Physiological aspects of growth and development of the preimplantation mammalian embryo.

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Annu. Rev. Physiol. 1976.38:95-119. Downloaded from www.annualreviews.org Access provided by University of California - Davis on 01/25/15. For personal use only.

PHYSIOLOGICAL ASPECTS OF

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GROWTH AND DEVELOPMENT OF THE PREIMPLANTATION MAMMALIAN EMBRYO John D. Biggers and Raymond M Borland Department of Physiology and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts 02115

INTRODUCTION This review deals with the physiology of the preimplantation stages of pregnancy, with particular emphasis on the mammalian embryo between fertilization and im plantation. Fertilization takes place in the ampullary region of the oviduct and implantation takes place in the uterus. The passage of the embryo from the ampulla to the uterus is called the tubal journey; throughout this period the embryo can easily be flushed from the female genital tract without disrupting any fixed morpho logical relationship between it and its mother. During this free-living period, the embryo undergoes first the cleavage divisions and then becomes regionalized into the trophoblast and inner cell mass, which enclose the blastocoele fluid to form the blastocyst. The general background for this area is provided by several recent textbooks and monographs (4, 8, 19, 68, 69, 71, 115). The time of the tubal journey varies according to species (17). Moreover, the free-living period and tubal journey are not necessarily coincident because of the phenomenon of delayed implantation which occurs in some species (55, 138). The embryo is the hub of this system. An analysis of the physiology of the preimplantation embryo, however, is complicated; during its journey to the uterus, the embryo follows an internally programmed sequence of development while mov ing from one environment to another. Thus the potential for exchange between the embryo and its environment depends both on its stage of development and its location. The developmental sequence followed by the embryo is an irreversible process. Studies on the embryos of oviparous species indicate that the developmental se95


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quence follows a program determined partly by the transcription of the maternal genome during oogenesis and partly by the reading of the new genome after fertiliza tion (50, 67, 1 20). The available evidence also suggests that qualitatively similar processes occur in mammals (19, 36). A major part of this review deals with recent biochemical work on these developmental processes in the preimplantation embryo. The literature covered is primarily that published since January 1973. However, it has had to be selective. Until recently, this area has been largely descriptive, first in morphological and then in biochemical terms ( 1 7). We have tried to present the information in· functional terms, raising, for example, questions concerning the nature of the control mechanisms, the role of transport mechanisms, and the effects of metabolic pools and compartmentalization. We feel that such approaches are essential if further fundamental progress is to be made in understanding the initial stages of pregnancy. ANABOLIC AND CATABOLIC PROCESSES

The volume of the preimplantation embryo of several species falls during the initial cleavage divisions (17, Table I). In contrast, during the growth of the blastocyst, the volume increases (14); in some species, such as the rabbit, the increase in volume is large and rises exponentially. Table I also shows that a significant fall in total protein occurs during the initial cleavage divisions of the mouse in parallel with the decrease in volume. It has been argued from this data that catabolic processes predominate during the early cleavage stages. Recent studies on the total dry weight of preimplantation mouse embryos using a quartz-fiber balance (74, Table 1) and interference microscopy ( 1) indicate that more complex changes occur during the initial cleavage stages. The absolute values observed differ considerably between methods. Nevertheless, both studies show that no change in dry weight occurs after the first cleavage division. It is clear that if proteins are lost during this period other substances must replace them. One conTable 1 The volume, dry weight, total protein, DNA, and RNA per embryo in several stages of preimplantation development of the mouse. (The stages have been approximated

for the dry weight and nucleic acid data as the authors timed the stages in terms of the

time of HCG injection.)

Stage

Volu me (nl) a

Dry weight b (ng)

Total protein (ng)C

DNA

RNA

(ng)d

(ng)d 0.55

Unfertilized ovum

I-cell fertilized

0.192

23

27.8

29.3

2-cell

0.158

24.2

26.1

41.1

8-cell

0.138

morula - early blastocyst

0.219

a From

22.6

measurements of projections (86).

bFrom measurements with a quartz fiber balance (74). CUsing Lowry's method (26). dUsing fluorometric assays (100).

0.40

23.4

155

0.46

20.6-23.9

439

1.37


PHYSIOLOGY OF PREIMPLANTATION EMBRYOS

97

tributor is glycogen, which is rapidly accumulated between the 1- and 8-cell stages (101, 121). The contribution of other substances has yet to be determined. A significant fall in dry weight of the ovum has been demonstrated between fertilization and the first cleavage division ( 1 ). The initial loss could be caused by the cortical reaction and the extrusion of the first polar body, which follow sperm penetration. These possibilities emphasize that loss of cytoplasm may also contrib ute to changes in the volume and mass of embryos during the initial cleavage divisions. A phenomenon known to classical embryologists. called deutoplasmolysis -meaning elimination of yolk (24)-should always be considered in accounting for the gross loss of material during the cleavage stages. The contents of several macromolecules change during preimplantation develop ment (Table I). The list includes complex, heterogeneous mixtures such as total protein, and RNA; substances that may be compartmentalized, such as DNA found in both the nucleus and cytoplasm ( 1 00); and specific substances such a glycogen and enzymes, which may or may not be compartmentalized. Perhaps the amount

of RNA is the simplest case to consider. The mean values of RNA measured in the unfertilized ovum, 1-, 2-, 8- 1 6-cell and blastocyst stages of the mouse demonstrate that no significant change in RNA per embryo occurs until after the 8-cell stage (100). Similarly, in the rabbit, the amount of RNA per embryo changes little until the 16-cell stage (90). In both species, the RNA per embryo then rapidly increases. Thus the increase in RNA occurs at about the same stage in both species. A simple interpretation of these results is that RNA synthesis is low during the early cleavage stages and that it increases rapidly at the early morula stage. The amount of several enzymes has been measured in several preimplantation stages of the mouse ( 1 7, 27, 57). Some of these enzymes increase, others decrease, some decrease and then increase, some increase and then decrease, and some do not change. The results summarized by Epstein (57) are based on the total enzyme activity assayed under optimal conditions. The results are therefore estimates of the total specific protein at each stage and do not reflect the functional activity of the enzymes. Nevertheless, the results are significant physiologically in that they demonstrate that simulta neously some proteins may accumulate whereas others may be lost. Thus major shifts in the patterns of accumulation and loss of proteins may occur during develop ment; presumably such changes are correlated with the amounts of different The balance between synthesis and breakdown of macromolecules in a preimplan tation embryo can be usefully analyzed by compartmental analysis (5). At present a two-compartment model may be appropriate (Figure I). where QI is the amount Exogenous preformed product

M ---��

Figure 1 embryo.

\3;

lIk" 1 2 ----==-=--)o� ---

lURU

�

�

20 IIkll

0

2

)0

Two-compartment model of macromolecule metabolism in the preimplantation

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of precursor, Q2 is the amount of product, R20 is the rate of transport of preformed exogenous product directly into the embryo, k21 is the rate constant for conversion of precursor to product, and k02 is the rate constant for removal of the substance from the embryo. The behavior of Q2 is described by the differential equation


I. The component represented by Rzo provides for the uptake by the embryo of a macromolecule present in the environment, for example by pinocytosis. There is ample evidence from ultrastructural studies that preimplantation embryos can as similate materials by this route (72). To what extent this process is important under normal conditions is unknown. However, it may be relevant that the isthmic region of the mouse oviduct secretes glycogen into its lumen at a time when resident 2-cell embryos rapidly accumulate this substance (109). Also uteroglobin (blastokinin), a protein of maternal origin, accumulates in the blastocoele fluid (10) after the differentiation of tight junctions that prevent the entry of macromolecules between the outer trophoblast cells (52, 73). The third term in equation 1, k02Q2, represents the rate of degradation of the particular substance. Little is known about such processes other than that they occur. The loss of several enzymes and glycogen during development in vivo of the mouse implies that degradation can become the dominant term. A particularly interesting example is the partial replacement of lactate dehydrogenase I by lactate dehydrogenase V during the cleavage stages of the mouse (7). Most attention has been focused on the second term of equation 1, with special reference to the overall rates of RNA and protein synthesis. In the earlier work in this area (17), embryos of different stages were exposed for short times to labeled precursors, usually pH]uridine for RNA and pH]amino acids for protein. The labeled macromolecules were detected by autoradiography or the uptakes quantita tively. measured by scintillation counting. The rate of incorporation of pH]uridine into RNA of mouse embryos is low until the 8-cell stage, at which time there is a marked increase. A major part of the precursor is incorporated into rRNA (54). In the rabbit embryo, the correspondence is less precise because uridine incorporation into RNA is low until the transition from morula to blastocyst (90). Such studies support the view that RNA synthesis is low until a critical stage of development. The results show in addition that synthesis of proteins occurs at all stages ·of preimplantation development of the mouse (58). Nevertheless, the rate of protein synthesis is low until the 8-cell stage, after which it rapidly increases. The reality of this pattern of protein synthesis is also supported by the observations that fertil ized ova contain very few ribosomes, whereas morulae and blastocysts are abun dantly supplied with ribosomes and polyribosomes (33). Similar studies on the preimplantation stages of the rabbit suggest a slightly different pattern in the rate of protein synthesis in that it remains low until the blastocyst has formed (day 3 postcoitum) after which a rapid increase occurs (80).



99

Recently, it has been pointed out that a comparison between stages of the uptake of precursors into macromolecules is not simple (21, 58). An estimate of the rate of incorporation of a labeled amino acid into protein is a measure of the rate of protein synthesis only if adequate concentrations are available at the intracellular site of synthesis. Should the supply of a labeled precursor be limited by the permea bility of the cell or by dilution by a large endogenous pool, the rate of protein synthesis estimated from the amount of isotope incorporation may be seriously in error. The observed differences in the incorporation of an amino acid between developmental stages could, in fact, be the result of complex differential changes in cell permeability, the size of the endogenous pool, and the rate of protein synthesis. The existence of a significant amino acid pool in preimplantation mouse embryos should be kept in mind because protein synthesis occurs in the absence of any exogenous amino acids (35), and single amino acids such as [lH]leucine and pH]methionine are incorporated linearly with time in the absence of any other endogenous amino acids or amino nitrogen source (21). Recent studies on the rate of protein synthesis in the preimplantation mouse embryo have tried to avoid these difficulties (21, 58) by determining for each stage the relation between accumulation of the labeled amino acid into the trichloroacetic acid-soluble pool and into the macromolecule. The uptake of the labeled amino acid into the acid-soluble pool continues to rise at very high concentrations of the precursor, whereas the incorporation into protein reaches a maximum at relatively low concentrations. It is thus possible to find a concentration of precursor that does not limit the inherent rate of protein synthesis. Using this method, Epstein & Smith (58) have shown in the mouse that, prior to the 8-16-cell stage, there is a low level of protein synthesis after which a marked increase occurs. Borland (21) has also demonstrated in the mouse a 2-8-fold increase in protein synthesis between the 4-cell and early blastocyst stage. By expressing incorporation rates at each stage on a per cell basis, it becomes clear that the early cleavage stages of the mouse, between the 1- and 8-16-cell stages, do not increase protein synthesis in proportion to the increase in cell number. This finding suggests that part of the synthesis taking place during this period is deter mined by preexisting messengers partitioned to the cells. In contrast, between the 8- l 6-cell stage and the early blastocyst stage, there is a 4-6-fold increase in synthesis and a 3-4-fold increase in cell number. Thus this burst of protein synthesis may represent little more than a relatively constant rate of synthesis per cell over a period of 2-3 divisions. Nevertheless, the overall change still represents a departure from the pattern at earlier stages (58). In the rabbit, no increase in incorporation occurs until blastocyst formation, at which time the rate of incorporation per cell increases 8-IO-fold (80). Despite an increase in cell number, the rate per cell remains constant during the blastocyst stage. The developmental changes in protein synthesis in early mammalian embryos differ considerably from the pattern present in lower organisms. In sea urchin embryos, within minutes after fertilization, there is a 2.5-5-fo1d increase in the rate of protein synthesis (56, 129). Similarly, in the mollusc, Iflyanassa, there is a 5-fold

./


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increase in leucine uptake and a 12.5-fold increase in incorporation into protein by two hours after fertilization (97). Recent work indicates that specific proteins are synthesized at certain stages of development (59), although the full spectrum is unknown. The electrophoretic pattern of proteins synthesized by preimplantation mouse embryos on days 1-4 has been analyzed using electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate (SDS). For comparisons between stages, the embryos were incubated pairwise in sufficiently high concentrations of PH]- and (14C]-labeled amino acids to minimize the effects of endogenous pools. The two groups of embryos were then combined and the proteins isolated and electrophoresed simultaneously. Although the resolving power of polyacrylamide gel electrophoresis is limited, 2()...25 labded protein components of apparent molecular weights 25,000-1- 15,000 were identified, five of which were dominant. The rates of synthesis of three proteins increased with development, one decreased, and the other did not change. The comparisons showed that the greatest changes occurred between the 2- and 8-16-cell stages. The finding is unexpected as this is a period when only a slight increase in the rate of protein synthesis occurs; however, it does coincide with the period when the morphology of the mitochondria undergoes major changes (122). There were fewer differences between the 8-16-cell stage and the early blastocyst stage, with an increase in a single protein being the only major event. Between the early blastocyst and late blastocyst (day 4) stages, there was again only one major change, a decrease in a protein peak at 67,500 daltons. Possibly these changes represent the synthesis of major, quantitatively important structural proteins such as microtubular protein and membrane constitutents. It is relevant that the synthesis of microtubular protein with a molecular weight of 52,000 has been demonstrated at the l 6-cell stage in sea urchins (78, 108). The quantitative patterns of protein synthesis in preimplantation rabbit embryos grown in vivo and in vitro have been measured using SDS polyacrylamide gel electrophoresis followed by autoradiography (131). These results show that most of the quantitative changes in the pattern of protein synthesis occur during cleavage (day 0.5-3, postcoitum), that the blastocyst period (day 3-6) is characterized by a uniform and constant pattern of protein synthesis, and that the quantitative pattern of protein synthesis in embryos cultured from the I-cell stage to the blastocyst stage is identical to the pattern in vivo. The most obvious changes in the pattern of protein synthesis occurs during the first few days of development, especially between days 0.5-1.5. Of particular interest are the protein bands at 200,000, 51,000, and 45,000 daltons that appear after day 1.5. The band at 45,000 daltons is quite dense by day 2.5 and represents several percent of the total protein synthetic activity of the embryo. Preliminary studies have indicated that these bands are probably myosin, tubulin, and actin, respectively (131). From days 3-6, during blastocyst expansion, the pattern of protein synthesis remains remarkably uniform and constant. with the exception .of two bands. Petzoldt (102) has separated the soluble proteins from early rabbit blastocysts and, in later stages, from the embryoblastic and trophoblastic parts. Twenty or more proteins were detected. At the beginning of blastocyst formation, there is an increase


101

in protein synthesis in the embryoblastic part. The protein pattern also changes qualitatively. These changes nearly coincide with the passage of the embryo through the uterotubal junction and thus with possible changes in the environment. No changes were observed in the spectrum of detectable proteins during the rapid growth and rapid accumulation of proteins that occurs in blastocysts developing in utero.


CONTROL OF PROTEIN SYNTHESIS

The control of protein synthesis in early mammalian embryos is not understood. A priori, there are several physiological conditions that could regulate protein syn thesis: (a) the synthesis and availability of mRNA, rRNA, and tRNA could be limited by low RNA polymerase activities, (b) the nature and availability oftRNAs may control both rRNA and protein synthesis, and/or (c) the supply of ribosomes may limit translation of existing mRNAs. There are at least three species of RNA polymerase having specific subcellular localizations (111): polymerase I is present primarily within the nucleolus and is involved in the synthesis of rRNA; polymerase II is localized in the nucleoplasm and is associated with the synthesis of DNA-like RNA; and a third minor polyme rase, whose transcriptive properties are still unknown, is present in the nucleoplasm. Total RNA polymerase activity as measured by total P H]UTP incorporation in vitro with an embryo homogenate is very low in unfertilized eggs and in I-cell mouse embryos ( l18). This activity increases in an exponential fashion during subsequent development. From the 2-cell to the early blastocyst stage, RNA polymerase activity increases 8-fold, in contrast to a 30-fold increase in number of cells per embryo. This difference indicates that the amount of RNA polymerase per cell actually decreases with embryonic development at the same time that the rate of RNA synthesis increases faster than the number of blastomeres (99). No apparent correlation can be detected between the enzymatic activity present at a certain stage and the amount of RNA synthesized. Two points must be remembered in evaluating this data: 1. total RNA polymerase activity was measured, and a totally different pattern may exist for each of the three polymerases, and 2. true RNA synthetic rates are not known for day 2 of development (45). An estimate of the relative activities of RNA polymerase I and II in mouse blastocysts has been obtained by measuring the RNA polymerase activity in vitro in the presence or absence of a-amanitin, an inhibitor of RNA polymerase II (137). The assays were performed at high ionic strength, which favors the detection of RNA polymerase II, and at low ionic strength. The enzymes are inhibited 6% at low ionic strength and 35% at high ionic strength, indicating that more RNA polymerase I relative to RNA polymerase II is present in blastocysts. This result, as well as the effect of a-amanitin on development in vitro (see below), provides evidence for the presence and function of both forms of polymerase in preimplanta tion mouse embryos. The role of newly synthesized RNA in early mammalian development is not clear. The sensitivity of the I-cell mouse embryo to inhibition by actinomycin D and a-


102

BIGGERS & BORLAND

amanitin has suggested that normal development depends upon immediate genomic activation following fertilization (66, 96, 128). Other studies show that a-amanitin can arrest development of mouse embryos at the 2-cell stage ( 1 37). Although 67% of the untreated embryos developed to the morula or blastocyst stages, less than 1 % of the embryos exposed t o I p- g ml-1 of a-amanitin developed to the morula or blastocyst stage. Thus 1- and 2-cell mouse embryos probably express their own genome; moreover, the class of RNA synthesized during the first cleavage division is probably mRNA, as there are no mature nucleoli present at the I -cell stage (76) and the earliest demonstration of rRNA synthesis has been at the 4-cell stage (8 1 ). In the cleaving rabbit embryo, actinomycin D causes an immediate arrest of cleavage, whereas a-amanitin allows limited further development (9 1 ). A decreasing ratio of amino acid incorporation caused by continuous exposure of cleaving rabbit embryos to a-amanitin suggests that a relatively homogenous embryonic mRNA supports early protein synthesis and turns over with a half-life of approximately 24 hr. Polysome associated mRNA containing polyadenylic acid sequences are detect able from the 1 6-cell stage through the blastocyst stage in preimplantation rabbit embryos ( 1 1 3). The metabolic stability of this polysomal poly(A) containing mRNA in 6-day rabbit blastocysts has been examined under conditions which do not involve the use of inhibitors of RNA synthesis ( 1 14). Because the rRNA is stable and increases linearly with the time, mRNA decay could be analyzed by comparing the ratio ofmRNA to 18S RNA labeling. Such a comparison has shown that mRNAs can be divided into two groups: those with a half-life of approximately 7 hr and those with a half-life of approximately 18 hr. Unfortunately, the 2-cell rabbit embryo does not synthesize rRNA (89) and this technique could not be applied to early cleavage stages. Because the half-life of mRNA molecules in rabbit blastocyst is long (as in growing mammalian cell lines), rapid changes in rates of protein synthesis probably depend on control mechanisms which operate at the translational level rather than at the level of transcription. The nature and synthesis of tRNAs could provide this translational control. Limited information is available on the presence of tRNAs in mouse embryos. Different stages have been incubated in the presence of labeled uridine and the classes of RNA separated on columns of methylated albumin keiselguhr (MAK) (54, 1 39). Several classes of RNA are separable by the 8-cell stage. The first major component eluted from the column contains soluble RNA (sRNA). The sRNA has been further separated on Sephadex G- 1 oo columns into 4S and 5S fractions. The sRNA from 8-cell mouse embryos was found to have a high proportion of 5-ribosyl uracil, which suggests that it contains tRNA. No further studies have been under taken on the nature of tRNAs and their synthesis in the mouse. In the rabbit, however, Manes & Sharma (92) have used simultaneous labeling of embryonic ' tRNA with [32Pl phosphoric acid and L-[methyPHlmethionine to faci litate a com parison between the rate of synthesis of the nucleic acid polymer and the rate and extent of its subsequent methylation in the intact embryo. Transfer RNA methyla tion decreases approximately 6-fold between the 52nd and 84th hour of embryonic development, a period coinciding with blastocyst formation, increased protein syn thesis, and increased ribosome production (80).



103

The hypermethylation of tRNA in the cleaving rabbit embryo resembles that demonstrated in transformed and neoplastic cells (20). The methylated bases of tRNA are known to be determinants in amino acid acceptance (117), codon re sponse (34), and ribosomal binding (61). Thus decreased methylation in rabbit embryos may be directly and causally related to the elevated protein and ribosome synthesis that occurs with blastocyst formation. Bernstein & Mukherjee (12) have accumulated evidence that a cytoplasmic factor is involved in the regulation of RNA synthesis in the early mouse embryo. By producing heterokaryons between the 2- and 4-cell stage mouse embryos and adult somatic cells using Sendai virus-mediated cell fusion, it was found that the cyto plasm of 2-cell blastomeres contains factor(s) that inhibit RNA synthesis in previ ously competent somatic cell mouse nuclei. Inhibitory factors were largely lost by the 4·cell stage. The nature of this cytoplasmic factor, however, is not known, but the possibility that it is a tRNA should be considered. An additional point of control of protein synthesis could be the availability of ribosomes. Highly ordered lattice-like structures in the cytoplasm of mouse oocytes are probably ribosomes stored in an inactive form (32). They may be released after fertilization to be used for protein synthesis until the embryo can make its own functional ribosomes. Some protein synthesis occurs in the mouse embryo shortly after fertilization, i.e. before rRNA synthesis begins, and this suggests that maternal ribosomes are functional during these very early stages of development. Although rRNA synthesis begins at the 4-8-cell stage in the mouse and rat (125), this does not, in itself, indicate that functioning ribosomes are being produced. Little is known concerning the synthesis of ribosomal proteins during the preimplantation period or how quickly new ribosomes are formed or are capable of functioning in protein synth�sis. Tasca & Willis (126) obtained biochemical evidence for the assemblage of ribo somes from newly synthesized rRNA and ribosomal proteins (rPr) during mouse cleavage. Large numbers of synchronously developing embryos in vitro were labeled with either [14C]amino acids or [5-3H]uridine to label rPr and rRNA respectively. Ribosomes of labeled embryos were coextracted with mouse liver ribosomes to provide optical density markers for 40S and 60S ribosomal subunits on sucrose gradients. The 40S and 60S marker regions were heavily labeled with both (I4C]amino acids and [5-3H]uridine within 6 hr, as early as the 8-cell stage. To ensure the removal of nascent proteins from the two subunits, 0.2 M NaCI was used in the extraction of the ribosomal subunits. The rate of rPr synthesis relative to general protein synthesis was expressed as the percentage of all incorporated labeled amino acid entering the two ribosomal subunits. In the early 8-cell stage, only 2% of the incorporated amino acids entered ribosomal proteins. In contrast to this low rate of synthesis, at the late 8·cell, morula, and blastocyst stages, 6-7% of the incorpo rated label entered ribosomal proteins. At these stages, the rate of rPr synthesis relative to the rate of general protein synthesis approaches that found in growing cell cultures, both mammalian and bacterial. The (l4C]amino acids are associated with both subunits and separation of the rPrs by acrylamide gel electrophoresis (R. J. Tasca and D. W. Willis, personal communi-


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cation) has indicated a low level of labeling throughout the gels. Presumably, rPr synthesis is coordinated with rRNA synthesis. These data correlate well with elec tron microscopical studies, which show an increase in the number of poly ribosomes as cleavage progresses (33) and as the nucleolus progressively differentiates (76). In the rabbit, Karp, Manes & Hahn (80) have shown that protein synthesis markedly increases at the morula stage of rabbit development (60-hr embryo). In the late morula, an increase in cytoplasmic ribosomes is observed, suggesting that ribosome availability may be rate-limiting for protein synthesis during cleavage. As shown by radioautography (3H]amino acids become highly localized within the nucleoli of late morulae that have been pulse labeled for 10 min. This localization indicates that rPr synthesis increases at the same time as rRNA synthesis increases. Concurrent synthesis of rRNA and rPr in preimplantation embryos is similar to the situation in L cells (37), where rPrs must be synthesized concurrently with rRNA in order to be used in the formation of ribosomal subunits, which are assembled in the nucleolus (116). Work with rat skeletal muscle and bacteria suggests that protein degradation, as well as protein synthesis, is regulated by the same mechanisms that control rRNA synthesis, possibly by a lack of complete complement of aminoacyl tRNA (65). Existing evidence indicates that the production of ribosomes in bacteria is dependent on the intracellular supply of aminoacyl tRNA (53), and that when a cell lacks a full complement of charged tRNA, synthesis of rRNA ceases (65). GENERATION OF ATP

One general measure of the overall metabolic activity of the preimplantation embryo is its adenylate content. The ATP and ADP contents of several stages of preimplan tation mouse embryos have been measured (62, 104) using the firefly luciferase assay. The I-cell embryo is characterized by a high concentration of ATP, which decreases gradually through the blastocyst stage. Most importantly, the ATP/ADP ratio is very high in early stages. In this respect, the mouse ovum resembles the ovum of the teleost fish (140) rather than that of the sea urchin or sea squirt (49) with regard to the levels of ATP present before and after fertilization. In the ovum of the sea squirt and sea urchin, a marked decrease in ATP occurs immediately after fertiliza tion. The effect of culturing mouse embryos on the ATP and ADP contents has been determined ( 1 05). Both S-cell and morula stage embryos cultured for 24-48 hr in medium containing pyruvate and lactate have greater amounts of ATP than em bryos cultured in either energy substrate-free medium or medium containing glu cose. At the 1 - and 2-cell stage, a combination of pyruvate and lactate is more effective than either energy substrate alone in maintaining the levels of ATP during a 6-hr culture period. As development progresses, the higher levels of ATP in embryos cultured in glucose than in embryos cultured in energy substrate-free medium indicate that glycolysis plays an increasingly important role in ATP syn thesis.



105

During short-term culture of the early mouse embryo, only pyruvate and lactate, but not glucose, can maintain levels of ATP equivalent to those in embryos in vivo. Therefore, it seems probable that oviducal mouse eggs derive most of their energy from the oxidative phosphorylation of pyruvate and lactate (70). Unlike the mouse, large increases in ATP do not occur until the blastocyst stage in the rabbit (28). Ginsberg & Hillman (62) have determined the rates of synthesis and turnover of ATP in 2-, 4-, 8-cell, late morula, and late blastocyst stage mouse embryos in vitro. Progressive decreases in total ATP and the ATP/ADP ratio from the 2-cell stage to the late blastocyst stage are accompanied by increases in the rates of ATP turnover and ATP synthesis. Significant differences in the levels of ATP synthesis between preimplantation stages suggest that an altered concentration or availability of energy coupling sites during development might account for the observed differ ences (63). The actual and the potential activities of the cytochrome system were studied in cleavage-stage mouse embryos by assaying for total ATP and rates of [32P] ATP synthesis both before and after incubation in medium supplemented either with an energy coupling site inhibitor: amytal (site I active), antimycin (site II active), cyanide (site III active), or with the FADH-linked substrate succinate. None of the coupling site-specific inhibitors produces more than a 20% inhibition of total ATP at the 2-cell stage. At the 4-8-cell stage, the inhibiting effects of the three agents rise markedly. Assuming that the permeability of the different-stage embryos to the inhibitors is constant, this rise could be caused by a higher rate of ATP metabolism at the late 4-cell stage compared to the 2-cell stage. The level of aerobic ATP synthesis at the 2-cell stage suggests that the activity of the citric acid cycle is low. Addition of succinate to the medium bypasses the NADH-Iinked isocitrate dehydrogenase (IDH) (EC 1 . 1. 1 .4 1 ) and stimulates ATP synthesis, in dicating that low citric acid cycle activity is due to a low level of IDH activity. Beginning at the late 4-cell stage and continuing through the 8-cell stage, the ATPIADP ratio decreases, IDH activity increases, and succinate no longer stimu lates ATP production. A second shift in metabolism occurs between the 8-cell and late morula stage of the mouse. Although a large increase in O2 uptake starts at the late morula stage (95), no accompanying increase in ATP synthesis occurs (62). These conditions of high O2 uptake and low ATP synthesis suggest that late morulae mitochondria are uncoupled. The three inhibitors are less effective at this stage than at the 8-cell stage (63) and ATP synthesis probably occurs predominantly by glycolysis. Net ATP synthesis and sensitivity to the three inhibitors increase at the late blastocyst stage, indicating that the citric acid cycle is again functionaL These changes in metabolism are well correlated with structural changes in the mitochondria of the preimplanta tion rat (93), rabbit (3), and mouse ( 1 22). In the mouse, mitochondria contain few transverse cristae and have a dense matrix up to the 4-8-cell stage, at which time they appear to undergo a transition to a more orthodox structure that is finally realized in the morula and maintained in the blastocyst. Although it is known that the turnover of ATP increases with development, the mechanisms by which ATP is utilized are obscure. Studies from other systems

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indicate that at least four means of utilizing ATP can be highly significant: I. for microtubule assembly (49); moreover tUbules may be synthesized in preimplantation rabbit embryos, 2. for amino acid activation (98), 3. for phosphorylation processes such as the synthesis ofNADP fromNAD+ (2), and 4. increased activity of various ATPases (112).


METABOLIC PATHWAYS AND THEIR CONTROL

The changes in the ATP content and ATP synthetic and turnover rates in the early mouse embryo suggest the occurrence of major changes in the activity of metabolic pathways. Recent work has helped to elucidate the mechanisms controlling these changes. Barbehenn et al (9) investigated the metabolite levels and enzyme activity in single mouse embryos using the techniques of amplification through enzymatic cycling (87). Hexokinase (Ee 2.7.2.2) is probably not the site of control of glucose metabolism because glucose-6-phosphate levels rise rapidly after refeeding starved embryos, indicating that neither low amounts of hexokinase nor glucose entry into the embryo is a direct cause of the failure of the early embryo to survive on glucose alone. This failure is probably due to inhibition of 6-phosphofructokinase (PFK) (Ee 2.7.1.11). There is little change in the absolute amount of this enzyme from the 2-cell stage to the blastocyst stage measured in vitro under optimal conditions (57). Lack of fulI functional activity of PFK in vivo could be due to the high concentra tions of A TP, a powerful allosteric inhibitor of this enzyme. Among the deinhibitors of PKF, ADP and fructose-6-phosphate probably increase during this period, whereas Pi does not change (134). These factors, and failure of hexokinase to generate sufficiently high levels of hexose monophosphate, may be the true causes of blockage of glucose utilization. The activity of the citric acid cycle has been partially assessed by the effects of succinate on ATP levels (63). Enzyme activity and substrate level measurements (9) indicate that restoration of pyruvate to embryos after starvation causes the citrate and 2-oxoglutarate levels to increase at all stages faster than the initial decline in citrate that follows starvation. In contrast, at the 2- and 8-celJ stage, malate fell transiently during refeeding with pyruvate, indicating a slow rate of conversion of 2-oxoglutarate to malate. At the morula stage, malate does not fall and presumably the slow step is removed.. Regulation of IDH may cause these shifts (63). The complex shifts in the function of metabolic pathways in the preimplantation embryo may be related to the energy charge (15), defined by Atkinson (6) as ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]). The energy charge can range from 0 to 1. When the energy charge is near unity, the proportion of ATP is very high and certain energy generating enzymes such as IDH and PFK are inhibited. The evi dence described previously indicates that both of these enzymes are inhibited in the preimplantation mammalian embryo. Unfortunately no measurements of AMP levels have been made in preimplantation embryos to test this hypothesis. Metabolic function is also controlled by other factors such as the redox states of various oxidation-reduction couples of the cell (84). The redox states of the NAD+ couple and the NADP couple are defined as [NAD+free]![NADHfree] and [NADP+free]![NADPHfree]' respectively. These ratios determine: (a) the direction

PHYSIOLOGY OF PREIMPLANTATION EMBRYOS of reversible reactions,

107

(b) the extent to which pyridine nucleotides are effective as

reducing agents in reactions such as the synthesis of fatty acids and nonessential amino acids, and

(c) the magnitude of the free energy changes of oxidation-reduc

tion reactions such as those involved in the transport of electrons along the respira tory chain. Little work

has been done on the role of NAD+/NADH in early mammalian Elias & van Beuningen (123) showed that NAD+ (0.23 mM) could improve the development of 2-cell embryos in culture in the presence of pyruvate, whereas higher concentrations appeared toxic. The NAD(H) (total NAD+ plus NADH) content was found to decrease during cleavage (85, 124). Recently, the reduced and oxidized forms of NAD in single embryos were mea sured by enzymatic cycling (134). The total content of NAD(H) falls from the I to 2-cell stage. This fall is apparently not caused by conversion to NADP (85). There does not appear to be any overall change in the redox state of NAD-NAD H during development, and 30-40% of the NAD+ pool is present in the reduced form at each


development. Streffer,

stage. These estimates, however, do not differentiate between bound and free nudeo tide or between mitochondrial and cytoplasmic pools. Significant shifts between these pools could be important in controlling metabolism. The values of the redox states are also related to the relative concentrations

of adenylates. Krebs (84) has shown that the phosphorylation state defined as [ATP]![ADPl [Pil is directly proportional to [NAD+rree]![NADHrrtel in the cyto plasm and indirectly proportional to the same ratio in the mitochondria. The study of these interrelations in preimplantation embryos may provide further insight into the control of energy generating and energy utilizing processes in early development. Fortunately techniques for approaching these problems are available (87).

ANABOLIC FUNCTIONS OF METABOLIC PATHWAYS Glucose The activity of the metabolic pathways also affects synthetic processes in the em bryo. For example, culture experiments with chemically defined media have shown that the mouse oocyte, fertilized ovum (18), and 2-cell embryo (25) cannot develop when glucose is the sole carbon source. The 8-cell and morula stages, however, can utilize glucose for

a number of synthetic processes (133). By characterizing the [U-14C]glucose, Wales showed that

labeled products derived from the metabolism of

14C incorporation increases with development, and that only 50% appears in the remainder found in protein lipids, and nucleic acids. Both the 8-cell and morula stage embryos cultured for 24 hr in [U 1 4C)glucose exhibit increasing isotope incorporation into RNA. Moreover, the relative incorpo ration of carbon from glucose into the various classes of RNA differs significantly

acid-soluble fraction, with the

,

-

from the pattern of incorporation of PH]uridine (54). Thus the existence of different precursor pools for the entry of glucose or uridine into the various classes of

RNA

needs to be considered along with differential entry of the compounds into the same precursor pools. The results seriously question the reliance on the incorporation of a single precursor as the criterion for estimating the relative rates of synthesis of

RNA species in the embryo. Subsequent experiments following the incorporation


108

BIGGERS & BORLAND

of CI and C6 labeled glucose into RNA (133) have demonstrated no difference in the incorporation of specifically labeled sugars and have confirmed the insignificant part the hexose monophosphate shunt plays in the metabolism of the mouse embryo (27). Nevertheless nonoxidative interconversion of hexose and pentose phosphates must occur to account for labeling of the sugar moiety in the nucleic acids. Preimplantation rabbit embryos in vitro cannot develop in the presence of glucose alone until the blastocyst stage (47). The uptake of glucose carbon by the embryo increases with development, with a substantial increase in the incorporation of carbon from glucose occurring after blastocyst formation (106). A considerable amount of the label incorporated is present in the embryos as the parent substrate, indicating that the uptake of glucose is greater than its conversion to other metabo lites. Throughout development, glucose carbon is incorporated into acidic and basic acid-soluble compounds, proteins, and lipids. The blastocyst tissue incorporates carbon from glucose into lactate, citrate, and malate, as well as glutamate, alanine, and aspartate, demonstrating the operation of glycolysis and the citric acid cycle. As glucose carbon was also detected in lactate, pyruvate, and acetate in the incuba tion medium prior to the blastocyst stage, glycolysis must occur throughout preim plantation development in the rabbit, contrary to earlier studies indicating little or no glycolytic activity prior to blastocyst formation (60).

Pyruvate and Lactate The ability of lactate to support development of 2-cell mouse embryos in vitro is less than that of pyruvate (25), and the incorporation of substrate carbon from lactate alone, as compared to that from pyruvate, is also lower at this stage (107). This difference occurs up to the morula stage, but after this stage the incorporation of· C2 of lactate is greater than that of pyruvate. The incorporation of both pyruvate and lactate carbon increases with the develop ment of the preimplantation mouse embryo (107). Before the morula stage, the presence of unlabeled lactate in the medium depresses incorporation of C2 of pyru vate, whereas the opposite effect occurs when embryos are cultured in [2-14C]Iactate in the presence of unlabeled pyruvate. The failure of 1- and 2-cell mouse embryos to develop in culture media containing lactate as the sole energy source may depend ultimately on the high ATPIADP ratio, which probably limits the flux of reducing equivalents through the electron transfer chair. This limitation may cause a gross imbalance in the redox state of the NAD+ and NADH couple when lactate is converted to pyruvate (107). The simulta neous production of excessive amounts of NADH by lactate dehydrogenase, and the inability of the electron transfer chain to regenerate adequate levels of NAD+, may limit the activity of the citric acid cycle (83) to such a degree that it prevents the metabolic conversion of adequate amounts of lactate to various essential intermedi ary metabolites needed for both biosynthetic and energy generating purposes. Carbon Dioxide The distribution of radioisotope fixed from 14C02 has been studied in various mac romolecular fractions of mouse embryos (16). At early stages, the degree of 14C02



109

fixation is small. As development progresses, an increasing amount of 14C02 is fixed direct ly into DNA and RNA (103). No label was found in RNA at the 2-cell stage. At all subsequent stages, all RNA subclasses were labeled. The purine, adenine, was most heavily labeled, but label was also found in the various other bases. Significant fixation of carbon dioxide for the de novo synthesis of RNA probably occurs via the enzymes, ATP:carbamate phosphotransferase (dephosphorylating) (EC 2.7.2.5) and phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21). The simi larity of the pattern of 14C02 incorporation into RNA to labeled uridine incorporation supports the claim that RNA synthesis increases with development. Intermediary Metabolites Although acetate, like citrate, will not support development of2-cell mouse embryos (25), its pattern of uptake is considerably different from that of citrate (135). Al though significant amounts of substrate carbon accumulate from acetate between the 2- and 8-cell stage, there is no change in incorporation. This observation is in contrast to the findings for citric acid cycle intermediates (82) and probably explains why these latter compounds rather than acetate can act as suitable energy sources at this stage (25). Because acetate can enter the cell (136), its inability to support development probably arises either from a limited conversion of acetate to acetyl CoA or from an insufficient endogenous pool of oxaloacetate to act as acceptor for the acetyl CoA that is formed. Malate and oxoglutarate are also unable to support the development of 2-cell embryos (25). The accumulation of citrate, malate, and oxoglutarate in vitro is significantly greater in 8-cell embryos than in 2-cell embryos (82). The low uptake of these three citric acid cycle intermediates by 2-cell mouse embryos compared to later stages may indicate that transport mechanisms in mitochondria are deficient at this stage and become active after the 2-cell stage. TRANSPORT PHENOMENA

Amino Acid Transport Epstein & Smith (58) reported that the uptake of leucine and perhaps lysine in preimplantation mouse embryos is saturable, temperature sensitive, and mediated by active transport systems. No developmental changes in the transport system were described, and the kinetic characteristics of amino acid transport were not exam ined. Borland & Tasca (22) analyzed the transport properties of methionine and leucine in late 4-cell, late morulae, and early blastocysts. Late 4-cell embryos and early blastocysts that had developed in vitro concentrated both amino acids by processes exhibiting saturable, Michelis-Menten-type kinetics, characteristic of car rier-mediated active transport. The uptake is temperature sensitive and inhibited by certain large neutral amino acids competing for the same uptake site. Kinetic analyses indicate that the uptake of methionine is mediated by a single transport system at the 4-cell stage, and by two distinct transport systems at the early blas tocyst stage. There is a 2-2.5-fold increase in Vma. values for methionine and leucine

1 10

BIGGERS & BORLAND

between the 4-cell and early blastocyst stage. Most importantly, leucine, methionine, and phenylalanine transport is Na+ independent at the 4-ceJl stage, methionine transport is partially Na+ dependent at the late morula stage, and the transport of all three amino acids is Na+ dependent at the early blastocyst stage. Specific amino acids are required for outgrowth of the mouse trophoblast cells in vitro. The increased uptake and activation of the Na+-dependent amino acid trans port system may serve to regulate growth mechanisms by controlling the internal


concentrations of nutrients

(77).

The fact that the Na+-dependent transport systems are activated at the late morula and early blastocyst stages suggests that they may be involved in normal blastocyst formation and expansion. The nature of the ion requirements and energy requirement was further investigated by determining if increased transport is energy dependent and the nature of the Na+ requirement for transport (23). Embryos were preincubated for 1 hr in inhibitor(s) and incubated an additional hour in inhibi tor(s) plus labeled methionine. Methionine transport was inhibited by oligomycin

(5 /-Lg ml-I) at the late 4-cell stage, indicating an energy requirement for transport. At the early blastocyst stage, iodoacetate (1 mM) and oligomycin (5 ,... g ml-I) effectively blocked methionine transport. Iodoacetate was ineffective as a transport inhibitor at the 4-cell stage. These data indicate that oxidative phosphorylation is necessary for transport at the 4-cell stage, but transport at the early blastocyst stage is energized by both glycolysis and oxidative phosphorylation. These results are not surprising considering the increasingly important role of glycolysis at the later stage in preimplantation mouse embryos. The cation dependency of pH]methionine transport was studied at the late morula and early blastocyst stage (23). The effect of Na+-depleted medium on (lH]methionine uptake by early mouse blastocysts indicates a competitive type of inhibition. This result suggests that Na+ serves to increase the affinity of methionine for the carrier. The effect of Na+-depleted medium is specific for Na+-dependent amino acid transport and does not appear to be a nonspecific effect on overall cell metabolism. For example, (3H]methionine transport is Na+ dependent in late moru lae, whereas pH]phenylalanine transport at this stage is not affected by Na+-dep leted medium. It was not shown if amino acids accumulated at the blastocyst stage of the mouse are accumulated in the blastocoele fluid as reported for the rabbit blastocyst

(79).

Ions and Blastocyst Formation Variations in the concentrations of several ions can affect the development of the 2-cell mouse embryos ( 1 32). Potassium and calcium were the only ions found to be essential for development to the blastocyst stage. Calcium may be needed for stabili zation of membranes and junctional complexes. Calcium and ferrous iron are neces sary for development of the preimplantation rabbit embryo, whereas K+ was not essential (48). The role of these ions in preimplantation development is not under stood. The role of some of these ions in mammalian blastocyst formation is, how ever, partially defined.


PHYSIOLOGY OF PREIMPLANTA nON EMBRYOS

111

The mammalian blastocyst develops in part by the internal accumulation of fluid consisting of inorganic and organic substances dissolved in water ( 1 4, 88). Almost all of the work has been done on the rabbit, a species espechdly suitable for pioneer ing studies because of the large increases in volume from about 2 nl between the third and fourth day postcoitum to about 2.5 ml on the tenth day. A transtrophoblastic electrical potential difference (TPD), presumably indicating active transport, was demonstrated in rabbit (42) and in mouse blastocysts (44). This TPD was greatly reduced by anoxia and metabolic inhibitors and could be altered by replacement of Na+ and CI- with other ions (43). Direct demonstration of active transport came when Cross (39) applied the Ussing and Zerahn short-circuit method to 6-day rabbit blastocysts. In the absence of electrochemical gradients, net sodium and chloride transport are both three times larger than the short-circuit current (SCC) and account for only 5% of the SCC. The average rate of bicarbonate transport is in good agreement with the nonsodium, nonchloride component of the SCC, suggesting that bicarbonate accumulation in the 6-day rabbit blastocoele is also due to active transport (41). H+ is passively distributed across the blastocoele . wall (40). R. M. Borland, J. D. Biggers, and C. P. Lechene (unpublished) have used electron probe microanalysis of picoliter samples of fluid to measure the concentration of Na, Cl, K, Ca, Mg, S, and P in rabbit blastocysts at 110, 135, and 159 hr postcoitum. Using these data and the calculated rate of accumulation of blastocoelic fluid (46), the rate of accumulation of fluid and solutes during the first 3 days of blastocyst development was estimated. The rates of accumulation of both fluid and solute show striking increases during each 24-hr interval. These increases in accumulation rates of both fluid and solutes are not proportional to the increase in cell number or embryo surface area and indicate that the capacity of transport mechanisms for solute and fluid movement increases with development. These increases may be a result of the development of carrier systems or of morphological changes in the rabbit blastocyst. Electron microscopical studies of the mouse embryo at the early morula stage show focal tight junctions near the cell apices (52). As the morula develops, these become zonular and exclude lanthanum, thereby separating the inner cells from the maternal environment. In rabbit embryos, both gap and tight junctions were visualized in freeze-fracture replicas of rabbit blastocysts (52, 73). The zonula occludens forms a lanthanum impermeability barrier consistent with the high transtrophoblastic electrical resistance of 2188 ohms cm2 found by Cross (38). Freeze fractures of 5- and 6-day rabbit blastocysts showed that the 5-day blastocysts averaged 2-3 ridges per tight junction lattice; 6-day blastocysts had lattices that averaged 5-6 ridges (73). These morphological changes could partially explain the increased fluid and solute accumulation rates during development. The mechanisms involved in nlOuse blastocyst formation remain obscure. Mouse blastocysts can be mechanically collapsed with a bevelled pipette and reexpansion will occur (64). Neither actinomycin D (0.1 iJ-g ml-I) nor puromycin (SO iJ-g ml-I) inhibited reexpansion, although both agents eventually destroyed the embryos. Other studies have used the mold metabolite cytochalasin B to collapse mouse


112

BIGGERS & BORLAND

blastocysts (5 1 ). Early mouse blastocysts incubated for 2 hr in a concentration of 5 f-Lg ml-1 of culture medium collapsed in 2 hr, but 95% of them recovered in 3 hr after removal of the drug. Na+-depleted medium (25 mM Na+), K+-free medium, and ouabain (I mM) all blocked reexpansion in the recovery medium. This blockage was reversible upon reincubation in normal medium. The mode of action of cyto chalasin B on the early mouse embryo is not known; one possibility is that it may weaken the junctional complexes. If this mode of action is correct, �he data support the suggestion that ouabain-sensitive sites may be localized on the blastocoelic surface of the trophoblast cells (23). A similar situation has been directly demon strated in early Xenopus laevis embryos in which ouabain did not influence the development of the embryos unless it was injected into the blastocoele ( 1 1 9). The elemental composition of the early mouse blastocoele fluid (8�87 hr post coitum) in embryos in vivo has recently been measured in our laboratory by electron probe microanalysis. The mouse blastocoele contains very high concentrations of K, Mg, and Ca (33, 5, and 6 mM, respectively). Mouse serum in comparison contains only 5.3 mM K and 3.8 mM Ca. The composition of mouse blastocoele fluid is very unlike that of the rabbit blastocyst. This difference may be due to the fact that rabbit embryos undergo rapid expansion from days 4 -8 postcoitum, whereas the mouse blastocyst expands very little ( 1 4). These elements in the mouse and rabbit blastocyst may be critical in creating a microenvironment for differentiation of thc inner cell mass (75). A possible role of ions in differentiation has recently been discussed by McMahon (94). Unfortunately, elemental analyses of the uterine fluids of the mouse and rabbit are not available during the time the embryos are present in the uterus. The mechanism of accumulation of fluid in the mouse and rabbit is not known. Tuft & Boving ( 1 30) have suggested from measurements of chemical potential that active water transport occurs on day 7-8 postcoitum. Active water transport is not widely accepted by physiologists, and this phenomena needs further exploration. PREIMPLANTA nON EMBRYO AND ITS ENVIRONMENT

Most of this review has focused upon the irreversible developmental aspects of the preimplantation embryo. The embryo, however, is located within the female genital tract whose local secretions contribute to its microenvironment. Because the embryo migrates down the oviduct during this period of development, theoretically the environment can change. As yet, relatively little is known about these microenviron ments because of the very low rates of secretion of oviductal fluid ( 1 10). As pointed out by Biggers & Bellve ( 1 6), the analysis of oviductal fluids collected by catheteriza tion over extended periods may give a poor indication of the tubal environment. Recently, it has been possible to sample the fluid surrounding mouse I -cell embryos in the ampUlla and 2-cell embryos in the isthmus using micropuncture techniques, and analyze the droplets for Na, Cl, K, Ca, Mg, S, and P by electron microprobe analysis (unpublished, L. Roblero, J. D. Biggers, and C. P. Lechene). The results show that the elemental compositions of ampullary and isthmal fluid are dissimilar and that both are very different in composition from blood plasma. A very striking feature is the very high level of potassium-up to 30 mM.


Figure

2

113

illustrates the relationship of a preimplantation embryo to its surround

ings and the factors that influence the composition of the environment. Three types of exchange can be recognized: 1 . those between the embryo and its environment,

2.

those between the microenvironment and the maternal tissues in its immediate

neighborhood, and

3.

those between the microenvironment and the neighboring

regions of the genital tract. As yet, we know very little about these processes. Quantitative aspects of exchanges between the embryo and its microenvironment are


unknown although, as discussed already, the nature of these exchange mechanisms is now under active study. For example, a start has been made in the study of the maternal contribution to the microenvironment by studies on the active transport of Na and Cl by the ampullary region of the oviduct

(29-3 1 )

and by studies of the

longitudinal flow of fluids along the length of the oviduct ( 1 1). The application of micropuncture techniques and the techniques of ultramicrochemistry show promise of opening up this field of investigation.

PROXIMAL ENVIRONMENT

��

M1CROEIi....IRONM(Nl

ll£ OI STAL ENVIRONMENT

Figure 2

The relationship of a preimplantation embryo to its microenvironment and the

factors influencing the microenvironment. (a) Exchanges between the embryo and its microen vironment; (b) exchanges between the microenvironment and maternal tissue; (c) exchanges between the microenvironment and adjacent regions of the female genital tract.

CONCLUSION The evidence presented in this review demonstrates that many complex processes occur during mammalian preimplantation development. The general pattern is similar to that found in nonmammalian forms. However, there are two major differences. First, the rate of development and biochemical change is much slower in mammalian forms. Second, the mammalian embryo is not completely self-con tained but undergoes important exchanges with its mother. These exchanges are of three types: 1 . those concerned with homeostatic functions that preserve the internal environment of the embryo, e.g. pH, osmotic pressure, 2. those involving the uptake


1 14

BIGGERS & BORLAND

of substances that can be metabolized, e.g. glucose and amino acids, and 3. specific signals that pass between mother and embryo whose role is to synchronize develop ment and maternal function ( 1 3). It is sometimes argued that the oviducal environment is not important because embryos can develop in ectopic sites and, in some species, in very artificial chemi cally defined media. These arguments, however, do not prove that exchanges be tween the embryo and microenvironment are not important or that the embryos are self-contained, but rather that the embryos concerned are very adaptable. The preimplantation stages of many species cannot be successfully cultured with present techniques and may require more specific conditions than the mouse and rabbit. As more is known about the composition of oviducal and uterine fluids, the in vitro development of these other species may soon be possible, as has been demonstrated with preimplantation sheep embryos ( 1 27). ACKNOWLEDGMENTS

The preparation of this review has been made possible by grants from the Rockefel ler Foundation RF65040, the NICHD grant HD-069 1 6-0 I A I , and the NIH Cen ters Grant 1 PO I HD 06645-03. Dr. Borland is a postdoctoral fellow supported by the Ford Foundation. Literature Cited I . Abramczuk, J., Sawicki, W. 1974. Vari

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Zool. 1 88:25-35 2. Aketa, K., Biachett, R., Marre, E. , Monroy, A. 1964. Hexose monophos

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the rabbit, Oryc tolagus cuniculus, with special refer ence to the structural changes of mito chondria. J. Morpho!. 1 30:67-92 Arey, L. B. 1974. Developmental Anatomy. Philadelphia: Saunders. 695 pp. Atkins, G. L. 1969. Multicompartment Models/or Biological Systems. London: Methuen. 1 5 3 pp. Atkinson, D. E. 1968. The energy charge of the adenylate pool as a regula tory parameter. Interaction with feed back modifiers. Biochemistry 7:4030-34 Auerbach, S., Brinster, R. L. 1967. Lac tate dehydrogenase isozymes in the early mouse embryo. Exp. Cell Res. embryogenesis

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