"The Carnegie Stages of Early Human Embryonic Development: Chart of all 23 Stages, Detailed Descriptions of Stages 1 - 6"




Part Four: Descriptions of First Six Embryonic Stages in The Carnegie Chart (Ronan O'Rahilly and Fabiola Muller)

National Museum of Health and Medicine [can use search engine on this page] http://www.nmhm.washingtondc.museum/

Human Developmental Anatomy Center http://nmhm.washingtondc.museum/collections/hdac/anatomy.htm

Carnegie Stages of Development http://nmhm.washingtondc.museum/collections/hdac/Select_Stage_and_Lab_Manual.htm


http://nmhm.washingtondc.museum/collections/hdac/stage1.pdf

(Developmental Stages in Human Embryos by Ronan O'Rahilly and Fabiola M0ller. Published by Carnegie Institution of Washington, Publication 637. 1987)

Stage 1


Approximately 0.1-0. 15 mm in diameter
Approximately 1 postovulatory day
Characteristic feature: unicellularity

Embryonic life commences with fertilization, and hence the beginning of that process may be taken as the point de depart of stage 1. Despite the small size (ca. 0.1 mm) and weight (ca. 0.004 mg) of the organism at fertilization, the embryo is "schon ein individual-spezifischer Mensch" (Blechschmidt, 1972). The philosophical and ethical implications have been discussed briefly by O'Rahilly and M0ller (1987).

Fertilization is the procession of events that begins when a spermatozoon makes contact with an oocyte or its investments and ends with the intermingling of maternal and paternal chromosomes at metaphase of the first mitotic division of the zygote (Brackett et al., 1972). Fertilization sensu stricto involves the union of developmentally competent gametes realized in an appropriate environment to result in the formation of a viable embryo capable of normal further development (Tesar0k, 1986).

Fertilization requires probably slightly longer than 24 hours in primates (Brackett et al., 1972). In the case of human oocytes fertilized in vitro, pronuclei were formed within 11 hours of insemination (Edwards, 1972).

Given the availability of a mature oocyte (first meiotic division completed) and capacitated spermatozoa (permitting the acrosomal reaction), the criteria for fertilization generally adopted are (1) the presence of two or more polar bodies in the perivitelline space, (2) the presence of two pronuclei within the ooplasm, and (3) the presence of remnants of the flagellum of the fertilizing spermatozoon within the ooplasm (Soupart and Strong, 1974).

Fertilization, which takes place normally in the ampulla of the uterine tube, includes (a) contact of spermatozoa with the zona pellucida of an oocyte, penetration of one or more spermatozoa through the zona pellucida and the ooplasm, swelling of the spermatozoalhead and extrusion of the second polar body, (b) the formation of the male and female pronuclei, and (c) the beginning of the first mitotic division, or cleavage, of the zygote. The various details of fertilization, including such matters as capacitation, acrosomal reaction, and activation, are dealt with in special works.

When cortical granules are released, their contents appear to reinforce the structure of the zona pellucida (Sathananthan and Trounson, 1982). This is thought to be the morphological expression of the zonal reaction, and the cortical and zonal reactions may provide a block to polyspermy.

The three phases (a, b, and c) referred to above will be included here under stage 1, the characteristic feature of which is unicellularity. The sequence of events before and during the first three stages is summarized in Table l-l.


Table 1-1
Tabulation of the First Three Stages
Stage Event Products/strong>
(mm)
Meiotic division 1 Oocyte 2 and polar body 1
Beginning of meiotic divison 2
and ovulation
Ovulated oocyte
1a Fertilization: Penetration Penetrated oocyte
1b Fertilization: Completion of
of Penetrated oocyte meiotic
division 2

Fertilization: Pronuclei
enter cleavage division
Penetrated occyte



Ootid and polar body 2
1c Fertilization Zygote
2 Cleavage continues 2 to about 16 cells
3 Formulation of blastocystic
cavity
Blastocyst, from about
32 cells onward


The term "ovum," which has been used for such disparate structures as an oocyte and a 3-week embryo, has no scientific usefulness and is not used here. Indeed, strictly speaking, "the existence of the ovum... is impossible" (Franchi, 1970). The term "egg" is best reserved for a nutritive object frequently seen on the breakfast table.

At ovulation, the oocyte is a large cell surrounded by a thick covering, the zona pellucida, which is believed to be produced (at least largely) by the surrounding follicular cells. Processes of the follicular cells and microvilli of the oocyte both extend into the zona. The diameter of such a mammalian cell, including its zona, ranges from 70 to 190 0m. In the human, the ooplasm measures about 100 0m, and the thickness of the zona ranges from 16 to 18 0m (Allen et al., 1930). Good photomicrographs and electron micrographs of human secondary oocytes are available (e.g., Baca and Zamboni, 1967, figs. 20 to 24; Kennedy and Donahue, 1969). The zona pellucida is covered externally by the corona radiata, which is a loose investment of granulosa cells from the ovarian follicle. On fixation and embedding, the oocyte undergoes shrinkage; this affects the cytoplasm more than the zona, so that a subzonal (or perivitelline) space becomes accentuated. The polar bodies are found within that space.

It is said that the first polar body may divide before the second is released, and it has been claimed that each of the three polar bodies is capable of being fertilized. Although it is not unusual for the second polar body to display a nucleus, the chromosomes of the first polar body are isolated and naked (Zamboni, 1971).

It is "likely that no more than one day intervenes between ovulation and fertilization, This time interval may be taken then as the possible error in age of [an] embryo when it is considered the same as ovulatory age" (Rock and Hertig, 1942).

(a) Penetrated oocyte. This term may conveniently be used once a spermatozoon has penetrated the zona pellucida and, strictly, "after gamete plasma membranes have become confluent" (Zamboni et al., 1966).

Penetration has been inferred from the presence of spermatozoa in the zona pellucida or in the subzonal space (Edwards, Bavister, and Steptoe, 1969). Moreover, in vitro examples showing portions of spermatozoa within the ooplasm are illustrated by Sathananthan, Trounson, and Wood (1986), in whose work are also detailed views showing the formation of the secondpolar body.

(b) Ootid The cell characterized by the presence of the male and female pronuclei is termed an ootid (figs, l and 1-2). Several examples of human ootids have been described. They are probably about 12-24 hours in age. The diameter, including the zona pellucida, is about 175 0m (Hamilton, 1946; Dickmann et al., 1965), and the diameter of the subzonal space is approximately 140 μm. The cytoplasm of the ootid has a diameter of about 100 μm (Hamilton, 1946; Noyes et al., 1965); each of the pronuclei measures about 30 μm (Zamboni et al., 1966). The various ultrastructural features of the ootid have been described and illustrated (Zamboni et al., 1966; Sathananthan, Trounson, and Wood, 1986). Although "in most mammalian species, the male pronucleus has been reported to be larger than the female pronucleus," the converse has been found in one human specimen and, in two others, the pronuclei appeared to be of equal size (Zamboni, 1971).

(c) Zygote. The cell that characterizes the last phase of fertilization is elusive. The first cleavage spindle forms rapidly and has been used in identification. Such cells have probably been seen in certain mammals, e.g., the pig, cow, hamster, rat, and mouse. Pronuclear fusion does not occur. Rather, the two pronuclear envelopes break down ("post-apposition envelope vesiculation," Szabo and O'Day, 1983), and the two groups of chromosomes move together and assume positions on the first cleavage spindle. Thus the zygote lacks a nucleus.

A human embryo "in syngamy just prior to cleavage" has been illustrated by Sathananthan and Trounson (1985, fig. 2). "The chromosomes, some associated in pairs, are located in an agranular zone in the central ooplasm."

In the human, the initial cleavage that heralds the onset of stage 2 occurs in the uterine tube "some time between twenty-four and thirty hours after [the beginning of] fertilization" (Hertig, 1968).


http://nmhm.washingtondc.museum/collections/hdac/stage_2.htm

Stage 2


Approximately 0.1-0.2 mm in diameter
Approximately 10 -3 postovulatory days
Characteristic feature: more than 1 cell but no blastocystic
cavity seen by light microscopy

Stage 2 comprises specimens from 2 cells up to the appearance of the blastocystic (or segmentation) cavity. The more advanced examples (from about 12 cells on) of stage 2 are frequently called morulae (L., morus, a mulberry). The term morula is not historically appropriate for mammals, however, because the amphibian morula gives rise to embryonic tissues only, whereas in mammals non-embryonic structures (such as the chorion and the amnion) are also derived from the initial mass of cells.

Size and Age

The diameter at stage 2 before fixation is of the order of 175 μm; after fixation, it is approximately 120 μm (Hertig et al., 1954). Indeed, shrinkage of as much as 50 percent may occur in some instances (Menkin and Rock, 1948). Whether before or after fixation, the diameter at stage 2 may be expected to lie between 75 and 200 0m.

The volume of the protoplasmic mass diminishes during cleavage (O'Rahilly, 1973, table 5). The age at stage 2 is believed to be approximately 10-3 postovulatory days. The range is probably l-5 days (Sundstrμm, Nilsson, and Liedholm, 1981). In vitro, 2 cells may be found at 10 days, 4 cells at 2 days, and 8 cells by about 20 days.

General Features

The organism proceeds along the uterine tube by means not entirely understood (reviewed by Adams, 1960). It leaves the tube and enters the uterine cavity during the third or fourth day after ovulation, when probably 8-12 cells are present, and when the endometrium is early in the secretory phase (corresponding to the luteal phase of the ovarian cycle).

It has been shown experimentally (in the mouse, rat, and rabbit) that a blastomere isolated from the mammalian 2-cell organism is capable of forming a complete embryo. Separation of the early blastomeres is believed to account for about one-third of all cases of monozygotic twinning in the human (Corner, 1955).

Such twins should be dichorial and diamniotic (fig. 5- 2). The fact that nearly 60 percent of dichorial twins (whether monozygotic or dizygotic) have two unfused placentae "indicates that the zona pellucida must have disappeared sufficiently long before implantation to allow the twins to become implanted in independent positions in the uterus" (Bulmer, 1970). Dizygotic twins, in contrast, are believed to arise from two oocytes, from a binucleate oocyte, or from a second polar body (Gedda, 1961).

The successive cleavage divisions do not occur synchronously, so that (in the pig) specimens of anywhere from 1 to 8 cells can be found. It has been suggested that the more precociously dividing cells may be those that give rise to the trophoblast. Moreover, differences in the size, staining, and electron density of the blastomeres are observed. There is reason to believe, however, that the blastomeres are not determined very early in development.

For example, it has been shown experimentally in the mouse that the ability to develop into trophoblastic cells is inherent in all blastomeres of the first two stages. Up to 16 cells, none of the blastomeres is yet determined to give rise to cells of the inner mass. It may be that the primary factor responsible for the determination of one of the two alternative routes of differentiation (trophoblast or inner cell mass) is simply the position (peripheral or internal) that a given cell occupies.

According to the "inside/outside hypothesis," micro-environmental differences influence the determination of blastomeres (between 8 and 16 cells in the mouse) so that those on the outside become more likely to form trophoblast (with more restricted potential) whereas those enclosed by other cells become more likely to form the inner cell mass. Another hypothesis accounting for early cellular diversity (in the mouse) is based on polarization of the larger, external cells, characterized by microvilli.

Furthermore, it has been possible in the mouse to unite two 16-cell organisms and obtain from them one giant, but otherwise perfectly normal, blastocyst. Fusion of mouse organisms with close to 32 cells each has also resulted in a single blastocyst. It has been stressed that it is dangerous readily to infer normality on purely morphological grounds.

In the human, two significant specimens of stage 2 (Hertig et al., 1954) will be cited. A 2-cell specimen (No. 8698) was spherical and surrounded by a transparent zona pellucida (fig. 2-1). Two polar bodies were present. Each blastomere was nearly spherical. It has been maintained that the larger blastomere would probably divide first and hence may perhaps be trophoblastic (Hertig, 1968). A 12-cell specimen (No. 8904) was perfectly spherical and surrounded by a clear zona pellucida. One blastomere, central in position and larger than the others, was presumed to be embryogenic, whereas the smaller cells were thought to be trophoblastic.

A number of human specimens of stage 2 found in atretic ovarian follicles were considered to be parthenogenetic by their authors (Häggström, 1922; Krafka, 1939; Herranz and Vázquez, 1964; Khvatov, 1968). Such a claim, however, has been disputed (Ashley, 1959), and it has been pointed out that polysegmentation, that is, cleavage-like conditions described as "pseudoparthenogenesis," are not infrequently encountered in moribund oocytes (Kampmeier, 1929). It is likely also that some instances of cleavage obtained in vitro may be pseudoparthenogenetic rather than caused by actual fertilization by spermatozoa. The presence of a Y chromosome in a "spread from a replicating blastomere" (Jacobson, Sites, and Arias-Bernal, 1970) has been claimed "but not convincingly demonstrated" (Brackett et al., 1972).

The embryonic genome probably becomes functionally active during stage 2. Activation of transcription of rRNA genes (contributed to the embryonic genome by the male and female gametes at fertilization) is indicated in vitro by the pattern of nucleologenesis, which changes in 6- to 8-cell embryos and becomes typical in 10- to 12-cell embryos (Tesar0k et al., 1986).


http://nmhm.washingtondc.museum/collections/hdac/stage3.pdf

Stage 3


Approximately 0.1-0.2 mm in diameter
Approximately 4 postovulatory days
Characteristic feature: free blastocyst

Stage 3 consists of the free (that is, unattached) blastocyst, a term used as soon as a cavity (the blastocystic, or segmentation, cavity) can be recognized by light microscopy. (The staging system is based on light microscopy and, in later stages, on gross structure also.)

The blastocyst is the hollow mass of cells from the initial appearance of the cavity (stage 3) to immediately before the completion of implantation at a subsequent stage. The blastocystic cavity, under the light microscope, begins by the coalescence of intercellular spaces when the organism has acquired about 32 cells. In in vitro studies, a cavity formed in some human embryos at 16-20 cells (Edwards, 1972).

It is necessary to stress that the cavity of the mammalian blastocyst is not the counterpart of the amphibian or the avian blastocoel. In the bird, the blastocoel is the limited space between the epiblast and the primary endoderm. The cavity of the mammalian blastocyst, however, corresponds to the subgerminal space together with the area occupied by the yolk (Torrey, personal communication, 1972).

The mammalian blastocyst differs from a blastula in that its cells have already differentiated into at least two types: trophoblastic and embryonic cells proper. Heuser and Streeter (1941) emphasized an important point by using stage 3 as an example: The blastocyst form is not to be thought of solely in terms of the next succeeding stage in development. It is to be remembered that at all stages the embryo is a living organism, that is, it is a going concern with adequate mechanisms for its maintenance as of that time.

It is no less true, however, that changes occur "in the growing organism and its environment which provide critically for the future survival of the organism" (Reynolds, 1954). Indeed, such morphological and functional changes during development "critically anticipate future morphological and functional requirements for the survival and welfare of the organism" (ibid.).

Sex chromatin has been "tentatively identified" in two in vitro human blastocysts (Edwards, 1972). Probably the first recognition of the inner cell mass of the mammalian (dog and rabbit) blastocyst was that by Pr0vost and Dumas in 1824. This and many other aspects of the blastocyst are considered in a book edited by Blandau (1971).

Size and Age

In the human embryo the maximum diameter increases from 100-200 μm at stages 2 and 3 to 300-450 0m at stage 5a. Embryos of stage 3 are believed to be about 4 days in age. In vitro embryos of stage 1 have been recorded at 9-32 hours after insemination; stage 2 at 22-40 hours (2 cells), 32-45 hours (4 cells), and 48 hours (8 cells); stage 3 at 100 hours, and extruding from the zona pellucida at 140-160 hours, at which time they show differentiation into trophoblast, epiblast, and hypoblast (Mohr and Trounson, 1984).

Histological Features

Zona pellucida In stage 3 the zona pellucida may be either present or absent. In vitro, the blastocyst emerges from the zona at about 6-7 days. The emergence is commonly referred to as "hatching."

Trophoblast. During stage 3 the trophoblastic cells, because of their peripheral position, are distinguishable from the embryonic cells proper. The trophoblastic cells that cover the inner cell mass are referred to as polar: i.e., at the embryonic pole or future site of implantation. The remaining trophoblast is termed mural.

Cavitation. It is believed that the blastomeres (in the mouse) attain the ability to secrete the blastocystic fluid after a definite number of cleavages, namely at the end of the fifth and at the beginning of the sixth mitotic cycle. In the mouse it has been shown that, when the organism consists of about 32 cells, small cavities unite to form the beginning of the blastocystic cavity. In other words, the solid phase of development (in the mouse) ends at about 28-32 cells, when fluid begins to accumulate beneath the trophoblastic cells.

As the blastocyst develops, it undergoes expansions and contractions. When contracted, a "pseudomorula" of about 100 cells in the mouse can be seen. Because no appreciable increase in size of the (cat) embryo occurs at first, it is thought that no mere flowing together of inter- or intra-cellular spaces or vacuoles is a sufficient explanation of the origin of the blastocystic cavity. Thus an additional factor, namely cytolysis of certain of the central cells, is also involved.

Electron microscopy has added further details. The formation of junctional complexes, which is regarded as the first sign of blastocystic formation, is found very early in the rat, when the embryo consists of only 8 cells, although the first indication of a cavity, as opposed to intercellular spaces, is not seen until after another series of cell divisions. In two human, 8-cell, in vitro embryos studied by electron microscopy, "a small cleavage cavity was already apparent within each embryo" (Sathananthan, Wood, and Leeton, 1982).

Inner cell mass. The embryonic cells proper become surrounded by the trophoblastic cells and form an inner mass. Studies of various mammals have indicated that the inner cell mass represents more than the embryo itself, insofar as it constitutes a germinal mass of various potentialities which continues for a time to add cells to the more precociously developed trophoblast.

The inner cell mass gives origin to the hypoblast, and its remainder (the "formative cells") constitutes the epiblast. The epiblastic cells soon become aligned into what was frequently described as the "germ disc."

These various relationships are summarized in figure 6-2. It has been found that hypoblastic differentiation in the macaque occurred at about the same time that a basal lamina was found under mural trophoblast and epiblast (but not polar trophoblast or hypoblast) (Enders and Schlafke, 1981).

Duplication of the inner cell mass probably accounts for most instances of monozygotic twinning (Corner, 1955; Bulmer, 1970). Such twins should be monochorial but diamniotic (fig. 5-2). In vitro, "many blastocysts fail to hatch fully from their zona pellucida," and "two separate embryos could form if the inner cell mass was bisected during hatching" (Edwards, Mettler, and Walters, 1986).

Two significant specimens of stage 3 (Hertig et al., 1954) are here cited. In a 58-cell specimen (No. 8794) 53 of the cells were trophoblastic whereas 5 were embryonic. The latter composed the inner cell mass, which was located eccentrically within the blastocystic cavity but had not yet assumed a truly polar position.

In a 107-cell specimen (No. 8663), 99 of the cells were trophoblastic, and, of these, 69 were mural in position and 30 were polar (i.e., covering the embryonic pole). Eight of the 107 cells were embryonic, and were characterized by their larger size and by the presence of intracytoplasmic vacuoles. Moreover, the 8 cells comprised three types: "obvious primitive, vacuolated ectoderm [ epiblast]; flattened primitive endoderm [ hypoblast]; and a large indifferent cell, presumably a [primordial] germ cell" (Hertig, 1968). In addition, of the 30 polar trophoblastic cells, 4 which were situated "ventral and lateral to the formative cells may actually be of primitive endodermal type" (Hertig et al., 1954).

Dorsoventrality. A comparison of stage 3 embryos with those of stage 5 makes it clear that the surface ofthe inner cell mass that is adjacent to the polar trophoblast represents the dorsal surface of the embryo, and the surface of the mass that faces the blastocystic cavity represents the ventral surface. In other words, "dorsalization," or "dorsoventrality," becomes apparent during stage 3 (O'Rahilly, 1970). The possibility should be kept in mind, however, that the inner cell mass can perhaps travel around the inside of the trophoblastic layer.

Rate of division. In the pig embryo it has been shown that, in general, "during the first seven days the cells undergo about eight divisions, that is, they divide about once a day" (Heuser and Streeter, 1929). A similar generalization may be made for the human embryo during stages l-3, and also for the baboon (Hendrickx, 1971). In the case of the baboon, "there is a close correlation between age and cell number," although "there is no consistent relationship between age and size for these stages of development" (ibid.).


http://nmhm.washingtondc.museum/collections/hdac/stage4.pdf

Stage 4


Probably approximately 0.1-0.2 mm in diameter
Approximately 5-6 postovulatory days
Characteristic feature: attaching blastocyst

Stage 4, the onset of implantation, is reserved for the attaching blastocyst, which is probably 5-6 days old.

Although the criteria for the first three stages are those of the first three horizons, it has not proved practicable in stages 4-10 to retain the criteria for horizons IV-X. This abandonment had already been begun by Hertig, Rock, and Adams (1956) and by Heuser and Corner (1957).

It should be noted that certain specimens that are now listed in stages 5a and 5b (Hertig, Rock, and Adams, 1956) were formerly included in horizon IV. In other words, because such specimens represent "significantly different stages of development" (ibid.), they have been transferred, and, as a result, stage 4 has been made more restricted. Healing of the uterine epithelium over the conceptus, for example, is too variable and has been eliminated as a criterion for stage 5; it usually occurs after horizon VI has begun (Böving, 1965).

Implantation is the specific process that leads to the formation of a specialized, intimate cellular contact between the trophoblast and the endometrium, or other tissue in the case of ectopic implantation (Denker, 1983).

Implantation is a highly complicated and ill-understood phenomenon "by which the conceptus is transported to its site of attachment, held there, oriented properly, and then attached by adhesion, trophoblastic penetration, spread, proliferation, envelopment of vessels, and other developments of the placenta, both conceptal and maternal parts" (Böving, 1963). In this broad sense, implantation includes at least stages 4 and 5.

Implantation, then, includes (1) dissolution of the zona pellucida, and contact and attachment (adhesion) between the blastocyst and the endometrium, (2) penetration, and (3) migration of the blastocyst through the endometrium. On the basis of comparative studies, it has been suggested (Boving, 1965) that stage 4 might be subdivided into these three phases. Human (but not macaque) implantation is interstitial in type: i.e., the blastocyst comes to lie entirely within the substance of the endometrium. In the human (as also in the macaque), implantation occurs into an edematous, non-deciduous endometrium. In other words, decidualization takes place at the end of implantation.

In his important study of the early development of the primates, Hill (1932) concluded as follows: The outstanding feature of the early human blastocyst is its extraordinary precocity as exemplified in the relations it very early acquires to the uterine lining and in the remarkably early differentiation of its trophoblast and its extraembryonal mesoderm. It is no longer content to undergo its development in the uterine lumen as does that of all the lower Primates, but, whilst still quite minute, burrows its way through the uterine epithelium and implants itself in the very vascular subepithelial decidual tissue of the uterus. Therein it forms for itself a decidual cavity and undergoes its subsequent development, completely embedded in the maternal tissue. In this way the Primate germ reaches the acme of its endeavour to maintain itself in the uterus and to obtain an adequate supply of nutriment at the earliest possible moment.

The mammalian stage 2 organism and the early blastocyst are surrounded by an intact zona pellucida, which disappears at the beginning of implantation. Hence, implantation "is taken as beginning when the zona pellucida is lost and the trophoblast is in contact with the uterine epithelium throughout its circumference" (Young, Whicher, and Potts, 1968). Although claims have been made that the blastocyst emerges from its zona pellucida by "shedding" or by "hatching," it has also been maintained that, at least in the mouse, the zona undergoes rapid dissolution all around the blastocyst in situ, that is, at the actual site of implantation.

After the blastocyst becomes attached at random to the uterine epithelium in the mouse, it is believed that the inner cell mass can travel around the inside of the trophoblastic shell (presumably somewhat like a satellite gear). Although the nature of the stimulus responsible for the final orientation of the inner cell mass is unknown, it is postulated that the final position is determined either by a morphogenetic gradient across the vertical axis of the uterus or by changes in thetrophoblast associated with its attachment to the underlying tissues, or by both.

The implantation site has been studied by electron microscopy in several mammals, such as the mouse. The cell membranes of the trophoblast and uterine epithelium become intimately related, and large cytoplasmic inclusions are found in the trophoblastic cells. The ultrastructural changes taking place at implantation suggest that there may be a high degree of permeability between maternal and embryonic cells.

In addition, there may be an exchange of cellular material between uterus and embryo. After the zona pellucida has become dissolved, the surface membranes of the trophoblast and uterine epithelium are separated by a very narrow interval (in the mouse). This first morphological sign of implantation can be detected only by electron microscopy.

In at least some macaque specimens a distinction between cytotrophoblast and syncytiotrophoblast can be made (Heuser and Streeter, 1941, e.g., No. C-520, their fig. 38). Moreover, amniogenic cells have been claimed to be "separating from the trophoblast above and distinct from the germ disk" (ibid., No. C-610, their fig. 53).

Finally, epiblastic cells and hypoblast can be distinguished (ibid., No. C-520, their fig. 40). Of the several macaque specimens of stage 4 that have been described, in one instance (No. C-560), the uterine epithelium at the site of attachment showed a disturbed arrangement of its nuclei. Moreover, the cytoplasm had become paler, which was taken to indicate beginning cytolysis. In the conceptus, the site of attachment was formed by syncytiotrophoblast, which is initially formed by the coalescence of polar trophoblast (Hertig, 1968). Some of the increased number of nuclei appeared to have been released from the uterine epithelium and then engulfed by the rapidly expanding trophoblast.

Within the cavity of the blastocyst, disintegrating embryonic cells were interpreted as a mechanical accident caused by displacement of the cells. At the site of attachment, fused multinucleated cells of the uterine epithelium constitute a "symplasma," which fuses with the syncytiotrophoblast (in macaque No. C-610, and also in the rabbit; in the latter it has been studied by electron microscopy by Larsen, 1970).

A specimen of stage 4 in the baboon has been illustrated (Hendrickx, 1971). The single layer of abembryonic trophoblast was continuous with the cytotrophoblast dorsally. The syncytiotrophoblast was in contact with the uterine epithelium, which had lost its columnar appearance. Moreover, as much as one-half of the surface portion of the uterine cells had disappeared at the site of attachment. The inner cell mass showed occasional "endoblastic" cells bordering the blastocystic cavity. The age of the specimen was estimated as 9 days.

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