autoradiographic studies of t12/t12 mouse embryos

Autoradiographic Studies of V 2 / f l 2 Mouse Embryos ' NINA HILLMAN Department of Biology, Temple Universi ty , Philadelphia, Pennsylvania 191 22

ABSTRACT Several studies have indicated that morula-staged t'Z/t'2 mouse embryos are deficient in nucleolar RNA synthesis and that this deficiency may be a causall factor of death. The present investigation was undertaken to see if decreased KNA synthesis in t*2/t'2 embryos occurs prior or subsequent to developmental arrest. In this study, embryos obtained from +/t" inter se matings were labeled with 3H-5-uridine at specific cleavage stages and then subjected to high resolution autoradiographic study. These staged embryos were either examined immediately following labeling or were reincubated in nonradioactive medium and then studied. Comparisons between autoradiographs of the phenotypically mutant embryos and their phenotypically wild-type littermates revealed no significant difFerence between these two groups in either the amount or distribution of the incorporated RNA precursor at any cleavage stage. Late morula t1z/t12 embryos, prior to the appearance of advanced degenerate changes following developmental arrest, did not differ significantly in their labeling patterns from the labeling patterns of phenotypically wild-type late morula. These findings indicate that the initial effect of the t'"/t12 genome is not on rRNA synthesis. The incorporation of the labeled precursor into the intranuclear fibrillo-granular bodies which characterize tZ2 homozygotes, was also studied.

The t'* allele is one of a series of mul- tiple alleles at the T (or brachyury) locus located on autosome IX of the mouse. This recessive allele results in a wild-type when heterozygous with the wild-type phenotype allele, in a tailless phenotype when heterozygous with the T allele, and in an embry- onic lethal phenotype when homozygous.

It has been suggested, on the basis of morphological, histochemical and low resolution autoradiographic studies, that the cause of lethality of the t'z/t'2 genome is related to abnormal nucleolar development and reduced RNA synthesis at the late morula stage (Smith, '56; Mintz, '64a,b,c). Recent studies indicate, however, that homozygous, developmentally arrested, t" embryos do not differ from their littermates either in total RNA content, or in nucleolar ultrastructure prior to advanced degenerative changes. For instance, Ca- larco and Brown ('68) were unable to find any differences in the amount of basophilia between azure B-stained late morula t'Z/t'2 embryos and azure B-stained late morula +/+ embryos. Hillman et al. ('70) found that nucleoli in tIZ homozygotes de-

AM. J. ANAT., 134: 411424.

velop normally up to the stage of developmental arrest. In those embryos which develop to the late morula stage, the nucleoli are elongate and have the same ultrastructural appearance as those found in their phenotypically normal late morulae littermates or in random-bred late morulae (Hillman and Tasca, '69). The elongated nucleoli become rounded only following arrested development at a time when numerous cytoplasmic organelles are losing their ultrastructural integrity.

Prior to arrested development the t I 2 homozygotes do not differ from their phenotypically wild-type +I + or +/t'* littermates in (1 ) numbers of primary or definitive nucleoli, (2) the degree of hypertrophy of the pars granulosae of the definitive nucleoli, and ( 3 ) the character- istic increases of cytoplasmic polyribosomes and rough endoplasmic reticulum at the successive cleavage stages (Hillman et al., '70). The combined findings from these latter studies suggest that deficient RNA synthesis in homozygous t I z late

1 Supported by a US. Public Health Research grant, HD-00827.

411

412 NINA HILLMAN

morulae, particularly rRNA synthesis as described in earlier works, had occurred subsequent rather than prior to developmental arrest and thus might be a result of cell degeneration rather than a causal factor of death. In order to test this hy- pothesis high resolution autoradiographic studies of cleavage staged tr2, t'" embryos were undertaken.

MATERIALS AND METHODS

Two-cell embryos were removed from the oviducts of + t'" superovulated fe- inales (Edwards and Gates, '59) mated to + t f2 males. The 30 females and 22 males used tor this series were tested for the presence of the 1'' allele (Smith, '56). The female + t'2 produces gametes in a ratio of 1 (+) : 1 ( t " ) while the male transmits the alleles in an average ratio of 0.24(+): 0.76( P ) (Smith, '56). Approximately 40% of the offspring from large numbers of + t'l inter se matings are therefore oS the genotype t'" t". Superovulatory hormones used in the present experiments resulted in an average litter size of 22 embryos. Their use has been found not to alter the transmission ratio of the females and the expected frequencies of t"/ ti2 embryos are tound in pooled litters from superovulated +/t" females mated inter se (Hillman et al., '70).

Entire litters of 2-cell embryos, removed from the oviducts of the superovulated females. were separately placed into culture (Brinster, '63). A t each successive cleavage stage (4-cell, 8-cell, early and late morulae) two litters were removed from the culture medium and each placed into medium containing !H-5-uridine (6 ,Ci ml of medium) for three hours. The uridine was purchased from Schwarz Bio- Research Inc. in a stock solution of 20-25 Ci mrnole and diluted to the desired con- centration with Brinster's medium.

Following the thr ee-hour labeling period one litter was removed from culture, washed in nonradioactive medium and fixed tor ultrastructural studies. The other litter. also removed from radioactive medium, was washed and reincubated in nonradioactive medium for an additional nine hours. In most cases, the cells of the embryo undergo one mitotic division during this reincubation time, advancing the em-

bryo to the next cleavage stage. These periods of time for labeling and reincubation were chosen because these times were previously used for autoradiographic studies of "H-uridine labeled random-bred cleavage staged embryos (Hillman and Tasca, '69). Using the same time schedule allows the labeling patterns of embryos obtained from +It1* inter se matings to be compared with the labeling patterns reported for their wild-type counterparts.

In the second series of experiments. staged litters were removed from the oviducts (4-, 8-cell) or uteri (early and late morulae) of superovulated +,"IL females mated inter se. They were immediately placed into medium containing 3H-5-uridine where they remained for three hours. Following the time in culture. the embryos were removed from this medium, one-half of the litter was pre- pared for ultrastructural examination and the other one-half returned to nonradioactive medium for an additional nine hours of incubation. Comparisons of autoradiographs of these staged embryos with those of similarly staged and similarly handled embryos developing from the 2- cell stage in vitro showed no significant differences in the patterns or numbers of silver grains between these two groups,

Following the desired treatment, all of the embryos were fixed for one hour in 3% glutaraldehyde in 0.1 M PO, buffer (pH 7.4), washed in 0.1 M PO1 buffer containing 5% sucrose for 24-48 hours, postfixed for one hour in 1% OsO, (Millonig's buffer, pH 7 . 3 ) , dehydrated and embedded in Epon. Thin sections were placed on un- coated copper grids and covered with 11- ford L-4 nuclear track emulsion. The emulsion-coated grids were stored in light-tight boxes for one week. The emulsion was then developed with Dektol, acid-fixed and rinsed with distilled water. The sections were stained with lead citrate (Venable and Coggeshell, '65) arid photographed with either an RCA EMU-36 or a Zeiss 9-A electron microscope.

Selected grids of phenotypically normal and mutant morulae were treated with either trichloroacetic acid or ribonuclease (Worthington). Examination of these embryos showed that the number and dis-

STUDIES OF t '2/t '2 MOUSE EMBRYOS 413

tribution of the silver grains did not differ in the trichloroacetic acid-treated embryos when compared with non-treated embryos but that the silver grains were almost com- pletely removed by ribonuclease treatment (for methods see Hillman and Tasca, '69).

Cleavage-staged, homozygous tZ2 embryos, obtained from either superovulated or spontaneously ovulated females, can be distinguished from their phenotypically normal (+/+ and +/t'") littermates on the basis of ultrastructural characteristics, i.e., nuclear lipid droplets, fibrillo-granular bodies and binucleate cells (Hillman et al., '70). Since the two phenotypes are dis- tinguishable, the areas of the nuclei and nucleoli of the two groups were separately measured, using a planimeter, and the distribution of label per unit area ( p z ) determined. The distribution of label per unit area of the small nuclear fibrillo- granular bodies, which characterize t"/t" embryos, was also determined. The signifi- cance of the differences between grain counts was determined by the Student "t- test." Grain counts were made only on those cells of the homozygous P2 embryos which did not show signs of advanced degeneration and in which individual silver grains could be distinguished. For the present report the t"/t'" cells which are described as nondegenerate are cells in

which there were no visual ultrastructural differences between those organelles which are common to both mutant and wild-type littermates.

OBSERVATIONS

Nondegenerate cells. The labeling patterns and the distribution of silver grains are the same both in cells of tlZ/t12 embryos which show no ultrastructural signs of arrested development or degeneration and in comparable cells of their phenotypically wild-type littermates (table l ) . These patterns do not differ from those reported for their randomly-bred counterparts (Hillman and Tasca, '69). Regard- less of the developmental stage of the embryos at the time of the three-hour labeling period, most of the incorporated labeled precursor is associated with the definitive nucleoli. The number of silver grains localized over these nucleoli is significantly greater than the amount found over the nucleoplasm at any stage examined (table 1). The amount of label localized over a unit area of these nucleoli increases between the 4- and 8-cell stages, whereas the amount per unit area remains rather constant between 8-cell embryos and early morula embryos as well as between early and late morulae. The total number of silver grains localized over both

TABLE 1 Grain counts /uni t area o f nucleoli and nucleoplasm in embryos labeled f o r

three hours with 3H-5-uridine

Stage labeled

Phenotype

Definitive nucleoli

Number Number of examined grains/pz

Nucleoplasm

Number examined

4 cell

4 cell I,*

8 cell

8 cell * Early Morula Early Morula * Late Morula Late Morula *

Wild-type (7) Mutant (8) Wild-type (15) Mutant (15) Wild-type (10) Mutant (8) Wild-type (10) Mutant (10) Wild-type (14) Mutant (16) Wild-type (9) Mutant (8) Wild-type (10) Mutant (11) Wild-type ( 11 ) Mutant

14 15 20 20 12 16 18 18 22 21 15 17 14 16 11 -

1.05 t 0.09 0.99t 0.06 0.83 t 0.10 0.79 t 0.08 1.94 t 0.21 1.87t0.18 0.95 C 0.15 0.98 t 0.09 2.00 t 0.23 1.94ir0.19 0.922 0.09 0.86ir 0.13 1.93t0.18 1.99 ir 0.24 0.72 t 0.05 -

10 8 17 15 12 13 15 16 20 21 14 8 12 14 11 -

Number of grains/p2

0.21 ir 0.06 0.19?0.05 0.61 f 0.05 0.57t 0.07 0.25 t 0.05 0.25f 0.03 0.49 & 0.08 0.46 -I- 0.06 0.45 t 0.07 0.41 f 0.04 0.35 t 0.06 0.33 & 0.07 0.36 ir 0.04 0.38 t 0.07 0.19 t 0.02

-~

-

1 The embryos with asterisks were labeled for three hours and reincubated in "cold" medium for an additional nine hours. The embryos without asterisks were examined immediately following the labeling period. Parentheses indicate number of embryos examined.

414 NINA HILLMAN

the nucleoli and nucleoplasm increases, however, at each successive cleavage stage with the exception of the late morula stage. Although the total number of nucleolar and nucleoplasmic silver grains in late morula embryos is not greater than the total found in early morula embryos, there is more cytoplasmic labeling in the three-hour labeled late morulae than in similarly labeled early morulae. There is, in fact. increased cytoplasmic labeling at each successive cleavage stage in those embryos which are examined immediately following the labeling period.

Nondegenerate cells of reincubated cleavage-staged t"jtZ2 embryos show no significant differences in the numbers of silver grains or in the distribution of these grains when cornpared with the numbers and distribution of grains in their reincubated littermates (table 1) . In the reincubated t" homozygotes, as in the reincubated phenotypically wild-type embryos, there is a significant decrease in the amount of label associated with the definitive nucleoli at each cleavage stage when compared with the nucleoli of their nonreincubated counterparts. In the reincubated 4- and 8-cell embryos of both phenotypes there is an increase in nucleoplasmic labeling per unit area when compared with the amount of labeling localized over the same unit area of nucleoplasm in the correspondingly staged nonreincubated embryos (table 1). In all early morulae and in phenotypically wild-type late morulae there is a decline in the numbers of silver grains localized over the nucleoplasms following reincubation (table 1 ) . Although cytoplasmic silver grains were not counted, there is an obvious increase in cytoplasmic labeling in reincubated staged embryos of both phenotypes when compared with similarly staged nonreincubated embryos. The amount of cytoplasmic labeling increases at each successive cleavage stage,

The Significant decrease in nucleolar labeling, the decline in nucleoplasmic labeling. and the increased cytoplasmic labeling found in phenotypically wild-type late morulae are also found in those cells of reincubated t'? t I 2 late morulae embryos which are not in advanced stages of degeneration. Although these numbers of

silver grains do not differ significantly from the numbers found in reincubated phenotypically wild-type late morulae, none of the t'2/t'2 cells are without some degenerate characteristics. These latter cells are therefore considered degenerate and are described in the following section.

Degenerate cells. The stages of morphological degeneration of t'2,/t*2 embryos have been described previously (Hillman et al., '70). Briefly, the first morphological difference noted between the developmentally arrested mutant cells and similarly staged healthy cells is the contracted appearance of the pars fibrosae of the definitive nucleoli. In succeeding stages of degeneration, the nucleoli are further contracted (elongated nucleoli become rounded) and the nucleolar granules and granules associated with the outer nuclear membrane are no longer distinct. Concomitantly, rough endoplasmic reticulum breaks down and ribosomes appear as monosomes rather than as polyribosomes. Lipid droplets, mitochondria and crystalline inclusions, begin to cluster within the cytoplasm leav- ing large areas devoid of organelles. Fur- ther degeneration results in the loss of structural integrity of most cytoplasmic organelles and in the vacuolization of the cytoplasm. Ultimately, the blastomeres separate from each other and remain as round cells within the zona pellucida. Al- though developmentally arrested embryos have been observed to contain cells all of which are in the same stage of degeneration, most of these embryos contain cells which range from highly degenerate to normal in appearance. Frequently these embryos contain extremely degenerate cells as well as normal dividing cells.

Since most t"/'t" embryos die during the morula stage, the labeling patterns of morula-staged mutant embryos were studied in order to determine if the patterns in degenerate morula cells can be correlated with the extent of degeneration. Excluded from these observations are embryos which were examined immediately after a three-hour labeling period and found to contain blastomeres which were separated or vacuolated. These embryos were discarded because the degenerative changes observed indicated that the blastomeres had been in developmental arrest

STUDIES OF tiZ/t'* MOUSE EMBRYOS 415

during the entire period of labeling. All embryos which had been labeled for three hours and reincubated for nine hours were studied regardless of their state of degeneration.

In those cells of three-hour labeled t"/t" morulae embryos which are developmentally arrested, the pattern of label ranges from normal (label found primarily localized over the nucleoli with some in the nucleoplasm and cytoplasm) to extremely atypical (label clumped over the nucleoli). In general it was found that the less degenerate the cell, the more normal the distribution of silver grains. Noted frequently were cells which had degenerated to the stage in which the nucleolar granules were no longer distinct. These cells contain ribosomes which exist mainly as monosomes and organelles which are clustered into some areas of the cytoplasm. However, the labeling pattern and the numbers of silver grains in these cells are normal (figs. 1, l a , 2).

Grain counts were made on 12 early morula cells and 16 late morula cells all of which were in initial stages of degeneration. There are 1.86 2 0.13 silver grains per unit area of the nucleolus and 0.39 I: 0.03 silver grains per unit area of the nucleoplasm in early morulae, and 1.90 t 0.10 silver grains per unit area of the nucleolus and 01.36 2 0.06 per unit area of the nucleoplasm in late morulae. In these cells the amount of label associated with the nucleoli and nucleoplasm does not differ significantly either from that found in nondegenerated t12/t1" cells or from the numbers found in the phenotypically wild-type cells at either the early or late morula stages (table 1). In cells which are in a more advanced stage of degeneration (small cytoplasmic vacuoles and loss of ultrastructural characteristics in most organelles), the label is localized almost entirely over the contracted nucleoli and is clumped (figs. 3, 3a). In these later embryos it is not possible to count silver grains.

In developmentally arrested reincubated early and late morula t'*/t*" embryos the pattern of labeling also ranges from normal to grossly atypical. As in the embryos examined immediately after labeling, the pattern of labeling can be correlated with

the extent of degeneration of the cell. In those cells showing initial signs of degeneration, the distribution of label does not differ from that found in healthy cells (figs. 4, 4a). Here the number of silver grains associated with 15 reincubated early morula t"/t12 cells averages 0.81 t 0.06 per unit area of nucleolus and 0.32 I: 0.04 per unit area of nucleoplasm while in ten cells of reincubated late morulae there are 0.68 0.04 silver grains per unit area of nucleolus and 0.21 +- 0.03 per unit area of nucleoplasm. These numbers do not differ significantly from those observed in similarly treated phenotypically mutant early morulae and phenotypically wild-type early and late morulae (table 1).

In cells which are in later stages of degeneration, the nucleoli and, in most cases, the nucleoplasms are heavily labeled. In most of these cells, the cytoplasms contain fewer silver grains than are found in nondegenerate cells of reincubated morulae (figs. 5, 5a). In a few, however, the cytoplasm is heavily labeled. In neither of these two groups of embryos is it possible to count the silver grains which are clumped over the nucleoli.

Fibrillo-granular bodies. Silver grain counts were made on 32 fibrillo-granular bodies located in nondegenerate cells of three-hour labeled early morula t" homozygotes. The number of silver grains average 0.33 2 0.02 per pz. This number is significantly less than the number of grains associated with a similar area of definitive nucleoli and in the same range as that found over the same unit area of nucleoplasm in three-hour labeled early morula tl"/t" embryos (table 1 ) .

DISCUSSION

The results reported here show that early and late morula t"/t'* embryos do not differ from their littermates in their incorporation of 3H-5-uridine into RNAase digestible material prior to developmental arrest. Individual cells, within developmentally arrested embryos, can be in the initial stages of degeneration and still have normal labeling patterns. Atypical labeling patterns are found only in those cells of morula tZ2/t'* embryos which are in advanced stages of degeneration, The usual pattern for these cells is the clumped ac-

416 NINA HILLMAN

cumulation of silver grains over the nucleolus, the pattern of labeling noted by Mintz ('64) in late morula tl'/tl' embryos. The present study shows that this nucleolar accumulation of labeled precursor is found in extremely degenerated cells both immediately following labeling and after labeling and reincubation in nonradioactive medium.

It has been reported that the rate of total RNA synthesis increases progressively from the 4-cell stage to the late morula stage in random-bred mouse embryos (Tasca and Hillman, '67, '70). On the basis of biochemical determinations as well as studies utilizing inhibitors of RNA synthesis, it is now well established that rRNA synthesis in mouse embryos begins as early as the 4-cell stage and accounts for increasing amounts of the total RNA synthesized at the older cleavage stages (Woodland and Graham, '69; Tasca and Hillman. '70; Monesi et al., '70; Piko, '70). In late morula embryos 60-80% of the total RNA synthesized is rRNA (Ellem and Gwatkin, '68). The increasing rRNA synthesis can be correlated with progressive hypertrophy of the pars granulosae of the definitive nucleoli and with the greater incorporation of 'H-5-uridine into nucleolar RNA as determined by high resolution autoradiographic studies (Hillman and Tasca, '69 ). The patterns of labeling found in nondegenerate t'"/t'" cells, either immediately following labeling or following labeling and reincubation, do not differ significantly from those found in either their phenotypically wild-type littermates nor from those reported for their randomly bred counterparts at any cleavage stage.

In the earlier studies, the late morulae tl' t'" embryos which were found to contain less basophilia than either their cavi- tating littermates (Smith, '56) or +/+ late morulae (Mintz, '64b), were described as containing round, condensed nucleoli. It has since been noted that rounded, condensed nucleoli are present only in those late morula mutant cells which have been in a state of developmental arrest suf- ficiently long for most cytoplasmic organelles, including rough endoplasmic reticulum and polyribosomes, to have lost their ultrastructural integrity (Hillman et al., '70). Since the degenerative changes ap-

pear to be progressive, the discrspancy ncted between the findings of Calarco and Brown ('68) and those of Smith ('56) and Mintz ('64b) on the intensity of basophilia in t"/t'" late morulae may rea- smably be related to the length of time the embryos had been in developmental arrest prior to the histochemical deter- mination of their RNA content. The deficiency of cytoplasmic basophilia described by both Mintz and Smith is most likely related to the breakdown of polyribosomes and solubilization of RNA within the mutant cells.

The small fibrillo-granular nuclear bodies which characterize t I 2 homozygotes are ultrastructurally and chemically similar to definitive nucleoli and like the nucleoli are most granular at the later cleavage stages (Hillman et al., '70). Unlike the definitive nucleoli, however, these structures do not actively incorporate "-5- uridine. The bodies examined in the present study were in early morulae t''//12 embryos which had been treated with "-5- uridine for three hours. In these cells the nucleoli had actively incorporated the labeled RNA precursor, but the number of silver grains associated with the small fibrillo-granular bodies was never greater than the number of silver grains randomly distributed throughout the nucleoplasm. If these bodies were actively synthesizing RNA, one would expect a greater localiza- tion of silver grains, particularly at the early morula stage when the bodies are quite granular. Since they do not appear to function as definitive nucleoli, then i t is possible, as previously suggested, that they are degenerate structures (Hillman et al., '70). Although their relationship to the t"/t'* genotype is not known. the fact that they are found at all cleavage stages, not only in early and late morulae, sug- gests that the t" homozygous genotype may express itself much earlier than the stage at which the embryos cease development.

LITERATURE CITED Brinster, R. L. 1963 A method for in uitro cul-

tivation of mouse ova from 2-cell to blastocyst. Exp. Cell Res., 32: 205-207.

Calarco, P. G., and E. H. Brown 1968 Cyto- logical and ultrastructural comparisons of t ' Z / t l z and normal mouse morulae. J. Exp. Zool., 168: 169-186.

STUDIES OF t”/t l2 MOUSE EMBRYOS 417

Edwards, R. G., and A. H. Gates 1959 Timing on macromolecular synthesis and early de- of the stages of the maturation divisions, velopment in the mouse embryo. Exp. Cell ovulation, fertilization and the first cleavage Res.. 59: 197-206. of eggs of adult mice treated with gonado- tropins. J. Endocr., 18: 292-304.

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

1970 Ultrastructural studies of cleavage stage t *Z/ t12 mouse embryos. Am. J. Anat., 128: 311-339.

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

Mintz, B. 1964a Gene expression in the morula stage of mouse embryos, as observed during development of t l ~ / t l ~ lethal mutants in nitro.

196413 Formation of genetically mosaic mouse embryos, and early development of “lethal ( t12/t‘2)-normal” mosaics. J. Exp. Zool.,

Synthetic processes and early development in the mammalian egg. In: Differen- tiation and Development. Little, Brown and Company, Boston, pp. 85-100.

Monesi, V., M. “olinaro, E. Spalletta and C. Davoli 1970 Effect of Metabolic inhibitors

Hillman, N., R. Hillman and G. Wileman

J. EXP. ZOO^., 157: 267-272.

167: 273-299. 1964c

Piko, L. 1970 Synthesis of macromolecules in early mouse embryos cultured in vitro: RNA, DNA, and a polysaccharide component. Devel. Biol., 21: 257-279.

Smith, L. J. 1956 A morphological and histochemical investigation of a preimplantation lethal ( P ) in the house mouse. J. Exp. Zool., 132: 51-83.

Tasca, R. J., and N. W. Hillman 1967 Uptake and incorporation of H3-uridine and Ci4-leucine in preimplantation mouse embryos: effects of inhibitors of RNA and protein synthesis. Am. Zool., 7: 170 (Abstr).

1970 Effects of actinomycin D and cyclohexamide on RNA and protein synthesis in cleavage stage mouse embryos. Nature, 225:

Woodland, H. R., and C. F. Graham 1969 RN.4 synthesis during early development of the mouse. Nature, 221: 327-331.

Venable, J. H., and R. Coggeshall 1965 A sim- plified lead citrate stain for use in elecirriii microscopy. J. Cell Biol., 25: 407-4”.

1022-1 025.

PLATE 1

EXPLANATION OF FIGURES

1 An autoradiograph of a developmentally arrested morula tr2//tr2 embryo which was labeled for three hours. This part of the embryo contains portions of cells in varying stages of degeneration. The cell which shows a normal distribution of label for a n early morula staged cell is degenerate. Silver grains are found over both the nucleolus and nucleoplasm as well as over the cytoplasm. )< 4,000.

A higher magnification of the delineated portion of the cytoplasm of the labeled cell shown in figure 1. Ribosomes exist mainly as monosomes and there is no rough endoplasmic reticulum. Mito- chondria still appear structurally intact. x 22,000.

2 Cytoplasm and cytoplasmic organelles of a nondegenerate P/ tr2 morula embryo. In this cell the ribosomes are in polyribosomal clusters and rough endoplasmic reticulum is present. A comparison of this micrograph with figure l a shows the degenerate state of the cytoplasmic organelles of the developmentally arrested embryo RER, rough endoplasmic reticulum. x 22,000.

l a

418

STUDIES OF t'e/t'r MOUSE EMBRYOS Nina Hillman

PLATE 1

419

PLATE 2

EXPLANATION O F FIGURES

3 A portion of a three-hour labeled morula embryo which is in a more advanced stage of degeneration than that shown i n figure 1. The silver grains are clumped over the contracted nucleolus and few are present i n either the nucleoplasm or cytoplasm. x 5,100.

A higher magnification of the delineated portion of a serial section of the labeled cell shown in figure 3. With the exception of a mito- chondrion ( M ) , cytoplasmic organelles are no longer identifiable. x 22,000.

3a

420

STUDIES OF t 1 2 / t * 2 MOUSE EMBRYOS Nina Hillman

PLATE 2

42 1

PLATE 3

EXPLANATION OF FIGURES

4 A cell of a late morula t J z / t J 2 embryo labeled at the early morula stage and reincubated for nine hours. This cell, in the first stage of morphological degeneration (contracted pars fibrosa of the nucleolus), has a normal distribution of label. Lipid droplets (arrow) are present in the nucleus. x 6,700.

A higher magnification of the delineated portion of figure 4 showing the normal appearance of the cytoplasmic organelles. A fibrillo- granular body (arrow) is present i n the nucleus. RER. rough endoplasmic reticulum. x 22,000.

5 A degenerate t*2/t*” cell of an embryo labeled at the late morula stage and reincubated in nonradioactive medium. In this degenerate cell most of the label is clustered over the contracted nucleolus. X 7,800.

5a A high magnification of the cytoplasm of a serial section of the cell seen i n figure 5. Comparison of this cytoplasm with that seen in figure 4a shows the extensive degeneration of the cytoplasmic organelles. x 22,000.

4a

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STUDIES OF t 1 2 / t * 2 MOUSE EMBRYOS Nina Hillman

PLATE 3

423

autoradiographic studies of t12/t12 mouse embryos

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