cytological and ultrastructural comparisons of t12/t12 and normal mouse morulae

Cytological and Ultrastructural Comparisons of f12/t'2 and Normal Mouse Morulae

PATRICIA G. CALARC02 AND EDWARD H. BROWN Department of Zoology, University of Illinois, Urbana, Illinois

ABSTRACT Cytological and ultrastructural observations were made of normal (BALB/c) mouse morulae and embryos homozygous for the tl2 mutation, which i s lethal at the late morula stage. Normal and t12/t12 embryos are indistinguishable cytologically until the late morula stage, at which time cytoplasmic and nuclear differences become apparent. Different cells within individual normal morulae exhibit differences in cytoplasmic basophilia, and the cells appear closely applied to one another. Differences in cytoplasmic basophilia are not apparent between different cells of t12/tz2 morulae, and the cells appear more rounded and less closely applied. Nucleoli of normal morulae are irregular in shape, with projections which often reach the nuclear envelope, while nucleoli of t 1 2 / P morulae exhibit extreme rounding.

Nucleoli of normal embryos exhibit a net-like nucleolonema, with fibrillar and granular elements, and several pars amorpha. Nucleoli of t 1 2 / P morulae possess the same structural elements but the nucleolonema appears contracted. Nuclei of mutant embryos often contain several agranular electron-dense bodies, 0.1 - 0.7 p in diameter. These are infrequently observed in normal morulae. Ribosomes exist primarily in clusters in normal morulae, but Iarge numbers of free ribosomes are observed in t'2/tz2 morulae. Many structures are common to both normal and mutant morulae, such as: fibrous strands, crystalloids, granular endoplasmic reticulum, vacuolated mitochondria, cytoplasmic vesicles, tight junctions, nuclear pores, and intranuclear annulate lamellae.

Since the abnormalities exhibited by t 1 * / t I C morulae can be interpreted as degenera- tive changes, they do not resolve the mode of action of the t locus or the primary cause of t 1 2 / t 1 2 lethality.

A genetically complex region, designated T (Brachug), on autosome IX in mice con- tains a number of genes which act on embryonic development. The dominant mutation T is lethal at placentation when homozygous, and when heterozygous (T/+) results in short-tailed mice. When T is combined with any of a large series of recessive t "alleles" (T/t"), a tailless mouse results. Recessive lethal t mutations fall into five complementation groups, each of which is distinguished by a characteristic range of developmental abnormalities and time of death. Cytogenetic investigations suggest that the T region covers most of the long arm of chromosome IX, that t mutations from different complementation groups are not unilocal, and that some of them are deletions (Geyer-Duszynska, '64). The mutation t" is particularly interesting because it has the earliest expression of any known mammalian lethal gene, caus- ing the t1'/tI2 embryo to arrest at the morula stage (Smith, '56).

Observations from light microscopy and autoradiography suggest that the arrest of J. EXP. ZOOL., 168: 169-186.

t" homozygotes as early morulae may be due to abnormalities in the development and function of the nucleoli. Mintz ('64b) has reported that t12/t1P morulae show less basophilia (with Azure B staining) before degenerating than the peak level reached by control morulae, and fail to show the increase in RNA synthesis characteristic of the late morula stage. Although nucleoli of t" homozygotes appear normal during cleavage stages, and nucleoli of mutant morulae incorporate tritiated uridine (Mintz, '64a), they do not change to the irregular elongate shape characteristic of blastocyst nucleoli. Instead, the nucleoli of the mutant embryos exhibit a more spherical shape (Smith, '56). Whether the nucleolar abnormality is a cause of lethality or simply an expression of cellular death is

1 This work was supported by a National Institutes of Health Predoctoral Fellowship, l-FI-GM-24,170-04, awarded to P.G.C. and (in part) by a grant to E.H.B. from the Graduate College Research Board of the University of Illinois.

SThis work is based on part of a dissertation sub- mitted by the senior author to the Graduate College of the University of Illinois in partial fulftllment of the requirements for the Ph.D. degree.

169

170 PATRICIA G. CALARCO AND EDWARD H. BROWN

not yet clear. Mutant morulae are able to synthesize protein from tritiated leucine, but this synthesis gradually declines (Mintz, '64b). In addition, mutant morulae fail to show the shift toward a less granular cytoplasm and the formation of "spherical bodies" reported to be characteristic of normal late morulae (Mintz, '64b). The mitotic rate of the cells of mutant morulae also appears to decline, along with a decline in DNA synthesis detected by tritiated thymidine incorporation (Mintz, '64c). However, the number of cells per mutant morula ranges from 20 to 38, with an average of 28.4, so it is probably not a lack of cells which causes the failure of blastulation in t" homozygotes (Smith, '56).

Studies of experimentally produced mosaic embryos, containing normal cells and mutant (ti2/t1') cells, suggest that the mutant cells are less mobile than normal cells, retard development of the mosaic embryos, and are less able than normal cells to with- stand the increasing pressure of the en- larging fluid-filled blastocoel (Mintz, '64c).

This paper presents ultrastructural observations of t"/t'" morulae, with emphasis placed on those structures which differ in t'' homozygotes as compared to normal morulae.

Since no ultrastructural descriptions of the normal mouse morula were available, an electron microscopic examination of developmental stages up to the blastocyst stage was carried out for purposes of com- parison. This developmental study will be reported in more detail in another paper.

The authors wish to express their appre- ciation to Dr. L. C . Dunn for supplying balanced lethal stocks (T/t") from those maintained in his laboratory.

A single T/PZ male was outcrossed to a Bagg Albino female, and the normal-tailed (+/P) offspring were mated inter se and with BALB/c mice to provide the required numbers of +/ti' males and females used for subsequent matings. Normal-tailed animals which were obtained from the above crosses and which produced a litter containing at least 25% tailless (T/t") offspring, when crossed with a Brachyury

MATERIALS AND METHODS

mate (T/+), were assumed to be of genotype +/P.

Segregation is distorted in males heterozygous for a lethal t mutation, the mutant allele being transmitted at a frequency greater than 50% ; transmission is normal in females (Dunn, '64). From data on 429 matings of +/t*' males, Smith ( '56) reported an average transmission ratio of 0.76 for the t'' allele. In the present study, only males which showed a transmission ratio of at least 0.86 on the basis of 2 or 3 test matings were used and a ratio of 0.76 was used for calculations.

The desired embryos (t*2/t'2) were obtained from crosses between +/ti' males and females and were expected in a frequency of approximately 38%. The t"/t" embryos discussed in this paper were from crosses averaging 38% morulae and 62% blastulae when flushed from the uteri. These morulae exhibited the rounded nucleoli characteristic of the t'' homozygote, when sectioned and examined with the light microscope, and were considered to be t"/t". Normal embryos obtained from crosses of BALB/c males and females were used for comparative purposes.

The technique of Edwards and Gates ('59) was used to induce superovulation in mature females. Six international units of pregnant mare's serum (Equinex, Ayerst Labs) were injected intraperitcneally and followed 40 hours later by an i.p. injection of 6 i.u. of chorionic gonadotropin (A.P.L., Ayerst Labs, or Follutein, Squibb). Males were introduced at the time of the second injection and matings occurred within 4 to 8 hours. Because cleavage in the mouse is asynchonous, it is difficult to identify a particular developmental stage (on the basis of cell number) beyond the 8-cell stage. The use of superovulation increased the number of embryos obtained and also permitted an approximation of staging on the basis of age. Since ovulation occurs from 11-14 hours after the second hor- mone injection (Edwards and Gates, '59), embryonic age (in hours) was figured from the time of ovulation, which was considered to have occurred 12 hours after the injection of chorionic gonadotropin. By this criterion, the t" homozygotes discussed in this paper varied from 70-77 hours of age.

ULTRASTRUCTURE OF t''/tl' MOUSE MORULAE 171

Pregnant females were killed and the ovaries, oviducts and uterine horns re- moved and placed in Brinster's culture medium without BSA (Brinster, '65), here- after designated BCM. Morulae were flushed from the uterus into a depression slide, washed twice with BCM, and examined at 500 X with phase contrast optics to determine the approximate number of cells present in each embryo. The eggs were then isolated, still in BCM, in a modified version of the microchamber devised by Izquierdo ('67), consisting of a center well drilled into a glass depression slide. All subsequent fluid changes were carried out while the embryos remained in this chamber.

A 6-minute incubation at room temperature in BCM containing 0.5% Pronase (Calbiochem) and heated to 37°C before use was employed to partially remove the zona pellucida (Mintz, '62). This prefixa- tion step was found to allow more rapid penetration of the fixatives and did not significantly alter the ultrastructure of the embryos.

Embryos were rinsed three times with BCM, fixed for 30 minutes in 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2-7.4), and postfixed for 15 minutes in 2% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2-7.4). The embryos were then rapidly dehydrated with a cold ethanol series: 35%, 50%, 70%, 95% and 100%. All of the above steps were carried out at 0°C. Replacement of 100% ethanol with propylene oxide was carried out at room temperature. Embryos were then embedded in EPON 812 by a modification of the procedure described by Luft. ('61).

Serial sections 2 I.I thick were cut on an LKB Ultratome. Individual sections were placed on drops of water on albuminized microscope slides and allowed to dry on a 70°C hotplate. Thin sections for electron microscopy were also cut on the LKE! U1- tratome, using a diamond knife, and picked up on 150 mesh parlodian-covered, carbon- coated copper grids.

Two-micron sections were routinely stained with 0.025% Azure B bromide at pH 4 (Flax and Himes, '52), either by em- ploying the method of King ('60) or by using longer incubation times at 60°C.

For electron microscopy, contrast was enhanced by treating the sections with saturated uranyl acetate (Watson, '58) followed by lead citrate (Reynolds, '63). Grids were examined with a Hitachi Hull-A electron microscope.

OBSERVATIONS

Light microscopy Azure B staining reveals no differences

in overall cytoplasmic basophilia between normal BALB/c morulae and P2/t1' morulae, or between morulae obtained from +/ti' matings. However, differences in overall basophilia between blastulae and morulae (normal or mutant), are quite pronounced. This increase in basophilia at the blastocyst stage reflects the increased concentration of cytoplasmic RNA (primarily ribosomal) occurring at the blastula stage (Enders and Schlafke, '65; Mintz, '64c; Smith, '56).

The most prominent cytoplasmic structures noted with the light microscope in normal and t"/P morulae are spherical vesicles which are usually greater than a micron in diameter and contain a substance of medium density (fig. 1). These are present in the unfertilized egg, where they are less than a micron in diameter, and increase in number and size as cleavage progresses. Many dark-staining masses are also observed in the cytoplasm of both normal and mutant morulae.

Several consistent cytological differences are noted between normal and mutant morulae :

1. Different cells within single normal morulae often show differences in cytoplasmic basophilia, i.e. Azure B staining, (fig. 1 ). This differential basophilia within single embryos is never observed in t"/tZZ embryos.

2. The cells of the Phomozygote appear more rounded and less closely applied to one another than the cells of normal embryos (compare figs. 1 and 2).

3. The nucleoli of normal late morulae are roughly spherical (fig. 1 ) and exhibit projections which often extend to the nuclear envelope. In contrast, the nucleoli of tIZ/tr2 morulae show extreme rounding, but the nuclei are not obviously pyknotic (fig. 2). Nucleoli of both normal and mu-

172 PATRICIA G . CALARCO AND EDWARD H. BROWN

tant morulae stain intensely with Azure B. Prior to the late morula stage the nucleoli of normal and mutant embryos are indistinguishable, as was previously observed by Mintz (’64b).

Electron microscopy Mutant morulae resemble normal mor-

ulae very closely with respect to the various classes of cytoplasmic and nuclear organ- elles present and in their structure.

Vacuolated mitochondria of the type described by Wischnitzer (’67) are found in both types of morulae (figs. 3, 6 ) and are often associated with strands of granular endoplasmic reticulum. The Golgi ap- paratus does not seem to be a prominent feature in cells of the morula stage. Al- though large numbers of sacs and vesicles are present throughout the cytoplasm, the typical aggregations of stacked cisternae and associated vesicles are rarely en- countered. Those Golgi regions that are observed usually occupy perinuclear posi- tions.

The spherical cytoplasmic vesicles noted with the light microscope are identified with the electron microscope by the medium-density material which they contain (fig. 4). The fixation procedure apparently causes the loss of some of this material. These vesicles are not membrane bound and are often found closely associated with mitochondria.

Two kinds of fibrous materials, exhibiting distinct but different periodicities, are found in the cytoplasm of both mutant and nomal morulae. These have been described by Enders and Schlafke (’65). One is composed of wisps of straight or curved fibrous strands exhibiting cross striations (see F in fig. 3), while the second exhibits a more compact crystalline structure composed of closely packed dense fibers (see C in fig. 3). The crystalloids correspond to the dark- staining masses observed with the light microscope. The crystalline material is present in very small amounts in the fertilized egg. As cleavage progresses, it is found in increasing amounts and in the morula it is often associated with granular endoplasmic reticulum. This material presumably corresponds to the “dark yolk” observed with the light microscope by Lewis and Wright (’35). In addition to the fibrous

strands and crystalline material, mutant morulae possess aggregates of material which are similar in density to the crystalloids but lack the characteristic periodicity (figs. 5, 6). This material is always in close association with many single ribosomes and may represent aberrant synthesis of the crystalline material or a stage in synthesis prior to the organization of the crystalline pattern. These disorganized aggregates are not observed in normal morulae. If these aggregates represent an intermediate stage in the synthesis of crystalloids, their absence in normal morulae implies that crystalloid synthesis is normally quite rapid. It may be that the arrested t‘e/t12 cells permit this intermediate stage to be seen.

Ribosomes in normal late morulae usually exist in clusters (presumably poly- somes) or as components of the rough-sur- faced endoplasmic reticulum, both of which are detected in increasing amounts throughout cleavage. Single (free) ribosomes are rarely observed. Obvious differences in the number or concentration of ribosomes in different cells, which might correspond to the differences in basophilia observed with the light microscope, are not evident. Mutant morulae also possess poly- somes and granular endoplasmic reticulum (fig. 6), but differ from normal morulae in possessing large numbers of single ribosomes (fig. 7).

‘Tight junctions” or “‘junctional complexes’’ are present between peripheral cells of normal late morulae just below the free surface. Their formation shortly precedes the appearance of the fluid-filled blastocoel. These junctions consist of an amorphous dense substance which is located in the intercellular space and extends across the cell membranes into the cytoplasm of both cells (fig. 8). Ribosomes are often observed adjacent to the cell membranes in these regions, before junctional complexes appear. Perhaps these ribosomes engage in the synthesis of a protein component of the terminal dense substance. Although the cells of the tz2/t’2 morula appear to be less closely joined, when examined by light microscopy, terminal complexes are present between most peripheral cells (fig. 7) . The time of their formation in the mutant morulae is not known, and they may not

ULTRASTRUCTURE O F tlZ/tl’ MOUSE MORULAE 173

be formed between all peripheral cells. The nucleoli of the normal morula (fig.

9) exhibit the two structural components typically observed in nucleoli: a net-like nucleolonema, consisting of filaments which contain fibrillar and granular elements, and one or more roughly spherical dense regions composed primarily of fine fibrils. The latter regions are generally re- ferred to as the pars amorpha (Fawcett, ’66). The nucleolonema is reported by Bernhard (’65) to consist of a protein matrix with RNA-containing fibrils and ribonucleoprotein granules embedded within it. Nucleoli of the t” homozygote (fig. 10) exhibit the nucleolonema, with its granular and fibrillar components, and an occasional pars amorpha, but differ from normal nucleoli in that the nucleolonema appears to be more contracted. Condensed chromatin is occasionally observed in association with the nucleoli of both normal and mutant morulae.

A characteristic of t” /P morulae is the presence of a number of dense, agranular, intranuclear bodies ranging from about 0.1 to 0.7 u in diameter. Fine fibrils radia- ting from these bodies give them a stellate appearance (fig. 11). Rarely a dense body of similar size is observed in a nucleus of a normal morula, but such bodies lack the stellate appearance and possess a peripheral zone of granules. The stellate intranuclear bodies of mutant nuclei are present in large numbers, never possess a granular coat, and can be found in the same nuclei as condensed nucleoli.

Intranuclear annulate lamellae (IAL), usually associated with condensed chromatin (fig. 12) or nucleolar material (fig. 13), are frequently observed in both normal and mutant morulae near the nuclear envelope. IAL have previously been observed in the tunicate oocyte (Hsu, ’63; Kessel, ’65), the pronuclear human egg (Zamboni et al., ’66), and re- cently in cleavage stages of the mouse (Hillman and Tasca, ’67). In the present study, IAL were observed in the fertilized egg, in all cleavage stages examined and in the blastocyst. The structure of the IAL is similar to that of the nuclear envelope. They consist of two parallel membranes, each about 75 A in thickness and separated by a space of about 400-700 A, and are

usually oriented perpendicularly to the nuclear envelope. In some sections they are seen to be continuous with the inner membrane of the nuclear envelope. The IAL can be several microns in length. In favorable sections the IAL exhibit “pores” similar to those observed in the nuclear envelope. Dense fibrous material extending outward at regular intervals from each membrane is often observed, and probably corresponds to the “pores,” along the IAL (fig. 13). The space which separates the two membranes is occasionally expanded into sac-like structures (fig. 12). The IAL have never been observed in the form of lamellar stacks and have never been observed in the cytoplasm of any stage. The micrographs do not provide evidence that the IAL are sheet-like structures; they may be tubular in nature. In this respect they do not resemble cytoplasmic annulate lamellae, first described by Swift (’56).

Nuclear ‘pores” were also found to be a regular feature of the nuclear envelopes in cells of both normal and mutant cleavage stages and morulae.

DISCUSSION

The reduced cytoplasmic basophilia (Azure B staining) of t”/t“ morulae, as compared to normal late morulae, reported by Mintz (’64c) was not observed in the present study. The reason for this discrep- ancy is not clear but may be due to differences in fixation and embedding tech- niques. It is also possible that in the mosaic embryos studied by Mintz (e.g. her fig. 8, ’64c), the cells of normal genotype ad- vanced to the higher level of RNA synthesis characteristic of the early blastula, but the presence of developmentally retarded mutant cells inhibited blastocoel formation. The differences in basophilia between normal and mutant cells in mosaic ‘knorulae” might then simply reflect the developmental retardation of the mutant cells.

Smith (’56) reported that some older (presumably normal) morulae are composed of an inner cell mass, consisting of larger rounded cells, and a surrounding layer of smaller flattened cells. Among embryos obtained approximately 83 hours after a +/t’” X +/ tXa mating, the blasto- cysts exhibited a layer of trophoblast cells which stained more intensely with the

174 PATRICIA G. CALARCO AND EDWARD H. BROWN

PA/S technique than did the inner cell mass (Smith, '56). The morulae (presumably ti2/t12) among the same group of embryos did not show this differential staining, suggesting that mutant embryos fail to differentiate into these two cell types. Per- haps the differences in cytoplasmic basophilia which were observed between different cells of some individual morulae in the present study reflect differential synthesis of RNA or protein related to an early stage of differentiation. Alternatively, this differential basophilia may reflect different rates of protein synthesis or ribosome formation due to asynchronous cell cycles, or it may be due to differences in cell volumes. How- ever, in morulae obtained from +/t" X +/t'" crosses, this differential basophilia within embryos was not observed in all embryos. Additional studies will be necessary to determine whether the number of morulae which exhibit this differential basophilia consistently approximates the expected number of normal morulae.

The number of ribosomes in the cytoplasm of the normal morula is much higher than the number present at the 2-cell stage (presumably before nucleolar function be- gins). The t"/t" embryos also show an increase in the number of ribosomes during cleavage and they possess cytologically normal nucleoli, at least during the early cleavage stages. It thus appears that ribosome synthesis occurs. The ribosomes synthesized by ti' homozygotes may be abnor- mal, however, since they exist primarily as free ribosomes in the cytoplasm.

Several explanations of the action of the t'" locus have been proposed. Smith ('56) suggested that the t'' allele prevents blastocyst formation either because a substance necessary for the formation of blastocoel fluid is not produced or because the outer layer of cells is not able to retain the fluid. Bennett ('64) proposed that the normal action of this locus is to cause the differentiation of the trophectoderm as part of blastocoel formation. Mintz ('64b) suggested that the function of the normal gene may be related to cytoplasmic structural reorganization.

The formation of the blastocoel is cur- rently under investigation. Work to date suggests that the spherical cytoplasmic vesicles (fig. 4) contain blastocoel fluid,

and a quantitation of these vesicles at suc- cessive developmental stages is now in progress. These vesicles, which presumably correspond to the 'light yolk globules" observed by Lewis and Wright ('35) and the "spherical bodies" reported by Mintz ('64b), are present in both normal and mutant embryos, suggesting that t"/t" embryos are capable of producing blastocoel fluid.

The retention of the blastocoel fluid is presumably dependent upon the presence of junctional complexes between the cells which border the blastocoel. According to Enders and Schlafke ('65), "the blastocyst stage can be considered to begin when the junctional complexes have formed between the cells." However, the t" homozygote pos- sesses junctional complexes but arrests before the appearance of a fluid-filled cavity. The term "blastocyst" as used in this paper refers only to those embryos which possess at least a small fluid-filled cavity. Prior to this time an embryo is termed a morula. It is not known if the junctional complexes are formed between all peripheral cells in mutant embryos, or if they are formed in time to retain the blastocoel fluid.

The intranuclear bodies observed in the cells of tl'/t'" morulae may be comparable to the bodies which are present in anucleolate nuclei of maize and Xenopus. In nuclei of Zea mays microspores which lack a functional nucleolus organizer, Swift and Stevens ('65) observed numerous, dense, spherical, RNA-containing inclusions, which never develop a peripheral zone of granules. The authors postulate that these nucleolus-like bodies represent accumula- tions of ribonucleoprotein synthesized by specific chromosomal loci other than the nucleolus organizer; in normal nuclei, these materials would be incorporated into the nucleolus, or products of the nucleolus would mobilize the transfer of this material to the cytoplasm. The nuclei of the anucleolate mutant of Xenopus laevis also possess multiple nucleolar-like bodies, some of which are less than a micron in diameter (Hay and Gurdon, '67).

The presence of nucleoli in addition to the intranuclear bodies in the nuclei of P/t'' morulae does not rule out a possible similarity between the intranuclear bodies and the inclusions observed in the nuclei

ULTRASTRUCTURE O F t'z/t12 MOUSE MORULAE 175

of anucleolate cells in maize and Xenqpus. While normal maize microspores possess single nucleolus organizers and somatic cells of Xenopus Zaevis possess two (homol- ogous) nucleolus organizers, there may be as many as six nucleolus organizers per diploid genome in the mouse (Shea and Leblond, '66). This is supported by the cytological identification of at least three pairs of chromosomes beasing secondary constrictions (Bennett, '65). The possibility thus remains that the intranuclear bodies in the cells of t12/t12 morulae reflect an abnormality in the function or control of one or more nucleolus organizers.

However, the abnormalities exhibited by t"/t" morulae can be interpreted as de- generative changes of metabolically declin- ing cells. These abnormalities include the rounding up of the nucleoli, the appearance of intranuclear bodies, the presence of large numbers of free ribosomes and the rounding up of the blastomeres and their apparent inability to maintain close intercellular contact. The reduced incorporation of precursors of DNA, RNA and protein (Mintz, '64c) also supports this interpreta- tion. At the present time it is impossible to decide which, if any, of these pleiotropic effects provide insight into the mode of action of the t locus and the primary cause of developmental arrest in the t" homozygote.

LITERATURE CITED Bennett, D. 1964 Abnormalities associated with

a chromosome region in the mouse. 11. Embry- ological effects of lethal alleles in the t-region. Science, 144: 263-267.

The karyotyre of the mouse with identification of a translocation. Proc. Natl. Acad. Sci., 53: 730-737.

Bernhard, W. 1965 Ultrastructural asFects of the normal and pathological nucleolus in mammalian cells. In: International Symposium On the Nucleolus - Its Structure and Function. W. S. Vincent and 0. L. Miller, eds. National Cancer Inst. Monograph No. 23, U. S. Govt. Printing Office, Washington, D. C., pp. 13-38.

Brinster, R. L. 1965 Studies of the development of mouse embryos in vitro: energy metabolism. In: Preimplantation Stages of Pregnancy. G. E. W. Wolstenholme and M. O'Connor, eds. Little Brown & Co., Boston, pp. 60-74.

Dunn, L. C. 1964 Abnormalities associated with a chromosome region in the mouse. I. Transmission and population genetics of the t-region. Science, 144: 260-263.

Edwards, R. G., and A. H. Gates 1959 Timing of the stages of the maturation divisions, ovula-

1965

tion, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophins. J. Endocrin., 18: 292-304.

Enders, A. D., and S. J. Schlafke 1965 The fine structure of the blastocyst : some comparative studies. In: Preimplantation Stages of Preg- nancy. G. E. W. Wolstenholme and M. O'Con- nor, eds. Little Brown & Co., Boston, pp. 29-54.

Fawcett, D. W. 1966 The Cell. W. B. Saunders Co., Philadelphia, 448 pp.

Flax, M. H., and M. H. Himes 1952 Microspec- trophotometric analysis of metachromatic staining of nucleic acids. Physiol. Zool., 25: 297-311.

Geyer-Duszynska, I. 1964 Cytological investigations on the T-locus in Mus musculus L. Chrom- osoma, 15: 478-502.

Hay, E. D., and J. B. Gurdon 1967 Fine structure of the nucleolus in normal and mutant Xenopus embryos. J. Cell Sci., 2: 151-162.

Hsu, W. S. 1963 The nuclear envelope in the deveIoping oocytes of the tunicate Boltenia vil- losa. Zeit. Zellforsch., 58: 660-678.

Hillman, N. W., and R. J. Tasca 1967 Ultra- structural studies of preimplanation mouse embryos. (Abstr.) J. Cell Biol., 35: 56A.

Izquierdo, L. 1967 A microchamber for process- ing mammalian eggs. Stain Tech., 42: 35-37.

Kessel, R. G. 1965 Intranuclear and cytoplasmic annulate lamellae in tunicate oocytes. J. Cell Biol., 24: 471488.

King, R. C. 1960 Oogenesis in adult Drosophila melanogaster. I X . Studies on the cytochemistry and ultrastructure of developing oocytes. Growth, 24: 265-323.

Lewis, W. H., and E. S. Wright 1935 O n the early development of the mouse egg. Carnegie Inst. Contrib. to Embryol., 25: 115-143.

Luft, J. H. 1961 Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol., 9: 409-414.

Mintz, B. 1962 Experimental study of the developing mammalian egg: removal of the zona pellucida. Science, 138: 594-595.

Synthetic processes and early development in the mammalian egg. J. Exp. Zool.,

1964b Gene expression in the morula stage of mouse embryos as observed during development of t'2/t12 lethal mutants in vitro.

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176 PATRICIA G . CALARCO AND EDWARD H. BROWN

Swift, H., and B. J. Stevens 1965 Nucleolar- Watson, M. 1958 Staining of tissue sections for chromosomal interaction in microspores of electron microscopy with heavy metals. J.

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PLATE 1

EXPLANATION OF FIGURES

1 Phase contrast photomicrograph of a 2 p section through a BALB/c morula fixed in glutaraldehyde, postfixed in osmium tetroxide and embedded in EPON. The section was stained with Azure B bromide. Note the differences in basophilia exhibited by different cells, the clusters of vesicles (V) and the dark-staining masses (C). x 750.

Phase contrast photomicrograph of a 2 p section through a t12/t12 morula, stained with Azure B bromide. Note the extreme rounding of the nucleoli, the enlarged intercellular spaces, the clusters of vesicles (V) and the dark-staining masses (C). X 750.

Electron micrograph of a section through a cell of a BALB/c morula. Note the vacuolated mitochondria (M), and the fibrous (F) and crystalline (C) material. x 30,150.

2

3

ULTRASTRUCTURE OF t12/t12 MOUSE MORULAE Patricia G. Calarco and Edward H. Brown

PLATE 1

177

PLATE 2


4 Spherical cytoplasmic vesicles, containing partially extracted material of medium density, i n a BALB/c morula. Note the association of two vesicles with a vacuolated mitochondrion (arrows). X 20,100.

5 Clusters of disorganized crystalloid material surrounded by free ribosomes in a t12/t1Z morula. A portion of a nucleus ( N ) is evident along the left side of this micrograph. x 30,150.

6 A tI2/t i2 morula. The prominent cytoplasmic constituents appear similar to those observed in normal morulae (see fig. 3), except for the presence of disorganized crystalloids (arrow). M, mitochondria; C, cyrystalline material. X 30,150.

178

ULTRASTRUCTURE OF t * z / t * z MOUSE MORULAE Patricia G. Calarco and Edward H. Brown

PLATE a

179

PLATE 3


7 Two adjacent peripheraI cells of a t12/t1z morula. Note the presence of a tight junction (arrow) and the numerous free ribosomes in the cytoplasm. X 30,150.

a A tight junction between two peripheral cells of a BALB/c morula. X 30,150.

9 A nucleolus of a BALB/c morula, exhibiting the nucleolonema ( N ) and several pars amorpha (P). X 15,075.

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ULTRASTRUCTURE OF t*2/t lz MOUSE MORULAE Patricia G. Calarco and Edward H. Brown

PLATE 3

181

PLATE 4


10 A nucleolus of a t 1 2 / t f 2 morula. Note the contracted state of the nucleolonema ( N ) as compared to figure 9. Several pars amorpha (P) are evident. x 25,125. Section through a nucleus of a t'z/t12 morula. Note the presence of dense agranular bodies (arrows) in addition to a nucleolus (Nu). X 25,125.

11

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ULTRASTRUCTURE OF t12 / t12 MOUSE MORULAE Patricia G. Calarco and Edward H. Brown

PLATE 4

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PLATE 5


12 Nucleus of a BALB/c morula, showing a n intranuclear annulate lamella (IAL) associated with condensed chromatin (Ch). Note the sac-like enlargement of the IAL (arrow). x 30,150.

13 Nucleus of a BALB/c morula, showing an intranuclear annulate lamella (IAL) associated with nucleolar material. Note the fibrous material a t regular intervals along the IAL (arrows). Several pores are evident in the nuclear envelope. x 30,150.

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ULTRASTRUCTURE OF t l Z / t l Z MOUSE MORULAE Patricia G. Calarco and Edward H. Brown

PLATE 5

185

cytological and ultrastructural comparisons of t12/t12 and normal mouse morulae

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