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Vassalli Infertility and Gynecologic Endocrinology Clinic, Department of Obstetrics and Gynecology, University Cantonal Hospital, 1211 Geneva 14, Switzerland *Institute of Histology and Embryology, Department of Morphology University Medical Centre, 1211 Geneva 4, Switzerland Introduction In mammals, the meeting of the oocyte and sperm, and subsequent fertilization, travels down the oviduct to the uterus, and prepares for implantation. present chapter.
take place in the ampulla of the oviduct. During the following days, the embryo Preimplantation development, which can also occur in vitro, is the subject of the
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 first events of cellular differentiation are observed. At the blastocyst stage the in the uterus.
embryo enters the uterine environment, developing into a blastocyst, in which the embryo hatches from the surrounding zona pellucida and subsequently implants
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.
Embryo cleavage 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. whereas the duration of G2 and M increases from 8 h to nearly 12 h (43). this has also allowed the cellular and genetic manipulation of early embryos from a number of species.43). The ability to culture embryos in simple serum-free media has greatly benefited subsequent studies on the mechanisms controlling early embryo development. energy requirements and amino acid uptake as it develops from a fertilized zygote to the blastocyst stage. The duration of the S phase increases from 4 h to 7 h from the pronuclear stage to the 2-cell stage. .23. The duration of certain phases of the cell cycle differ considerably between 1. Development of the preimplantation embryo The preimplantation embryo passes through distinct metabolic phases.cell cycle in different mouse strains (33). 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. depending on the strain of mice (7. undergoing changes in protein synthesis.to 2-) and 2nd (2to 4-) cell cycles of the mouse embryo take between 16-20h and 18-22h respectively.The following year Whitten (49) demonstrated that the addition of calcium lactate allowed late 2-cell embryos to develop to blastocysts. it also undergoes morphological changes. Furthermore.and 2-cell mouse embryos. Concurrently. 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. The 1st (1. particularly at compaction when the first differentiative process is observed.to 4.
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). may be related to one of the major events of preimplantation development. Brownell and Warner (10) demonstrated that the Ped gene phenotype of embryos cultured in vitro is maintained.e. In addition. (46) have described a H-2 linked gene. the control of embryo cleavage is largely dependent on the genes of the embryo itself and is not a function of the uterine environment.The rate of cleavage has also been linked to genetic influences: Warner et al. in particular. Burgoyne (11) has provided evidence that in certain strains of mice there is a Y chromosomal effect that accelerates the rate of preimplantation development. The activation of the mouse embryonic genome occurs at the late 2-cell stage (21).to 4-cell stage in mouse embryos. embryonic genome activation. The earliest developmental development can perhaps be linked to specific developmental events that occur at changes are under post-transcriptional maternal control. thus. In a more recent study. i.e. they rely on changes in the translation of mRNAs synthesized during oocyte growth. Embryonic genome activation The variations in the duration of the cell cycle during the early stages of embryo this time. Although the first sign of major transcription by the embryonic genome appears during the 2-cell stage. called the preimplantation embryo development (Ped) gene. with the fast allele being dominant. The lengthened cycle from the 2. 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 . corresponding with the long 2nd cell cycle. fast and slow. that influences the rate of cleavage divisions of preimplantation mouse embryos. and/or posttranslational protein modifications. The Ped gene has two functional alleles. i. as defined by the rate of development of preimplantation embryos.
for instance 18 SSP detected in single cell embryos had disappeared by the 4-cell stage. This dramatic switch to glucose utilization as development proceeds may be related to a number of metabolic requirements: .e.26). These changes ultimately lead to the appearance of tissue-SSP in the inner cell mass (ICM) and trophectoderm at the blastocyst stage (22. i.000 d) (3). Protein synthesis In conjunction with the activation of the embryonic genome. while during the morula-blastocyst transition there is also a change (8). glucose cannot support early embryo development until the 8-cell stage (4).to 8(30) demonstrated that certain stage-specific polypeptides (SSP) are expressed during preimplantation development of mouse embryos.differences between the transcriptional activity of the male and female pronuclei exist (36).and 8-cell stage new transcription is necessary to prepare the embryo for compaction. 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. Levinson et al. During the late 4. The first cell stage) of preimplantation mouse embryo development (13. proteins synthesized in the late 2-cell embryo coinciding with embryonic genome activation appear to be heat shock proteins (67. Embryo culture experiments have shown that the mouse oocyte and zygote have an absolute requirement for pyruvate (5).45). Around the time of compaction the mouse embryo switches from a dependence on the tricarboxylic acid cycle to a metabolism based on glycolysis. in transcriptional activity in line with the increase in the rate of protein synthesis Factors affecting embryo metabolism and development One of the most striking changes that the preimplantation embryo experiences is in its energy preferences.000-70.
24. in particular insulin and EGF. There is an increase in energy demand around the time of compaction. it is conceivable that this primes the embryo for the anoxic environment it encounters at implantation. in vitro it can be detrimental to the pre-compacted mouse embryo by causing cleavage arrest or retarding the cleavage rate (9. In conjunction with changes in preferences for specific energy metabolites the factors (19). 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. to have a mitogenic effect and to be present in the mouse oviduct (25). Studies examining the role of these factors when added to embryo culture medium show that some growth factors.1. IGF-I receptors. required for phospholipid and non-essential amino acid biosynthesis. The embryo displays a considerable capacity for anaerobic glycolysis by the blastocyst stage (17).40). Studies relating to factors affecting preimplantation embryo development of the embryo is also regulated by amino acids. Of the growth factors studied only embryo. protein synthesis increases when the blastocoele is formed. although glucose is present in the oviductal fluid (18). In addition streptozocin-induced diabetic mice produce blastocysts with lower cell . Glucose provides the pentose moieties for nucleic acid synthesis and is 3.39). as 2.25. EGF receptors and PDGF subunit receptors (37. fibroblast growth factor (FGF). The transcripts for transforming growth factor (TGF).50). For example. 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. vitamins and growth 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. stimulate metabolic and synthetic activities in the early mouse embryo as well as increase cleavage rates insulin has been reported to be endocytosed by the preimplantation mouse and cell numbers in blastocysts (1.
to areas of cell-cell contact and remains absent from the apical areas of the outer Once a cell acquires polarity the progeny of the cell will be influenced by the orientation of the subsequent cleavage plane. to form the trophectodermal lineage while the blastomeres inside the embryo are During the 8-. It is only in fully expanded blastocysts that ICM and trophectodermal cells cannot cross lineages. The trigger to the development of a intercellular contacts: polarization is suppressed when a cell is completely surrounded by other cells. hence either two polar or one polar and one non-polar cell will arise. 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 destined to form the ICM. The close cell contacts that develop are due to the presence of the cell adhesion molecule uvomorulin which progressively becomes distributed polarised cells (27). Polarization of the outer cells is evident by the basal migration of the nucleus and the apical accumulation of actin.numbers than do normal mice and this effect is reversed when the diabetic mothers are treated with insulin (1). endosomes and microvilli. with the outer cells being polarised and polarized phenotype in the outer cells may be related to the pattern of with the division of the 8-cell embryo generating an average of 9 " outside " and larger than the inner cells that remain apolar. This commences 7 " inside " cells in the embryo (28). 16. clathrin.and 32-cell stages. while when contact with other cells is incomplete polarity develops. specific cells are induced to change their morphological and functional phenotype to a polarized form. Morphological changes in the preimplantation embryo Compaction Compaction is the first event of morphogenic and cellular differentiation. This initial differentiation is also .
forming the blastocoelic cavity (6). when aggregated to a morula. It provides an impermeable seal allowing fluid polarization of the distribution of the Na. required to move these ions against their concentration gradients are thought to that has been localised to the basolateral domain of the trophectoderm (2). compact readily on each other Blastocyst formation The trophectodermal cells acquire the characteristics of epithelial cells in being flattened and joined together by tight junctional complexes (12). Trophectodermal cells therefore are polar.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. The active transport mechanisms involve the transport of Na+ and Cl-. regulates paracellular transport (31) and contributes to a The blastocyst contains two distinct cell types: the ICM cells which go on to form the embryo proper. K+. When the mouse embryo has about 32 cells. . (12. The accumulation. Ca2+ and Mg2+ have shown that all these ions are concentrated within the blastocoelic fluid (6). enveloping and fluid transporting and. The main contributor is the Na. and thereby in providing the force that drives water into the blastocoelic fluid. Cl-. The trophectoderm ion transport systems play an important role in establishing ion concentration gradients across the epithelium. 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. trophectodermal cells begin to pump fluid into intracellular spaces and later into extracellular spaces. move to its center (28. Electron probe microanalyses of Na+.44).20) whereas ICM cells are highly adhesive.K-ATPase presence of the tight junctional complex is also necessary and plays a multifunctional role.K-ATPase (47).
The greatest diversity between species exists at the peri-implantation stage. early mammalian embryos are easily accessible to experimental manipulation. whose blastocysts undergo dramatic increases in cell number and size (reviewed by 15). The mouse remains a valuable tool for research largely due to its size and the ease of obtaining many oocytes and embryos. the duration of the cell cycles and the there is a synchronous cleavage in the first few cell cycles followed by timing of specific events differ. normally after the 8-cell stage. precise modifications in their genetic composition. and their subsequent development. Most work in this embryos into foster mothers. Many of the above-described events that occur during preimplantation development have also been observed in other mammalian embryos. However. can yield area to date has been performed on mouse embryos. Experimental manipulation of mammalian preimplantation development Since the first week of development can take place in vitro. Most species also show similarities in the events of compaction and in a switchover in their preferences for specific energy metabolites. 1) has been widely studied and has served as an excellent model for other species. forming either minimal or maximal expanding type blastocysts. for instance. Another difference is in the activation of the embryonic genome. This is particularly applicable to farm animals. In nearly all mammalian species studied to date asynchronous divisions. where many embryos experience long periods at the blastocyst stage. This fact in particular has lead to the use of the mouse model in experimental research using embryo micromanipulation techniques. such as the sheep and pig.Interspecies differences The development of the mouse embryo (Fig. especially in studies involving the generation of transgenic mice. The transfer of manipulated animals with. . which occurs during a different cell cycle in most species (Table 1) but is still linked to a lengthened cleavage cycle.
the relative contribution of the two partners is unpredictable. and therefore each one contain the entire body of genetic information present in the chimera. the exploration in a given individual and thereby under the influence of identical environmental factors. clones and transgenics represent extraordinarily powerful tools to part in experimental biology. can develop to term. Finally. This can be achieved by placing two morula-stage embryos in direct contact (following removal of their zona pellucida). Thus. but a unique individual. nor a hybrid.Chimeras. of the fate of cells having different genetic compositions. or be very markedly skewed in favor of one or the other. and each chimeric individual is therefore different. It can be expected that chimeras will also be of importance in the study of aspects of the pathogenesis of genetic diseases: they allow. the contribution of the two partners can be approximately which. the generation of chimeras is one step in the production of transgenic mice by . and thereby in understanding how adult tissues are built during development. and does not that chimerism is not hereditary: each cell of the chimera. Furthermore. or by introducing cells from a " or in the blastocoelic cavity (in contact with the ICM) of a blastocyst-stage donor " embryo either underneath the zona pellucida of a morula-stage recipient. Depending on the balanced. recipient. The cells from the two embryos assemble to form a single chimera circumstances. derives from either one or the other partner. It is important to realize of its germ cells. Chimeras can be very useful in defining cell lineages. unravel multiple aspects of physiology and pathology. the generation of a chimera does not produce a member of a new species. for instance. when placed in a foster mother. and thus play a growing Chimeric embryos Chimeric embryos (also known as allophenic or tetraparental) are generated by " mixing " cells obtained from genetically different embryos (for reviews see 29). with a comparable proportion of cells from each origin in all tissues.
It is possible because of the totipotency of nuclei from morula stage embryos in these species. in which nuclear potency appears to become more rapidly restricted.38). and the mitochondrial genes that will be required throughout life) brought by the oocyte is in each case different. Two distinct experimental approaches have been used to produce animal embryos in multiple genetically identical copies.or 16-cell stage are separated from each other. including mice. for instance by mixing cells from Clones Clones are groups of cells or of individuals that are genetically identical. which can which uses a procedure of nuclear transfer. This genetic identity is however not complete. The nuclear transfer cloning procedure allows the generation of multiple twins. produced between animals of different species. Monozygotic twins thus form a clone. from embryos at the 8.32. and does not seem possible for other species. the restriction of . the mechanisms of development compensate for the initially small size of the two resulting embryos. Multiple cloning has been achieved for sheep (51). the cells of an early embryo are separated into two groups. The resulting fused cell can develop into an entire animal. which will be with other enucleated unfertilized eggs. cattle (35. In any event. Chimeras have been goat and sheep embryos (14. while the first procedure is in this respect more limited. In the first such approach. that is technically quite simple and that mimics the " natural " mode of generation of monozygotic twins. The second approach. is technically more complex: cells grow into two distinct but genetically identical individuals.homologous recombination. since the maternal contribution (the maternal mRNAs that are genetically identical to its twins generated by the fusion of the sister blastomeres necessary for the early stages of development. and this will be detailed later. and each of these individual blastomeres is made to fuse with an enucleated unfertilized egg.41) and rabbit (42) embryos.
see 52). Clearly the socio-ethical aspects of such cloning Transgenic animals Transgenic animals carry an alteration of their genetic information. procedures should be carefully evaluated. Among the different approaches used to generate transgenic animals. and is thus heritable. or due to the insertional modification of an endogenous gene. that will be present only in the progeny of the transfected cells but not in the germ generation of a transgenic animal. Similarly. with more uniformly predictable properties. herds of genetically identical farm animals. transgene injection into the zygote. including the germ cells. may have economic advantages. and homologous recombination in embryonic . which would be much more difficult to explore by the usual inbreeding techniques. this difference is precisely that which line and therefore that will not be transmitted to the animal’s offspring. This alteration is most often In this respect there is a crucial difference between the grafting to an adult present in all cells of the organism.nuclear potency occurs early in mammalian development. It is easy to envision the potential of this approach in the study of multigene disorders. 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. both technically and ethically. 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. somatic gene therapy and germ line gene therapy. and the separates. two are at present most relevant to experimental mammalian biology: stem cells (for a detailed review on these issues. and it is thus not at present possible to perform cloning with cells from later embryos or from adults. animal of a cell population that has been transfected with a foreign gene.
The embryos are then transferred to foster mothers.Transgene injection is performed in one (generally the male) of the pronuclei present in 1-cell embryos. that is encountered to different degrees depending on the transgene. One major difficulty. and will direct expression in multiple cell types. and will thus also be transmitted to the progeny. This implies that multiple transgenic families carrying the same provided. with cells that do and cells that Although a transgene can be present in all cells of the organism. 10-20% of the injected embryos that survive the manipulation being transgenic. and allowed to develop. In a limited number of transgenes. and tissue-specific promoters allows to direct transgene expression to selected cell perhaps also on additional post-transcriptional regulatory influences. the cells in which it is expressed depend on the promoter that drives its transcription. at the same chromosomal site. that allow transgene expression to be modulated by exogenous influences. but do not contain the transgene. in all cells of the organism. In certain cases the transgene appears not to integrate in chromosomal DNA at the zygote stage. later. certain promoters have a broad specificity. including the germ cells. and often in multiple tandemly-organized copies. It is also important to note that the site of integration of a transgene can . probably because large enough regions of the transferred gene had been used so that appropriate regions of attachment to the nuclear scaffold were be within a normally expressed gene. this can be due to cases it has been possible to obtain position-independent expression of the site of integration or to other epigenetic phenomena. hence the resulting animal may be chimeric. The currently-used procedures result in approximately The transgene usually integrates in the chromosomal DNA at the 1-cell stage. is a marked variability in its’ level of expression or penetrance. it is present. and integration may then have phenotypic effects that do not result from transgene expression but from insertional mutagenesis. The use of types. probably at a random site. A limited number of inducible promoters are available.
and can be achieved by culture in the presence of fibroblast feeder reason. has attracted enormous attention. see 34). since this produce and select mutations that can subsequently be propagated to generate lines of transgenic animals (for a review. ES cells can have a normal karyotype. but.transgene must be studied to unambiguously identify the effects of transgene expression. New-born mice entirely derived from ES cells have been obtained. despite its relative ease and great experimental potential. under conditions that prevent their differentiation. can contribute to all tissues. of conditioned culture medium from certain cell lines. occurs only with very low frequency. transgenesis by pronuclear injection has severe limitations. These limitations are akin to those that. opens the possibility of using transfection in culture and somatic cell genetics to The possibility of achieving the specific alteration of a targeted gene by homologous recombination between the endogenous gene and an introduced DNA sequence. The uncertainties underlined above indicate that. sequence in the targeted gene. and. or of the cytokine LIF. render for the time being germ line gene therapy a totally Embryonic stem (ES) cell lines Embryonic stem (ES) cell lines can be established by culturing cells collected from peri-implantation blastocysts. Thus. The capacity of ES cells to colonise the germ line is their most relevant quality in the context of transgenesis. selection . undergo early postnatal death. together with the development of ES cell lines and techniques to Homologous recombination that can lead to insertion of an exogenous DNA produce germ line chimeras from these ES cells. in addition to the critical unrealistic proposition. ethical issues involved. for an undetermined This is critical. layers. or to the replacement of a fragment of the targeted gene by the exogenous DNA. when introduced into normal embryos to generate chimeras.
The changes brought about by natural evolution take place over thousands of years. in principle. 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 after generation of chimeric animals containing ES derived cells in their germ homozygotes. their already been studied in this way. The overall procedure is technically demanding and labour intensive. It is the possibility of working with cell lines that renders selection possible. Animal breeding . 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. makes it statistically impossible to obtain Homologous recombination in ES cells has until now mostly been used to generate mutations that result in preventing the production of given gene products. at targeted genetic alterations by the pronuclear injection approach. heterozygous mice can be obtained. and breeding allows the production of information on the role of any given gene in vivo. i.procedures are necessary to identify and isolate the cells that have undergone the desired recombination event.e. at least in the setting of the laboratory. the low frequency of homologous recombination. line. Several dozen genes have genes which were expected to be essential are in fact dispensable. allow the study of any desired mutation in any gene. but it can provide unique of the gene. and requires many months of work. Improvements in the vectors used for homologous recombination should. one surprising result has been that quite a few function is not required for the development and reproduction of mice. least with the presently used techniques. The transfected ES cells are in general heterozygous for the mutation. Conclusion 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.
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