317
Development 105, 317-322 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Involvement of oocyte-coded message in cell differentiation control of early
human embryos
JAN TESARIK*
Centre for Reproductive Medicine, Purkyne University Medical Faculty, ndm. Sov. hrdinii 11, CS-65677 Brno,
Czechoslovakia
* Address for reprint requests: Unite INSERM 187, Service de Gynecologie-Obstetrique, Hopital Antoine-Beclere, 157, Rue de la Porte-deTrivaux, 92141 Clamart, France
Summary
Considerable evidence indicates that the first phenotypical diversification of embryonic cells during mammalian
preimplantation development is achieved in two successive steps: (i) generation of cell asymmetry and (ii)
unequal cell division. This paper shows that ultrastructural signs of blastomere surface regionalization in
human preimplantation embryos are evident as early as
the 2-cell stage when modifications of the plasma membrane (loss of microvilli and endocytotic activity, formation of cell junctions) are induced in places of blastomere
contact. The capacity of the plasma membrane to
undergo these cell-contact-dependent changes precedes
any detectable activity of the embryonic genome. The
area of the modified plasma membrane shows a continuous increase during the first three cleavage stages. The
progression of these membrane modifications is the same
in embryos that have properly enhanced their transcriptional activity at the 8-cell stage and in those that have
not. In spite of the failure of this early-cleavage-
progressed-cleavage transition of gene activity, the formation of zonula adherens and gap junctions goes on
apparently normally in the respective embryos and
morphologically distinct inner cell mass and trophectoderm cell lineages are subsequently segregated in 16-cell
morulae. However, tight junctions do not develop under
these conditions. The occurrence of the progressedcleavage pattern of gene activity in the majority of
embryonic cells is a necessary prerequisite for the
appearance of the blastocyst cavity. Thus, oocyte-coded
message is apparently involved in the control of relatively late stages of human preimplantation development
including the differentiation of the first two embryonic
tissues, but the embryonic genome is required for the full
achievement of this early differentiative event.
Introduction
cytoplasmic membranous organelles (Maro et al. 1985;
Batten et al. 1987) appear for the first time at the 8-cell
stage of the preimplantation development. These morphological changes are preceded by regionalization of
the plasma membrane at the molecular level, involving
polarized distribution of membrane-associated enzymes
(Izquierdo et al. 1980; Izquierdo & Ebensperger, 1982),
membrane lipids (Pratt, 1985) and antibody- and lectinbinding sites (Handyside et al. 1987), which can be
traced back up to the 2-cell stage.
As it is known that the early animal embryonic
development is controlled by proteins/enzymes coded
for by two different genomic sources: the products of
oocyte genome transmitted to the embryo via the
oocyte cytoplasm and the products of the embryonic
genome proper (reviewed in Tesafik, 1988), a question
arises about the genetic origin of factors that are
responsible for these very early differentiative events.
This question can hardly be answered by studies carried
Fertilization of the mammalian oocyte triggers a series
of mitotic divisions which, at the beginning, give rise to
phenotypically identical progeny, followed by a period
during which cell diversity is created by means of
unequal blastomere divisions (Davidson, 1986; Johnson, 1986; Johnson et al. 1986). This mechanism of cell
diversification obviously requires the previous development of a clear asymmetry of cell organization. In the
mouse embryo, which represents the most studied
subject in this regard, structural asymmetries involving
the distribution of microvilli (Ducibella et al. 1977;
Handyside, 1980; Reeve & Ziomek, 1981) and cell
junctions (Ducibella & Anderson, 1975; Magnuson et
al. 1977; McLachlin et al. 1983; Goodall & Johnson,
1984), polarization of the endocytotic system (Reeve,
1981; Fleming & Pickering, 1985), the position of nuclei
(Reeve & Kelly, 1983) and distribution of various
Key words: cell differentiation control, embryonic genome
activation, blastomere surface regionalization, cell lineage
segregation, preimplantation development, human.
318
/. Tesafik
out on mouse embryos, since the activation of the
mouse embryonic genome, largely occurring at the 2cell stage (e.g. Flach et al. 1982), precedes the appearance of the first signs of blastomere polarization in this
species. While the oocyte-derived message in the form
of maternal mRNA may be rapidly eliminated after the
onset of embryonic genome transcription (Bachvarova
& De Leon, 1980; Piko & Clegg, 1982), longevity of
some oocyte gene products, such as glucose phosphate
isomerase (West & Green, 1983), in the cytoplasm of
embryonic cells is remarkable, which makes the possibility of oocyte-derived influences on some relatively
late embryonic processes quite real.
It is evident that the study of the impact of oocytecoded factors on the early embryonic development
would be more feasible in those mammalian species in
which the activation of the embryonic genome occurs
relatively late in development, so as to avoid misinterpretations relating to the superposition of oocyte- and
embryo-coded factors. As demonstrated recently, the
human preimplantation embryo represents such a kind
of material. The first limited transcription can be
detected in human embryos at the 4-cell stage, followed
by a major outburst of transcriptional activity at the 8cell stage (Tesafik et al. 1986a,/?; Tesafik, 1987). The
first signs of human embryonic genome expression can
also be seen during the transition between the 4- and 8cell stages (Tesafik, 1987; Tesafik et al. 1988; Braude et
al. 1988). Moreover, the existence of two possible
patterns of gene activity in blastomeres of human 8- to
16-cell embryos (Tesafik etal. 1986a), each reflected by
distinct qualitative (Tesafik, 1987) and quantitative
(Tesafik et al. 1988) ultrastructural cytological characteristics, enables a direct study of correlations between
the activity of embryonic genome and the progression
of differentiation.
Here we use a morphometric ultrastructural analysis
of human preimplantation embryonic cells to examine
the relationship between the development of cellcontact-dependent surface regionality and the activity
of the embryonic genome. It is shown that the generation of cell surface heterogeneity precedes any overt
embryonic gene expression and that the persistence of
the limited gene activity characterizing the early-cleavage transcriptional pattern is compatible with the differentiation of the first two embryonic cell lineages. We
conclude that cytoplasmic factors inherited from the
oocyte contribute a message utilized in the control of
these early differentiative events in human preimplantation embryos.
The decision concerning embryo allocation to the experimental group was made on day 3 after in vitro insemination (day 1
was the day of insemination) when the embryos were at the 2or 4-cell stages; some of the selected embryos were then
cultured for an additional 1 to 3 days to achieve more
advanced developmental stages, from 8-cell to blastocyst.
When the culture was finished, embryos were fixed and
processed for electron microscopy as described (Tesafik et al.
1987). Individual embryonic cells were examined in thin
sections for the presence of the qualitative (Tesafik, 1987) and
quantitative (Tesafik et al. 1988) ultrastructural markers of
embryonic gene activity. Details of morphometric measurements of fractional volumes of cell organelles were reported
earlier (Tesafik et al. 1988). For determination of the fractional surface of different plasma membrane domains thin
sections were photographed and printed at the final magnification of x 10000. The prints were plotted against a lattice
consisting of test lines whose intersections served as test
points in a coherent stereological test system (Weibel, 1969).
Fractional surfaces (Sv) of different types of the plasma
membrane (fractional surface is the surface of a membrane
constituting unit cell volume) were calculated using the
formula
S v = 2IL
where IL represents intersection density of membrane profiles
on unit test line length (Weibel, 1969). Practically, IL values
were determined as fractions obtained by dividing the number
of intersections of test lines with the plasma membrane of a
given type by the number of test points of the coherent
stereological system falling on intracellular areas of measured
blastomere sections. The plasma membrane surfaces were
calculated from the fractional surfaces using the values of
fixed embryo volume determined as described earlier (Tesafik
et al. 1986a).
Beginning with the 4-cell stage, when the embryonic
genome transcription is first detectable (Tesafik et al.
1986a,b), all embryonic cells were classified into two categories, based on the character of their gene activity evaluated using the qualitative and quantitative ultrastructural
markers. These two types, denoted as the early-cleavage and
progressed-cleavage patterns, were characterized in a previous
study (Tesafik et al. 1988). Briefly, the early-cleavage-type
blastomeres show a low level of extranucleolar RNA synthesis
and the absence of nucleolar transcriptional activity, whereas
the progressed-cleavage-type blastomeres, first appearing in
the fourth cell cycle after fertilization, are characterized by
intense transcription of both nucleolar and extranucleolar
genes. Morphologically, the transition from the early-cleavage to the progressed-cleavage pattern is signalled by the
development of nucleolar structure, disappearance of vesicles
from the perinuclear space, increase in the number of tubules
of the endoplasmic reticulum and lysosomes and reduction of
the Golgi apparatus and vesicular endoplasmic reticulum
profiles. Fractional surfaces of different types of the plasma
membrane were expressed separately for the two types of
blastomeres. Significance levels were ascertained by r-test.
Materials and methods
Results
Embryos used in this study were 55 supernumerary concepti
from an in vitro fertilization programme. Criteria for embryo
selection for experimental purposes were those reported
earlier (Tesaffk et al. 1986a,b). All of the embryos had
developed at normal rates and showed normal morphology
when examined under a dissecting microscope. Delayed
embryos and those with irregularly sized blastomeres or
abundant cell fragments were excluded from this analysis.
Definition of basic plasma membrane patterns
From the topographical viewpoint, the cell surface of
blastomeres of cleaving embryos shows two well-defined regions: the free surface - denoted as apical, and
that making contact with adjacent embryonic cells denoted as basolateral. From the morphological view-
Cell differentiation in early embryos
319
Fig. 1. Electron micrographs showing the morphology of the two basic patterns of the plasma membrane in human 2-cell
(C) and 4-cell (A,B) embryos. The microvillar type shows the presence of numerous microvilli (mv) and characteristic pits
(arrows) giving rise to endocytotic vesicles; it can be observed at both the apical (A) and basolateral (B) blastomere
surfaces. The junctional type (C) lacks microvilli and signs of endocytosis, develops focal submembrane densities
(arrowheads) and is restricted to the basolateral blastomere surface. (A) X25000; (B and C) X60000.
point, two distinct patterns of the blastomere surface
can be distinguished again, based on the ultrastructural
properties of the corresponding plasma membrane
(Fig. 1). The microvillar type of the plasma membrane
is characterized by the presence of microvilli and signs
of active endocytotic membrane transport. This type of
membrane can be observed at both the apical (Fig. 1A)
and basolateral (Fig. IB) surfaces. On the other hand,
the junctional type of the plasma membrane is free of
microvilli, lacks the signs of endocytotic activity and
shows a tendency to develop focal submembrane densities and cell junctions. Under normal circumstances it
appears at the basolateral blastomere surfaces only
(Fig. 1C).
Plasma membrane modifications and cell
differentiation during preimplantation development
Fig. 1C shows that the junctional type of the plasma
membrane can be recognized as blastomere basolateral
surfaces as early as the 2-cell stage of human preimplantation development. The microvillar and junctional
plasma membrane domains display a proportional
growth at the basolateral surfaces between the 2- and 8cell stages, followed by an isolated marked increase in
the junctional type at the 16-cell stage, when the
segregation of the inner cell mass and trophectoderm
cell lineages begins (Fig. 2). The area of the microvillar
plasma membrane at the apical blastomere surfaces
remains the same throughout the first four cleavage
stages.
Beginning with the 8-cell stage, the differentiation of
the cell surface was evaluated separately for the blastomeres having undergone the early-cleavage-progressed-cleavage transition of gene activity and for
those in which this process had not occurred. A clear
difference in the microvillar/junctional membrane ratio
70
60-
I
50-
I
40
2
30
| 2010
AB
2-cell
AB
AB
4-cell
8-cell
Developmental stage
AB
16-cell
Fig. 2. Quantitative representation of the microvillar (open
columns) and junctional (hatched columns) types of plasma
membrane at the apical (A) and basolateral (B) blastomere
surfaces at different stages of human preimplantation
development. The bars above columns indicate standard
deviations. At least 10 embryos were analysed for each
stage.
was found between outer and inner cells of 16-cell
morulae (Table 1). However, the extents of the microvillar and junctional types of the plasma membrane in
blastomeres of 8-cell embryos and in both the outer and
inner cells of 16-cell morulae did not depend on the
proper stage-specific enhancement of embryonic gene
activity (Table 1), as was previously shown to be the
case for the process of compaction of human morulae
(Tesaffk et al. 1986a). These findings indicate that
morphologically distinct (as to their plasma membrane
at least) populations of inner and outer cells are
320
/. Tesaflk
Table 1. Quantitative representation of different types of plasma membrane (PM) in relation to actual pattern of
gene activity
Fractional surface of microvillar and junctional PM at different developmental stages (xlO""2) (/an"1)
16-cell
8-cell
Pattern of gene activity
Early-cleavage
Progressed-cleavage
P
Outer cells
Inner cells
Microvillar
Junctional
Microvillar
Junctional
Microvillar
Junctional
10-1 ±1-2
9-7 ±0-9
>O05
3-4 ±0-4
3-6 ±0-4
>0-05
12-3 ±1-4
12-5 ±1-1
>0-05
4-7 ±0-5
4-9 ±0-4
>0-05
5-4 ±0-6
5-3 ±0-6
>0-05
7-6 ±0-9
7-9 ±0-7
>0-05
Values are means ± standard deviations. Each type of blastomere was represented by at least 25 cells.
Table 2. Relationship between formation of specialized cell junctions, cavitation and actual pattern of gene activity
Occurrence of gap junctions (GJ), tight junctions (TJ)
and development of blastocyst cavity
32-cell
GJ
TJ
GJ
TJ
GJ
TJ
Cavity
+
-
I
++
Pattern of gene activity
Early-cleavage
Progressed-cleavage
16-cell
++
8-cell
+
+
Fig. 3. Electron micrograph showing the contact of two 8-cell blastomeres both of which have failed to assume the
progressed-cleavage pattern of gene activity. Note a structurally well-developed gap junction (arrows). X65000.
segregated in human 16-cell embryos, regardless of the
previous acquisition of the progressed-cleavage transcriptional pattern.
The same approach was used for the examination of
the relationship between the pattern of blastomere gene
activity and the ability to form specialized cell junctions
and to support the development of the blastocyst cavity
(Table 2). The formation of zonula-adherens-type junctions and gap junctions was observed in 8-cell and older
embryos, irrespective of the actual pattern of gene
activity (Fig. 3). By contrast, the presence of the
progressed-cleavage pattern in both neighbouring cells
was necessary for the development of tight junctions in
the nascent trophectoderm layer and this pattern of
gene activity was always detected in cells facing the
blastocyst cavity. When the progressed-cleavage pattern had not developed in the majority of embryonic
cells, a normal blastocyst cavity was never formed
(Table 2). It is worth mentioning that, on the other
hand, cavitation was observed in all five embryos that
succeeded in developing the progressed-cleavage pattern in >50 % of cells and that were cultured for the
appropriate time (>5 days after in vitro insemination).
Discussion
The appearance of distinct plasma membrane domains
in blastomeres of preimplantation embryos represents
the first step towards the development of cell asymmetry, a necessary prerequisite for future establishment
and diversification of cell lineages. The development of
uneven distribution of microvilli and cell junctions on
the blastomere surface are two aspects of this process
commonly denoted as blastomere surface polarization.
Originally, the term 'polarization' was used for gener-
Cell differentiation in early embryos
ation of cell polarity in situ (e.g. Ducibella et al. 1977).
Later this term was usually reserved for the designation
of changes that were not dependent upon continuing
cell contact and were maintained when embryos were
disaggregated to single cells (e.g. Handyside, 1980;
Reeve & Ziomek, 1981). In order to avoid misinterpretation, we therefore use the term plasma membrane
'regionalization' for the changes described in this study
as no attempt was made to examine the stability of these
changes after embryo disaggregation. Nevertheless, as
judged by analogy with the murine system, the asymmetries in the distribution of microvilli, endosomes and
cell junctions described here are most probably stable
ones which may have been preceded by less stable
asymmetries at the molecular level, similar to those
described in mice (Izquierdo et al. 1980; Izquierdo &
Ebensperger, 1982; Pratt, 1985; Handyside et al. 1987).
In mouse embryos, morphological polarization of the
blastomere surface is first evident at the 8-cell stage.
Thus microvilli (Ducibella etal. 1977; Handyside, 1980;
Johnson & Ziomek, 1981; Reeve & Ziomek, 1981) and
endosomes (Reeve, 1981; Fleming & Pickering, 1985)
become localized apically, whereas cell junctions develop at apposed cell surfaces (Ducibella & Anderson,
1975; Magnuson et al. 1977; McLachlin et al. 1983;
Goodall & Johnson, 1984). At the same time, blastomeres gain the capacity to respond to contact between
blastomeres by changes in cytocortical organization
(Johnson et al. 1988). Fluorescent ligand (Handyside,
1980; Reeve & Ziomek, 1981) and electron microscopic
studies (Calarco & Epstein, 1973; Ducibella etal. 1977;
Van Blerkom & Motta, 1979; Reeve & Ziomek, 1981)
of intact mouse embryos did not reveal any asymmetries in the distribution of microvilli prior to the 8cell stage. Also the distribution of the endocytotic
system in the cortical cytoplasm of blastomeres of 2and 4-cell mouse embryos is random (Fleming &
Pickering, 1985). Surprisingly, in human embryos, morphological regionalization of the plasma membrane,
corresponding to that occurring at the 8-cell stage of the
mouse development, is evident as early as the 2-cell
stage. Together with the delayed activation of human
embryonic genome transcription (Tesaffk etal. 1986a,b\
Tesafik, 1987) and expression (Tesaffk, 1987; Tesaffk et
al. 1988; Braude et al. 1988), as compared with the
mouse, this finding is proof of a role of oocyte-coded
message in the control of this process. Information
concerning the stage at which morphological heterogeneity of the plasma membrane first appears in embryos of
other mammalian species is actually lacking; neither is
it known, except for human and mouse embryos, how
these changes temporally relate to the embryonic gene
activation.
It is believed that the development of cell surface
polarity is induced by interactions occurring in places of
blastomere contact (Ziomek & Johnson, 1980). We
have shown here that the process of cell-contactmediated transformation of the plasma membrane from
the microvillar to the junctional type and the segregation of the first two embryonic cell lineages are
propagated at virtually constant rates in embryos show-
321
ing different patterns of gene activity. This finding
represents a further argument supporting the view that
the message necessary for the adequate reaction of
preimplantation embryonic cells to positional stimuli is
apparently coded for, at least in part, by the maternal
genome and transmitted to the early embryo from the
oocyte. It is not clear whether this information is stored
in the form of maternal RNA or other cytoplasmic
components. Informational signals stored in the egg
cytocortex, whose persistence during cleavage of mouse
embryos up until the blastocyst stage has been suggested (Pratt, 1985), are also possible candidates for this
regulatory role.
On the other hand, the embryonic transcripts newly
appearing at the 8-cell stage of human development
seem to be implicated in the activation of vectorial
transport mechanism and/or tight junctional assembly
necessary for establishing a permeability seal capable of
sequestering the nascent blastocyst fluid within the
embryo. These two conditions are known to be
required for blastocyst formation (Magnuson et al.
1978; Wiley, 1984; Fleming & Pickering, 1985). In this
study, we observed no blastocysts among embryos in
which most blastomeres failed to undergo the earlycleavage-progressed-cleavage transition of gene activity by 5 days in culture. It is possible that the failure
of proper gene activation at the 8-cell stage of human
embryogenesis, which represents a rather frequent
anomaly in cultured human embryos (Tesafik et al.
1986a; Tesaffk, 1988), may be the main cause of the
generally poor development of human blastocysts in
vitro.
In conclusion, the present analysis performed on
human embryos, in which the activation of the embryonic genome is delayed as compared with mouse
embryos, has shown the persistence of an oocytederived regulatory mechanism involved in the control
of early cellular differentiative events up to relatively
late stages of preimplantation development. Whether
this delayed maternal effect is a specific feature of
human embryos or whether it is shared by embryos of
other mammalian species remains to be elucidated.
References
BACHVAROVA, R. & D E LEON, V. (1980). Polyadenylated RNA of
mouse ova and loss of maternal RNA in early development. Devi
Biol. 74, 1-8.
BATTEN, B. E . , ALBERTINI, D . F. & DUCIBELLA, T. (1987). Patterns
of organelle distribution in mouse embryos during
preimplantation development. Am. J. Anal. 178, 204-213.
BRAUDE, P., BOLTON, V. & MOORE, S. (1988). Human gene
expression first occurs between the four- and eight-cell stages of
preimplantation development. Nature, Lond. 332, 459-462.
CALARCO, P. G. & EPSTEIN, C. J. (1973). Cell surface changes
during preimplantation development in the mouse. Devi Biol. 32,
208-213.
DAVIDSON, E. H. (1986). Gene Activity in Early Development, 3rd
Edition. New York: Academic Press.
DUCIBELLA, T. & ANDERSON, E. (1975). Cell shape and membrane
changes in the eight-cell mouse embryo: prerequisites for
morphogenesis of the blastocyst. Devi Biol. 47, 45-58.
DUCIBELLA, T., UKENA, TV, KARNOVSKY, M. & ANDERSON, E.
(1977). Changes in cell surface and cortical cytoplasmic
322
J. Tesafik
organization during early embryogenesis in the preimplantation
mouse embryo. /. Cell Biol. 74, 153-167.
distribution of membrane components on two-cell mouse
embryos. Wilhelm Roux' Arch, devl Biol. 196, 273-278.
IZQUIERDO, L. & EBENSPERGER, C. (1982). Cell membrane
regionalization in early mouse embryos as demonstrated by 5'nucleotidase activity. J. Embryol. exp. Morph. 69, 115-126.
PRATT, H. P. M. (1985). Membrane organization in the
preimplantation mouse embryo. J. Embryol. exp. Morph. 90,
101-121.
REEVE, W. J. D. (1981). Cytoplasmic polarity develops at
compaction in rat and mouse embryos. /. Embryol. exp. Morph.
62, 351-367.
REEVE, W. J. D. & KELLY, F. P. (1983). Nuclear position in the
cells of the mouse early embryo. /. Embryol. exp. Morph. 75,
117-139.
REEVE, W. J. D. & ZIOMEK, C. A. (1981). Distribution of microvilli
on dissociated blastomeres from mouse embryos: evidence for
surface polarization at compaction. J. Embryol. exp. Morph. 63,
339-350.
TAYLOR, K. D. & PIK6, L. (1987). Patterns of mRNA prevalence
and expression of Bl and B2 transcripts in early mouse embryos.
Development 101, 877-892.
TESASIK, J. (1987). Gene activation in the human embryo
developing in vitro. In Future Aspects in Human In Vitro
Fertilization (ed. W. Feichtinger & P. Kemeter), pp. 251-261.
Berlin & Heidelberg: Springer-Verlag.
TESAMK, J. (1988). Developmental control of human
preimplantation embryos: a comparative approach. /. In Vitro
Fert. Embryo Transfer 5, 347-362.
IZQUIERDO, L., LOPEZ, T. & MARTICORENA, P. (1980). Cell
TESAMK, J., KOPECNY, V., PLACHOT, M. & MANDELBAUM, J.
FLACH, G., JOHNSON, M. H., BRAUDE, P. R., TAYLOR, R. A. S. &
BOLTON, V. N. (1982). The transition from maternal to
embryonic control in the 2-cell mouse embryo. EM BO J. 1,
681-686.
FLEMING, T. P. & PICKERING, S. J. (1985). Maturation and
polarization of the endocytotic system in outside blastomeres
during mouse preimplantation development. J. Embryol. exp.
Morph. 89, 175-208.
GOODALL, H. & JOHNSON, M. H. (1984). The nature of intercellular
coupling within the preimplantation mouse embryo. J. Embryol.
exp. Morph. 79, 53-76.
HANDYSIDE, A. H. (1980). Distribution of antibody- and lectinbinding sites on dissociated blastomeres from mouse morulae:
evidence for polarization at compaction. J. Embryol. exp.
Morph. 66, 99-116.
HANDYSIDE, A. H., EDIDIN, M. & WOLF, D. E. (1987). Polarized
membrane regions in preimplantation mouse embryos. J.
Embryol. exp. Morph. 59, 89-102.
JOHNSON, M. H. (1986). Manipulation of early mammalian
development: what does it tell us about cell lineages? In
Developmental Biology: a Comprehensive Synthesis (ed. R. B. L.
Gwatkin), pp. 279-296. New York & London: Plenum Press.
JOHNSON, M. H. & ZIOMEK, C. A. (1981). The foundation of two
distinct cell lineages within the mouse morula. Cell 24, 71-80.
JOHNSON, M. H., CHISHOLM, J. C , FLEMING, T. P. & HOULISTON,
E. (1986). A role for cytoplasmic determinants in the
development of the mouse early embryo? /. Embryol. exp.
Morph. 97 Supplement, 97-121.
JOHNSON, M. H., PICKERING, S. J., DHIMAN, A., RADCLIFFE, G. S.
& MARO, B. (1988). Cytocortical organization during natural and
prolonged mitosis of mouse 8-cell blastomeres. Development 102,
143-158.
MAGNUSON, T., DEMSEY, A. & STACKPOLE, C. W. (1977).
Characterization of intercellular junctions in the preimplantation
mouse embryo by freeze-fracture and thin-section electron
microscopy. Devi Biol. 61, 252-261.
MAGNUSON, T., JACOBSON, J. B. & STACKPOLE, C. W. (1978).
Relationship between intercellular permeability and junction
organization in the preimplantation mouse embryo. Devi Biol.
67, 214-224.
MARO, B., JOHNSON, M. H., PICKERING, S. J. & LOUVARD, D.
(1985). Changes in the distribution of membranous organelles
during mouse early development. J. Embryol. exp. Morph. 90,
287-309.
MCLACHLIN, J. R., CAVENEY, S. & KIDDER, G. M. (1983). Control
of gap junction formation in early mouse embryos. Devi Biol. 98,
155-164.
PIK6, L. & CLEGG, K. B. (1982). Quantitative changes in total
RNA, total poly(A), and ribosomes in early mouse embryos.
Devi Biol. 89, 362-378.
(1986a). Activation of nucleolar and extranucleolar RNA
synthesis and changes in the ribosomal content of human
embryos developing in vitro. J. Reprod. Fert. 78, 463-470.
TESAWK, J., KOPECNY, V., PLACHOT, M., MANDELBAUM, J., DA
LAGE, C. & FLECHON, J. E. (19866). Nucleologenesis in the
human embryo developing in vitro: ultrastructural and
autoradiographic analysis. Devi Biol. 115, 193-203.
TESAMK, J., KOPECNY, V., PLACHOT, M. & MANDELBAUM, J. (1987).
High-resolution autoradiographic localization of DNA-containing
sites and RNA synthesis in developing nucleoli of human
preimplantation embryos: a new concept of embryonic
nucleologenesis. Development 101, 777-791.
TESARIK, J., KOPECNY, V., PLACHOT, M. & MANDELBAUM, J. (1988).
Early morphological signs of embryonic genome expression in
human preimplantation development as revealed by quantitative
electron microscopy. Devi Biol. 128, 15-20.
VAN BLERKOM, J. & MOTTA, P. (1979). The Cellular Basis of
Mammalian Reproduction. Munich & Baltimore: Urban &
Schwarzenberg.
WEIBEL, E. R. (1969). Stereological principles for morphometry in
electron microscopic cytology. Int. Rev. Cytol. 26, 235-302.
WEST, J. D. & GREEN, J. F. (1983). The transition from oocytecoded to embryo-coded glucose phosphate isomerase in the early
mouse embryo. J. Embryol. exp. Morph. 78, 127-140.
WILEY, L. M. (1984). Cavitation in the mouse preimplantation
embryo: Na/K-ATPase and the origin of the nascent blastocoele
fluid. Devi Biol. 105, 330-342.
ZIOMEK, C. A. & JOHNSON, M. H. (1980). Cell surface interactions
induce polarization of mouse 8-cell blastomeres at compaction.
Cell 21, 935-942.
{Accepted 30 September 1988)