co.) for autoradiography. Exposure times ranged between 1 and 4 months
depending upon the amounts of TCA-precipitable counts per minute applied
to gels. Details are given in the legends to the figures. For each particular
preimplantation stage, two or more batches of embryos were independently
prepared, labeled with [35S]-methionine and electrophoresed. All conclusions
were based on these repeated experiments.
RESULTS
Cytoplasmic proteins of preimplantation embryos
The cytoplasmic proteins synthesized by embryos at the various preimplantation stages were analyzed by high resolution two-dimensional gel electrophoresis, and the results are shown in Fig. 1. Several hundred spots were
detectable on these autoradiographs. The polypeptides synthesized in the zygote
were nearly identical to those synthesized in the unfertilized egg (Fig. \A, B).
Significant changes in the pattern of cytoplasmic protein synthesis can be
seen in the course of the zygote's development from early cleavage to early
blastulation (Fig. \B-E). In order to illustrate these changes more clearly,
the polypeptides synthesized at the various stages of preimplantation development were grouped according to the stage at which they were first observed and
only proteins appearing as distinct spots in the autoradiographs in Fig. 1 were
used in the illustration. Fig. 2(a) shows the proteins that were first observed in
the unfertilized egg while Fig. 2(b-d) show those that continued to be synthesized
at the 2-cell stage, the 4- to 8-cell stage and the blastocyst or ICM stage,
respectively. Progressively fewer of the proteins observed in an unfertilized egg
or zygote were present in the 2-cell embryo, the 4- to 8-cell embryo and the
blastocyst or ICM, which clearly shows the disappearance of oocyte stagespecific proteins as the embryos developed from zygotes to blastocysts. In
particular, a large number of oocyte-specific proteins disappeared between the
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212
C. C. HOWE AND D. SOLTER
zygote and the 4- to 8-cell stage, and relatively few disappeared during the
blastocyst formation.
Many proteins appeared for the first time in 2-cell embryos (Fig. 2e). The
fate of these 2-cell-stage-specific proteins at later stages is shown in Fig. 2(/ 3 g).
Again, a greater number of 2-cell-stage-specific proteins disappeared at the 4- to
8-cell stage than during blastulation. Fig. 2(h, i), respectively, show the appearance of proteins specific to the 4- to 8-cell embryos and the subsequent disappearance of some of them at the blastocyst or ICM stage. Figure 2(j) shows
the new proteins appearing for the first time in the blastocyst or ICM. Significantly, a large number of stage-specific proteins (i.e. proteins appearing for
the first time at a certain stage) appeared between the zygote and the 4- and
8-cell stages, and only a small number appeared during blastulation. Actin,
the major cytoplasmic protein for embryos between the 4- to 8-cell and the
blastocyst stages, was barely detectable in the unfertilized egg and the zygote
and began to appear at the 2-cell stage. On the other hand, tubulin did not
appear to be stage specific but was abundant in all stages.
The blastocyst, consisting of trophectodermal cells and ICM, showed a
profile of protein synthesis similar to that of the ICM with the exception of a
few polypeptides. These polypeptides appeared as spots of greater intensity in
the blastocyst (indicated by arrows in Fig. IE) than in the ICM (indicated by
arrows in Fig. IF) and are, therefore, presumably trophectoderm specific.
Nuclear proteins of preimplantation embryos
Nuclear proteins from various stages of preimplantation embryos are shown
in the autoradiograms in Fig. 3. More than one hundred spots were detectable
FIGURE 1
Autoradiographs of two-dimensional polyacrylamide gels of cytoplasmic proteins
from (A) unfertilized eggs, (B) zygotes, (C) 2-cell embryos, (D) 4- to 8-cell embryos,
(E) blastocysts and (F) ICM. The number of embryos used in labeling, amount of
TCA-precipitable radioactivity applied to the gels and duration of X-ray film
exposure are as follows: (A) 420, 52000 cpm, 4 months; (B) 340, 140000 cpm, 3
months; (C) 300, 140000 cpm, 3 months; (D) 250, 630000, cpm, 1 month; (E) 115,
680000 cpm, 1 month; and (F) 170, 380000 cpm, 1 month. The basic end of the
isoelectrofocused gel is on the left and the direction of SDS electrophoresis is from
top to bottom. The pH gradient and the molecular weight scale were determined
as described in Materials and Methods. The molecular weight scale was based on the
positions of the following marker proteins: myosin heavy chain (200000 daltons),
phosphorylase A (94000 daltons), bovine serum albumin (68000 daltons), actin
(45000 daltons) and carbonic anhydrase (29000 daltons). In this figure, actin (three
forms) is indicated by the letter 'A' (and by arrows in Fig. 1 C) and tubulins (55000
and 53 000 daltons) by the letter'T' (and by arrows in Fig. 1 A). The actin and tubulin
were identified by electrophoresis of purified actin and tubulin from mouse cells.
The arrows in Fig. 1 (E) point to trophectoderm-specific proteins in the blastocyst
and those in Fig. 1 (F) point to faint traces of trophectoderm-specific proteins in the
ICM.
213
Protein synthesis in mouse embryos
4-5
pH 6-7
200-1
94-
68.
45-
29-
Unfertilized egg
94 -
68
45-
29-
Fig. 1
214
C. C. HOWE AND D. SOLTER
PH6-7
4-5
200
94 H
68
45 ^
29 4
2 cell
?. 200
94
68-1
45
29
4-8 cell
Fig. 1 (cont.)
215
Protein synthesis in mouse embryos
4-5
pH6-7
200 -,
94 -
68 -
45 -
29
Blastocyst
I
200 -«
94 -
68 -
45 -
29 -
ICM
Fig. 1 {cont.)
216
C. C. HOWE AND D. SOLTER
(a)
(e)
-A
(c)
A
(f)
A
(d)
6u
:C . _
T
A
111
IV
Fig. 2. Diagrams showing the changes in the pattern of cytoplasmic proteins synthesized from the unfertilized egg stage up to early blastulation. Only proteins clearly
identifiable as stage specific in the autoradiographs in Fig. 1 are shown. The heavy
bordered boxes show the proteins that were observed for the first time at the stages
indicated in the extreme left column. The proteins are grouped according to the stage
at which they were first observed. Column 1 presents proteins first observed at the
unfertilized egg or zygote stage (a) and their subsequent fate at: the 2-cell stage (b),
the 4- to 8-cell stage (c), and the blastocyst or ICM stage (d). Column 2 presents proteinsfirstobserved at the 2-cell stage (e) and their fate at: the 4- to 8-cell stage (/) and
the blastocyst or ICM stage (g). Column 3 presents proteins first observed at the 4to 8-cell stage (h) and their fate at the blastocyst or ICM stage (/). Column 4 presents
proteins that appeared for thefirsttime in the blastocyst or ICM stage. In this figure,
the position of actin corresponds to the top of the letter 'A' and that of tubulin to
the left of the letter 'T'.
217
Protein synthesis in mouse embryos
4-5
pH6-7
200 T
94 -
68 -
Unfertilized egg
200 -i
94 -
68-
45-
29-
Zygote
Fig. 3
218
C. C. HOWE AND D. SOLTER
pH6-7
4-5
200 n
94-
68-
45-
29-
2 cell
200 n
94-
68-
45-
29'
4 - 8 cell
Fig. 3 (cont.)
Protein synthesis in mouse embryos
219
4-5
pH6-7
200 -i
94 -
68 -
\
45-
29
o
X
Blastocyst
g>
1
I
200 n
94 -
68-
45-
49-
F
ICM
Fig. 3 (cont.)
220
C. C. HOWE AND D. SOLTER
on these autoradiograms. A comparison of the patterns of nuclear and cytoplasmic proteins from a given embryonic stage showed that while many proteins
were common to both the nuclear and cytopJasmic fractions, a significant
number of proteins (designated as nuclear-specific proteins and indicated by
arrows in Fig. 3) were clearly more abundant in nuclear fractions than in cytoplasmic fractions. Many proteins specific to cytoplasmic fractions were also
detected.
In general, the trend of changes in the pattern of protein synthesis in the
nucleus during preimplantation development was comparable to that in the
cytoplasm: little change in the pattern of nuclear protein synthesis was observed
between the unfertilized egg and the zygote stages, but changes did occur
during development from the zygote stage to blastocyst stage. Figure 4 illustrates
in detail the appearance and disappearance of stage-specific nuclear proteins
according to the stages of embryonic development. Many oocyte-specific nuclear
proteins disappeared between the zygote and the 4- to 8-cell stage (Fig. 4a-c).
A large number of stage-specific nuclear proteins (Fig. 4e, /?), including many
nuclear-specific ones (indicated by arrows in Fig. 4/?), appeared between the
zygote and the 4- to 8-cell stages and some of these disappeared during blastulation (Fig. 4/, g and /). Only a few polypeptides, some of them nuclear
specific, appeared during blastulation (Fig. 47). As in the case of cytoplasmic
proteins, nuclear proteins from the intact blastocyst were nearly identical to
those from the ICM.
DISCUSSION
In this study, we have used two-dimensional gel electrophoresis to follow
the changes in stage-specific cytoplasmic and nuclear proteins in preimplantation mouse embryos. The trends of the changes in the nuclear protein synthesis
parallel those in the cytoplasmic protein synthesis: major changes in the pattern
of cytoplasmic and nuclear protein synthesis were observed between the zygote
and 4- to 8-cell stages and limited changes between 4- to 8-cell and blastocyst
stages. These results are consistent with previous results from one-dimensional
electrophoretic analysis of mouse (Epstein & Smith, 1974; Van Blerkom &
Brockway, 1975; Martin, et al. 1978) and rabbit embryos (Van Blerkom & Manes,
FIGURE 3
Autoradiographs of two-dimensional polyacrylamide gels of nuclear proteins from
(A) unfertilized eggs, (-B)zygotes, (C) 2-cell embryos, (D)A- to 8-cell embryos, (E) blastocysts, and (F) ICM. The number of embryos used in labeling, amount of TCAprecipitable radio-activity applied to the gels and duration of X-ray film exposure
are as follows: (A) 4.20, 53000 cpm, 4months; (5)340,170000 cpm, 3 months; (C)
300, 110000 cpm, 3 months; (D) 250, 560000 cpm, 1 month; (E) 115, 590000 cpm,
1 month; and (F) 170,440000 cpm, 1 month. The arrows indicate the nuclear-specific
proteins. Other details are as described in the legend to Fig. 1.
221
Protein synthesis in mouse embryos
(a)
T5 «
-T
A
-*r""r
(e)
-T
."
= -
A
A
T
(c)
J}
* *
T
A
A
V
W)
0)
(g)
•
.2 o
pa
A
•"•
-
A
T
*t
T.., T
A . ;
1
^ \ »- ' T
A
t 111
IV
Fig. 4. Diagrams showing the changes in the pattern of nuclear proteins synthesized
from the unfertilized egg stage up to early blastulation. Stage-specific proteins clearly
identifiable in Fig. 3 are shown. Details of the diagram are as described in the legend
to Fig. 2. Nuclear-specific proteins are indicated by arrows.
1974) and also with a more recent two-dimensional study of mouse (Levinson
et ah 1978) and rabbit embryos (Van Blerkom & McGaughey, 1978). However,
unlike earlier studies in which proteins from whole embryos were analyzed,
embryos in the present study were separated into cytoplasmic and nuclear
fractions so that changes in their patterns of protein synthesis could be separately
analyzed and compared.
The gels of cytoplasmic fractions are similar to those of Levinson et al. (1978)
detailing the changes in protein synthesis throughout all stages of preimplantation mouse embryo development. We believe our results complement those of
Levinson et al. since different molecular weight ranges were used in the two
studies. Our gels have better resolution of higher molecular weight proteins
while those of Levinson et al. provide more detailed resolution in the lower
molecular weight range. Since only a few low molecular weight proteins were
222
C. C. HOWE AND D. SOLTER
present, we chose to run the gels longer to resolve the abundant high molecular
weight proteins.
In a comparison of ICM and blastocyst, a few trophectoderm-specific polypeptides, similar to those reported by Van Blerkom et al. (1976), Handyside &
Barton (1977), and Dewey et al. (1978), were detectable in the cytoplasmic
fraction of blastocyst (arrows, Fig. IE). However, differences in the pH and
molecular weight ranges employed preclude a detailed comparison of our work
with those cited above.
Our observation that nuclear and cytoplasmic fractions share many of the
same proteins is in agreement with reported results (Peterson & McConkey,
1976<7, b; Howe, Gmiir & Solter, 1979). However, in view of these results, it is
important to establish that the observed nuclear proteins truly represent in
vivo nuclear proteins and not simply contaminating cytoplasmic proteins. In
this study, NP-40 was used in the fractionation to minimize contamination of
the nuclear fraction by cytoplasmic proteins (Berkowitz et al. 1969). Phasecontrast and electron microscopic examination of the nuclear preparations
from pluripotent teratocarcinoma cells by the same fractionation procedures,
confirmed that contamination was minimal. Treatment of cells with NP-40
in the absence of sucrose is known to cause certain freely diffusable nuclear
proteins to leak from the nucleus (Kellermayer et al. 1976; Loeb, Ritz, Creuzet
& Jami, 1976). Thus, with our fractionation procedures, the cytoplasmic fraction
consists of cytoplasmic and nucleoplasmic proteins, whereas the nuclear fraction
consists of nuclear proteins that are not freely diffusable or are tightly bound
to the chromosomes as well as NP-40-insoluble extranuclear proteins. Our
observation that many proteins are more abundant in the nuclear fractions and
others in the cytoplasmic fractions along with the microscopic evidence make
it clear that cytoplasmic contamination of nuclei was not significant.
Some spots showing charge heterogeneity were observed in all of the gels
examined. Since precautions were taken to avoid degradation of proteins, these
proteins probably represent in vivo modified glycoproteins, phosphorylated
proteins, acetylated proteins (O'Farrell, 1975) or protein-ampholyte complexes
(Hare, Stimpson & Cann, 1978). In addition, when the gels of cytoplasmic
fractions were compared to those of Levinson et al. (1978), numerous identical
clusters of spots can be identified indicating that the charge heterogeneity is
not caused by sample preparation.
The fate of nuclear-specific proteins in the developing embryo, in particular
the stage-specific, nuclear-specific proteins which appear in 4- to 8-cell embryos
and subsequently disappear, is most interesting in view of available evidence
suggesting a regulatory function for non-histone chromosomal proteins (Gilmour
& Paul, 1971). Whether these stage-specific, nuclear-specific, proteins indeed have
any regulatory functions remains to be elucidated.
Our observation that the pattern of cytoplasmic or nuclear protein synthesis
did not change significantly immediately after fertilization of the oocyte (Fig.
Protein synthesis in mouse embryos
223
\A, B) is consistent with the absence of new mRNA in the zygote (Woodland &
Graham, 1969; Graham, 1973) and with the interpretation that stored mRNA
in the unfertilized egg is utilized for the development of the fertilized egg up to
the first cleavage (Young, 1977). On the other hand, it is possible that some of
the new stage-specific proteins appearing before 4- to 8-cell stage (in both
cytoplasmic and nuclear fractions) are synthesized from newly transcribed
mRNA since transcription of polyadenylated RNA occurs as early as the 2-cell
stage (Levey, Stull & Brinster, 1978). Actin is present in large quantities in both
fractions at the 4- to 8-cell stage but actually begins to appear at the 2-cell stage.
Thus, its appearance occurs somewhat earlier than previously reported (Van
Blerkom & Brockway, 1975). Another structural protein, tubulin, previously
thought to appear at the 4- to 8-cell stage (Van Blerkom & Brockway, 1975),
was actually observed by us in both fractions of the unfertilized egg and in
somewhat larger amounts at the 4- to 8-cell stage. Thus, with respect to both
actin and tubulin, our results are not inconsistent with those of earlier workers
but the use of two-dimensional electrophoresis has allowed us to detect more
accurately when these proteins first appear and to follow their fate through the
various developmental stages.
Compaction of embryos occurs at the 8-cell stage. At the same time the
formation of intercellular junctions, such as gap junctions and tight junctions,
becomes visible via electron microscopy (Ducibella & Anderson, 1975). The
dramatic increase in the synthesis of actin, one of the structural proteins in
these intercellular junctions, at the 4- to 8-cell stage suggests that this protein
is associated with the structural changes that occur during the early cleavage
period. Tubulin, another structural protein of the intercellular junctions, also
increased in amount at the 4- to 8-cell stage. Presumably, many other proteins
appearing during this stage are likewise correlated to the structural changes
during this period. Early embryos use pyruvate as an energy source (Biggers,
Whittingham & Donahue, 1967) but cannot utilize glucose for this purpose
until about the 4- to 8-cell stage (Brinster, 1965). These facts may account for
the disappearance and appearance of some proteins between the zygote and the
4- to 8-cell stages.
In conclusion, our observation of changes in the pattern of protein synthesis,
characterized by the appearance and disappearance of stage-specific proteins,
shows that regulation of protein synthesis and presumably differential gene
expression is very much in evidence during the early development of the mouse
embryo. With a knowledge of the basic pattern of polypeptides synthesized at
each developmental stage, we should be able to identify and determine the
function of some stage-specific proteins using various developmental mutants
(McLaren, 1976). Examination and comparison of protein patterns of individual
embryos produced by heterozygous mating and isolated before gross abnormalities are visible would indicate which polypeptides are missing or altered in an
embryo homozygous for a lethal recessive gene. In this way we hope to identify
224
C. C. HOWE AND D. SOLTER
the products of developmental genes and start to elucidate the genetic control
of development.
This work was supported by USPHS research grants CA 10815, CA 17546 and CA 21069
from the National Cancer Institute and HD 12487 from the National Institute of Child
Health and Human Development, and by PCM 78-16177 from the National Science Foundation. We thank David Lugg for his excellent assistance in some of these experiments.
REFERENCES
D. M., KAKEFUDA, T. & SPORN, M. B. (1969). Simple and rapid method for the
isolation of enzymatically active HeLa cell nuclei. /. Cell Biol. 42, 851-855.
BIGGERS, J. D., WHITTINGHAM, D. G. & DONAHUE, R. P. (1967). The pattern of energy
metabolism in the mouse oocyte and zygote. Proc. natn Acad. Sci. U.S.A. 58, 560-567.
BRINSTER, R. L. (1965). Studies on the development of mouse embryos in vitro: IV. Interaction of energy sources. /. Reprod. Fertil. 10, 227-240.
DEWEY, M. J., FILLER, R. & MINTZ, B. (1978). Protein patterns of developmentally totipotent mouse teratocarcinoma cells and normal early embryo cells. Devi Biol. 65, 171-182.
DUCIBELLA, T. (1977). Surface changes of the developing trophoblast cell. In Development
in Mammals (ed. M. H. Johnson), vol. 1, pp. 5-30. Amsterdam: Elsevier North-Holland
Biomedical Press.
DUCIBELLA, T. & ANDERSON, E. (1975). Cell shape and membrane changes in the 8-cell mouse
embryo: pre-requisites for morphogenesis of the blastocyst. Devi Biol 41, 45-58.
EPSTEIN, C. J. (1975). Gene expression and macromolecular synthesis during preimplantation
embryonic development. Biol. Reprod. 12, 82-105.
EPSTEIN, C. J. & SMITH, S. A. (1974). Electrophoretic analysis of proteins synthesized by
preimplantation mouse embryos. Devi Biol. 40, 233-244.
GILMOUR, R. S. & PAUL, J. (1971). Tissue specific transcription of the globin gene in isolated
chromatin. Proc. natn. Acad. Sci. U.S.A. 70, 3440-3442.
GRAHAM, C. F. (1973). Nucleic acid metabolism during early mammalian development.
In The Regulation of Mammalian Reproduction (ed. S. J. Segal, R. Crozier, P. A. Corfman
& P. G. Condliffe), pp. 286-289. Springfield, 111.: Thomas.
HANDYSIDE, A. H. & BARTON, S. C. (1977). Evaluation of the technique of immunosurgery
for the isolation of inner cell masses from mouse blastocysts. J. Embryol. exp. Morph. 37,
217-226.
HANDYSIDE, A. H. & JOHNSON, M. H. (1978). Temporal and spatial patterns of the synthesis
of tissue-specific polypeptides in the preimplantation mouse embryo. /. Embryol. exp.
Morph. 44, 191-199.
HARE, D. L., STIMPSON, D. 1. & CANN, J. R. (1978). Multiple bands produced by interaction
of a single macromolecule with carrier ampholytes during isoelectric focusing. Archs
Biochem. Biophys. 187, 274-275.
HOWE, C, GMUR, R, & SOLTER, D. (1979). Cytoplasmic and nuclear protein synthesis during
in vitro differentiation of murine ICM and embryonal carcinoma cells. Devi Biol. (in
press).
JOHNSON, M. H., HANDYSIDE, A. H. & BRAUDE, P. R. (1977). Control mechanism in early
mammalian development. In Development in Mammals (ed. M. H. Johnson), vol. 2,
pp. 67-97. Amsterdam: Elsevier North-Holland Biomedical Press.
KELLERMAYER, M., TALAS, M., WONG, C, BUSCH, H. & BUTEL, J. S. (1976). The solubility
characteristics of SV-40 tumor antigen. Expl Cell Res. 99, 456-460.
LEVEY, I. L., STULL, G. B. & BRINSTER, R. L. (1978). Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo. Devi Biol. 64, 140-148.
LEVINSON, J., GOODFELLOW, P., VADEBONCOEUR, M. & MCDEVITT, H. (1978). Identification
of stage-specific polypeptides synthesized during murine preimplantation development.
Proc. natn. Acad. Sci. U.S.A. 75, 3332-3336.
BERKOWITZ,
Protein synthesis in mouse embryos
225
J. E., RITZ, E., CREUZET, C. & JAMI, J. (1976). Comparison of chromosomal proteins
of mouse primitive teratocarcinoma, liver and L cells. Expl Cell Res. 103, 450-454.
MANES, C. (1975). Genetic and biochemical activities in preimplantation embryos. In
The Developmental Biology of Reproduction (ed. C. L. Markert & J. Papaconstantinou),
33rd Symposium, pp. 133-163. New York: Academic Press.
MARTIN, G. R., SMITH, S. & EPSTEIN, C. j . (1978). Protein synthetic patterns in teratocarcinoma stem cells and mouse embryos at early stages of development. Devi Biol. 66,
LOEB,
8-16.
A. (1976). Genetics of the early mouse embryo. Ann. Rev. Genet. 10, 361-388.
(1962). Experimental study of the developing mammalian egg: removal of the
zona pellucida. Science, N.Y. 138, 594-595.
MULNARD, J. G. (1967). Analyse microcinematographique du developpment de 1'oeuf de
souris du stade 11 au blastocyst. Archs Biol. 78, 107-138.
O'FARRELL, P. H. (1975). High-resolution, two-dimensional electrophoresis of proteins. /.
biol. Chem. 250, 4007-4021.
PETERSON, J. L. & MCCONKEY, E. H. (1976a). Non-histone chromosomal proteins from
HeLa cells: A survey by high resolution, two dimensional electrophoresis. /. biol. Chem.
251, 548-554.
PETERSON, J. L. & MCCONKEY, E. H. (19766). Proteins of Friend leukemia cells: Comparison
of hemoglobin-synthesizing and noninduced populations. /. biol. Chem. 251, 555-558.
SOLTER, D. & KNOWLES, B. B. (1975). Immunosurgery of mouse blastocyst. Proc. natn. Acad.
Sci. U.S.A. 72, 5099-5102.
SOLTER, D. & SCHACHNER, M. (1976). Brain and sperm cell surface antigen (NS-4) on
preimplantation mouse embryos. Devi Biol. 52, 98-104.
VAN BLERKOM, J. & BROCKWAY, G. O. (1975). Qualitative patterns of protein synthesis in
the preimplantation mouse embryo. 1. Normal pregnancy. Devi Biol. 44, 148-157.
VAN BLERKOM, J. & MANES, C. (1974). Development of preimplantation rabbit embryos in
vivo and in vitro. II. A comparison of qualitative aspects of protein synthesis. Devi Biol. 40,
40-51.
VAN BLERKOM, J. & MCGAUGHEY, R. W. (1978). Molecular differentiation of the rabbit
ovum. Devi Biol. 63, 151-156.
VAN BLERKOM, J., BARTON, S. C. & JOHNSON, M. H. (1976). Molecular differentiation in the
preimplanation mouse embryo. Nature, Lond. 259, 319-321.
WHITTEN, W. K. (1971). Nutrient requirements for the culture of preimplantation embryos
in vitro. Acta Biosci. 6, 129-141.
WOODLAND, H. R. & GRAHAM, C. F. (1969). RNA synthesis during early development of
the mouse. Nature, Lond. Ill, 327-332.
YOUNG, R. J. (1977). Appearance of 7-methylguanosine-5'-phosphate in the RNA of mouse
1-cell embryos three hours after fertilization. Biochem. Biophys. Res. Commun. 76, 32-39.
MCLAREN,
MINTZ, B.
{Received 25 January 1979, revised 12 March 1979)