Cytochemistry and fine structure of elimination chromatin in Dytiscidae

0

1968 by Academic

Experimental

Press Inc.

507

Cell Research 52, 507-522 (1968)

CYTOCHEMISTRY AND FINE STRUCTURE OF ELIMINATION CHROMATIN IN D YT1SClDAE KAORI KATO Department

of Zoology,

University

Received

of California, January

Berkeley,

Calif. 94720, USA

25, 1968

has been known for a long time that Dyfiscus and some of the related water beetles produce extrachromosomal chromatin (elimination chromatin) during the oocyte differentiation. The elimination chromatin first appears in the oogonium nucleus prior to the first of the four differential mitoses and it goes to only one of the daughter cells after each mitosis, thus resulting in one large oocyte nucleus with the elimination chromatin and 15 small nurse cell nuclei without it (Fig. 1) [lo, 13, 14, 17, 301. The elimination chromatin is Feulgen-positive and accumulates progressively [28] and contains RNA [27]. A similar chromatic body has been observed in the whirligig beetle, Dineufes [15], Tipula (Diptera) [2] and the fleas, Nosopsyllus and Xenopsyllus [3]. The chromatic body has often been referred to as the oocyte determinant, on the assumption that its presence in the cell leads to the development of an oocyte. In the vast majority of animals, no such structure exists, but differentiation of oocytes takes place. With our great wealth of knowledge about DNA and its behavior, it is a challenging question why in certain forms such a phenomenon takes place. In the present research, the synthetic activity and the tine structure of the elimination chromatin were studied by means of H3-thymidine and 3Huridine incorporation, cytochemical staining, microspectrophotometry and electron microscopy. IT

MATERIALS The adult Agabus

lutosus,

predaceous

water

A. disintegratus

AND

METHODS

beetles, Dytiscus marginicollis, Rhantus consimilis, and Acilius semisulcatus abbreviatus (Family Dytis-

cidae) from Inverness, Richmond and San Francisco, California were used. The ovaries were fixed with acetic alcohol (1:3) for Feulgen reaction, azure B staining and autoradiography. Histones were stained according to the method of Alfert

and Geschwind

[l]. Experimental

Cell Research 52

W-Thymidine (;i.30 c/mmole. New E11gla11d Nlrclcar~ Corp.): I jr\, ‘I‘(: llol~sc (Difco Laboratories. Detroit) was dissolved in ac~~~eous 311-thymitlinc solution (1 me/ml f-1,0) to establish the original serum concentration. Using this solrllion. 20 /I(’ of W-thymidinc was injected into lhc abdomen of II’. co~xinrilis !yilh :I I’inc glass capillary connected to a 0.25 ml tuberculin syringe. i\fter 2.5 minutes, about 0.003 ml of a horse serum solution containing an excess amount of cold t hymitlinc (al)proximately 100 i’ hot) was injected to displace the labeled precursor. The room Icniperature during the treatment was %%26”C. Sections 3 1, in thickness were coated with Kodak Nuclear Emulsion type NTN 2 after Feulgen reaction. The slices of a single nucleus were carefully identified and the number of grains FOLIII~ in thcsc serial sections was taken as the total for the nucleus. 3H-Yridine (2.0!) c/m mole, New England Nuclear Corp.): The procedure was the same as for 3H-thymidinc except that an excess amount of cold uridine was in]rcted after 5 minutes. Preparations were made by mounting adjacent sections alternately on two slides to facilitate comparison. One of them was coated directly with the liquid emulsion after rernoval of paraffin. After the autoradiographs were tleveloped, they were stained with Azure-Rosin Giemsa according to the method of McClure and I’elc. The other (control) was digested for 3 h at 37°C with I would be done. Using these photographs as templets, diaphragrns to mask everything but the nucleus were painted with lamp black lacquer on clean 18 mm round coversfxuin

Experimentcrl

Cdl Reserrrch52

Cytochemistry

and fine structure of elimination

chronmtin

509

slips for each oocyte nucleus. Thus extinction for whole nuclei was obtained. As a reference, the nuclei of the peritoneal epithelium were used. The shape of these nuclei is very irregular and the distribution of chromatin is quite inhomogeneous. Therefore, a fixed, round diaphragm of 3 mm diameter was used to include the whole nucleus. In order to make sure that these nuclei are diploid, some telophase nuclei of early nurse cells were measured using individually fitted masks. The extinctions cannot be compared with each other when a different-sized diaphragm is used for each nucleus. The difference in the diaphragm area was corrected by dividing the extinction with a size factor derived from the blank reading of a fixed, 6 mm round diaphragm when the blank reading of a specific mask was brought to the standard galvanometer deflection of 400 or 200 mm. Measurements were taken at 566 m,u using an interference filter with a half band width of 10 m/L (Jena Glaswerk, Mainz). Since the “entrance window” of the multiplier phototubes of the lP21 type is highly nonuniform (to be published in a separate report), the image of the nucleus was sent into an integrating sphere through a fine ground glass window. A multiplier phototube (RCA 11’21) was attached to the side of the integrating sphere, at 90 degrees to the image window. Thus at least the errors caused by the interaction between the inhomogeneities of the phototube and the specimen and by the different areas of the entrance window were avoided.

RESULTS DNA

The early period of the oocyte development may be divided into 3 stages for convenience: in stage 1 DNA synthesis takes place, in stage 2 DNA synthesis has been completed but the elimination chromatin remains as an intact mass, and in stage 3 the elimination chromatin begins to fragment hut is not completely eliminated (Fig. 1). Seventy seconds after the injection of 3HTdk, labeling appeared on both the elimination chromatin and the chromosomes, or on the elimination chromatin alone, of some of the undividing preoocytes III and I\’ and the oocytes of stage 1. The dividing preoocytes first became labeled 4 h and 10 min after the treatment. The ovaries which were examined after 2 h had no labeling on the dividing cells. This shows that the DNA synthesis is completed sometime between 2 and 4 h before division under the experimental conditions. The number of dividing preoocytes (both labeled and unlabeled) observed at various treatment times are given in Table 1. There \\-ere dividing preoocytes which were labeled only on the elimination chromatin or on both the elimination chromatin and the chromosomes (Figs 3 and 4). But there mere no dividing cells with labeling only on the chromosomes. It map be inferred that the synthesis period for the DNA of the elimination chromatin is longer than that for the chromosomes, as shown in Fig. 2. Between 14 and 24 h, all Experimental

Cell Research 52

h .- i d

k

Fig. l.-Schematic

drawing

of

the

ovariole.

a, Terminal filament. b, Tunica propria. C, Preoocyte I. d, Preoocyte III (dividing). e, Oocyte stage 1. I, Oocyte stage 2. g, Oogonia. h, Prefollicular nucleus. i, Preoocyte II. j, Preoocyte IV. k, Peritoneal epithelium. I, Kurse cells. m, Oocyte stage 3.

d-

-~. .

Div.

1

~~~

S(elimination -

i i Labeling on both : / 1 ^-and elimination : 11

‘\

‘Labeling

Experimental

only

chromatin)

-

S(chromosomes)p-~-~

Cell Research 52

*

chromosomes chromatin

on elimination

.-

chromatin

/ pa\

j fl j

1 Div. 2

Fig. Z.-Span thesis.

in time of DNA

syn-

Cyfochemisfry

and fine structure of elimination

511

chromafin

dividing preoocytes were labeled but after 28 h some unlabeled preoocytes appeared again. Thus it may be concluded that the period (S) is about 24 h for both preoocytes III and IV. Preoocytes showed the same tendency but the number of dividing cells observed small to draw a conclusion.

dividing synthesis I and II was too

TARLE 1. Dividing

freafmenf

Preoocyte Time (h)

EC

E*C

4 h 10’ 6h 8 11 10 h 12 h 14 11 16 h 18 h 20 h 22 h 24 h 28 h 32 h 10’ 48 h 10’

5

1

Total

5

preoocyfes of R. consimilis I

Preoocyte

E*C*

affer 3H-fhymidine Preoocyte

II

Preoocyte

III

EC

E*C

E*C*

EC

E*C

E*C*

9

1

2 3

4

1 5 3 2

11

2

2

4 2 2

1

9

3 1

2

2

2 3

1

7 2 2

2 6 2 1

3

11

11

14

33

1 1 1

9

14

2 3

4 4 15 6 3 2 4

1

2

EC

IV

E*C

E*C*

1 6

13 1 1

1 1

3 3 4

3

3 3

2 4 5 4 5 5 12 5 5

59

8

15

62

EC, No label on either the elimination chromatin or the chromosomes. E *C, Label only on the elimination chromatin. E*C*, Label on both the elimination chromatin and the chromosomes.

Among the undividing nuclei of the same stage, a considerable difference was observed in the degree of labeling on the elimination chromatin. Grain counts showed that there is no correlation between the degree of labeling on the elimination chromatin and the chromosomes. Therefore the possibility that the elimination chromatin may have a definite pattern of DNA synthesis during its development was examined. Labeled metaphase preoocytes II, III or IV which are found in the same ovary (sections from each ovary were all placed on a single slide) may be assumed to have been at the same phase of the cell cycle when 3HTdk was injected. Grain counts were made on 42 metaphase elimination chromatin “rings” which were divided into 9 groups depending on the preoocyte stage and the slide they belonged to. It was Experimental

Cell Research 52

512

Iitrori

Iicrto

Cytochemistry

and fine structure of elimination

513

chromatin

observed that a preoocyte and all of its accompanying nurse cells are always in the same phase of the nuclear cycle. They seem to develop in complete synchrony. For the control, therefore, 41 groups of either dividing or undividing nurse cells IV and 20 groups of nurse cells III were used. The total number of these nurse cells were 342. All cells which belong to a single group were on one slide and related to one preoocyte. If the relative variance for the experimental group agrees with that of the control group within certain limits, we may conclude that the elimination chromatin has a prescribed course of DNA synthesis, just as the chromosomes do in a nucleus. This was tested at 10 per cent level of significance. The pooled relative variance of grain counts for the elimination chromatin was found to be 0.215. That for the nurse cell nuclei was 0.219. F = 0.215lO.219 = 0.982 Since the obtained F value is smaller than F.,, (33, 280) = 1.33, it is highly probable that the DNA synthesis follows a fixed schedule. The elimination chromatin remained labeled as long as it was followed (48 h). The microspectrophotometry of Feulgen-stained nuclei of D. marginicollis showed that the average extinction for 21 diploid nuclei of the peritoneal epithelium is 0.0185 while the average extinction for 14 oocyte nuclei of stage 2 is 0.415. 0.415/0.0185 = 22.43. C, Chromosomes; E, elimination NM, nuclear membrane.

chromatin;

N, nucleolar

material;

NC, nurse cell chromosomes;

Fig. S.---Dividing preoocyte IV, IO h after 3HTdk injection. The elimination is heavily labeled but the chromosomes show no labeling. Feulgen reaction.

chromatin

“ring”

Fig. 4.-Dividing preoocyte III, 12 h after treatment with 3HTdk. Both the elimination chrotnatin and the chromosomes are labeled. The chromosomes of the accompanying nurse cell also show labeling. Fig. 5.--Early stage 3 oocyte, 5 min after 3H-uridine injection. and the chromosomes are labeled. Azure-Eosin Giemsa staining. Fig. 6.--Stage 2 oocyte, tion chromatin. Fig. 7.--Late labeling.

10 min after 3H-uridine

injection.

stage 2 oocytes, 20 min after 3H-uridine

Both the elimination

The labeling

treatment.

is mostly

chromatin

on the elimina-

The chromosomes

show a heavy

Fig. S.--Early stage 2 oocyte, 8 h after 3H-uridine treatment. Most of the labeling on the oocyte chromosomes has gone to the cytoplasm, causing some nuclei to look empty. Nurse cell nuclei are still heavily labeled. The elimination chromatin remains labeled. Experimental

Cell Research 52

The hislones in the elimination chromatin are found to ac~umulatc~ gratlually from its early stages in preoocyte I. The elimination chromatin 01 all stages gave a progressively positive reaction, including that of di\~itling l)re”ocytcs. Ho\verer, even at the final stage of elimination chromatin I’ovmation, the amount of histones per unit amount of DNA in the elimination chromatin never reaches as high a value as in the chromosomes. This can lx> c*c~nclutietl from a comparison bet\veen .5 ,U sections of the elimination chromatin mass and of the polyploid nurse cell ~~uc*leus 01 stage 3 or later. The Ialter stainctl much ~norc darkly \vith Fast green hut nluch mow \\,ealily \vith l:eulgcn reaction than the former.

The nuclei of the preoocyte I in which the elimination chromatin has not formed a mass stained pale purple with azure 13, showing the l~resencc of RNA. III the later stages the elimination chromatin stained dark bluish purple. The incorporation of 3H-uridine took place 2.5 min after injection mostly in the chromosomes of the oocytes of stages 2 and 3 and the nuclei of their attendant nurse cells. The elimination chromatin of some stage 2 antl most stage *3 oocytes w-as also labeled. After .5 min preoocyte I nuclei hecame slightly labeled. ;\fter 10 min light labeling appeared OJI both the elimination chromatin and the chromosomes, or o111yon the former, in most of lhc preoocytes II1 and I\‘. After 20 min practically all nuclei of every stage \vere labeled. In preoocytcs II, III and I\‘, the elimination chromatin was fairl? heavily labeled but the chromosomes showed only light or no labeling, \vhilc the accompanying nurse cell nuclei \\-ere al\vays labeled. In all of the oocytcs chromatin was labeled more heavily Ihan of stages 2 and 3 the elimination in preoocytes, and the chromosomes \\-ere much more heavily labeled than those in the accompanying nurse cells. After one hour, the cytoplasm of stage 2 and 3 oocytes and of the preoocyte I became lightly labeled. The elimination chromatin of dividing preoocytes I\’ first became labeled after 2 h. ,411 the incorporation so far mentioned \vas confirmed to be in RNA b? the use of Fig. Y.--Electron micrograph of a preoocytc II nucleus of Il. marginicollis. The nucleolar material is scattered all over the nucleus. DNA-containing component of the elimination chromatin is not apparent. The nuclear membrane appears irregularly scalloped. Experimental

Cell Research 52

Cytochemistry

and fine structure of elimination

chromatin

Experimental

515

Cell Research 52

,316

Iirrori

licrlo

RKase. Ho\vevcr, the RiKase-trcatcd, IJeulgcn-stainctl slicles show.ctl that som(’ 3H-uridine is incorporated into I>S,i, 1 h or more after the trcatlncnt. The labeling of this class \\-as remorcd by a 3 h digestion \\-ith 1)Nasc (0.1 nlg/ml, pH 6.5) at 35’C. The process \vas follo\ved up to 3 days. Labcling incrcasccl \yith time in the cytoplasm and labeling on the chromosomes of oocvtcs became lighter. As a result, after 8 h or later the chromosome region of’thc oocyte nuclei of stages 1 and 2 looked “empty” in contrast to the heavil! labeled cvtoplasm. Up to 4 days the elimination chromatin retained its labeling which was confirmed to be in RNA (Figs (i-8). Fine

structure

In the oogonium nuclei of both D. marginicollis and A. lutosus, the nucleolar material appears as small bodies with random sizes and distribution. The association of the nucleolar material to chromatin is quite conspicuous. The nucleus becomes larger in the preoocyte I and it contains much more nucleolar material as prominent, small, irregularly-shaped bodies. These bodies are made of dense particles about 200 A in diameter, similar to the particles found in the nurse cell nucleoli. In the preoocyte II, the nucleus becomes very much enlarged. The nucleolar material takes about the same shapes and distribution as in the preoocyte I and increases in amount roughly proportionally to the nuclear size (Fig. 9). Its association with chromatin is not very clear-cut. In the preoocyte 111, the nucleus becomes smaller again. The nucleolar bodies are gathered to one side of the nucleus. In the preoocytc I\‘, a DNA-containing component of the elimination chromatin becomes prominent. It fills the space between the small nucleolar bodies and has the same structure as the chromosomes. Although the nucleolar bodies remain scattered more or less evenly in the elimination chromatin mass of 1). murginistructure in the mass in the case of collis, they fuse to form a single “spongy” A. lutosus. In the latter, a part of the nucleolar material remains distributed as small fragments throughout the elimination chromatin (Fig. 10). In the the nucleolar material which has snch a dividing preoocpte of A. lutosns, prominent appearance in the interphase nuclei is not seen. Instead, dense particles which are similar to ribosomes form a loose network surrounding small masses of the DNA-containing component lvithin the elimination chromatin “ring” (Fig. 11). The elimination chromatin mass has no limiting membrane at any phase of the cell. In the oocyte nuclei of stages 1 and 2, the DNA-containing component of the elimination chromatin becomes denser. As they approach the stage 3, the nucleus grows in size and some spaces develop within the elimination chromatin mass. In D. nmrginicollis, the nuExperimental

Cell Research 52

Cytochemistry

and fine structure of elimination

chromrrtin

517

Fig. IO.-Nucleus of a stage 2 oocyte of A. lutosus. The elimination chromatin is seen as a large mass in the upper half of the nucleus. The DNA-containing component appears quite dense and similar to chromosomes. A larger part of the nucleolar material forms a “spongy” mass. There are always some groups of dense particles with a diameter around 450 A in the preoocyte and oocyte nuclei of this species, generally in association with the elimination chromatin. Two groups are seen in this section (arrom). 34 - 681812

Experimental

Cell Research 52

Experimental

Cdl

Reseccrch 52

Cytochemistry

nnd fine structure of elimination

chromatic

519

cleolar material forms at this stage larger bodies which are distributed throughout the mass (Fig. 12). They become even larger and more round in shape as the elimination chromatin starts to fragment, while the DNA-containing of the elimination chrocomponent becomes less dense. The fragmentation matin proceeds further and in some cases fugures are seen which look like a beginning of the disintegration of the nucleolar bodies (Fig. 13). ,4 part of the nueleolar material remains scattered as very small fragments throughout this process. DISCUSSION

According to Zalokar, the formation time of RNA is of the order of a few minutes [31]. It may be assumed to be the same, if not longer, for DNA. It can be concluded, then, that DNA is replicated and RNA is synthesized in the RNA is actively synthesized even in the stage 2 elimination chromatin. oocyte, when the elimination chromatin becomes the densest structure in the anterior o\-ariole, or in the stage 3 oocyte when it starts to fragment. Unlike nuclear RNA which moves to the cytoplasm, RNA synthesized by the elimination chromatin stays in place. Hoxvever, the possibility of transference of some RSA and DNA from the chromosomes to the elimination chromatin is not excluded. It is quite probable that DNA of the elimination chromatin is synthesized according to a fixed schedule, but it must be stated that pulse labeling in the strict sense was impossible with this material. In order to produce an optimum grain count, the ovaries of the beetles that were sacrificed during the first several hours had to be in contact with the emulsion for a longer periods of time, approximately inversely related to the length of time that elapsed between the injection and fixation. There seems to be no way at present to determine the exact amount of DNA in the oocyte nucleus by means of microspectrophotometry. When the reference nucleus is reasonably stained, the dye concentration in the stage 2 oocpte nucleus is so high that the departure from Reer’s law becomes too Fig. Il.-Dividing preoocyte IV of A. Zufosus. The elimination chromatin consists of small masses of the DNA-containing component surrounded by a loose network which is made of dense particles similar to ribosomes (arrows). The DNA-containing component of the elimination chromatin appears less dense than the chromosomes. Fig. I%.-Elimination chromatin of a stage 3 oocyte of U. marginicolfis. The whole elimination chromatin becomes loose and the process of fragmentation proceeds. The nucleolar material forms larger and larger bodies. Later, they become even larger and round in shape. Fig. l:l.-Stage the beginning

3 oocyte of D. marginicollis. of disintegration.

This particular

nucleolar

body shows what is possibly

Experimental

Cell Research 52

320

lirrori

lirrlo

great to give any c~losc cstiniaie 01’ Ihc c[tianLity. In an c~xf)~~rir~l~lll:il 1~91 01’ Ilrc. t\\o-\\a\-clcngth method. Van l)ui.jn and his co-\~orkcrs f’oltlt~l a I’.\?;-sl;~inetl film of kno\vn rslinction lo :~~l~ir~~c~aIi ir~lionio~cneo~~s clislrihuliori. ‘l‘lic eslinc*lion heca~nc about half the original value \vhen Ihc tlcnwst l):~r~ hacl ;III extinction of about 1.6 Ill ,. II is very likely that the aoiounl 01’ I).\.4 linall\ accumulalcd in the elimination chromatin is mow than 10 times 111~ cliploitl value. The amount of extrachromosonlal I)SA in Il’iprrlo ooc~ylrs is ~~portctl to be ahout 59 per cent of the total 1)N.A in the n~~~lcus [l 8 ,. ‘I’hr lo\\- Ie\.cl ol histonrs in the elimination chromatin may be rclatetl to the latter’s high syrlthetic activity. The elimination chromatin contains all ol’the nuclcolar nlalcrial in the nucleus. In fact, the first prominent changes to take place in Ihr prcoocylc I nucleus are hightened RNAsynthesis and the increase of the nuc~lcolar inalcrial. The elimination chromatin maybe regarded as a n~rc~leolrrsextreuwly rich ill 1),\:4. It seems that KKA stays in the elimination chromatin during mitosis in lhr form of RNP-particles although the nucleolar structure is dissol\ccl ,jusl as in ordinary cells. In this w.ay, the total amount of’ its l due to the duplication of effective nucleolar chromosomes !20!. For genetic reasons, oocgtes cannot become polyploid to increase the cell volume. There must he some other mechanism to increase the amount of either 1)N.i OI RNA or both in order that the cells can become larger. The estra amount of nucleic acids could be present in the nucleus or the cytoplasm. Later it ma! be utilized for building nuclei in the developing embryo. In the ovarioles of the large milkweed bug, free DNA droplets are transferred from the nurse cells to the oocyte [3]. The maturing oocyte of the amphibians contains several hundred nucleoli. These nucleoli are endowed with an autonomous capacity to synthesize HNA [8]. Considerable evidence has been accumulating that the nucleolus is a source of ribosomes and that Experimental

Cell Research 52

Cytochemistry

and fine structure of elimination

chromatin

521

the nucleolar apparatus synthesizes ribosomal RNA [4, 7, 9, 12, 24, 291. According to Brachet and Quertier, oocytes and unfertilized eggs of amphibians contain a large excess of DNA, 5000 times the diploid value, in the cytoplasm. This DNA is located around the germinal vesicle when nucleoli begin to disappear [6]. The presence of DNA in the nucleoli has been demonstrated in the amphibian oocyte [23]. There is evidence that this DNA is replicated at and detached from the nucleolar organizer region of the lampbrush chromosomes in the form of rings [16]. The formation of the elimination chromatin in Dytiscidae seems to represent a similar mechanism. It could well be that at least a part of the extra DNA is the multiplicated nucleolar organizers and that the elimination chromatin synthesizes and stores ribosomal RNA along with the extra amount of DNA for the later development of the oocyte and the embryo. It is interesting to note that the oocyte has large nucleoli in the case of Cybister tripunctatus (Dytiscidae) which does not produce any elimination chromatin [28]. In relation to the extrachromosomal chromatin of Tipula, Lima-de-Faria and Moses offered a similar suggestion that the chromosome region that takes part in forming the DNA body is either the nucleolar organizing region or a chromosomal segment involving it [19].

SUMMARY

The elimination chromatin of Dytiscidae contains the whole nucleolar material. It synthesizes both DNA and RNA independently of the chromosomes. Once synthesized, these nucleic acids stay within the elimination chromatin mass throughout the differentiation process until the whole structure is eliminated from the oocyte nucleus. The total amount of DNA thus synthesized is at least 20 times the diploid value, most probably more than 40 times. This research was carried out to fulfill a part of the requirement for a Ph.D. at the University of California, Berkeley, in 1961-64, supported by University Fellowship, Abraham Rosenberg Research Fellowship and U.S.P.H.S. Grant RG-6025. I wish to express my deep appreciation to Professors M. Alfert, D. Mazia, J. E. Gullberg, R. I. Smith, and other faculty members at the campus who were helpful and generous in offering valuable suggestions. REFERENCES 1. ALFERT, 11. and GESCHWIND, I. I., Proc. Natl Acad. Sci. US 39, 991 (1953). 2. BAYREUTHER, K., Chromosoma 7, S. 508 (1956). 3. --~ Z. Naturforsch. 12b, 458 (1957). 4. BIRNSTIEL, M. L., CHIPCHASE, 11. I. H. and HYDE, B. B., Biochim. Biophys. (1963).

Experimental

Acta 76, 454

Cell Research 52

173 (1962).

12.

J.-E., GKAMPP, \V. and Sctrotc, S., .I. liiophys. Z~iochern. (T!ylol. A., Inlern. Monutssckr. And. Physiol. 18, -117 (1901).

EDSTRBM, 13. GIARDIXA,

11, .TJ 10 I IWil ).

11. G~~NTHERT, T., Zool. Jakrb. Abf. Anal. Onfog. l’ierr 30, 301 (1910). 15. HGONER, R. W. and RUSSIXL, C:. P., Pm-. Xutl Arm!. Sri. l.‘S 2, X6 (1916). 16. liezmr, J., I’npublished. 17. ~~OHSCIIEI.T, E., z. Wiss. Zool. 43, 33i (1886). 18. LIMA-HE-FARIA, A., Chromosomn 13, .47 (1962). 19. LIMA-LIE-FARIA, A. and MOSES, M. J., .J. Cell Jjiol. 30, 177 (1966). 20. LOSVC.~VEI,I,, A. C. and SVIHIA, G., Ezpfl Cell Res. 20, 294 (1960). 21. LUFT, J. H., .I. Jjiophys. Riochem. Cytol. 9, 409 (1961). 22. MAZIA, D., Am. Sci. 44, 1 (1956). 23. ,~IILLEH, 0. I,., .JH., Safl Cancer Inst. Monograph 23, 53 (1966). 24. R~UR.~JIATSU, M., HODNETT, .J. I,. and BUSCH, H., Riockim. Biopkys. Acta 91, 592 (1961). 25.

PAIA~E.,

26.

REYNOI.DS,

27. 28. 29.

30.

31.

(;.

E.,

J.

b'xptl

&fed.

95,

285 (1952).

Biol. 17, 208 (1963). I.7~sax~, E:., Afti Accad. Naz. Lincei, Rend. Classe Sci. J’is. Mat. ,Yal. 8, 153 (1950). VARD~~:. Y. P., Arcwrnia Riologica 1930, 5 (1930). \%'ALI.ACE, H. and ~IRSSTIEL, hr. I,., ~~iOChiI% Hiophys. rtCfC7 114, 296 (1966). \VILI., I,., Z. Ii’iss. 2001. 43, 329 (1886). ZALOKAR, M., Bzpfl Cell Res. 19, 559 (1960).

Experimental

E. S., J. Cdl

Cell Research 52