a comparison of the fine structure of late mouse blastocysts developed in vivo and in vitro

A Comparison of the Fine Structure of Late Mouse Blastocysts Developed In Vivo and In Vitro

HAL D. McREYNOLDS AND ROBERT HADEK Department of Histology, School of Dentistry, Loyola University of Chicago, Maywood, Illinois 601 53 a n d Department of Anatomy, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 601 53

ABSTRACT The ultrastructure of the in uiuo developed late preimplantation mouse blastocyst has been compared with the fine structure of mouse blastocysts developed in uitro for 72 and 92 hours from the late two-cell stage, as well as those grown in uztro for 26 hours from the late morula or early blastocyst stage. Very little difference was observed between the in uiuo developed blastocysts and those cultured 26 hours in uitro. The differences between 72 and 92 hour-embryos and the in uiuo grown ones were significant. The in uiuo specimen and those grown 26 hours in uitro had differentiated a layer of endoderm cells while the ones cultured in uitro from the two-cell stage did not. Nuclear and cytoplasmic structures in the in uiuo developed blastocysts are well-differentiated while those of the embryos grown in uitro from the two-cell stage were less advanced. Possible reasons for the apparently less differentiated stage of blastocysts developed in uitro from the two-cell stage are discussed.

Study of mammalian preimplantation development in vitro has been greatly advanced by Brinster’s (‘63) formulation of a method which allows two-cell mouse ova to grow to the blastocyst stage in a simple, chemically defined culture medium. Searching for a single source of energy, Brinster (’65) found that pyruvate will support the in vitro development of the largest number of embryos. As a matter of fact, their development (as observed by the light microscope) appears to advance at the same rate in vitro as in vivo (Brin- ster, ’63).

Although ultrastructural studies have been completed on in vivo developed preimplantation blastocysts of several species (Enders and Schlafke, ’65), and on mouse embryos developed to the morula stage in vitro (Hillman and Tasca, ’69) and to the early blastocyst stage in uivo (Calarco and Brown, ’69), to our knowledge this is the first study which compares the in vitro developed “late” mouse blastocyst with those grown entirely within the maternal environment. Therefore, the purpose of this investigation was (1) to study in detail the fine structure of “late” preimplantation mouse blastocysts developed in vivo

J . EXP. ZOOL., 182: 95-118.

for 100 to 108 hours after ovulation, and (2) to compare them with mouse blastocysts of the same post-ovulatory age which had been developed in vitro for 72 hours from the late two-cell stage in Brinster’s simple culture medium.

MATERIALS AND METHODS

Sexually mature, random-bred Swiss- Webster female mice were superovulated with pregnant mare serum (PMS, Gestyl) and human chorionic gonadotropin (HCG, Pregnyl), then mated according to the method of Brinster (‘63). Since ovulation in the laboratory mouse occurs 11 to 14 hours after the injection of HCG (Edwards and Gates, ’59), the embryonic age of the preimplantation embryos used in this investigation was calculated by estimating the total time which had elapsed since ovulation. At the desired stage of development the specimens were collected and were either (1) fixed immediately or (2) cultured in vitro.

In vivo developed late preimplantation 1 A portion of this material was presented at the

eighty-third session of the American Association of Anatomists, April 2. 1970, and is abstracted in the Ana- tomical Record, 166: 341, 1970.

95

96 HAL D. McREYNOLDS AND ROBERT HADEK

blastocysts were collected by flushing the uteri of post copulatory females with fix- ative 100 to 108 hours after estimated ovulation. For cultivation in vitro, preimplantation embryos were flushed from the oviducts or uteri with warm culture medium, rinsed in the same several times, then evaluated under a light microscope (Brinster, '63). Eggs were discarded if they did not appear normal or if they were not at the desired stage of development. Ova were then cultured in small drops of Brinster's pyruvate medium (Brinster, '65) and kept under liquid paraffin oil in small Petri dishes. The dishes were transferred to an incubator (National Incuba- tor Model 3221-7) and maintained in a humid atmosphere of 5% COz in air at 37" c.

The following experiments were per- formed: (A) two-cell ova were collected from the oviducts 33 to 36 hours after estimated ovulation and cultured in vitro for 72 hours (72-hour-embryos); (B) two- cell ova were collected from the oviducts 33 to 36 hours after estimated ovulation and cultured 92 hours in vitro (92-hour- embryos); and (C) ova were collected at the late morula or early blastocyst stage (7S82 hours after estimated ovulation) and cultured 26 hours in vitro (26-hour- embryos).

The following number of specimens were studied by electron microscopy: (A) 17 embryos cultured for 72 hours, (B) 14 embryos cultured for 92 hours, (C) 13 embryos cultured 26 hours. In addition, 26 in vivo developed specimens were examined for ultrastructural details.

All specimens were fixed in chilled 3.15% glutaraldehyde adjusted to pH 7.4 with 0.1 M phosphate buffer (Sabatini, Bensch and Barnett, '63). After fixation for one hour, the specimens were rinsed in chilled buffer for three hours and post- fixed for 30 minutes in chilled phosphate buffered 1% osmium tetroxide at pH 7.4 (Millonig, '61). The specimens were de- hydrated in chilled ethanol baths of in- creasing strengths, passed through pro- pylene oxide, and embedded in Epon 812 (Luft, '61). Thin sections were cut with glass knives on a Porter-Blum MT-1 ul- trathin sectioning microtome and contrast was enhanced with uranyl acetate (Wat- son, '58), followed by lead citrate (Reyn-

olds, '63). The sections were examined with an RCA EMU-3F-2 electron microscope.

RESULTS

General observations

Observed with the light microscope, in vivo grown mouse blastocysts look similar to the in vitro grown ones. Both types of specimens display polarity and are cov- ered by a single layer of trophoblast cells. At the embryonic poles, disc-shaped groups of cells (inner mass cells) are observed between the trophoblast and the blastocele.

The fine structure of trophoblast and inner mass cells in blastocysts developed for 26 hours in vitro (from the late morula-early blastocyst stage) is remarkably similar to that of the same cell types in the in vivo developed specimens. One striking difference between these specimens is the presence of crystalloids in cells of embryos grown 26 hours in vitro. Both specimens differentiate a layer of endoderm cells.

The fine structure of blastocysts grown 72 hours in vitro (from the late 2-cell stage) differs significantly from that of the in vivo developed late preimplantation blastocyst. Blastocysts cultured for an ad- ditional 20 hours underwent further ex- pansion and may have escaped from their zonae pellucidae. However, most inner mass cells and a large number of trophoblast cells seem to be undergoing autoly- sis. Differentiation of endoderm cells does not occur in blastocysts grown 72 hours or 92 hours i n vitro.

I. Trophoblast and inner mass cells

A, Nuclei

In vivo developed blastocysts. Nuclear fine structure is essentially similar in trophoblast and inner mass cells of the in vivo developed specimens. Nuclei as a rule are centrally located and they appear well- differentiated (fig. 1). The inner and outer membranes of the nuclear envelope are usually separated by distended perinuclear cisternae, filled with a lightly electron- dense material (figs. 2, 3). Dense, granu-

ULTRASTRUCTURE OF LATE MOUSE BLASTOCYSTS 97

lar chromatin (heterochromatin) forms an irregular outline along inner nuclear membranes (figs. 1, 3, 6) , foci of condensed chromatin also appear scattered throughout the nucleoplasm (figs. 3, 6), and numerous ribosomes line the outer nuclear membranes (figs. 1, 2, 3) . Nucle- oli are large, compact, and well-differentiated in both cell types. They are elongate or spherical in shape (figs. 1, 7), consist of fibrillar and granular areas, and they may approach the nuclear envelope (fig. 1).

Blastocysts grown 72 hours in vitro. Intranuclear membranes (intranuclear annulate lamellae) are present in nuclei of trophoblast and inner mass cells (figs. 9, 13). Similar structures are not observed in nuclei of the in uiuo developed blastocyst. Very little condensed chromatin is associated with the inner nuclear membrane and heterochromatin is sparse within the nucleoplasm (figs. 9, 10). A small perinuclear cisterna is usually present, ribosomes incompletely line outer nuclear membranes, and connections with tubules of rough endoplasmic reticulum are not observed. Nucleolar appearance varies from undifferentiated (spherical agranular) to moderately differentiated (figs. 9, 10). The latter ones consist of fibrillar and granular components, but, as a rule, they are more reticular than nucleoli of the in uiuo grown blastocyst (fig. 1). Occasionally, several nucleoli may be observed in different stages of differentiation within the same nucleus (fig. 9).

Blastocysts grown 92 hours in vitro. Nuclei of normal-appearing trophoblast and inner mass cells appear to contain more condensed chromatin but intranuclear annulate lamellae were not observed.

Blastocysts grown 26 hours in vitro. Nuclei of the trophoblast and inner mass cells are essentially similar to those found in in uiuo developed specimens. Intranu- clear annulate lamellae are not observed.

B. Cytoplasmic organelles and inclusions

In vivo developed blastocysts. Ribo- somes are ubiquitous in inner mass and trophoblast cells (figs. 1, 7) and elements of rough endoplasmic reticulum are also often observed (fig. 2). The Golgi complex

is usually juxtanuclear in position and consists of one or two stacks of four or five flattened cisternae with slightly di- lated ends (figs. 1, 3). Mitochondria are elongate and many contain intracristal vacuoles (figs. 2, 4). As a rule, cristae are perpendicular to the longitudinal axis of the organelle and the matrix is relatively electron-dense (figs. 2, 3, 4). Electron- dense lysosome-like structures, measuring 1 to 1.6 p in diameter (fig. 5), are common throughout the cytoplasm of trophoblast and inner mass cells. Fibrous plaques and crystalloids are rarely observed in cells of the blastocyst grown in uiuo.

Blastocysts grown 72 hours in vitro. Ribosomes in the 72 hour specimens appear to be less numerous than in cells of the in uiuo grown ones (compare figs. 11, 12 with fig. 1). Granular endoplasmic reticulum is also less abundant but the Golgi complex is slightly enlarged and distended. Mitochondria are usually spherical or elongate and they may contain several intracristal vacuoles. However, the mitochondria1 matrix is usually less electron-dense and there are fewer cristae per organelle (fig. 14) than in those of the in uiuo developed specimens. As opposed to the solidly electron-dense ones observed in the in uiuo developed blastocysts (fig. 5), lysosome-like structures in 72 hour specimens have a greater diameter (up to 2 p ) and they contain patches of floccu- lent, electron-dense material (fig. 15).

Fibrous strands are prevalent throughout the cytoplasm of trophoblast and inner mass cells, and they are located between small groups of ribosomes (figs. l l , 12). The fibrous material usually forms parallel arrays of curved or straight strands with cross-striations at every 120 to 150 8, (fig. 11). These structures are often associated with granular endoplasmic reticulum. Crystalloids (fig. 11) are often associated with granular endoplasmic reticulum.

Blastocysts grown 92 hours in vitro. Lysosome-like structures characteristic of those found in in viuo specimens (fig. 16, inset), as well as in 72 hour specimens, are found in blastocysts grown 92 hours in vitro. Although fibrous strands are also present (fig. 16) these inclusions are less numerous than in cells of blastocysts grown 72 hours in vitro. Crystalloids are

98 HAL D. McREYNOLDS AND ROBERT HADEK

and Graham, ’69). Hillman and Tasca (’69), employing electron microscope auto- radiography to localize the uptake of tritiated uridine, have shown that there is no labeling of the fibrillar undifferentiated nucleoli present at the early two-cell stage of mouse preimplantation development. As cleavage proceeds the nucleoli differentiate further and incorporation of tritiated uridine increases substantially (Hillman and Tasca, ’69). Hence, it seems reasonable to expect a greater amount of ribosomal RNA synthesis in the more differentiated nucleoli of the in vivo developed blastocysts and those grown 26 hours in vitro than in specimens developed 72 hours in vitro. It is also assumed that little, if any, ribosomal RNA synthesis is taking place in the primary, undifferentiated nucleoli sometimes observed in cells of blastocysts grown 72 hours in vitro

Intranuclear annulate lamellae have been observed in tunicate oocytes (Kessel, ’65) and fertilized ova of the rabbit (Zam- boni and Mastroianni, ’66), but they decrease in number in the developing mouse embryo (Calarco and Brown, ’69; Hillman and Tasca, ’69). Kessel has suggested that the lamellae are involved in a special- ized nuclear-cytoplasmic interaction related to differentiation. While their func- tional significance in preimplantation mouse embryos is not understood, their disappearance at the late blastocyst stage of the in uivo developed specimen may in- dicate that an important aspect of cellular differentiation has been completed.

The observations in this study of moderate amounts of heterochromatin lining inner nuclear membranes and dispersed throughout the nucleoplasm of cells of in vivo developed blastocysts and those grown 26 hours in vitro could be an early morphological manifestation of gene repression (Brown, ’66). If so, this would tend to support the concept that “heterochroma- tization of chromosomal segments is al- ready instrumental in directing and con- trolling differentiation of development in the mouse during early preimplantation development” (Fraccaro, Hansson, Hul- ten, Linsten and Tiepolo, ’69). The limited amounts of condensed chromatin found in nuclei in embryos cultured 72 hours in vitro could be ultrastructural evidence

(fig. 9).

found in inner mass cells where two or three of the lattice-like structures are often surrounded by membranes of rough endoplasmic reticulum. (fig. 16).

Blastocysts developed 26 hours in vitro. The morphological pattern of free ribosomes, rough endoplasmic reticulum, lysosome-like structures and mitochondria in 26 hour cultured specimens is similar to that in the in vivo grown embryo. Few fibrous strands or cytoplasmic vesicles are observed, but the Golgi complex is moderately distended (fig. 17) and crystalloids are abundant (figs. 17, 20).

11. E n d o d e r m cells

Endoderm cells were observed in both the in vivo developed specimens (fig. 8) and in blastocysts grown 26 hours in vitro (fig. 18). Their fine structure is essentially the same as that of trophoblast and inner mass cells except that wide, branch- ing and anastomosing channels of rough endoplasmic reticulum are present. Dis- tension of perinuclear cisternae is usually pronounced (fig. 8). There was no evidence of endoderm cell differentiation in specimens cultured 72 or 92 hours in vitro from the two-cell stage.

DISCUSSION

Brinster (‘63) assumed that preimplantation development in the mouse occurs at the same rate in vitro as in vivo and that blastocele formation begins late on the second day of culture. While his inter- pretation was based on evidence that two- cell ova grown in culture form normal- appearing blastocysts, when viewed by light microscopy, this study has shown that certain salient ultrastructural features of blastocysts developed 72 hours in vitro differ from those of the “late” blastocysts grown entirely in vivo.

A. Nuclei

Biochemical (Monesi and Salfi, ’67; Ellem and Gwatkin, ’68; Tasca and Hill- man, ’70) and autoradiographic (Mintz, ’64; Hillman and Tasca, ’69) studies indi- cate that synthesis of RNA may begin as early as the late two-cell stage (Woodland

ULTRASTRUCTURE OF LATE MOUSE BLASTOCYSTS 99

that gene repression is less advanced in these specimens.

B. Cytoplasmic organelles

Large numbers of free ribosomes are characteristic of the rapidly proliferating embryonic cells which synthesize protein for growth (Fawcett, ’59). Accordingly, there is a general increase in the number of free ribosomes during preimplantation development in the mouse (Calarco and Brown, ’69; Hillman and Tasca, ’69). The large number of free ribosomes, the rough endoplasmic reticulum, and the connections between channels of rough endoplasmic reticulum and ribosome studded outer nuclear membranes support the claims of Weitlauf and Greenwald (‘67; ’68a,b; ’69) that the in vivo developed late mouse blastocyst is actively engaged in protein synthesis immediately prior to implantation. In these aspects, embryos grown 26 hours in vitro are similarly differentiated. However, the machinery for protein synthesis (nucleoli, free ribosomes, rough endoplasmic reticulum) does not appear to be equally developed in cells of mouse embryos cultured 72 hours in vitro. Nevertheless, one cannot make quantita- tive statements about cell function on the basis of ultrastructural evidence alone. It would be interesting to compare the protein synthesizing capabilities of in vitro and in vivo developed late blastocysts by biochemical methods.

Cells with important secretory functions usually display an elaborate Golgi complex. Why the organelle was somewhat more developed in cultured specimens than in those developed entirely within the maternal environment is not clear, and its function cannot be ascertained from the results presented in this study.

The metabolic rate of preimplantation mouse embryos, based on the rate of oxygen consumption, has been measured by Mills and Brinster (’67). These investigators found that oxygen consumption increases sharply at the eight-cell stage, and correlative electron microscopical studies have shown that the number of cristae per mitochondrion increases from the eight-cell stage onward (Hillman and Tasca, ’69; Stern, Biggers and Anderson, ’71). The large number of cristae found

in specimens cultured 26 hours in vitro, as well as in blastocysts developed in vivo, correlates well with the increased oxygen utilization by mouse embryos which occurs during the late stages of preimplantation development. Most mitochondria of embryos grown 72 hours in vitro contain fewer cristae and a less dense matrix. That these mitochondria could be less active metabolically is sup- ported by the recent demonstration by Menke and McLaren (’70) that COz production by in vitro developed blastocysts is significantly less than in those embryos developed entirely in vivo. Vacuolate mitochondria, similar to those observed in all blastocysts examined in this study, have been seen by other investigators in mouse ovarian oocytes (Wischnitzer, ’67; Odor and Blandau, ’69a, b) and they are considered regular cell components in preimplantation mouse embryos (Calarco and Brown, ’69; Hillman and Tasca, ’69; Stern, Biggers and Anderson, ’71).

C. Cytoplasmic inclusions

Fibrous inclusions have been described in rat (Szollosi, ’65) and hamster oocytes (Szollosi, ’65; Weakley, ’67; Hadek, ’69), and Enders and Schlafke (‘65) reported a general decrease in the amount of “fibrous material” during preimplantation development in mammals. In hamster oocytes, the fibrous strands are preserved by glutaraldehyde but they are labile to pepsin digestion (Weakley, ’67). Zamboni (’70) has shown that similar “fibrillar lat- tices” are a product of ribosomes in the cytoplasm of maturing mouse oocytes. Al- though these studies suggest that the lamellar structures described are largely protein, their function remains unknown. Szollosi (’65) suggests that the material may be a “yolky substance,” and Weakley (‘67) claims it may be sites for enzyme lo- calization. On the other hand, Brinster (’67) has shown that there is a 25% decrease in the total amount of embryonic protein between the morula and blastocyst stages. Since the fibrous material dis- appears at the late blastocyst, it may serve as an endogenous source of protein during the late preimplantation stages of development. The fact that there is still a large amount of fibrillar material re-

100 HAL D. McREYNOLDS

maining in cells of 72 hour embryos suggests that these specimens have not reached the stage of differentiation where the fibrous material can be utilized.

Even though crystalloids are numerous in the early blastocyst (Enders and Schlaf- ke, ’65; Calarco and Brown, ’69), they decrease with advancing development. Nev- ertheless, due to their intimate association with the rough endoplasmic reticulum, it is possible that they too are storage forms of protein. It is also possible that crystalloids are simply compacted masses of fibrous elements since they both share in common the fact that they are not membrane-bound.

Calarco and Brown (’69) have related “cytoplasmic vesicles” to the “dark yolk’ originally described in mouse eggs by Lewis and Wright (’35). They also resemble the lipid-like inclusions found in blastocysts of several species (Enders, ’71).

The function of the membrane-bound vesicles which resemble lysosomes has not been determined. However, an attempt to localize acid phosphatase in these structures could help establish their identity. Hillman and Tasca (’69) have described lysosome-like structures in mouse morulae which are very similar in appearance to those observed in the cells of 72 hour embryos. Hence, the compact, solidly electron-dense structures found at the late blastocyst stage of in vivo development actually may be more differentiated forms of the organelle. Alternatively, the very large lysosome-like bodies seen in cells of embryos cultured 72 and 92 hours in vitro may actually be artifacts result- ing from deleterious culture conditions.

D. Endoderm cells

The wide, interconnecting cisternae of rough endoplasmic reticulum found in endoderm cells of the in vivo grown specimens and those cultured 26 hours in vitro could be the first ultrastructural indica- tions of differentiation amongst the three cell types found in the mammalian blastocyst (Enders and Schlafke, ’65). On the other hand, the swollen endoplasmic reticulum could be a feature of differentiation, not unique to endoderm cells, which first manifests itself in cells of endoderm origin. In this respect, blastocysts grown

AND ROBERT HADEK

in vitro from the two-cell stage are clearly less differentiated since they did not develop endoderm cells.

E. Influence of the maternal environment on preimplantation development

General remarks

Maternal environment appears to be of paramount importance during mammalian preimplantation development. Mc- Laren (’68) suggests that the maternal reproductive tract has a regulatory effect on the developing embryo and it may influence the evolution of necessary enzyme systems since there are changes in metabolic pathways which are critical to successful embryonic development. For example, as cleavage proceeds, there is an increase in the number of compounds which can serve as energy sources (Brin- ster, ’68), and there is also an increase in oxygen consumption by the embryos (Mills and Brinster, ’67). Therefore, it may be more difficult for the early embryos to alter their basic metabolic patterns in order to accommodate changes when development occurs in vitro. As a result, ultrastructural differentiation at the blastocyst stage is less advanced than when development occurs entirely in vivo. In support of this hypothesis, Menke and McLaren (‘70) reported that “normal looking” in vitro cultured blastocysts, even when transferred to uteri of foster mothers, have only one-half the viability of in vivo developed ones.

Influence of ovarian hormones during preimplantation development

In normal pregnancy estrogen and progesterone are necessary to induce implantation (Smith and Biggers, ’68; Weitlauf and Greenwald, ’68a, b), and it is possible that they do this by acting directly on the preimplantation mouse blastocysts. For example, Smith (‘68) has shown that blastocysts cultured in vitro, and subsequent- ly treated with estradiol in vitro, implant- ed in significantly higher numbers in progesterone-primed foster mothers than when untreated control blastocysts were transferred. Similarily, incorporation of radioactive amino acids by mouse blasto-

ULTRASTRUCTURE O F LATE MOUSE BLASTOCYSTS 101

cysts in vivo is triggered by synergism of estrogen and progesterone and it does not occur in the absence of either hormone (Weitlauf, ’69). On the other hand, low levels of estrogen result in delayed implantation in the mouse (Whitten, ’55). Cells of delayed implanting rat blastocysts have been shown by electron microscopy to contain less granular endoplasmic reticulum than those of normal blastocysts (Schlafke and Enders, ’63). Moreover, there is a general increase in the number of free ribosomes in cells of delayed implanting rat blastocysts following stimulation by estrone (Wu and Meyer, ’70). Thus, it is clear that estrogen stimulation is related to the amounts of ribosomes and rough endoplasmic reticulum which are available for protein synthesis in the preimplantation mammalian conceptus. Blas- tocysts in this study which were grown completely in vivo were exposed to endogenous ovarian hormones. Since specimens cultured 26 hours in vitro were recovered from the uterus on the fourth day of pregnancy when there is a “surge” in estrogen secretion (Finn, ’65), it is possible that they also were “activated’ by ovarian hormones prior to their removal from the maternal environment. Thus, maternal hormonal stimulation may accelerate the pace of ultrastructural differentiation in the cleaving preimplantation mouse embryo.

Finally, since we did not actually compare in vivo grown specimens at earlier stages of development (i.e., early blastocyst stage) with those cultured in vitro from the two-cell stage, it is possible that embryos cultured 72 and 92 hours in vitro may not be “normal” (even for cultured specimens) and therefore do not repre- sent retarded stages of differentiation. However, we believe that this is the least likely alternative because most of the fine structural features particular to embryos cultured in vitro from the two-cell stage (intranuclear annulate lamellae, fibrous material, etc.) have also been observed in in vivo grown mouse morulae and early blastocysts by Hillman and Tasca (‘69).

In summary, preimplantation development in mammals is a complex biological phenomenon, there are many parameters involved in the process of embryonic differentiation, and exposure to the mater-

nal milieu seems to be extremely important. It could be that blastocysts grown 26 hours in vitro from the late morula- early blastocyst stage are ultrastructur- ally as differentiated as those developed in vivo only because they were allowed to complete most of their preimplantation development within the maternal reproductive tract. Besides possible “activa- tion” by ovarian hormones, they were allowed to develop in vivo at a time when critical changes were taking place in their basic metabolic patterns (Brinster, ’68) and they were also exposed to several important maternal “factors” (McLaren, ’68). Blastocysts grown 72 hours in vitro from the two-cell stage had only limited exposure to the maternal environment and this could be a major reason for their less advanced ultrastructural differentiation.

LITERATURE CITED

Brinster, R. L. 1963 A method for in vitro cultivation of mouse ova from two-cell to blastocyst. Exp. Cell Res., 32: 205-208.

___ 1965 Studies on the development of mouse embryos in vitro 11. The effect of energy source. J. Exp. Zool., 158: 5-8.

Protein content of the mouse embryo during the first five days of development. J. Reprod. Fert., 13: 413420.

1968 Mammalian embryo culture. In: The Mammalian Oviduct. E. S. E. Hafez and R. J. Blandau, eds. University of Chicago Press, DD. 419-444.

1967

BrbfYn, S. W. 1966 Heterochromatin. Science,

Calarco, P. G., and E. H. Brown 1969 An ultra- 151 : 417425.

structural and cytological study of preimplantation development of the mouse. J. Exp. Zool., 171: 253-284.

Edwards, R. G., and A. H. Gates 1959 Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophins. J. Endocrinol., 18: 292-304.

Ellem, K. A. O., and R. B. L. Gwatkin 1968 Patterns of nucleic acid synthesis in the early mouse embryo. Devel. Biol., 18: 311-330.

Enders, A. C. 1971 The fine structure of the blastocyst. In: The Biology of the Blastocyst. R. J. Blandau, ed. University of Chicago press,

Enders, A. C., and S. J. Schlafke 1965 The fine structure of the blastocyst: some comparative studies. In: Preimplantation Stages of Pregnan- cy, Ciba Foundation Symposium. G. E. W. Wolstenholme and M. O’Connor, eds. Little, Brown, and Company, Boston, pp. 2!3-59.

Fawcett, D. W. 1959 Changes in the fine structure of the cytoplasmic organelles during differentiation. In: Developmental Cytology. Doro-

pp, 71-94.

102 HAL D. McREYNOLDS

thea Rudnick, ed. Ronald Press Company, New York, pp. 161-189.

Finn, C. A. 1956 Oestrogen and the decidual cell reaction of implantation in mice. J. Endo- crinol., 32: 223-229.

Fraccaro, M., K. Hansson, M. Hulten, J. Linsten and L. Tiepolo 1969 Heterochromatin in preimplantation mouse embryos. Exp. Cell Res., 55: 427-430.

Hadek, R. 1969 Mammalian Fertilization, An Atlas of Ultrastructure, 295 pages, Academic Press, Inc., New York.

Hillman, N., and R. J. Tasca 1969 Ultrastruc- tural and autoradiographic studies of mouse cleavage stages. Am. J. Anat., 126: 151-174.

Kessel, R. G. 1965 Intranuclear and cytoplasmic annulate lamellae in tunicate oocytes. J. Cell Biol., 24: 471487.

Lewis, W. H., and E. S. Wright 1935 On the early development of the mouse egg. Contr. Em- bryol., 25: 115-143.

Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophysic. Biochem. Cytol., 9: 404-414.

McLaren, A. 1968 Mechanisms affecting embryo development. In: The Mammalian Oviduct. E. S. E. Hafez and R. J. Blandau, eds. Univer- sity of Chicago Press, pp. 477-490.

Menke, T. M., and A. McLaren 1970 Mouse blastocysts grown in vivo and in uitro: carbon dioxide production and trophoblast outgrowth. J. Reprod. Fert., 23: 117-127.

Millonig, G. 1961 Advantages of a phosphate buffer for Os04 solutions in fixation. J. Appl. Phys., 32: 1637 (abstract).

Mills, R. M., and R. L. Brinster 1967 Oxygen consumption of reimplantation embryos. Exp. Cell Res., 47: 337-344.

Mintz, B. 1964 Synthetic processes and early development in the mammalian egg. J. Exp. Zool., 157: 85-100.

Monesi, V., and V. Salfi 1967 Macromolecular synthesis during early development in the mouse embryo. Exp. Cell Res., 46: 632-635.

Odor, D. L., and R. J. Blandau 1969a Ultra- structural studies on fetal and early postnatal mouse ovaries I. Histogenesis and organogen- esis. Am. J. Anat., 124: 163-186.

196913 Ultrastructural studies on fetal and early postnatal mouse ovaries 11. Cytodif- ferentiation. Am. J. Anat., 125: 177-216.

Reynolds, E. S. 1963 The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212.

Sabatini, D. D., K. G. Bensch and R. 3. Barnett 1963 Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J. Cell Biol., 17: 19-58.

Schalfke, S., and A. C. Enders 1963 Observa- tions on the fine structure of the rat blastocyst. J. Anat., 97: 353-360.

AND ROBERT HADEK

Smith, D. M. 1968 The effect on implantation of treating cultured mouse blastocysts with oestrogen in uit-ro and the uptake of (3H) oestradiol by blastocysts. J. Endocrinol., 41: 17-29.

Smith, D. M., and J. D. Biggers 1968 The oestrogen requirement for implantation and the effect of its dose on the implantation response in the mouse. J. Endocrinol., 41: 17-29.

Stern, S., J. D. Biggers and E. Anderson 1971 Mitochondria and early development of the mouse. J . Exp. Zool., 176: 179-192.

Szollosi, D. 1965 Development of “yolky sub- stances” in some rodent eggs. Anat. Rec., 151: 424 (Abstract).

Tasca, R. J., and N. Hillman 1970 Effects of actinomycin D and cycloheximide on RNA and protein synthesis in cleavage stage mouse embryos. Nature (London), 225: 1022-1025.

Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478.

Weakley, B. S. 1967 Investigations into the structure and fixation properties of cytoplasmic lamellae in the hamster oocyte. 2. Zellforsch. Mikrosk. Anat., 81: 91-99.

Weitlauf, H. M., and G . S. Greenwald 1967 A comparison of the in uiuo incorporation of S35 methionine by two-celled mouse eggs and blastocysts. Anat. Rec., 159: 249-254.

1968a Influence of estrogen and progesterone on the incorporation of S35 methionine by blastocysts in ovariectomized mice. J. Exp. Zool., 169: 463-470.

1968b Survival of blastocysts in the uteri of ovariectomized mice. J. Reprod. Fert., 17: 515520.

Weitlauf, H. M. 1969 Temporal changes in protein synthesis by mouse blastocysts transferred to ovariectomized recipients. J. Exp. Zool., 171: 481486.

Whitten, W. K. 1955 Endocrine studies on delayed implantation in lactating mice. J. Endo- crinol., 13: 1-43,

Wischnitzer. S. 1967 Intramitochondrial trans- formations during oocyte maturation in the mouse. J. Morph., 121: 29-46.

Woodland, H. R., and C. F. Graham 1969 RNA synthesis during early development in mouse. Nature (London), 221: 327-332.

Wu, J. T., and R. K. Meyer 1970 Ultrastructur- a1 changes in the delayed implanting rat blastocyst following estrogen administration. Abstract presented at the Third Annual Meeting of the Society for the Study of Reproduction, Septem- ber 9-11, 1970, at the Ohio State University, Columbus.

Zamboni, L., and L. Mastroianni, Jf. 1966 Electron microscopic studies on rabbit ova 11. The penetrated tuba1 ovum. J. Ultrastruct. Res.,

Zamboni, L. 1970 Ultrastructure of mammalian oocytes and ova. Biol. of Reprod., 2 , Suppl. 2: 44-63.

14: i i a i 3 2 .

PLATES

PLATE 1

EXPLANATION O F FIGURE

1 Section of an inner mass cell of an in vivo developed late blastocyst containing abundant ribosomes (R) and a stack of four to five cisternae representing the Golgi complex (G). Note the fibrillar (F) and granular (G) areas of the nucleolus, the condensed chromatin (C) lining the inner nuclear membrane, and the ribosomes attached to the outer nuclear membrane. M, mitochondrion. X 30,0000.

104

ULTRASTRUCTURE OF LATE MOUSE BLASTOCYSTS Hal D. McReynolds and Robert Hadek

PLATE 1

105

PLATE 2

EXPLANATION O F FIGURES

2 In vivo developed late blastocyst. The ribosome studded outer nuclear membrane of this inner mass cell is continuous with channels of rough endoplasmic reticulum. In addition, the perinuclear cisterna of the inner mass cell is distended at several locations. N, nucleus; M, mitochondrion. X 30,000.

I n vivo developed late mouse blastocyst. Condensed chromatin lines the inner nuclear membrane and the outer one is heavily lined with ribosomes. G , Golgi complex; N, nucleus; M, mitochondrion. X 22,000.

Junction between two trophoblast cells a t the abembryonic pole of an in vivo developed blastocyst. A typical junctional complex is visible as well as microvilli at the basal end of the lateral trophoblast cell bor- ders (arrow). V , vacuolated mitochondrion; N, nucleus. X 9,700.

In vivo developed late mouse blastocyst. C, condensed chromatin of nucleus; L, solidly electron-dense lysosome-like structure. X 20,500.

3

4

5

106

ULTRASTRUCTURE OF LATE MOUSE BLASTOCYSTS H a l D. McReynolds and Robert Hadek

PLATE 2

PLATE 3


6 In vivo developed blastocyst. The arrow indicates the distended perinuclear cisterna of one trophoblast cell. The endoderm cell contains tubules of rough endoplasmic reticulum (ER). N, nucleus; M, mitochondrion. X22,OOO.

In vivo developed late blastocyst. Both cells contain numerous ribosomes and some tubules of endoplasmic reticulum. The trophoblast cell at the top contains a definitive nucleolus (DN), as well as a vacuolated mitochondrion (V). M, mitochondrion. X 11,200.

An endoderm cell of an in vivo developed late mouse blastocyst. Note the wide cisternae of rough endoplasmic reticulum in the endoderm cell (arrow). The cell is heavily laden with ribosomes. N, nucleus; M, mitochondrion; V, vacuolated mitochondrion. X 20,000.

7

8

108


PLATE 3

PLATE 4


9 Inner mass cells of a blastocyst cultured 72 hours in uiho. Nuclear morphology appears less differentiated than that observed in the in vivo developed late blastocyst. Two primary, undifferentiated nucleoli (P) and two reticulated, moderately differentiated nucleoli (MD) con- sisting of fibrillar and granular areas are observed in the nucleoplasm. An intranuclear lamella, not found in cells of the in uivo developed late blastocyst, is seen (arrow). Little heterochromatin lines the inner nuclear membrane or is found in the nucleoplasm. C, cytoplasmic vesicle; L, lysosome-like structure. X 11,900.

Trophoblast cell of a blastocyst grown from the two-cell stage after 72 hours cultivation in vitro. The nucleolus (MD) is moderately differentiated but is reticulated. Little heterochromatin lines the inner nuclear membrane or is found in the nucleoplasm. The perinuclear cisternae is not distended. L, lysosome-like structure. X 14,000.

10

110


PLATE 4

PLATE 5


11 Trophoblast cell of a blastocyst grown 72 hours in nitro from the late two-cell stage, Ribosomes are found in small groups interspersed amongst the fibrous strands (arrow). Note the crystalloid inclusions (Cr) which are surrounded by elements of rough endoplasmic reticulum. X 24,000.

12 Trophoblast cell of a 72 hour cultured specimen. Note the fibrous strands (arrow) interspersed amoungst the small groups of ribosomes. C, cytoplasmic vesicle; Z, zona pellucida. X 25,000.

This inner mass cell of an embryo cultured 72 hours in nitro contains an intranuclear lamellar structure (IAL). X 80,000.

Trophoblast cell of a blastocyst cultured 72 hours in nitro. The fully visible mitochondrion has relatively few cristae and its matrix is not par- ticularly electron dense. The granular material could possibly be mitochondria] ribosomes. X 75,000.

Inner mass cells of a blastocyst developed 72 hours in nitro from the late two cell stage. Notice the fibrous strands (arrow). L, lysosomelike structure; C, cytoplasmic vesicles. X 21,000. Inset shows a cluster of ribosomes and fibrous strands. X 60,000.

Normal appearing inner mass cell of a blastocyst developed 92 hours in nitro. Note the large number of crystalloids (Cr) and fibrous strands (arrow). X 22,000. The inset shows the electron-dense, lysosome-like structures which are typically found in cells of blastocysts grown 92 hours in nitro. x 20,000.

13

14

15

16

112


PLATE 5

113

PLATE 6


17 Trophoblast (T) and inner mass (IM) cells of an embryo grown 26 hours in vitro. A moderate amount of heterochromatin lines the inner nuclear membranes and is found in foci throughout the nucleoplasm. Mitochondria have an electron-dense matrix, and several contain intracristal vacuoles (V). As in inner mass and trophoblast cells of the in vivo developed late blastocyst these cells are heavily laden with ribosomes. G , Golgi Complex. X 11,000.

Endoderm cell of 26 hour cultured embryo. Note the wide cisternae of rough endoplasmic reticulum (ER). X 16,000.

Mitochondria found in a trophoblast cell of a blastocyst grown 26 hours in vitro. X 48,000.

Crystalloid inclusion in an inner mass cell of a blastocyst grown 26 hours in vitro. Note the close association with the elements of rough endoplasmic reticulum. X 22,500.

18

19

20

114

ULTRASTRUCTURE OF LATE MOUSE BLASTOCYSTS H a l D. McReynolds and Robert Hadek

PLATE 6

PLATE 7


21 Nucleolus of an inner mass cell of an embryo cultured 26 hours in vitro from the late morula stage. The well differentiated nucleolus found in this cell consists of fibrillar (F) and granular (G) areas. X 29,700.

Trophoblast cell of a blastocyst grown 26 hours in nitro from the late morula stage. The nucleus contains an elongate, well-differentiated nucleolus as well as condensed chromatin. The perinuclear cisterna is distended and the outer nuclear membrane is studded with ribosomes. X 18,000.

22

116


PLATE 7

117

a comparison of the fine structure of late mouse blastocysts developed in vivo and in vitro

Documents