THE PREIMPLANTATION EMBRYO: DEVELOPMENT AND EXPERIMENTAL MANIPULATION
D. Sakkas and *J.D. Vassalli
In mammals, the meeting of the oocyte and sperm, and subsequent fertilization, take place in the ampulla of the oviduct. During the following days, the embryo travels down the oviduct to the uterus, and prepares for implantation. Preimplantation development, which can also occur in vitro, is the subject of the present chapter.
In all mammalian species studied the preimplantation stages are characterized by a relatively synchronous doubling of cell numbers until the 8-cell stage followed by asynchronous cell divisions after compaction. At the 8- to 16-cell stage the embryo enters the uterine environment, developing into a blastocyst, in which the first events of cellular differentiation are observed. At the blastocyst stage the embryo hatches from the surrounding zona pellucida and subsequently implants in the uterus.
In 1956, Whitten (48), using a serum-free medium based on Krebs Ringer’s bicarbonate buffer, supplemented with glucose, bovine plasma albumin and antibiotics, successfully cultured mouse 8-cell embryos to the blastocyst stage. The following year Whitten (49) demonstrated that the addition of calcium lactate allowed late 2-cell embryos to develop to blastocysts. The ability to culture embryos in simple serum-free media has greatly benefited subsequent studies on the mechanisms controlling early embryo development. Furthermore, this has also allowed the cellular and genetic manipulation of early embryos from a number of species. In this chapter we will discuss the metabolic and developmental changes that take place during preimplantation development and conclude with a discussion of specific experimental techniques that can be applied to embryos in vitro.
Development of the preimplantation embryo
The preimplantation embryo passes through distinct metabolic phases, undergoing changes in protein synthesis, energy requirements and amino acid uptake as it develops from a fertilized zygote to the blastocyst stage. Concurrently, it also undergoes morphological changes, particularly at compaction when the first differentiative process is observed.
Mouse embryos take about three and a half days to develop from the 1-cell stage to the blastocyst stage containing 32 or more cells. The 1st (1- to 2-) and 2nd (2- to 4-) cell cycles of the mouse embryo take between 16-20h and 18-22h respectively, depending on the strain of mice (7,23,43). The duration of certain phases of the cell cycle differ considerably between 1- and 2-cell mouse embryos. The duration of the S phase increases from 4 h to 7 h from the pronuclear stage to the 2-cell stage, whereas the duration of G2 and M increases from 8 h to nearly 12 h (43). The duration of the G2 and M phase of the second cell cycle is strain-specific leading to differences in the length of the 2- to 4- cell cycle in different mouse strains (33).
The rate of cleavage has also been linked to genetic influences: Warner et al. (46) have described a H-2 linked gene, called the preimplantation embryo development (Ped) gene, that influences the rate of cleavage divisions of preimplantation mouse embryos. The Ped gene has two functional alleles, fast and slow, as defined by the rate of development of preimplantation embryos, with the fast allele being dominant. In a more recent study, Brownell and Warner (10) demonstrated that the Ped gene phenotype of embryos cultured in vitro is maintained; thus, the control of embryo cleavage is largely dependent on the genes of the embryo itself and is not a function of the uterine environment. In addition, Burgoyne (11) has provided evidence that in certain strains of mice there is a Y chromosomal effect that accelerates the rate of preimplantation development.
Embryonic genome activation
The variations in the duration of the cell cycle during the early stages of embryo development can perhaps be linked to specific developmental events that occur at this time. The lengthened cycle from the 2- to 4-cell stage in mouse embryos, in particular, may be related to one of the major events of preimplantation development, i.e. embryonic genome activation. The earliest developmental changes are under post-transcriptional maternal control, i.e. they rely on changes in the translation of mRNAs synthesized during oocyte growth, and/or post-translational protein modifications. The activation of the mouse embryonic genome occurs at the late 2-cell stage (21), corresponding with the long 2nd cell cycle. The embryonic genome is activated in two phases, a limited activation occurring between 18 and 21h post-insemination and a major activation occurring between 26 and 29h post-insemination (16). Although the first sign of major transcription by the embryonic genome appears during the 2-cell stage, recently a more sensitive assessment of the 1-cell mouse embryo has led to the suggestion that zygotic gene activation may begin in the 1-cell embryo and that differences between the transcriptional activity of the male and female pronuclei exist (36).
In conjunction with the activation of the embryonic genome, conventional one dimensional SDS polyacrylamide gel electrophoresis has shown that major changes occur in protein synthesis between days 1 (2-cell stage) and 2 (4- to 8-cell stage) of preimplantation mouse embryo development (13,45). Levinson et al. (30) demonstrated that certain stage-specific polypeptides (SSP) are expressed during preimplantation development of mouse embryos; for instance 18 SSP detected in single cell embryos had disappeared by the 4-cell stage. The first proteins synthesized in the late 2-cell embryo coinciding with embryonic genome activation appear to be heat shock proteins (67,000-70,000 d) (3). During the late 4- and 8-cell stage new transcription is necessary to prepare the embryo for compaction, while during the morula-blastocyst transition there is also a change in transcriptional activity in line with the increase in the rate of protein synthesis (8). These changes ultimately lead to the appearance of tissue-SSP in the inner cell mass (ICM) and trophectoderm at the blastocyst stage (22,26).
Factors affecting embryo metabolism and development
One of the most striking changes that the preimplantation embryo experiences is in its energy preferences. Around the time of compaction the mouse embryo switches from a dependence on the tricarboxylic acid cycle to a metabolism based on glycolysis. Embryo culture experiments have shown that the mouse oocyte and zygote have an absolute requirement for pyruvate (5); i.e. glucose cannot support early embryo development until the 8-cell stage (4). This dramatic switch to glucose utilization as development proceeds may be related to a number of metabolic requirements:
In conjunction with changes in preferences for specific energy metabolites the development of the embryo is also regulated by amino acids, vitamins and growth factors (19). Studies relating to factors affecting preimplantation embryo metabolism and development are however limited to in vitro culture conditions and may not necessarily identify the actual factors that are critical to the embryo in vivo. For example, although glucose is present in the oviductal fluid (18), in vitro it can be detrimental to the pre-compacted mouse embryo by causing cleavage arrest or retarding the cleavage rate (9,40).
The rapid increase in cell numbers post compaction and the changes in metabolic activity have also lead to the question of whether growth factors are involved in preimplantation embryo development. The transcripts for transforming growth factor a (TGFa), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and insulin-like growth factor II (IGF-II) have all been detected in cleavage stage mouse embryos as well as the transcripts for insulin receptors, IGF-I receptors, EGF receptors and PDGFa subunit receptors (37,50). Studies examining the role of these factors when added to embryo culture medium show that some growth factors, in particular insulin and EGF, stimulate metabolic and synthetic activities in the early mouse embryo as well as increase cleavage rates and cell numbers in blastocysts (1,24,25,39). Of the growth factors studied only insulin has been reported to be endocytosed by the preimplantation mouse embryo, to have a mitogenic effect and to be present in the mouse oviduct (25). In addition streptozocin-induced diabetic mice produce blastocysts with lower cell numbers than do normal mice and this effect is reversed when the diabetic mothers are treated with insulin (1).
Morphological changes in the preimplantation embryo
Compaction is the first event of morphogenic and cellular differentiation. The most significant event occurring at compaction is the emergence of 2 distinct cell populations: the blastomeres remaining in contact with the outside are destined to form the trophectodermal lineage while the blastomeres inside the embryo are destined to form the ICM.
During the 8-, 16- and 32-cell stages, specific cells are induced to change their morphological and functional phenotype to a polarized form. This commences with the division of the 8-cell embryo generating an average of 9 " outside " and 7 " inside " cells in the embryo (28), with the outer cells being polarised and larger than the inner cells that remain apolar. The trigger to the development of a polarized phenotype in the outer cells may be related to the pattern of intercellular contacts: polarization is suppressed when a cell is completely surrounded by other cells, while when contact with other cells is incomplete polarity develops. The close cell contacts that develop are due to the presence of the cell adhesion molecule uvomorulin which progressively becomes distributed to areas of cell-cell contact and remains absent from the apical areas of the outer polarised cells (27). Polarization of the outer cells is evident by the basal migration of the nucleus and the apical accumulation of actin, clathrin, endosomes and microvilli.
Once a cell acquires polarity the progeny of the cell will be influenced by the orientation of the subsequent cleavage plane, hence either two polar or one polar and one non-polar cell will arise. It is only in fully expanded blastocysts that ICM and trophectodermal cells cannot cross lineages. This initial differentiation is also the first decrease in cell totipotency in the embryo whereby the internalized apolar cell subpopulation in the morula will preferentially form the ICM of the blastocyst and the outer polar cells develop trophectodermal characteristics.
Trophectodermal cells therefore are polar, enveloping and fluid transporting (12,20) whereas ICM cells are highly adhesive, compact readily on each other and, when aggregated to a morula, move to its center (28,44).
The trophectodermal cells acquire the characteristics of epithelial cells in being flattened and joined together by tight junctional complexes (12). When the mouse embryo has about 32 cells, trophectodermal cells begin to pump fluid into intracellular spaces and later into extracellular spaces, forming the blastocoelic cavity (6). The trophectoderm ion transport systems play an important role in establishing ion concentration gradients across the epithelium, and thereby in providing the force that drives water into the blastocoelic fluid. Electron probe microanalyses of Na+, Cl-, K+, Ca2+ and Mg2+ have shown that all these ions are concentrated within the blastocoelic fluid (6). The active transport mechanisms required to move these ions against their concentration gradients are thought to involve the transport of Na+ and Cl-. The main contributor is the Na,K-ATPase that has been localised to the basolateral domain of the trophectoderm (2). The presence of the tight junctional complex is also necessary and plays a multifunctional role. It provides an impermeable seal allowing fluid accumulation, regulates paracellular transport (31) and contributes to a polarization of the distribution of the Na,K-ATPase (47).
The blastocyst contains two distinct cell types: the ICM cells which go on to form the embryo proper, and the trophectodermal cells which are involved in the initial contact with and the infiltration of the uterine wall and eventually contribute to the placenta and the extraembryonic membranes.
The development of the mouse embryo (Fig. 1) has been widely studied and has served as an excellent model for other species. Many of the above-described events that occur during preimplantation development have also been observed in other mammalian embryos. However, the duration of the cell cycles and the timing of specific events differ. In nearly all mammalian species studied to date there is a synchronous cleavage in the first few cell cycles followed by asynchronous divisions, normally after the 8-cell stage. The greatest diversity between species exists at the peri-implantation stage, where many embryos experience long periods at the blastocyst stage, forming either minimal or maximal expanding type blastocysts. This is particularly applicable to farm animals, such as the sheep and pig, whose blastocysts undergo dramatic increases in cell number and size (reviewed by 15). Another difference is in the activation of the embryonic genome, which occurs during a different cell cycle in most species (Table 1) but is still linked to a lengthened cleavage cycle. Most species also show similarities in the events of compaction and in a switchover in their preferences for specific energy metabolites.
The mouse remains a valuable tool for research largely due to its size and the ease of obtaining many oocytes and embryos. This fact in particular has lead to the use of the mouse model in experimental research using embryo micromanipulation techniques, especially in studies involving the generation of transgenic mice.
Experimental manipulation of mammalian preimplantation development
Since the first week of development can take place in vitro, early mammalian embryos are easily accessible to experimental manipulation. Most work in this area to date has been performed on mouse embryos. The transfer of manipulated embryos into foster mothers, and their subsequent development, can yield animals with, for instance, precise modifications in their genetic composition. Chimeras, clones and transgenics represent extraordinarily powerful tools to unravel multiple aspects of physiology and pathology, and thus play a growing part in experimental biology.
Chimeric embryos (also known as allophenic or tetraparental) are generated by " mixing " cells obtained from genetically different embryos (for reviews see 29). This can be achieved by placing two morula-stage embryos in direct contact (following removal of their zona pellucida), or by introducing cells from a " donor " embryo either underneath the zona pellucida of a morula-stage recipient, or in the blastocoelic cavity (in contact with the ICM) of a blastocyst-stage recipient. The cells from the two embryos assemble to form a single chimera which, when placed in a foster mother, can develop to term. Depending on the circumstances, the contribution of the two partners can be approximately balanced, with a comparable proportion of cells from each origin in all tissues, or be very markedly skewed in favor of one or the other. It is important to realize that chimerism is not hereditary: each cell of the chimera, and therefore each one of its germ cells, derives from either one or the other partner, and does not contain the entire body of genetic information present in the chimera. Thus, the generation of a chimera does not produce a member of a new species, nor a hybrid, but a unique individual. Furthermore, the relative contribution of the two partners is unpredictable, and each chimeric individual is therefore different.
Chimeras can be very useful in defining cell lineages, and thereby in understanding how adult tissues are built during development. It can be expected that chimeras will also be of importance in the study of aspects of the pathogenesis of genetic diseases: they allow, for instance, the exploration in a given individual and thereby under the influence of identical environmental factors, of the fate of cells having different genetic compositions. Finally, the generation of chimeras is one step in the production of transgenic mice by homologous recombination, and this will be detailed later. Chimeras have been produced between animals of different species, for instance by mixing cells from goat and sheep embryos (14,32,38).
Clones are groups of cells or of individuals that are genetically identical. Monozygotic twins thus form a clone. Two distinct experimental approaches have been used to produce animal embryos in multiple genetically identical copies. In the first such approach, that is technically quite simple and that mimics the " natural " mode of generation of monozygotic twins, the cells of an early embryo are separated into two groups; the mechanisms of development compensate for the initially small size of the two resulting embryos, which can grow into two distinct but genetically identical individuals. The second approach, which uses a procedure of nuclear transfer, is technically more complex: cells from embryos at the 8- or 16-cell stage are separated from each other, and each of these individual blastomeres is made to fuse with an enucleated unfertilized egg. The resulting fused cell can develop into an entire animal, which will be genetically identical to its twins generated by the fusion of the sister blastomeres with other enucleated unfertilized eggs. This genetic identity is however not complete, since the maternal contribution (the maternal mRNAs that are necessary for the early stages of development, and the mitochondrial genes that will be required throughout life) brought by the oocyte is in each case different.
The nuclear transfer cloning procedure allows the generation of multiple twins, while the first procedure is in this respect more limited. Multiple cloning has been achieved for sheep (51), cattle (35,41) and rabbit (42) embryos. It is possible because of the totipotency of nuclei from morula stage embryos in these species, and does not seem possible for other species, including mice, in which nuclear potency appears to become more rapidly restricted. In any event, the restriction of nuclear potency occurs early in mammalian development, and it is thus not at present possible to perform cloning with cells from later embryos or from adults.
The possibility of obtaining large numbers of identical twins represents a powerful tool to understand the relative contributions of genetic and environmental influences on health and disease. It is easy to envision the potential of this approach in the study of multigene disorders, which would be much more difficult to explore by the usual inbreeding techniques. Similarly, herds of genetically identical farm animals, with more uniformly predictable properties, may have economic advantages. Clearly the socio-ethical aspects of such cloning procedures should be carefully evaluated.
Transgenic animals carry an alteration of their genetic information, due to the introduction of a gene (or the fragment of a gene) obtained by recombinant DNA technology from another individual of the same or another species, or due to the insertional modification of an endogenous gene. This alteration is most often present in all cells of the organism, including the germ cells, and is thus heritable. In this respect there is a crucial difference between the grafting to an adult animal of a cell population that has been transfected with a foreign gene, that will be present only in the progeny of the transfected cells but not in the germ line and therefore that will not be transmitted to the animal’s offspring, and the generation of a transgenic animal; this difference is precisely that which separates, both technically and ethically, somatic gene therapy and germ line gene therapy. Among the different approaches used to generate transgenic animals, two are at present most relevant to experimental mammalian biology: transgene injection into the zygote, and homologous recombination in embryonic stem cells (for a detailed review on these issues, see 52).
Transgene injection is performed in one (generally the male) of the pronuclei present in 1-cell embryos. The embryos are then transferred to foster mothers, and allowed to develop. The currently-used procedures result in approximately 10-20% of the injected embryos that survive the manipulation being transgenic. The transgene usually integrates in the chromosomal DNA at the 1-cell stage, probably at a random site, and often in multiple tandemly-organized copies; it is present, at the same chromosomal site, in all cells of the organism, including the germ cells, and will thus also be transmitted to the progeny. In certain cases the transgene appears not to integrate in chromosomal DNA at the zygote stage, but later, hence the resulting animal may be chimeric, with cells that do and cells that do not contain the transgene.
Although a transgene can be present in all cells of the organism, the cells in which it is expressed depend on the promoter that drives its transcription, and perhaps also on additional post-transcriptional regulatory influences. The use of tissue-specific promoters allows to direct transgene expression to selected cell types; certain promoters have a broad specificity, and will direct expression in multiple cell types. A limited number of inducible promoters are available, that allow transgene expression to be modulated by exogenous influences. One major difficulty, that is encountered to different degrees depending on the transgene, is a marked variability in its’ level of expression or penetrance; this can be due to the site of integration or to other epigenetic phenomena. In a limited number of cases it has been possible to obtain position-independent expression of transgenes, probably because large enough regions of the transferred gene had been used so that appropriate regions of attachment to the nuclear scaffold were provided. It is also important to note that the site of integration of a transgene can be within a normally expressed gene, and integration may then have phenotypic effects that do not result from transgene expression but from insertional mutagenesis. This implies that multiple transgenic families carrying the same transgene must be studied to unambiguously identify the effects of transgene expression.
The uncertainties underlined above indicate that, despite its relative ease and great experimental potential, transgenesis by pronuclear injection has severe limitations. These limitations are akin to those that, in addition to the critical ethical issues involved, render for the time being germ line gene therapy a totally unrealistic proposition.
Embryonic stem (ES) cell lines
Embryonic stem (ES) cell lines can be established by culturing cells collected from peri-implantation blastocysts, under conditions that prevent their differentiation. This is critical, and can be achieved by culture in the presence of fibroblast feeder layers, of conditioned culture medium from certain cell lines, or of the cytokine LIF. ES cells can have a normal karyotype, and, when introduced into normal embryos to generate chimeras, can contribute to all tissues. New-born mice entirely derived from ES cells have been obtained, but, for an undetermined reason, undergo early postnatal death. The capacity of ES cells to colonise the germ line is their most relevant quality in the context of transgenesis, since this opens the possibility of using transfection in culture and somatic cell genetics to produce and select mutations that can subsequently be propagated to generate lines of transgenic animals (for a review, see 34).
The possibility of achieving the specific alteration of a targeted gene by homologous recombination between the endogenous gene and an introduced DNA sequence, together with the development of ES cell lines and techniques to produce germ line chimeras from these ES cells, has attracted enormous attention. Homologous recombination that can lead to insertion of an exogenous DNA sequence in the targeted gene, or to the replacement of a fragment of the targeted gene by the exogenous DNA, occurs only with very low frequency. Thus, selection procedures are necessary to identify and isolate the cells that have undergone the desired recombination event. It is the possibility of working with cell lines that renders selection possible; the low frequency of homologous recombination, at least with the presently used techniques, makes it statistically impossible to obtain targeted genetic alterations by the pronuclear injection approach.
Homologous recombination in ES cells has until now mostly been used to generate mutations that result in preventing the production of given gene products; this " knock out " of a gene can for instance be achieved by inserting into the chromosomal DNA a sequence that disrupts or replaces a critical region of the gene. The transfected ES cells are in general heterozygous for the mutation; after generation of chimeric animals containing ES derived cells in their germ line, heterozygous mice can be obtained, and breeding allows the production of homozygotes. The overall procedure is technically demanding and labour intensive, and requires many months of work, but it can provide unique information on the role of any given gene in vivo. Several dozen genes have already been studied in this way; one surprising result has been that quite a few genes which were expected to be essential are in fact dispensable, i.e. their function is not required for the development and reproduction of mice, at least in the setting of the laboratory. Improvements in the vectors used for homologous recombination should, in principle, allow the study of any desired mutation in any gene.
The spectacular advances that have taken place over the last 15 years in the fields of experimental embryology and molecular genetics lead us into a new era in the study of mammalian biology and in medicine. Procedures that were initially developed to study development play a decisive part in allowing man to modify at will the genetic composition of living mammalian species. The changes brought about by natural evolution take place over thousands of years. Animal breeding has already allowed man to very significantly accelerate the emergence and selection of desired traits. But the unprecedented power afforded by the techniques that allow direct intervention on the genetic heritage places us in a different type of relationship with the living world. Man is not anymore the innocent spectator of evolution that he used to be. The responsibility that this gives us should not be underestimated.
Edited by Aldo Campana,