THE ANATOMICAL RECORD 226237-248 (1990)
Differentiation of the Inner Cell Mass of the Baboon Blastocyst ALLEN C. ENDERS, KATHERINE C. LANTZ, AND SANDRA SCHLAFKE Department of Human Anatomy, School of Medicine, Uniuersity of California, Dauis, California 9561 6
ABSTRACT
During the blastocyst stage of development in the baboon, the inner cell mass changes from a n irregular accumulation of cells within the cavity of the blastocyst to a disk at one side of the blastocyst and finally to a spherical mass of epiblast cells exhibiting a distinct polarity. The cells that will become the primitive endoderm are first seen as flattened but undifferentiated cells on the cavity side of the disk-shaped inner cell mass. After endoderm cells develop their typical cytological characteristics, they extend well beyond the inner cell mass to form parietal endoderm. A basal lamina develops associated with the epiblast cells and mural trophoblast, but not with either parietal or visceral endoderm. Cytological differentiation of inner cell mass cells includes increased numbers of polyribosomes and a change in mitochondria from long, convoluted structures to short, more typical shapes. Evidence that epiblast is polarized is seen by the late zonal blastocyst stage. Apical junctional complexes develop within the center of the epiblast. These junctions presage the development of the potential amniotic cavity. Large vacuoles containing cell debris, some of which contain nuclear fragments, are present a t all stages. Extensive cell death occurs during growth of the blastocyst, but the pattern appears to be random and products of cell death are readily phagocytized by adjacent cells.
The typical blastocyst of eutherian mammals undergoes a series of maturational changes after formation of the blastocyst cavity prior to implantation. These changes include formation of primary endoderm (hypoblast), penetration or loss of the zona pellucida, modification of mitochondria and endoplasmic reticulum, and development of a n endocytic complex within trophoblast cells (Enders, 1989). Less general changes that may occur include activation from a delay of implantation (e.g., spotted skunk: Mead and Rourke, 1985; Enders et al., 1986a) and loss of the polar trophoblast overlying the inner cell mass (cow: Betteridge and Flechon, 1988; pig: Geisert e t al., 1982; Stroband et al., 1984; horse: Enders e t al., 1988). In some species pronounced shape changes occur; in the pig for example a change in cell shape is brought about by cytoskeletal redistribution (Albertini et al., 1987). Localized modifications of trophoblast that presage adhesion to the uterine epithelium also tend to be species-specific (Enders, 1989). In a chronological study of development of the baboon by Hendrickx (1971), the general development of the baboon blastocyst was described, including loss of the zona pellucida. Panigel et al. (1975) reported on aspects of the cytology of the blastomeres during cleavage, but the most advanced embryo in their material was a n early blastocyst with a small crescentic cavity. Flechon et al. (1976) described a few features of the surface morphology of the cleavage stage, using scanning electron microscopy. Although preimplantation stages from nonhuman primates are becoming more commonly available (Dukelow and Yorozu, 1986; Summers e t al., 19881, 0 1990 WILEY-LISS, INC.
studies of the cytology and differentiation of these stages remain sparse, particularly for monotocous species. Most studies using primate blastocysts examine these embryos as whole specimens prior to their utilization in embryo transfers, in vitro outgrowth, etc. Many aspects of blastocyst development, differentiation, normality, and cell death cannot be analyzed without the information concerning cellular structures that can be obtained from sectioned material. Blastocysts from the rhesus monkey, which are usually obtained by surgical collection techniques, have been used for a number of cytological studies (Hurst et al., 1978, 1980; Enders and Schlafke, 1981; Enders et al., 1982). Preimplantation stages of the baboon can be collected by using a nonsurgical method of flushing the uterus (Pope et al., 1980). Although the number of embryos that can be obtained in this way is relatively small compared to using polytocous laboratory species, a series of blastocysts has been obtained and studied to examine differentiation from the early blastocyst stage to preimplantation blastocysts. The study focussed on cellular structure and interactions during organization of the inner cell mass, polarization and segregation of cell layers, and formation of the first extracellular matrix materials during the early blastocyst stage to the late preimplantation blastocyst. Several abnormal blastocysts were also obtained in the course of this study but are not included here.
Received January 10, 1989; accepted May 31, 1989.
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MATERIALS AND METHODS
Sexually mature baboons (Papio sp.) were housed individually in compliance with the regulations of the Federal Animal Welfare Act and the Institution for Laboratory Animal Resources. Animals were maintained in air-conditioned quarters with artificial lighting 12 hrlday. Menstrual cycle data were recorded daily by observation of sex skin turgescence. Females were placed with males on alternate days during the time of maximum turgescence (usually beginning days 10-12) and were subsequently checked for the presence of sperm. The first day of deturgescence was designated day 2 of pregnancy, since recent studies in our laboratory and by others have indicated that ovulation occurs most frequently on the second day before detectable deturgescence (Shaikh et al., 1982; Pope et al., 1982; Enders e t al., 1989a). Collection of Blastocysts
On days 6-8 after ovulation, animals were anesthetized lightly with ketamine and placed in a prone position; a vaginal speculum was inserted; and the exocervix was located. A Curity Isaacs endometrial cell sampler (Kendall Co., Boston), modified as described by Pope et al. (1980) to allow continuous inflow and outflow, was extended into the uterine lumen. Then 10 cc of sterile Earle’s balanced salt solution (Grand Island Biological Company, Santa Clara, CA) was flushed slowly (2-4 ml/min) through the catheter; the effluent was collected in 30 mm petri dishes and examined immediately. Normal preimplantation stages examined for this study included seven blastocysts that had retained their zonas (one on day 5, two on day 6, three on day 7, and one on day 8) and three blastocysts that no longer had a zona pellucida (two on day 7, one on day 8). A number of unfertilized eggs, abnormal cleavage stages, morulas, and morula blastocyst transition stages were also obtained but are not included in this study. All blastocysts were fixed in freshly prepared 2% formaldehyde-2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 30 to 45 min. They were subsequently washed overnight in 0.1 M phosphate buffer, pH 7.3, for 30-60 min, rapidly dehydrated in ethanol, equilibrated in propylene oxide/Araldite, and embedded in Durcupan Araldite epoxy resin. Six of the blastocysts were incubated in cationized ferritin (Enders et al., 1989b) prior to fixation, and two blastocysts were treated with 1%tannic acid after fixation prior to dehydration. Blastocysts were carefully thick-sectioned, stained with Azure B, and examined by light microscopy, so that orientation of cavity and inner cell mass could be noted. Thin sections were stained with uranyl acetate and lead citrate and observed in either a Philips 400 or a Zeiss 10 electron microscope. OBSERVATIONS General Description ofBlastocyst Stages The blastocysts that constitute the basis of this report varied appreciably in stage of development, both in relationship to the organization of the cells in the inner cell mass (ICM) and in the structure of the cellular constituents.
There is a progressive change in shape and distribution of ICM cells during the blastocyst stage, consisting in sequence of: 1) segregation of the ICM cells to one pole of the blastocyst and reduction in cell size; 2) formation of a layer of flattened cells adjacent to the cavity of the blastocyst; 3) differentiation of primary endoderm (hypoblast) and a n epiblast-associated basal lamina; and 4) compaction of the ICM with concomitant establishment of epiblast cell polarity. Since these changes parallel the calculated ages of the blastocysts, it proved convenient to describe them as early zonal, mature, or late zonal and azonal stages. Early zonal blastocysts were collected on days 5 and 6 and ranged from 150 to 183 pm in diameter after fixation, with thickness of the zona pellucida of 11 pm to 15 pm. Mature zonal blastocysts were collected on day 7, except for a single expanded blastocyst on day 8. Mature zonal blastocysts showed considerable size variation, ranging from a small blastocyst (185 pm in diameter) with a zona pellucida thickness of 11 pm, to large expanded blastocysts 300 pm in diameter, with a zona pellucida only 7 pm thick. Azonal blastocysts, which were collected on days 7 and 8, are usually expanded (200-300 pm in diameter), but readily collapse during processing. The zona pellucida thus appears to be lost on the seventh or eighth day of gestation, and the zona thins from a maximum of 15 pm to 7 pm prior to loss. The overlap in stages by day of collection suggests that either timing based on deturgescence or development rate, or both, showed variation of about 1 day. Organization of the Inner Cell Mass ofthe Early Zonal Blastocyst
When the blastocyst cavity first forms, the inner blastomeres are large and irregularly distributed with incomplete segregation to one pole of the blastocyst (Enders et al., 1989a). Even after the ICM occupies a discrete segment of the blastocyst interior, some of the ICM cells adjacent to the cavity contact mural trophoblast cells (Fig. 1). At the same time polar trophoblast cells are irregularly interposed between ICM cells and do not a t this stage constitute a separate layer. At the margin of the ICM trophoblast cells are often pyramidal in shape, with surfaces associated with ICM cells bordering the blastocyst cavity, adjacent trophoblast, and internal ICM cells. Some of the intercellular spaces between the ICM cells are initially large, and there are few cell processes within these spaces (Fig. 2). When the ICM becomes more compact, intercellular spaces are reduced and microlamellae appear between regions of cell contact. At this stage there is little cytological difference between ICM cells and trophoblast cells (Fig. 31, and there is no apparent polarization in the ICM cells. Junctions between ICM cells or between ICM and polar trophoblast are small and most commonly consist of primitive junctions (areas of close association with only slight increase in density on the inner surface of the plasmalemma). During this stage, gap junctions become common and a few desmosomes are also seen between ICM cells and between ICM cells and trophoblast cells. In this and subsequent stages, there is evidence of cell death in the form of vacuoles, and phagolysosomes
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Fig. 1. Three stages in segregation of the ICM. a: Some polar trophoblast cells are cuboidal (arrow). ICM cells extend in irregular fashion into the blastocyst cavity and in other sections are attached to mural trophoblast. Note the large size of the blastomeres. b The ICM cells constitute a disk of cells at one pole of the blastocyst. Trophoblast
cells (arrow) at the margin of the ICM extend toward the d i s k c: The ICM cells of this expanded zonal blastocyst now constitute a n aggregated somewhat spherical cluster. The primary endodermal layer has formed and extends beyond the ICM as parietal endoderm (arrows). a: Day 5; b: Day 6; c: Day 7. X 320.
Abbreviations
that contain cell debris are present in several of the cells (Fig. 2). From the large size of some of the vacuoles and the occasional degenerating nuclei, it is clear that whole cells have been ingested. Smaller vacuoles that could be the result of earlier ingestion of cell fragments or autophagy are also present.
BL E ER G M PE
m
Z
basal lamina endoderm endoplasmic reticulum Golgi complex mitochondria parietal endoderm polar trophoblast zona pellucida
Organization of the ICM in Mature and Expanded Zonal Blastocysts
During the zonal stage, the blastocyst expands (Figs. l c , 4), and the ICM is consequently relatively smaller. It becomes more spherical, and the individual blastomeres decrease in size and begin to be polarized. The
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first indication of the acquisition of epiblast polarity is the formation of a basal lamina underlying some of the epiblast cells of the ICM, thus establishing a basal surface. Junctions resembling fascia adherens appear for the first time toward the center of the ICM (Fig. 5). Since the cells are in a flattened spheroidal arrangement, the junctional complexes are positioned toward the now-central apices of the epiblast cells. This arrangement is similar to that seen in the ICM of the implanting rhesus monkey embryo, thus presaging formation of the amniotic cavity (Enders et al., 1986b). In highly expanded zonal blastocysts (c. 300 Fm in diameter), evidence of epiblast polarity is seen in the form of extensive central apical junctional complexes. During this stage the flattened cells lining the cavity of the blastocysts first show cytological evidence of differentiation into primitive endoderm, and differentiated endodermal cells come to underlie both ICM and mural trophoblast (see below). Desmosomes and gap junctions link ICM cells to one another, to the polar trophoblast cells, and to endoderm (Fig. 7). Inner Cell Mass of Blastocysts After Loss of the Zona Pellucida
In many respects the ICM of azonal blastocysts resembles that of large zonal blastocysts. The ICM is restricted to a small portion of the circumference of the blastocyst, and apical junctional complexes define a central region within the epiblast (Fig. 8). Some isolated cells (large cells that retain a more primitive type of mitochondria) are seen. A pronounced basal lamina separates epiblast from the underlying endoderm, which lacks a basal lamina. Organelles of ICM Cells During Preimplantation Differentiation
A distinctive characteristic of ICM cells, especially in early blastocysts, consists of the empty-appearing areas of cytoplasm without discernible organelles (Figs. 3-7). These areas can occasionally be seen to contain a substructure of flocculent or finely granular material (Figs. 6, 7). Formed organelles are largely excluded from these areas of the cells, resulting in restriction of mitochondria and polyribosomes to perinuclear and peripheral regions and to columns. The organelle-free areas are seen in ICM cells of expanded zonal and azonal blastocysts, as well as earlier stages, and are also apparent in endodermal cells (Fig. 9). Mitochondria1 profiles in blastomeres of early blastocysts are characteristically clustered (Fig. 3). By following profiles through several sections, it is clear that the sections comprise multiple cuts through single long andlor branched mitochondria. The large convoluted
Fig. 2.
Fig. 2. Montage of electron micrographs of the ICM of the same zonal blastocyst as seen in Figure lb. Although portions of one cell near the cavity are flattened, no endodermal layer is formed here. Large vacuoles containing cell debris are seen both in the ICM and in polar trophoblast (arrows). At the right it can be seen that a trophoblast cell extends a process to one of the innermost cells of the ICM. Note also the pronounced clustering of the mitochondria1 profiles in the blastomeres. The area in the bracket is seen in Figure 3. Day 6. x 2,700.
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24 1
Fig. 3.Portions of two blastomeres of polar trophoblast and underlying ICM cell. Note that in both cell types the mitochondria1 profiles are clustered. From the convolutions seen, mitochondria such as those seen in the ICM cell are believed to be individual convoluted structures. Day 7. x 15,000.
mitochondria with few cristae are similar to those seen in the morula-blastocvst transitional stages (Enders et al., 1989a). Smooth o r sparsely granular membranes of are Often adjacent to mitochonendoplasmic dria. Mitochondria of later blastocysts are shorter and
Fig. 4. Montage of the ICM of a n expanded zonal blastocyst. There is now a complete layer of flattened squamous cells, the primary endoderm, underlying the ICM. The area in brackets is illustrated in Figure 9. Day 8. x 3,000.
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Fig. 5. Compact ICM from the blastocyst shown in Figure lc. The epiblast cells begin to form apical junctions toward the center of the ICM (see area in brackets and inset).Note the mitotic figure (MF) to the right. A necrotic cell (NC) lies between the polar trophoblast and
the zona pellucida, the density of which is increased with cationized ferritin. A thin layer of endoderm (E) underlies the epiblast. Day 7. x 6,100.
more evenly dispersed, are less frequently associated with endoplasmic reticulum, and contain an increased number of cristae (Figs. 6, 7). The endoplasmic reticulum is increased in amount and is distinctly granular; ribosomes, both free and associated with endoplasmic reticulum, are associated into polyribosomal clusters. The large Golgi complexes are usually perinuclear and contain numerous elements and many associated vesicles. There is a decrease in organelle-free areas and an increase in numbers of lipid droplets within ICM cells in large azonal blastocysts.
Differentiationof Endoderm Primitive endoderm, which is first detected during the zonal blastocyst stage, is seen initially as segregation of a few flattened cells on the inner surface of the ICM (Figs. 4, 9). Small junctions form between these cells, and they develop a microvillous surface toward the cavity of the blastocyst (Figs. 5, 6). Subsequently, the differentiation of characteristic dilated granular endoplasmic reticulum and the separation of this layer from the rest of the epiblast indicate the formation of
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Fig. 7. Higher magnification of ICM cells, showing the presence of many polysomes and, in the upper right, a region devoid of polysomes and other organelles (margin designated by arrowheads). Note the irregular-shaped mitochondrion (M) in the cell (probably endoderm) containing lipid (L). A caveolus is seen in one of the cells of the primitive endoderm (arrow). Day 7. x 28,000.
Fig. 6. Higher magnification of the margin of an ICM from a n expanded zonal blastocyst. A small amount of basal lamina is formed beneath the polar trophoblast. The mitochondria1matrix is less dense than in previous stages. Note that in the area of granular material (arrowheads), thought to be a mass of stored proteinaceous material, there are no polysomes (see Fig. 7). Day 7. x 12,900.
visceral endoderm (Figs. 9, 10). Phagolysosomes containing cell debris are seen frequently within visceral endodermal cells. In expanded zonal and azonal blastocysts, visceral endodermal cells have highly irregular folded basal surfaces, with no subjacent basal lamina. Occasional intercellular junctions (primitive, gap, and desmosomal) remain between visceral endodermal cells and ICM cells. Endodermal cells extend beyond the margin of the ICM in expanded zonal blastocysts, illustrating that differentiation of the parietal endoderm can occur prior to loss of the zona. Parietal endodermal cells may be closely apposed to mural trophoblast, including apparent regions of intercellular adhesion and fascia1 gap junctions; the cells often contain spherical gap junctions. in azonal b~astocysts, the parietal endodermal layer extends as a complete epithelium well beyond the margin of the ICM, to surround approximately 213 of
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terrupted where cells of epiblast and endoderm are contiguous. Frequently basal laminas form in places that are inappropriate. For example, basal lamina material is seen between epiblast cells as well as beneath the epiblast. Basal lamina material occurs as patches and folds in intercellular spaces between polar trophoblast and ICM, but does not form a complete lamina in this position (Fig. 6). A basal lamina underlies mural trophoblast near the junction of visceral endoderm with parietal endoderm and in marginal areas where mural trophoblast is underlain by parietal endodermal cells. However, mural trophoblast is often subtended by a basal lamina in regions without subjacent endodermal cells. Basal processes of mural trophoblast cells that project toward the endoderm usually have a continuous basal lamina, and occasionally small patches of basal lamina are present on the basal side of the endoderm per se. When such patches are found, they are continuous with the basal lamina of mural trophoblast. DISCUSSION Establishment of Polarity
The establishment of polarity in the epiblast is a striking feature of differentiation in the ICM prior to implantation. Since this polarization of cells follows the formation of the endoderm and coincides with the formation of a basal lamina on the basal surface of the epiblast, it seems possible that the endoderm may be responsible for the establishment of polarity within the epiblast. Should this be the case, there would be yet another situation in embryonic development where establishment of polarity plays an important role in differentiation of a cell layer (Ziomek and Johnson, 1980; Johnson, 1986; Fleming, 1987). The initial differentiation leading to formation of the amnionic cavity occurs not only prior to implantation but even prior to loss of the zona pellucida. The cellular mechanism of this differentiation, by cavitation of the ICM, is similar to that described in the rhesus monkey in an early implantation stage (Enders et al., 1986b). Cytoplasmic Differentiation of Inner Cell Mass Fig. 8. ICM from azonal blastocyst. Epiblast cells contain more lipid than previously but otherwise are similar to those of the expanded zonal blastocyst. An apical junctional complex is seen at the top of the micrograph (arrow), which is toward the center of the ICM. Day 7. x 7,820.
the circumference of the blastocyst. The parietal endoderm is sometimes adherent to trophoblast in regions lacking a trophoblast-associated basal lamina, but more frequently is separated from trophoblast by the basal lamina. In areas where the endodermal layer terminates and is adherent to trophoblast, the basal lamina also terminates abruptly a t this point. Formation of Basal Laminas
Extracellular material similar in appearance to that of the lamina densa of basal laminas is interspersed between the ICM and flattened presumptive endoderma1 cells, but does not form a complete layer in early zonal blastocysts. A discrete basal lamina is seen under the epiblast of expanded zonal blastocysts, but it is in-
Storage materials have been described in the oocytes and preimplantation embryos of rodents, where they aggregate in macromolecular arrays that are readily visible with the electron microscope (Enders and Schlafke, 1965; King and Tibbits, 1977; Parkening et al., 1985). A recent study of hamster and rat blastocysts indicated that the fibrous material may represent a source of cytokeratins (Capco and McGaughey, 1986). The large areas that lack organelles in the baboon blastocyst have also been reported in the rhesus monkey (Enders and Schlafke, 1981) and seem likely to be manifestations of masses of residual stored proteinaceous materials. The fact that such regions remain longest in the epiblast cells may be further evidence of the relatively slow differentiation of these cells compared to either trophoblast or endoderm. Differentiation of Primary Endoderm
It is interesting that the cytological features of endodermal differentiation do not occur until after ICM cells have been isolated to a cluster at one side of the cavity of the blastocyst for some time. Only then do
BABOON BLASTOCYST DIFFERENTIATION
Fig. 9. Primary endodermal cells. a: Enlargement of a portion of the
ICM illustrated in Figure 4.When a complete hypoblast layer is first formed, the cells are flattened and have junctional complexes and a few sparse microvilli toward the blastocyst cavity. b: Higher magni-
apical junctional complexes and microvilli facing the cavity of the blastocyst indicate formation of an endodermal layer. Even the dilated endoplasmic reticulum that has come to be the hallmark of endodermal cells (Enders, 1971; Rossant, 1987) is seen only after flattened cells are present in the appropriate position. Indeed the endoplasmic reticulum is more sparse in endodermal cells of the baboon blastocyst than in those of other species. The arrangement of the trophoblast cells at the margin of the ICM is such that one of the two daughter cells of a dividing trophoblast cell could become endoderm rather than mural trophoblast. On the other hand, flanges of trophoblast cytoplasm extend under part of the ICM subsequent to the stage of triangular cells but prior to differentiation of endoderm. Such an arrangement has been suggested as a way by which trophoblast can modify ICM potential (Fleming et al., 1984).
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fication of the cytoplasm of an endodermal cell, showing the granular endoplasmic reticulum with a flocculent content typical of endoderma1 cells. a , x 8,900. b, x 31,200.
Cell Death During Preimplantation Stages
Throughout the blastocyst stage large phagolysosomes containing cell debris are seen within cells of both epiblast and endoderm. Although the smaller vacuoles may contain debris from autophagy or from fragments of cells, many vacuoles contained both nuclei and recognizable organelles, suggesting death of whole cells. The distribution and stage in which they were found appeared random and therefore did not suggest programmed cell death or a specific role for cell death in any given morphogenic process. There appear to be fewer isolated cells in blastocysts of the baboon than in those of the rhesus monkey or human. Illustrations of Lopata et al. (1983), Sathananthan (19841, and others have indicated numerous regions of cell sequestration and isolation in human blastocysts in culture. An increased amount of cell death
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Fig. 10. Endodermal cells from an azonal blastocyst. Note that the basal lamina is associated with the epiblast (EPI), not with the endodermal cells. The endodermal cells are characterized by microvilli and granular endoplasmic reticulum. Day 8, X 12,900.
and cytoplasmic fragmentation seen in the human may be the result of disadvantageous culture conditions, but no human blastocysts from in vivo development have been studied by electron microscopy with which appropriate comparisons can be made. There is some indication, however, from the admittedly limited observations of Buster et al. (1985), that blastocysts from donor women are more viable than blastocysts from in vitro culture. There appears to be more cell death in the primate blastocyst than in laboratory rodents, and this cell loss during development may be accentuated by adverse culture conditions (Boatman, 1987). It is clear from the work of Willadsen (1981) and others t h a t only a portion of the cytoplasm of the original egg is necessary for development of a blastocyst. An apparently normal early baboon blastocyst was collected that lacked a t least a third of the original ooplasm (Enders e t al., 1989a). However, cell death at the wrong time or concentrated in location within the developing blastocyst might be deleterious to further development. Fixation obviously prevents following the subsequent fate of a given abnormal-appearing cleavage stage. It should be possible, however, to recognize individual abnormal blastomeres and to follow them for one or two cleavages in vitro. To date individual abnormal blastomeres have not been followed in vitro, although there is information from the human suggesting that “ugly” early cleavage stages (with considerable debris) can produce normal offspring (Veeck, 1986). Mitochondria of the cleavage stages and early blastocysts are particularly interesting because they undergo changes in shape and in distribution during preimplantation development, and because it may be possible to monitor distribution of these organelles in vitro by using fluorescent markers (Albertini et al., 1987). Both in human embryos in vitro (Nilsson and
Sundstrom, 1982; Lopata e t al., 1983; Johnson, 1986) and in the developing baboon embryo (Enders et al., 1989a), there is a tendency for mitochondria to aggregate near the nucleus in some of the blastomeres of morula stages. Later in development, not only are mitochondria more dispersed and less complex, but in addition other changes occur including a decrease in matrix density, which has also been reported in the human (Lopata et al., 1982). Johnson (1986), in analyzing results of cell marker experiments, pointed out the difficulties of using a cytoplasmic marker like peroxidase in the mouse, where residual midbodies sometimes link daughter cells for considerable time. It should also be pointed out that markers like fluorescent spheres that would not pass through midbodies might be ingested as cell debris, also providing non-lineage localization, whereas the genetic markers used extensively by Rossant (1987) would not be subject to either of the above difficulties. Basal Lamina Formation
It is perhaps ironic t h a t one of the best-studied basal laminas is Reichert’s membrane, which has been used as a source of studies of the way in which endodermal cells synthesize basal lamina constituents (Hogan et al., 1984; Mazariegos et al., 1987). Yet the parietal endoderm initially lacks a basal lamina, and the only basal lamina present is that subjacent to trophoblast. Surprisingly, the formation of the first basal lamina, that of the epiblast and trophoblast, has not been widely studied. Preliminary observations using antibodies to entactin and laminin showed intracellular localization of laminin in early cleavage stages (Wu et al., 1983). However, there is not a correlation between different studies, in that these authors show earlier synthesis of laminin than did Leivo e t al. (1980), and the former authors do not report trophoblast cell pro-
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duction of laminin whereas Wartiovaaro et al. (1980) did. Calarco-Gillam (1986) has summarized studies on formation of extracellular matrix materials by preimplantation embryos. ACKNOWLEDGMENTS
These studies were supported by grants HD 10342 and RR00169 from the National Institute of Child Health and Human Development. The authors would like to thank Drs. Mark Cukierski, Andrew Hendrickx, and Ross Tarara, and Pamela Binkerd, Carolyn Booher, and J i m Utz for assistance with the endocrine data, animal preparation, and collection techniques. LITERATURE CITED Albertini, D.F., E.W. Overstrom, and K.M. Ebert 1987 Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo. Biol. Reprod., 37,441-451. Betteridge, K.J., and J.E. Flechon 1988 The anatomy and physiology of pre-attachment bovine embryos. Theriogenology, 29r155-197. Boatman, D.E. 1987 In vitro growth of nonhuman primate pre- and peri-implantation embryos. In: The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation in vitro. B.D. Bavister, ed. Plenum Press, New York, pp. 273-303. Buster, J., M. Bustillo, I. Rodi, S. Cohen, M. Hamilton, J. Simon, I. Thorneycroft, and J. Marshall 1985 Biologic and morphologic development of donated human ova recovered by non-surgical uterine lavage. Am. J . Obstet. Gynecol., 15
Differentiation of the Inner Cell Mass of the Baboon Blastocyst ALLEN C. ENDERS, KATHERINE C. LANTZ, AND SANDRA SCHLAFKE Department of Human Anatomy, School of Medicine, Uniuersity of California, Dauis, California 9561 6
ABSTRACT
During the blastocyst stage of development in the baboon, the inner cell mass changes from a n irregular accumulation of cells within the cavity of the blastocyst to a disk at one side of the blastocyst and finally to a spherical mass of epiblast cells exhibiting a distinct polarity. The cells that will become the primitive endoderm are first seen as flattened but undifferentiated cells on the cavity side of the disk-shaped inner cell mass. After endoderm cells develop their typical cytological characteristics, they extend well beyond the inner cell mass to form parietal endoderm. A basal lamina develops associated with the epiblast cells and mural trophoblast, but not with either parietal or visceral endoderm. Cytological differentiation of inner cell mass cells includes increased numbers of polyribosomes and a change in mitochondria from long, convoluted structures to short, more typical shapes. Evidence that epiblast is polarized is seen by the late zonal blastocyst stage. Apical junctional complexes develop within the center of the epiblast. These junctions presage the development of the potential amniotic cavity. Large vacuoles containing cell debris, some of which contain nuclear fragments, are present a t all stages. Extensive cell death occurs during growth of the blastocyst, but the pattern appears to be random and products of cell death are readily phagocytized by adjacent cells.
The typical blastocyst of eutherian mammals undergoes a series of maturational changes after formation of the blastocyst cavity prior to implantation. These changes include formation of primary endoderm (hypoblast), penetration or loss of the zona pellucida, modification of mitochondria and endoplasmic reticulum, and development of a n endocytic complex within trophoblast cells (Enders, 1989). Less general changes that may occur include activation from a delay of implantation (e.g., spotted skunk: Mead and Rourke, 1985; Enders et al., 1986a) and loss of the polar trophoblast overlying the inner cell mass (cow: Betteridge and Flechon, 1988; pig: Geisert e t al., 1982; Stroband et al., 1984; horse: Enders e t al., 1988). In some species pronounced shape changes occur; in the pig for example a change in cell shape is brought about by cytoskeletal redistribution (Albertini et al., 1987). Localized modifications of trophoblast that presage adhesion to the uterine epithelium also tend to be species-specific (Enders, 1989). In a chronological study of development of the baboon by Hendrickx (1971), the general development of the baboon blastocyst was described, including loss of the zona pellucida. Panigel et al. (1975) reported on aspects of the cytology of the blastomeres during cleavage, but the most advanced embryo in their material was a n early blastocyst with a small crescentic cavity. Flechon et al. (1976) described a few features of the surface morphology of the cleavage stage, using scanning electron microscopy. Although preimplantation stages from nonhuman primates are becoming more commonly available (Dukelow and Yorozu, 1986; Summers e t al., 19881, 0 1990 WILEY-LISS, INC.
studies of the cytology and differentiation of these stages remain sparse, particularly for monotocous species. Most studies using primate blastocysts examine these embryos as whole specimens prior to their utilization in embryo transfers, in vitro outgrowth, etc. Many aspects of blastocyst development, differentiation, normality, and cell death cannot be analyzed without the information concerning cellular structures that can be obtained from sectioned material. Blastocysts from the rhesus monkey, which are usually obtained by surgical collection techniques, have been used for a number of cytological studies (Hurst et al., 1978, 1980; Enders and Schlafke, 1981; Enders et al., 1982). Preimplantation stages of the baboon can be collected by using a nonsurgical method of flushing the uterus (Pope et al., 1980). Although the number of embryos that can be obtained in this way is relatively small compared to using polytocous laboratory species, a series of blastocysts has been obtained and studied to examine differentiation from the early blastocyst stage to preimplantation blastocysts. The study focussed on cellular structure and interactions during organization of the inner cell mass, polarization and segregation of cell layers, and formation of the first extracellular matrix materials during the early blastocyst stage to the late preimplantation blastocyst. Several abnormal blastocysts were also obtained in the course of this study but are not included here.
Received January 10, 1989; accepted May 31, 1989.
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MATERIALS AND METHODS
Sexually mature baboons (Papio sp.) were housed individually in compliance with the regulations of the Federal Animal Welfare Act and the Institution for Laboratory Animal Resources. Animals were maintained in air-conditioned quarters with artificial lighting 12 hrlday. Menstrual cycle data were recorded daily by observation of sex skin turgescence. Females were placed with males on alternate days during the time of maximum turgescence (usually beginning days 10-12) and were subsequently checked for the presence of sperm. The first day of deturgescence was designated day 2 of pregnancy, since recent studies in our laboratory and by others have indicated that ovulation occurs most frequently on the second day before detectable deturgescence (Shaikh et al., 1982; Pope et al., 1982; Enders e t al., 1989a). Collection of Blastocysts
On days 6-8 after ovulation, animals were anesthetized lightly with ketamine and placed in a prone position; a vaginal speculum was inserted; and the exocervix was located. A Curity Isaacs endometrial cell sampler (Kendall Co., Boston), modified as described by Pope et al. (1980) to allow continuous inflow and outflow, was extended into the uterine lumen. Then 10 cc of sterile Earle’s balanced salt solution (Grand Island Biological Company, Santa Clara, CA) was flushed slowly (2-4 ml/min) through the catheter; the effluent was collected in 30 mm petri dishes and examined immediately. Normal preimplantation stages examined for this study included seven blastocysts that had retained their zonas (one on day 5, two on day 6, three on day 7, and one on day 8) and three blastocysts that no longer had a zona pellucida (two on day 7, one on day 8). A number of unfertilized eggs, abnormal cleavage stages, morulas, and morula blastocyst transition stages were also obtained but are not included in this study. All blastocysts were fixed in freshly prepared 2% formaldehyde-2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.3, for 30 to 45 min. They were subsequently washed overnight in 0.1 M phosphate buffer, pH 7.3, for 30-60 min, rapidly dehydrated in ethanol, equilibrated in propylene oxide/Araldite, and embedded in Durcupan Araldite epoxy resin. Six of the blastocysts were incubated in cationized ferritin (Enders et al., 1989b) prior to fixation, and two blastocysts were treated with 1%tannic acid after fixation prior to dehydration. Blastocysts were carefully thick-sectioned, stained with Azure B, and examined by light microscopy, so that orientation of cavity and inner cell mass could be noted. Thin sections were stained with uranyl acetate and lead citrate and observed in either a Philips 400 or a Zeiss 10 electron microscope. OBSERVATIONS General Description ofBlastocyst Stages The blastocysts that constitute the basis of this report varied appreciably in stage of development, both in relationship to the organization of the cells in the inner cell mass (ICM) and in the structure of the cellular constituents.
There is a progressive change in shape and distribution of ICM cells during the blastocyst stage, consisting in sequence of: 1) segregation of the ICM cells to one pole of the blastocyst and reduction in cell size; 2) formation of a layer of flattened cells adjacent to the cavity of the blastocyst; 3) differentiation of primary endoderm (hypoblast) and a n epiblast-associated basal lamina; and 4) compaction of the ICM with concomitant establishment of epiblast cell polarity. Since these changes parallel the calculated ages of the blastocysts, it proved convenient to describe them as early zonal, mature, or late zonal and azonal stages. Early zonal blastocysts were collected on days 5 and 6 and ranged from 150 to 183 pm in diameter after fixation, with thickness of the zona pellucida of 11 pm to 15 pm. Mature zonal blastocysts were collected on day 7, except for a single expanded blastocyst on day 8. Mature zonal blastocysts showed considerable size variation, ranging from a small blastocyst (185 pm in diameter) with a zona pellucida thickness of 11 pm, to large expanded blastocysts 300 pm in diameter, with a zona pellucida only 7 pm thick. Azonal blastocysts, which were collected on days 7 and 8, are usually expanded (200-300 pm in diameter), but readily collapse during processing. The zona pellucida thus appears to be lost on the seventh or eighth day of gestation, and the zona thins from a maximum of 15 pm to 7 pm prior to loss. The overlap in stages by day of collection suggests that either timing based on deturgescence or development rate, or both, showed variation of about 1 day. Organization of the Inner Cell Mass ofthe Early Zonal Blastocyst
When the blastocyst cavity first forms, the inner blastomeres are large and irregularly distributed with incomplete segregation to one pole of the blastocyst (Enders et al., 1989a). Even after the ICM occupies a discrete segment of the blastocyst interior, some of the ICM cells adjacent to the cavity contact mural trophoblast cells (Fig. 1). At the same time polar trophoblast cells are irregularly interposed between ICM cells and do not a t this stage constitute a separate layer. At the margin of the ICM trophoblast cells are often pyramidal in shape, with surfaces associated with ICM cells bordering the blastocyst cavity, adjacent trophoblast, and internal ICM cells. Some of the intercellular spaces between the ICM cells are initially large, and there are few cell processes within these spaces (Fig. 2). When the ICM becomes more compact, intercellular spaces are reduced and microlamellae appear between regions of cell contact. At this stage there is little cytological difference between ICM cells and trophoblast cells (Fig. 31, and there is no apparent polarization in the ICM cells. Junctions between ICM cells or between ICM and polar trophoblast are small and most commonly consist of primitive junctions (areas of close association with only slight increase in density on the inner surface of the plasmalemma). During this stage, gap junctions become common and a few desmosomes are also seen between ICM cells and between ICM cells and trophoblast cells. In this and subsequent stages, there is evidence of cell death in the form of vacuoles, and phagolysosomes
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Fig. 1. Three stages in segregation of the ICM. a: Some polar trophoblast cells are cuboidal (arrow). ICM cells extend in irregular fashion into the blastocyst cavity and in other sections are attached to mural trophoblast. Note the large size of the blastomeres. b The ICM cells constitute a disk of cells at one pole of the blastocyst. Trophoblast
cells (arrow) at the margin of the ICM extend toward the d i s k c: The ICM cells of this expanded zonal blastocyst now constitute a n aggregated somewhat spherical cluster. The primary endodermal layer has formed and extends beyond the ICM as parietal endoderm (arrows). a: Day 5; b: Day 6; c: Day 7. X 320.
Abbreviations
that contain cell debris are present in several of the cells (Fig. 2). From the large size of some of the vacuoles and the occasional degenerating nuclei, it is clear that whole cells have been ingested. Smaller vacuoles that could be the result of earlier ingestion of cell fragments or autophagy are also present.
BL E ER G M PE
m
Z
basal lamina endoderm endoplasmic reticulum Golgi complex mitochondria parietal endoderm polar trophoblast zona pellucida
Organization of the ICM in Mature and Expanded Zonal Blastocysts
During the zonal stage, the blastocyst expands (Figs. l c , 4), and the ICM is consequently relatively smaller. It becomes more spherical, and the individual blastomeres decrease in size and begin to be polarized. The
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first indication of the acquisition of epiblast polarity is the formation of a basal lamina underlying some of the epiblast cells of the ICM, thus establishing a basal surface. Junctions resembling fascia adherens appear for the first time toward the center of the ICM (Fig. 5). Since the cells are in a flattened spheroidal arrangement, the junctional complexes are positioned toward the now-central apices of the epiblast cells. This arrangement is similar to that seen in the ICM of the implanting rhesus monkey embryo, thus presaging formation of the amniotic cavity (Enders et al., 1986b). In highly expanded zonal blastocysts (c. 300 Fm in diameter), evidence of epiblast polarity is seen in the form of extensive central apical junctional complexes. During this stage the flattened cells lining the cavity of the blastocysts first show cytological evidence of differentiation into primitive endoderm, and differentiated endodermal cells come to underlie both ICM and mural trophoblast (see below). Desmosomes and gap junctions link ICM cells to one another, to the polar trophoblast cells, and to endoderm (Fig. 7). Inner Cell Mass of Blastocysts After Loss of the Zona Pellucida
In many respects the ICM of azonal blastocysts resembles that of large zonal blastocysts. The ICM is restricted to a small portion of the circumference of the blastocyst, and apical junctional complexes define a central region within the epiblast (Fig. 8). Some isolated cells (large cells that retain a more primitive type of mitochondria) are seen. A pronounced basal lamina separates epiblast from the underlying endoderm, which lacks a basal lamina. Organelles of ICM Cells During Preimplantation Differentiation
A distinctive characteristic of ICM cells, especially in early blastocysts, consists of the empty-appearing areas of cytoplasm without discernible organelles (Figs. 3-7). These areas can occasionally be seen to contain a substructure of flocculent or finely granular material (Figs. 6, 7). Formed organelles are largely excluded from these areas of the cells, resulting in restriction of mitochondria and polyribosomes to perinuclear and peripheral regions and to columns. The organelle-free areas are seen in ICM cells of expanded zonal and azonal blastocysts, as well as earlier stages, and are also apparent in endodermal cells (Fig. 9). Mitochondria1 profiles in blastomeres of early blastocysts are characteristically clustered (Fig. 3). By following profiles through several sections, it is clear that the sections comprise multiple cuts through single long andlor branched mitochondria. The large convoluted
Fig. 2.
Fig. 2. Montage of electron micrographs of the ICM of the same zonal blastocyst as seen in Figure lb. Although portions of one cell near the cavity are flattened, no endodermal layer is formed here. Large vacuoles containing cell debris are seen both in the ICM and in polar trophoblast (arrows). At the right it can be seen that a trophoblast cell extends a process to one of the innermost cells of the ICM. Note also the pronounced clustering of the mitochondria1 profiles in the blastomeres. The area in the bracket is seen in Figure 3. Day 6. x 2,700.
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24 1
Fig. 3.Portions of two blastomeres of polar trophoblast and underlying ICM cell. Note that in both cell types the mitochondria1 profiles are clustered. From the convolutions seen, mitochondria such as those seen in the ICM cell are believed to be individual convoluted structures. Day 7. x 15,000.
mitochondria with few cristae are similar to those seen in the morula-blastocvst transitional stages (Enders et al., 1989a). Smooth o r sparsely granular membranes of are Often adjacent to mitochonendoplasmic dria. Mitochondria of later blastocysts are shorter and
Fig. 4. Montage of the ICM of a n expanded zonal blastocyst. There is now a complete layer of flattened squamous cells, the primary endoderm, underlying the ICM. The area in brackets is illustrated in Figure 9. Day 8. x 3,000.
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Fig. 5. Compact ICM from the blastocyst shown in Figure lc. The epiblast cells begin to form apical junctions toward the center of the ICM (see area in brackets and inset).Note the mitotic figure (MF) to the right. A necrotic cell (NC) lies between the polar trophoblast and
the zona pellucida, the density of which is increased with cationized ferritin. A thin layer of endoderm (E) underlies the epiblast. Day 7. x 6,100.
more evenly dispersed, are less frequently associated with endoplasmic reticulum, and contain an increased number of cristae (Figs. 6, 7). The endoplasmic reticulum is increased in amount and is distinctly granular; ribosomes, both free and associated with endoplasmic reticulum, are associated into polyribosomal clusters. The large Golgi complexes are usually perinuclear and contain numerous elements and many associated vesicles. There is a decrease in organelle-free areas and an increase in numbers of lipid droplets within ICM cells in large azonal blastocysts.
Differentiationof Endoderm Primitive endoderm, which is first detected during the zonal blastocyst stage, is seen initially as segregation of a few flattened cells on the inner surface of the ICM (Figs. 4, 9). Small junctions form between these cells, and they develop a microvillous surface toward the cavity of the blastocyst (Figs. 5, 6). Subsequently, the differentiation of characteristic dilated granular endoplasmic reticulum and the separation of this layer from the rest of the epiblast indicate the formation of
BABOON BLASTOCYST DIFFERENTIATION
243
Fig. 7. Higher magnification of ICM cells, showing the presence of many polysomes and, in the upper right, a region devoid of polysomes and other organelles (margin designated by arrowheads). Note the irregular-shaped mitochondrion (M) in the cell (probably endoderm) containing lipid (L). A caveolus is seen in one of the cells of the primitive endoderm (arrow). Day 7. x 28,000.
Fig. 6. Higher magnification of the margin of an ICM from a n expanded zonal blastocyst. A small amount of basal lamina is formed beneath the polar trophoblast. The mitochondria1matrix is less dense than in previous stages. Note that in the area of granular material (arrowheads), thought to be a mass of stored proteinaceous material, there are no polysomes (see Fig. 7). Day 7. x 12,900.
visceral endoderm (Figs. 9, 10). Phagolysosomes containing cell debris are seen frequently within visceral endodermal cells. In expanded zonal and azonal blastocysts, visceral endodermal cells have highly irregular folded basal surfaces, with no subjacent basal lamina. Occasional intercellular junctions (primitive, gap, and desmosomal) remain between visceral endodermal cells and ICM cells. Endodermal cells extend beyond the margin of the ICM in expanded zonal blastocysts, illustrating that differentiation of the parietal endoderm can occur prior to loss of the zona. Parietal endodermal cells may be closely apposed to mural trophoblast, including apparent regions of intercellular adhesion and fascia1 gap junctions; the cells often contain spherical gap junctions. in azonal b~astocysts, the parietal endodermal layer extends as a complete epithelium well beyond the margin of the ICM, to surround approximately 213 of
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terrupted where cells of epiblast and endoderm are contiguous. Frequently basal laminas form in places that are inappropriate. For example, basal lamina material is seen between epiblast cells as well as beneath the epiblast. Basal lamina material occurs as patches and folds in intercellular spaces between polar trophoblast and ICM, but does not form a complete lamina in this position (Fig. 6). A basal lamina underlies mural trophoblast near the junction of visceral endoderm with parietal endoderm and in marginal areas where mural trophoblast is underlain by parietal endodermal cells. However, mural trophoblast is often subtended by a basal lamina in regions without subjacent endodermal cells. Basal processes of mural trophoblast cells that project toward the endoderm usually have a continuous basal lamina, and occasionally small patches of basal lamina are present on the basal side of the endoderm per se. When such patches are found, they are continuous with the basal lamina of mural trophoblast. DISCUSSION Establishment of Polarity
The establishment of polarity in the epiblast is a striking feature of differentiation in the ICM prior to implantation. Since this polarization of cells follows the formation of the endoderm and coincides with the formation of a basal lamina on the basal surface of the epiblast, it seems possible that the endoderm may be responsible for the establishment of polarity within the epiblast. Should this be the case, there would be yet another situation in embryonic development where establishment of polarity plays an important role in differentiation of a cell layer (Ziomek and Johnson, 1980; Johnson, 1986; Fleming, 1987). The initial differentiation leading to formation of the amnionic cavity occurs not only prior to implantation but even prior to loss of the zona pellucida. The cellular mechanism of this differentiation, by cavitation of the ICM, is similar to that described in the rhesus monkey in an early implantation stage (Enders et al., 1986b). Cytoplasmic Differentiation of Inner Cell Mass Fig. 8. ICM from azonal blastocyst. Epiblast cells contain more lipid than previously but otherwise are similar to those of the expanded zonal blastocyst. An apical junctional complex is seen at the top of the micrograph (arrow), which is toward the center of the ICM. Day 7. x 7,820.
the circumference of the blastocyst. The parietal endoderm is sometimes adherent to trophoblast in regions lacking a trophoblast-associated basal lamina, but more frequently is separated from trophoblast by the basal lamina. In areas where the endodermal layer terminates and is adherent to trophoblast, the basal lamina also terminates abruptly a t this point. Formation of Basal Laminas
Extracellular material similar in appearance to that of the lamina densa of basal laminas is interspersed between the ICM and flattened presumptive endoderma1 cells, but does not form a complete layer in early zonal blastocysts. A discrete basal lamina is seen under the epiblast of expanded zonal blastocysts, but it is in-
Storage materials have been described in the oocytes and preimplantation embryos of rodents, where they aggregate in macromolecular arrays that are readily visible with the electron microscope (Enders and Schlafke, 1965; King and Tibbits, 1977; Parkening et al., 1985). A recent study of hamster and rat blastocysts indicated that the fibrous material may represent a source of cytokeratins (Capco and McGaughey, 1986). The large areas that lack organelles in the baboon blastocyst have also been reported in the rhesus monkey (Enders and Schlafke, 1981) and seem likely to be manifestations of masses of residual stored proteinaceous materials. The fact that such regions remain longest in the epiblast cells may be further evidence of the relatively slow differentiation of these cells compared to either trophoblast or endoderm. Differentiation of Primary Endoderm
It is interesting that the cytological features of endodermal differentiation do not occur until after ICM cells have been isolated to a cluster at one side of the cavity of the blastocyst for some time. Only then do
BABOON BLASTOCYST DIFFERENTIATION
Fig. 9. Primary endodermal cells. a: Enlargement of a portion of the
ICM illustrated in Figure 4.When a complete hypoblast layer is first formed, the cells are flattened and have junctional complexes and a few sparse microvilli toward the blastocyst cavity. b: Higher magni-
apical junctional complexes and microvilli facing the cavity of the blastocyst indicate formation of an endodermal layer. Even the dilated endoplasmic reticulum that has come to be the hallmark of endodermal cells (Enders, 1971; Rossant, 1987) is seen only after flattened cells are present in the appropriate position. Indeed the endoplasmic reticulum is more sparse in endodermal cells of the baboon blastocyst than in those of other species. The arrangement of the trophoblast cells at the margin of the ICM is such that one of the two daughter cells of a dividing trophoblast cell could become endoderm rather than mural trophoblast. On the other hand, flanges of trophoblast cytoplasm extend under part of the ICM subsequent to the stage of triangular cells but prior to differentiation of endoderm. Such an arrangement has been suggested as a way by which trophoblast can modify ICM potential (Fleming et al., 1984).
245
fication of the cytoplasm of an endodermal cell, showing the granular endoplasmic reticulum with a flocculent content typical of endoderma1 cells. a , x 8,900. b, x 31,200.
Cell Death During Preimplantation Stages
Throughout the blastocyst stage large phagolysosomes containing cell debris are seen within cells of both epiblast and endoderm. Although the smaller vacuoles may contain debris from autophagy or from fragments of cells, many vacuoles contained both nuclei and recognizable organelles, suggesting death of whole cells. The distribution and stage in which they were found appeared random and therefore did not suggest programmed cell death or a specific role for cell death in any given morphogenic process. There appear to be fewer isolated cells in blastocysts of the baboon than in those of the rhesus monkey or human. Illustrations of Lopata et al. (1983), Sathananthan (19841, and others have indicated numerous regions of cell sequestration and isolation in human blastocysts in culture. An increased amount of cell death
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Fig. 10. Endodermal cells from an azonal blastocyst. Note that the basal lamina is associated with the epiblast (EPI), not with the endodermal cells. The endodermal cells are characterized by microvilli and granular endoplasmic reticulum. Day 8, X 12,900.
and cytoplasmic fragmentation seen in the human may be the result of disadvantageous culture conditions, but no human blastocysts from in vivo development have been studied by electron microscopy with which appropriate comparisons can be made. There is some indication, however, from the admittedly limited observations of Buster et al. (1985), that blastocysts from donor women are more viable than blastocysts from in vitro culture. There appears to be more cell death in the primate blastocyst than in laboratory rodents, and this cell loss during development may be accentuated by adverse culture conditions (Boatman, 1987). It is clear from the work of Willadsen (1981) and others t h a t only a portion of the cytoplasm of the original egg is necessary for development of a blastocyst. An apparently normal early baboon blastocyst was collected that lacked a t least a third of the original ooplasm (Enders e t al., 1989a). However, cell death at the wrong time or concentrated in location within the developing blastocyst might be deleterious to further development. Fixation obviously prevents following the subsequent fate of a given abnormal-appearing cleavage stage. It should be possible, however, to recognize individual abnormal blastomeres and to follow them for one or two cleavages in vitro. To date individual abnormal blastomeres have not been followed in vitro, although there is information from the human suggesting that “ugly” early cleavage stages (with considerable debris) can produce normal offspring (Veeck, 1986). Mitochondria of the cleavage stages and early blastocysts are particularly interesting because they undergo changes in shape and in distribution during preimplantation development, and because it may be possible to monitor distribution of these organelles in vitro by using fluorescent markers (Albertini et al., 1987). Both in human embryos in vitro (Nilsson and
Sundstrom, 1982; Lopata e t al., 1983; Johnson, 1986) and in the developing baboon embryo (Enders et al., 1989a), there is a tendency for mitochondria to aggregate near the nucleus in some of the blastomeres of morula stages. Later in development, not only are mitochondria more dispersed and less complex, but in addition other changes occur including a decrease in matrix density, which has also been reported in the human (Lopata et al., 1982). Johnson (1986), in analyzing results of cell marker experiments, pointed out the difficulties of using a cytoplasmic marker like peroxidase in the mouse, where residual midbodies sometimes link daughter cells for considerable time. It should also be pointed out that markers like fluorescent spheres that would not pass through midbodies might be ingested as cell debris, also providing non-lineage localization, whereas the genetic markers used extensively by Rossant (1987) would not be subject to either of the above difficulties. Basal Lamina Formation
It is perhaps ironic t h a t one of the best-studied basal laminas is Reichert’s membrane, which has been used as a source of studies of the way in which endodermal cells synthesize basal lamina constituents (Hogan et al., 1984; Mazariegos et al., 1987). Yet the parietal endoderm initially lacks a basal lamina, and the only basal lamina present is that subjacent to trophoblast. Surprisingly, the formation of the first basal lamina, that of the epiblast and trophoblast, has not been widely studied. Preliminary observations using antibodies to entactin and laminin showed intracellular localization of laminin in early cleavage stages (Wu et al., 1983). However, there is not a correlation between different studies, in that these authors show earlier synthesis of laminin than did Leivo e t al. (1980), and the former authors do not report trophoblast cell pro-
BABOON RLASTOCYST DIFFERENTIATION
duction of laminin whereas Wartiovaaro et al. (1980) did. Calarco-Gillam (1986) has summarized studies on formation of extracellular matrix materials by preimplantation embryos. ACKNOWLEDGMENTS
These studies were supported by grants HD 10342 and RR00169 from the National Institute of Child Health and Human Development. The authors would like to thank Drs. Mark Cukierski, Andrew Hendrickx, and Ross Tarara, and Pamela Binkerd, Carolyn Booher, and J i m Utz for assistance with the endocrine data, animal preparation, and collection techniques. LITERATURE CITED Albertini, D.F., E.W. Overstrom, and K.M. Ebert 1987 Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo. Biol. Reprod., 37,441-451. Betteridge, K.J., and J.E. Flechon 1988 The anatomy and physiology of pre-attachment bovine embryos. Theriogenology, 29r155-197. Boatman, D.E. 1987 In vitro growth of nonhuman primate pre- and peri-implantation embryos. In: The Mammalian Preimplantation Embryo: Regulation of Growth and Differentiation in vitro. B.D. Bavister, ed. Plenum Press, New York, pp. 273-303. Buster, J., M. Bustillo, I. Rodi, S. Cohen, M. Hamilton, J. Simon, I. Thorneycroft, and J. Marshall 1985 Biologic and morphologic development of donated human ova recovered by non-surgical uterine lavage. Am. J . Obstet. Gynecol., 15
