Serum induced changes in the fine structure of primary chick embryo cultures

667

Protein synthesis in Cerebratulus

the fluctuations of the rate of protein synthesis in the sea urchin egg during cleavage. The two conditions observed, namely of no change in the rate of protein synthesis during the first cell cleavage [3] and of a decline at the onset of the first cleavage [9] have been demonstrated in the same material independently of the species and of the experimental conditions [lo].

REFERENCES 1. BELL, E. and REEDER, R., Biochim. Biophys. Ada 142, 500 (1967). 2. COSTELLO, D. P., DAVIDSON, H. E., EGGERS, A., Fox, RI. H. and HENLEY, C., Methods for obtaining and handling marine eggs and embryos. lRIarine Biological Laboratory, Woods Hole, Mass., 1957. 3. Gross, P. R. and FRY, B. J., Science 153, 749 (1966). 4. LOWRY, 0. H., ROSENBOROUGH, N. .J., FARR, L. A. and RAKDALL, R. J., J. Bid. Chem. 193, 265 (1951). 5. MANS, R. J. and NOVELLI, G. D., Biochem. Biophys. Res. Comm. 3, 540 (1960). 6. MONROY, A., Chemistry and Physiology of Fertilization. Holt, Rinehart and Winston, New York, 1965. 7. MONROY, A. and TOI.IS, H., Bio!. Bun. 126, 456 (1964). 8. SS~ITH, D. L., ECKF,R, R. E. and SUBTELNY, S., Proc. 1Vatl Acad. Sci. US 56, 1724 (1966). 9. SOFER, W. H., GEORGE, J. F., and IVERSOS, R. M., Science 153, 1644 (1966). 10. TI~~O~RIAN, H., Science 154, 1055 (1966).

SERUM

INDUCED

CHANGES

OF PRIMARY

CHICK

J. D. LEVINTHAL Department

of Molecular

IN THE FINE EMBRYO

CULTURES

and H. RUBIN

Biology and Virus Laboratory, Berkeley, Calif. 94720, USA Received

STRUCTURE

University

of California,

May 6, 1968

STUDY of the stimulating effect of serum on primary chick embryo cultures by Rubin and HatiC [3] has shown that cells increase markedly in size upon cultivation and do so about 5 times more rapidly in the presence of serum than in its absence. In order to determine what changes in fine structure accompany the serum-stimulated increase in size, several series of cells grown at 37” in humidified 5 per cent CO, atmosphere with and without serum were taken for electron microscopy at intervals from 2 to 70 h after plating. Ten or 11 day chick embryos were harvested, eyes, brain and internal organs discarded, and the remaining tissue trypsinized successively for 10, 5 and 5 min in 0.25 per cent trypsin before stopping trypsin action in medium 199 and 20 per cent calf serum. The pool was resuspended in medium 199 with 2 per cent tryptose phosphate broth, with or without 1 per cent calf and I per cent chicken serum, and 8 x lo6 viable cells were plated on 100 x 20 mm plastic petri dishes. Beginning at Experimental

Cell Research 52

MS

.I. 1). Ixrfi~~llfrrl

rrrltl

11. Rubill

2 11, when attachment had taken place, specimens were fixed as ;I monol:~~ VI’, al’tcr discarding the medium, in 1.(i per cent glutaraldehgde in phosphate buffc~tl saline, pH 7.2 with 5 10P3 111magnesium chloride and 1 1OW 31 calciunl chloritic ( I’HS MgCa). The cells were then scraped, centrifuged in XcNaught protein tubes and postfixed as a pellet in 1 per cent osmic acid in PBS MgCa. After dehydration in a graded ethanol 0.15 ‘VI KaCl series, infiltration with propylene dioside and embedding in Epon X12, thin sections were cut on an Ll cultures, a total of 4,5 preparations were studied. The greatest number of preparations were taken at 11 h and at the 20 24 h period, after determining that these were the periods in which differences were consistently present in the cells grown \villl and without serum. Cells examined immediately after disaggregation and trypsinization showed extensive vacuolization, margination of nuclear chromatin, and loss of clear definition of cytoplasmic structures. The cell outline was highly irregular and often showed microspikes. Gradual recovery occurred in both types of preparations during the first 8 h to about the same degree. Cells lost their vacuoles and assumed either a round or an elongated shape with a high nucleus to cytoplasmic ratio. Ill-defined, single ribosomes were scattered at random in the scanty cytoplasm. The endoplasmic reticulum was limited to a few simple channels nearly devoid of attached ribosomes. Mitochondria were small and dense with poorly defined cristae. However, by 11 h a definite change had occurred in the serum-grown cultures. Free polyribosomes appeared in the cytoplasm of some of the cells, and the mitochondria were larger than at 8 h. By 20 h a remarkable difference in the appearance of the two types ol cultures was evident. The serum-grown cells had increased enormously in size compared with those grown without serum, and this change affected both the clongated as well as the round cells (Fig. 1 (I vs. Fig. 1 b). The increase in size was associated with notable changes in the fine structure of the cells. All serum-grown cells, whether elongated or round, showed many well-defined polyribosomes, both free and mernbrane associated. Microtubules and elaborate, loose networks of fine fibrils appeared in the cytoplasm. Small opaque masses, probably representing glycogen deposits, were occasionally observed. Some cells showed densely packed, parallel arrays of fine fibrils and microtubules, either parallel or at an angle to the plasma membrane. If at an angle, bundles of these fibrils were often interspersed with polyribosomes. Mitochondria were large with sharply and delicately defined details, and mitochondrial ribosomes were easily detected. The channels of the endoplasmic reticulum were now larger and more widespread through the greatly enlarged cytoplasm; their sur-

Fig. l.-(a)

Primary

Lrypsinized

chick cells grown with 2 y0 serum, 20 h ( ): 1000). (h) Same grown

without serum, 23 h ( x 4000). Fig. 2.-(a) Primary trypsinized chick cells grown with 2% serum, 11 h. Kale polyribosomes, large mitochondrion (M) ( x 30,000). (h) Same, grown without serum, 11 h. Note dense, small mitochondria (M) with poorly defined cristae, ribosomes scattered without clustering. Nucleus (N) occupies large proportion of cell ( x 30,000). Experimental

Cell Research 52

Serum and primary

chick embryo cultures

Experimental

669

Cell Research 52

face was studded with ribosomes. At this time (20 h), the cells grown withoul strum showed no obvious difference from their state at 8 h. Serum-grown cells taken after -10 h showed no further progression in their tlcgrer, of differentiation, and continued to show the elements described for the 20 h samples. On the other hand, changes were observed in the cells grown without serum. some of which now began to show free and membrane-associated polyribosomes. cytoplasmic microtubules and fibrils in networks and parallel arrays, well defined. eillarged mitochondria, and enlargement of the whole cell. Other cells appeared inactive or in the process of degeneration as evidenced by their general poverty of cell organelles, and poor definition. At 70 h this mixture of growin g and differentiating cells and small, often stringy, poorly defined cells was found to have persisted, while the cells grown with serum were again seen to appear as they had at 20 h. It should be emphasized that in both groups there was considerable individual variation. and that although growth and differentiation had occurred at 40 h in some cells grown without serum, it did not occur to the degree evident in those grown with serum. Comparative aspects of the fine structure of the cells of both groups, fised at different times after plating, are illustrated in Figs 2, 3 and 1. The earliest morphologic evidence of the effect of the serum-stimulating factor, the appearance of polyribosomes in cells of the cultures grown with 2 per cent serum, shows that synthesis of these elements must take place prior to the production of proteins required in growth and differentiation. This finding recalls the fact that ribosomes similarly increase in bacteria shifted from a minimal to a rich medium [2]. In accord with the work of Lieberman et nl. [lJ, who demonstrated that in newly cultured rabbit kidney cells the first macromolecular synthesis is of ribosomal RNA, we would conclude that the formation of large quantities of polyribosomes found as an early event in our preparations of primary cultured chick cells is evidence of a similar synthesis of ribosomal RNA. The fact that polyribosomes eventually appear after a lag period in the cells grown without serum, followed by growth and differentiation, probably means that the serum factor is produced slowly by these cells and, after 10 11, has accumulated in quantities adequate to induce changes similar to those found in serum-stiriiul:lted cells. That cells in culture do in fact release proteins which migrate like serum proteins upon electrophoresis in polyacrylamide gels has been demonstrated in recent studies (R. Williams and H. Rubin, unpublished). Summary. -Trypsinized chick embryo cells grown in medium 199, 2 per cent tryptose phosphate, with and without 2 per cent serum (1 per cent calf, 1 per cent

Fig. 3.-(a) Same, grown with 2 “6 serum, 20 h. Note parallel arrays of fibrils (ii) and irregular network of fibrils, microtubulcs (mf), clearly defined mitochondrion (M), numerous clustcrcd ribosomes ( x 50,000). (h) Same, grown without serum, 20 h. hlitochondria (AI) still dense, ribosomes scattered and poorly defined. Nucleus still occupies large proportion of cell. Endoplasmic reticulum (er) distended ( Y 50,000). Fig. J.--((I) Same, grown with 2% serum, 13 h. Note microtubule (mf), distended cndoplasmic reticulum (er) with many associated and well-defined ribosomes, large milochondrion (dl), and fibrils (fi) running parallel to plasma membrane ( Y 50,000). (h) Same grown without strum. 43 II. Recovery of delicate fine structure of mitochondria and appearance of cluslcrs of well-defined ribosomes ( y 50,000). Experimental

Cell Keseurch 52

Serum and primary

chick embryo cultures

Experimental

671

Cell Research 52

chicken) were examined by electron microscopy during t IIV first ‘it) II or cult urt’. Cells grown with serum showed formation of many polyribosomcs by 11 II. .A striking increase in size occurred by 20 h, accompanied by formation of differentiated cytoplasmic structures, fibrils, and microtubules in the serum-stirnul~ltctl cf~lls. Thfw changes were considerably delayed and of lesser magnitude in the cells growl1 \vi t Ilou t serum. This investigation CA02245, CA04774 Science Foundation

was supported in part by US l’ublic Health research grants and Ca05619 from the National Cancer Institute and Sational research grant, GB 6919. REFERENCES

1. LIEBERMAS, I. hnn~~s,

Ii. and OVI:, I’., J. Rio!.

Chem.

238, 21 11 (1963).

2. MAALOE, 0. and KJEI.D(~AARD,N. O., (:ontrol of hfacromolecular Synthesis, p. XX. Ilcnjanrin, New York,

1966.

3. HUBIN, H. and IH.ATII?, C., Deuelo~). I1iol. 17, 603 (1968).

MORPHOLOGICAL

OBSERVATIONS AND CYTOTOXIC I’. BIBERFELD,

Department

of Pathology Department

of

at Sabbatsberg Immunology,

ON LYMPHOCYTE ACTION

PERIPOLESIS

IN VITRO’

G. HOLM and P. PERLMANN Hospital, Weenner-Gren

Knrolinska Institute,

Institutet

Medical

Stockholm,

School

and

Sweden

Received May 13, 1968

P REVIOUS

studies have shown a rapid and strong damage of Chang cells (human liver, established cell strain) incubated with normal human blood lymphocytes and phytohaemagglutinin (PHA) [5]. This reaction seems to depend on the mixed agglutination of the cell types and on the stimulation of lymphocytic effector cells by PHA [4]. Similar observations have also been reported for other target cell systems in vitro [6, 7-91. The following report is concerned with light and electron microscopic observations, which suggest that peripolesis around target cells is a characteristic behaviour of lymphocytes in this in vitro system, and possibly of importance for the expression of their cytotoxicity. 1 This investigation \vas supported by grants from the Swedish National Heart and Chest Diseases, and the Lotten Bohman Foundation.

Association

against

Fig. I.--Phase-microscopy of Chang cells in monolayer incubated with lymphocytes and PH.%. (a) Still confluent monolayer with attached lymphocytes. Through focus examination did not disclose if lymphocytes (arrows) were within or beneath the Chang cells. 1,ymphocytes on the free surface of Chang cells were easy to localize (arrow heads). x 1000. (b) Extensive destruction of Chang cell monolayer after approx. 17 h incubation. Remaining Chang cells are spindleshaped and covered with aggregated lymphocytes. x 640. Experimental

Cell Research

52