A Cytologieal Study of the CentrifugedOocyte of the Mouse.

By

R. A. B. Gresson, P1.B.

Department of Zoology, University of Edinburgh.

With Plates 34^35 and 1 Text-figure.

CONTENTS.

PAGE1. INTRODUCTION 569

2. MATERIAL AND METHODS 572

3. OBSERVATIONS 572

4. DISCUSSION 577

5. SUMMARY 581

1. INTRODUCTION AND P R E V I O U S W O B K

THE cytoplasmic inclusions of the oocyte of the mouse havebeen described by the writer in a previous contribution (1933).The present investigation was undertaken in the hope that thestudy of oocytes which had been centrifuged, in the air-drivenultra-centrifuge, would throw further light on the cytoplasmicinclusions of the egg of the mouse. As is now well known,material so treated has proved to be of special value for thestudy of the inclusions and components of the cytoplasm inother cells.

According to the writer's previous findings, the Golgi materialof the young oocyte of the mouse is in the localized condition.In the older eggs the Golgi material becomes distributed, asrods and granules, throughout the cytoplasm. In the youngoocytes the granular mitochondria are most numerous in thevicinity of the nucleus, but in the older eggs they are collectedinto small clumps which are distributed through the cytoplasm.Extrusion of nucleolar material into the cytoplasm takes place.A small number of yolk-globules are situated towards the peri-phery, but fat is not present.

570 B. A. E. GEBSSON

Apart from a contribution by Beams and King (1938) on theovary of the guinea-pig, and a short note by the present writer(1938) on the ovum of the mouse, there does not appear to beany published work on the ultra-centrifuged mammalian ovum.

Beams and King (1938) have shown that the Golgi materialof the young oocyte of the guinea-pig, situated in the germinalepithelium, possesses a 'more or less concentrated type ofGolgi apparatus'. As the oocyte passes into the stroma of thecortex 'a well defined "yolk-nucleus" in the form of a diplo-some-idiosome-Golgi apparatus complex makes its appearancein a juxtanuclear position'. Later, the 'yolk-nucleus' breaksup and is dispersed in the form of irregular masses, rods, andgranules. Just before the appearance of the follicular cavitythere is a slight1 concentration of Golgi material at the peripheryof the cell. The mitochondria are granular and are fairly evenlydistributed throughout the cytoplasm. It will be seen from theabove account that the distribution of the cytoplasmic inclu-sions during the growth of the egg of the guinea-pig is similarto that of the mouse, except that in the mouse there is no con-centration of Golgi material at the periphery, and that in theolder oocytes the mitochondria tend to clump.

For references to other contributions on the cytoplasmicinclusions of the mammalian egg see the papers by Gresson(1933), Beams and King (1938), and Aykroyd (1938).

Beams and King (1938) have centrifuged the ovaries of theguinea-pig at 150,000 times gravity for thirty minutes. In thecentrifuged egg the cytoplasmie inclusions and components arestratified, according to their decreasing specific gravity, in thefollowing order: mitochondria in the centrifugal half of thecell; the nucleus; material of the 'yolk-nucleus' in the middleor in the centripetal half of about 75 per cent, of the eggs, inthe other 25 per cent, this material may be displaced among themitochondria; yolk-granules when present; a layer of clearcytoplasm; fat-globules, when present, at the centripetal pole.The authors are unable to state whether the variation in thedistribution of the material of the 'yolk-nucleus' is due todifference in specific gravity within the cell, or to mechanicalreasons. The nucleus is usually stratified with the nucleolus and

CBNTEIFUGBD OOCYTB OF MOUSE 571

chromatin at the centrifugal pole; it may or may not showmarked displacement.

Beams, King, and Eisley (1934) have ultra-centrifuged theeggs of E a n a a u r o r a , and have found that the contents arestratified into three layers: a centripetal layer of fat; a middlelayer of clear protoplasm; a centrifugal layer of yolk containingpigment granules. Beams and King (1936) have obtainedsimilar results for the fertilized eggs of Asear is suum.These authors did not determine whether or not the cytoplasmicinclusions of the egg of B a n a and of Asear is were stratified.

Norminton (1987) ultra-centrifuged the oocytes of Lumbr i -cus t e r r e s t r i s for four minutes at a force of about 40,000times gravity. The egg was stratified into four layers: fat-globules at the centripetal pole; Golgi bodies and the nucleus;mitochondria; a layer of non-granular substance at the centri-fugal end of the cell. •

Singh (1938) ultra-centrifuged the oocytes of the pigeon forfifteen to twenty minutes at pressures varying from 45 to 75 lb.per square inch. The cytoplasmic inclusions and componentsare arranged in the following layers: fat and cholesterol at thecentripetal end of the cell; a layer of granular Golgi elements;a layer of yolk; a layer of mitochondria at the centrifugal endof the cell. Singh states that the nucleus is sometimes in theGolgi layer and sometimes centrifugal to it. 'This depends tosome extent on the pressure of centrifuging, and on the stateof the oocyte.' In very young oocytes the nucleus is displacedbut little.

It •will be seen, from the above account, that fat is thelightest substance in the cell, and always occupies the centri-petal pole. There is, however, a certain amount of variationin the disposition of the other layers. In the pigeon and inL u m b r i c u s the fat is followed by the Golgi layer, while inthe guinea-pig a layer of clear cytoplasm and a layer of yolkintervenes between the fat and the Golgi elements. In thepigeon the yolk is stratified centrifugally to the Golgi layer.In the guinea-pig and in the pigeon the mitochondria aresituated in the centrifugal end of the cell, while i n L u m b r i c u sa layer of non-granular substance occurs at the centrifugal pole.

572 B. A. B. GEBSSON

2. MATERIAL AND METHOPS.

The material used in the present investigation consisted of theovaries of the mouse (Mus musculus) . The ultra-centrifugeused was one employed by Professor Gatenby and his students,and is similar to that described by Beams, Weed, and Pickles(1934).

The ovaries were dissected out, placed in saline solution inthe rotor of the centrifuge, and immediately centrifuged. Thecentrifuge was run at a pressure of 50 lb. per square inch fortwelve to fourteen minutes: this gives a force of about 130,000times gravity. After centrifuging, the ovaries were removed fromthe rotor, as speedily as possible, and placed in the fixing fluid.It was found that material fixed according to the method ofMann-Kopsch gave very good results. Some of the centrifugedovaries were fixed in formol, and subsequently stained withSudan IV according to the method of Kay and Whitehead(1935) for the demonstration of fat.

Uncentrifuged ovaries were fixed and used as control material.A few uncentrifuged ovaries were fixed in formol, sectioned,and stained with Sudan IV.

The work with the ultra-centrifuge and the preliminaryobservations were carried out in the University Department ofZoology, Trinity College, Dublin. The work was completed inthe Department of Zoology, University of Edinburgh. It isa pleasure to express my thanks to Professor J. Bronte Gatenbyfor granting me facilities to work in his Laboratory, and for theinterest he has shown in the work. I wish to thank Mr. E. J.Fant, Department of Zoology, University of Edinburgh, fortaking the photomicrographs.

3. OBSERVATIONS.

In the very young oocytes the cytoplasmic inclusions andcomponents are stratified into three layers. The centripetalthird of the cell is filled with lightly stained cytoplasm whichmay contain a few small granular Golgi elements. The middleregion of the cell is occupied by a layer of Golgi bodies whichare in the form of curved rods, small spherical bodies withdeeply impregnated rims, and of granules. The greater part

CBNTEIFUGBD OOCYTE OF MOUSE 573

of the nucleus lies in the Golgi layer, its centripetal pole,however, is free of Golgi elements and lies in the lightly stainedcytoplasm of the centripetal end of the cell. The region betweenthe Golgi layer and the centrifugal pole is filled •with granularmitochondria. Each of the three layers occupies, at this stage,approximately one-third of the volume of the cell.

In many of the ooeytes surrounded by a single layer of follicle-cells, the three layers, described for the younger oocytes, occupythe same relative positions; there is, however, variation in thesituation of the nucleus. In many cases the centripetal poleof the nucleus is free of Golgi material (fig. 1, PL 34), while inother oocytes the nucleus is completely surrounded by Golgielements. In a few eggs a small area of non-granular cytoplasmis present at the centrifugal pole of the cell.

In oocytes with about two layers of follicle-cells the nucleususually lies in the Golgi layer. Non-granular cytoplasm occupiesthe centrifugal pole of the cell. In some of the eggs, at thisstage of development, the centripetal end of the nucleus lies inthe clear cytoplasm, while in a few a narrow layer of deeplystained material is situated centripetally to the Golgi layer.The narrow layer is identified as yolk (fig. 4, PL 34).

In larger oocytes (three to four layers of follicle-cells) thecentripetal end of the cell is filled with lightly stained cyto-plasm, which usually contains a few small • Golgi elements.The Golgi layer, as in the earlier stages, occupies the middlepart of the cell; it tends, however, to be differentiated into tworegions. In the centripetal region of the Golgi layer mitochon-dria are few or absent, while in the centrifugal part mitochondriaare numerous. Non-granular cytoplasm still occupies the centri-fugal end of the cell, and the layer of mitochondria lies betweenthis area and the Golgi material. Yolk is situated between theGolgi layer and the centripetal cytoplasm (figs. 6, 7, and 8,PL 34; Text-fig. 1). The position of the nucleus varies slightly.In the majority of cases it is completely surrounded by the Golgielements, but in a few instances the centrifugal pole lies in themitochondrial layer.

In oocytes with more than four layers of follicle-cells, nongranular cytoplasm is absent from the centrifugal end of the

574 R. A. R. GRESSON

cell, so that mitochondria extend from the Golgi layer to thecentrifugal pole.

In the mature oocyte the centripetal end of the cell ib occupiedby lightly stained cytoplasm, which may contain a few small

c.c.y.

G.L.

TEXT-FIG. 1.

Generalized diagram of centrifuged oocyte with three to fourlayers of follicle-cells.

LETTEBING.CO., lightly stained centripetal layer with a few small granular

Golgi elements; O.L., Golgi layer; M.L., mitoohondrial layer;JV., nucleus; N.O., centrifugal non-granular layer; Y., yolk.

Golgi elements. This is followed by a thin layer of yolk. TheGolgi material occupies the middle region, but the division ofthe Golgi layer is more marked than in the preceding stage.The Golgi elements are closely packed together to form a dense

CENTRIPUGED OOOYTE OF MOUSE 575

layer next to the yolk. The centrifugal part of the layer con-tains fewer Golgi bodies, while mitochondria are numerous.The mitochondrial layer occupies, approximately, the centri-fugal third of the cell (figs. 9 and 10, PI. 35). In some of thecells examined a few Golgi elements were identified in themitochondrial layer. There is, therefore, a more gradualtransition between the mitochondrial and Golgi layers than inthe younger eggs. The nucleus of the mature oocyte is alwayssituated in the layer of mitochondria, and is usually in theextreme centrifugal end of the cell. The nucleolus, or nucleoli,are always close to, or at the centrifugal pole of the nucleus.In many of these oocytes the nucleus is dumb-bell shaped; alarge single nucleolus lies against the nuclear membrane at thecentrifugal pole (fig. 14, PI. 35). In other cases a large nucleolusoccupies a similar position, but the nucleus is very irregularin outline, and the periphery is frequently drawn out intoprocesses (fig. 13, PI. 35). In a few of the mature oocytes thenucleolus is thrown out of the nucleus and is present at thecentrifugal pole of the cell. In these cases the nucleus is situated,in the mitochondrial layer, centripetally to the nucleolus.

Centrifuged ovaries were fixed in formol and subsequentlystained with Sudan IV. Fat was not present in the oocytes,although fat-globules in other parts of the ovary gave a positivereaction. An examination of uncentrifuged oocytes failed toreveal the presence of globules or granules which stained withSudan IV. ' ;

Owing to the stratification of the cytoplasmic inclusionscentrifuged oocytes are favourable material in which to studythe Golgi elements and mitochondria. In the young oocytes,at the stage when the Golgi material of the normal oocyte islocalized, the Golgi elements are in the form of curved rods,granules, and irregularly shaped bodies. Some of the irregularlyshaped bodies have a deeply impregnated rim, so that theyappear to be somewhat ring-shaped. In the mature oocytesthe Golgi bodies are smaller, and are present chiefly as granulesand short rods; a few ring-shaped bodies may, however, bepresent. The mitochondria are granular throughout all stagesof the growth of the oocyte. In the older eggs the granules

NO. 324 p p

576 E. A. B. GBBSSON

are more numerous, and tend, for the most part, to be largerthan in the young oocytes.

The examination of preparations of uncentrifuged eggsshowed that the Golgi elements are, like those of the centrifugedmaterial, in the form of rods, irregularly shaped bodies, andgranules (figs. 2, 3, and 5, PI. 34; fig. 11, PI. 35). The Golgibodies of the older oocytes are larger than those of the centri-fuged eggs; this appears to be due to two or more elementscoming together and forming a clump.

A number of follicles with degenerating oocytes were observedin sections of the centrifuged ovaries. Two of these folliclescontained abnormal eggs which were unsegmented. Thecytoplasm contained numerous fat-globules of various sizes.Osmiophil granules were present scattered amongst the fat-droplets; these granules are probably Golgi elements. Mito-chondria were not identified with certainty. Some of theglobules and granules were present in groups, but were notstratified. Certain follicles contained eggs which had fragmentedto form several bodies. In most of these bodies the cytoplasmwas filled with fat-droplets, while Golgi elements were few orabsent. As in the earlier stages of degeneration, the cytoplasmiccomponents were not stratified. Degenerating oocytes were notidentified in the centrifuged material fixed in formol and stainedwith Sudan IV. Examination of uncentrifuged ovaries showedthat degenerating oocytes contain numerous globules whichstain with Sudan IV. In unsegmented eggs the globules aresmall and may not be very numerous; in eggs which haveundergone fragmentation the fat-globules are larger and morenumerous (fig. 12, PI. 35). They may be in clumps or distributedthroughout the cytoplasm.

An examination of centrifuged ovaries showed that there wasconsiderable variation in the distribution of the cytoplasmicinclusions of the follicle-cells. In some of the centrifugedfollicles the Golgi elements and mitochondria were not appre-ciably moved from their normal position, while in others theGolgi material lies between the nucleus and the centripetal endof the cell, or is situated laterally to the nucleus. In these cellsthe mitochondria occupy the centrifugal end of the cell. The

CENTBOTJGED OOCYTE OF MOUSE 577

variation in the position of the cytoplasmie inclusions indicatesthat the force necessary to bring about stratification of theoocyte is not always sufficient to produce a correspondingchange in the follicle-cells.

4. DISCUSSION.

The present account of the ultra-centrifuged oocyte of themouse agrees in general with that of Beams and King (1938)for the guinea-pig; there are, however, certain differences. Inthe eggs of the earthworm, pigeon, and guinea-pig, fat is thelightest substance and occupies the centripetal pole. It isremarkable that fat is absent from the ovarian egg of the mouse;the present findings, as the result of tests with Sudan IV, con-firm previous observations (Gresson, 1933). In the egg of themouse and of the guinea-pig a layer of yolk is situated betweenthe Golgi layer and the clear centripetal cytoplasm. Singh(1938) has shown that in the egg of the pigeon yolk is stratifiedbetween the mitochondria and the Golgi material. He claimsthat the yolk is mitochondrial in origin; this would explainthe stratification of the yolk close to the layer of mitochondria.

In the oocyte of the mouse a few small granular Golgi bodiesusually lie in the clear cytoplasm in the centripetal end of thecell. A similar condition has not been recorded for the egg ofthe earthworm, pigeon, or guinea-pig. Daniels (1938), describingultra-centrifuged gregarines, states that Golgi bodies, in theform of small granules and short rods, are present among thefat-globules at the extreme centripetal pole, while irregularring-like Golgi elements are situated centrifugally to the granulesand rods.

Beams and King found that the yolk nucleus (Golgi) materialin about 75 per cent, of the guinea-pig ova examined wassituated near the middle or in the centripetal half of the cell.In the remaining 25 per cent, it is found amongst the mito-chondria. It is of interest that in some of the mature eggs ofthe mouse Golgi elements were identified in the mitochondriallayer, and, further, that in the older cells the Golgi layer isdifferentiated into two parts. This division of the layer ofGolgi material into two regions would appear to be due in part

578 B. A. E. GEESSON

to changes in the specific gravity of some of the mitochondria.In the young oocytes a few granular mitochondria can usuallybe identified scattered amongst the Golgi elements. In the olderoocytes some of the Golgi material forms a narrow band centri-fugal to the layer of yolk: a few mitochondria may be presentin this region. In the centrifugal part of the Golgi layergranular mitochondria are numerous. It is evident, therefore,that many of the granules are less dense than in the youngeroocytes.

In a previous contribution the writer (1938) stated that theappearance of a non-granular area at the centrifugal pole ofoocytes of a certain age, and its disappearance from the oldereggs, is due to changes in viscosity correlated with the growthof the ovum. A centrifugal non-granular layer is not presentin the egg of the guinea-pig, but is described by Normington(1937) for the oocyte of the earthworm. In osmic and silverpreparations this region is composed of highly refringentglobular masses. Normington concludes that as this substanceis not visible in control material, it must be a fluid present inthe form of minute droplets which are heavier than the cyto-plasm and, when centrifuged, collect at the centrifugal pole.The appearance of droplets of a similar nature, at a certainstage in the growth of the mouse oocyte, may account for thecentrifugal non-granular layer. The droplets must disappear,or undergo a change in composition, at a later stage.

The nucleus of the young oocyte of the mouse, in centrifugedmaterial, is either completely surrounded by Golgi material orlies with its centripetal pole in the clear cytoplasm. At thisstage the nucleus is not displaced to any great extent from itsnormal position; its specific gravity, therefore, differs but littlefrom that of the surrounding cytoplasm. With the growth ofthe oocyte the nucleus becomes relatively increasingly heavy, •so that in the mature egg it lies in the mitochondria! layer at,or close to, the centrifugal pole of the cell. The nucleolus, ornucleoli, are always the heaviest structures in the nucleus. Inthe mature oocyte the nucleus is often dumb-bell shaped, orelse is very irregular in outline. In these cases the appearanceof the nucleus suggests that it is thrown towards the centrifugal

CENTRIFUGES OOCYTE OF MOUSE 579

pole by the weight of the nucleolus. Further evidence in supportof this view is afforded by the examination of eggs in whichthe nucleolus is thrown out of the nucleus. The nucleolus isalways at the centrifugal pole, while the nucleus is situatedmore centripetally in the mitochondrial layer. It would appearthat the position of the nucleus, in the centrifuged matureoocyte of the mouse, is largely determined by the nucleolus, andthat the nucleolus increases in density with the growth of theegg. It is. of interest that Singh, figures the nucleus of theoocyte of the pigeon as being somewhat dumb-bell shaped andwith chromatin at its centrifugal pole.

Beams and King (1935), working on centrifuged mammalianspinal ganglion cells, found that the nucleus is often elongateand sometimes dumb-bell shaped. This, they state, suggeststhat some of the nuclear contents are slightly heavier thanothers. They found that the nucleus is stratified into threelayers of which the nucleolar material is the heaviest. Theypoint out that the parts of the nucleus do not appear to differgreatly in density from the cytoplasm, and that the differentpositions of the nucleus in centrifuged cells 'are probablyexplained by the assumption that the specific gravity of thenucleus does not differ from cell to cell in the same degree asdoes that of the cytoplasm'.

Beams and King (1938) found that the nucleus of the guinea-pig egg may or may not show marked displacement. Thenucleolus and chromatin are situated at the centrifugalpole.

In a previous contribution the writer (1933) states that thejuxta-nuclear Golgi material of the young oocyte of the mouseconsists of rods and granules. The present observations on theultra-centrifuged oocyte add further evidence in support of theview that the localized Golgi material of the mouse egg is notin the form of a network. In the young oocyte the elementsof the Golgi layer consist of rods, irregularly shaped bodies,and granules. It is reasonable to suppose that if the localizedGolgi material be in the form of a network, then it might,sometimes, be displaced as a whole, or if broken up, by thecentrifugal force, some of the strands of the network would be

580 B. A. E. GKESSON

present as filamenta. That the Golgi bodies of the youngoocytes are always in the form of rods, irregularly shapedbodies, and of granules, and that long filaments have not beenidentified argues against the existence of a network. As thegranules are, at first, not numerous, it is possible that thelocalized Golgi material consists of rods and irregularly shapedbodies only, and that, in both the normal and the centrifugedoocyte, the granules are broken from the rods and irregularbodies during dispersal. The absence of large rods from theGolgi layer of the mature oocyte shows that fragmentation ofthe Golgi elements does take place. It is of interest that Daniels(1938), working on gregarines, has seen small black granulesattached to irregular ring-like Golgi elements, and believes thatthe granules are broken off to form the granular Golgi bodieswhich, in the centrifuged gregarine, lie centripetally to thelarger Golgi elements.

Beams and King, in their recent paper on the guinea-pigovary, state that it is difficult to determine the structure of thelocalized Golgi material. It appears to be in the form of rodletsor of a loose network which surrounds and encloses the idio-some; occasionally, all the material does not collect around theidiosome.

In a previous contribution the writer (1933) has shown thatyolk is present in the oocyte of the mouse in the form of globulessituated chiefly at the periphery. Nucleolar extrusions arepresent in the cytoplasm. Yolk-globules or nucleolar extrusionswere not identified in the centrifuged eggs. A layer of deeplystained material, centripetal to the Golgi layer, is present inthe older oocytes. As this layer could not be identified with anyof the other components of the cell, and as globules were notpresent in other parts of the egg, it is concluded that it consistsof yolk. If this be correct, the globules, due to the centrifugalforce, must have broken down and run together. If, as has beensuggested (Gresson, 1933), the nucleolar extrusions supply someof the material for yolk-formation, it is probable that theextrusions are stratified together with the yolk. The examina-tion of the centrifuged material has not produced evidence toshow that the Golgi elements, or the mitochondria, play a direct

CENTBIEUGED OOCYTE OF MOUSE 581

part in the formation of yolk. The egg of the mouse, however, isnot favourable material for the study of yolk-formation.

The writer has (1933) given an account of the degeneratingegg of the mouse. The present investigation adds further evi-dence in support of the view that the Golgi bodies of degeneratingeggs give rise to fat. In eggs which had fragmented into severalpieces Golgi elements are few or absent, while fat-globules aremore numerous than in the early stages of atresia. It is worthyof note that the cytoplasmic inclusions and components werenot visibly affected by the ultra-centrifuge. It is evident thatthe viscosity of the degenerating ovum differs considerablyfrom that of the normal egg, and that a much higher force isnecessary to stratify the cytoplasmic inclusions and components.

5. SUMMAKT.

1. The ovaries of the mouse were ultra-centrifuged at a forceof about 130,000 times gravity.

2. The young oocyte is stratified into three layers: lightlystained cytoplasm, usually containing a few small granularGolgi elements, in the centripetal end of the cell; a middle layerof Golgi elements containing the nucleus; a centrifugal layer ofmitochondria.

3. In the larger oocytes a thin layer of yolk is present betweenthe Golgi layer and the centripetal pole, while a layer of non-granular substance is present at the centrifugal pole.

4. The mature oocyte is stratified into four layers: a lightlystained centripetal layer; a layer of yolk; the Golgi layer; acentrifugal layer of mitochondria. The Golgi layer is nowdivided into two parts; the centrifugal part contains numerousmitochondria amongst the Golgi elements.

5. The nucleolar material is the heaviest substance in thenucleus; it increases in density with the growth of the egg. Inthe mature oocyte the nucleus lies in the mitochondrial layer;the nucleus is thrown towards the centrifugal pole by theweight of the nucleolus.

6. The Golgi elements are in the form of granules, rods, andof irregularly shaped bodies. It is probable that the localizedGolgi material of the mouse egg is not in the form of a network.

682 R. A. E. GRBSSON

7. Fat was not identified in the normal ovarian eggs, but ispresent in degenerating ova. The Golgi elements give rise tothe fat of degenerating eggs.

8. The inclusions and components of the follicle-cells werenot always affected by the force necessary to bring aboutstratification of the oocyte. The degenerating eggs were not{stratified.

EBFERENOES.

Aykroyd, 0. E., 1038.—"Cytoplasmio Inclusions in the Oogenesis ofMan", 'Zeiteoh. f. Zellforseh. u. mikr. Anat.', 27.

Beams, H. W., and King, R. L., 1938.—"Effects of UltraeentrifugingSpinal Ganglion Colls of the Rat, with Special Reference to Nissl Bodies",' Journ. Corap. Nour.', 61.

—— 1080.—"Survival of Auoaria Eggs after Centrifuging", 'Science', 84.— 1938.—"Cytoplasmio Components and Inclusions of the Developing

Guinea Pig Egg", 'Cytologia', 8.Beams, H. W., King, R. L., and Risley, P. L., 1934.—"Centrifuged Frog

Eggs", 'Proo. Soc. Exp. Biol. and Med.', 82.Beams, H. W., Weed, A. W., and Pickles, E. G., 1934.—"The Ultracentri-

fuge",'Science', 78.Daniels, M. L, 1938.—"Cytological Study of the Gregarine Parasites of

Tenebrio molitor, using the Ultra-centrifuge", 'Quart. Journ. Mier.Soi.', 80.

Gresson, R. A. R., 1933.—"Cytoplasmic Inclusions and Nucleolar Pheno-mena during Oogenesis of the Mouse', ibid., 75.

— 1 9 3 8 . — " Effect of Ultra-centrifuging Oocytes of the Mouse", 'Nature',142, no. 3604.

Kay, W. W., and Whitehead, R., 1935.—" Staining of Fat with Sudan IV ",'Journ. Path, and Bact.', 41.

Normington, G. M., 1937.—"Effects of Ultra-centrifuging the Oocytes ofLumbricus terrestris", 'Quart, Journ. Micr. Sci.', 79.

Singh, B. N., 1938.—"Cytoplasmic Bodies in the Oogenesis of the Vulture(Neophron Percnopterus Ginginianus) and the Effect of Ultra-centri-fuging the Oocytes of the Pigeon", 'Proo. Roy. Irish Acad^', 45.

Quart. Journ. Micr. Sci. Vol 81, N. 8., PI 34

Nu.4

O^

c-c.-.m.

iJ. A. B. Oresson

Quart. Journ. Micr. Sci. Vol. 81, N. 8., PL 35

C.C.

R. A. R. Qresson

CENTBIFUGED OOCYTE OF MOUSE 583

DESCEIPTION OF PLATES 34 AND 35.

C.C, lightly stained centripetal cytoplasm; F., fat-globules; O.A.,localized Golgi material; O.E., Golgi elements; O.L., Golgi layer; M.L.,layer of mitochondria; JV., nucleus; N.G., centrifugal non-granular layer;Nu., nucleolus; Y., yolk.

PLATE 34.

Photomicrographs. Figs. 2, 3, and 5 from material treated accordingto the method of Aoyama. All other figures from osmic preparations.

Kg. 1.—Centrifuged young oocyte.Fig. 2.—Uncentrifuged oocyte at about the same stage as fig. 1. Localized

Golgi material; few Golgi elements scattered through the cytoplasm.Fig. 3.—Uneentrifuged oocyte; slightly later stage than fig. 2. Golgi

material becoming distributed through the cytoplasm.Fig. 4.—Centrifuged oocyte. Later stage than fig. I. Layer of yolk and

small non-granular area at centrifugal pole.Fig. 5.—Uncentrifuged. Slightly older than oocyte in fig. 4. Golgi

material distributed through the cytoplasm.Fig. 6.—Centrifuged. Oocyte with 3-4 layers of follicle-cells. The

centrifugal pole of the cell is not shown.Figs. 7 and 8.—Two sections through the same egg. Centrifuged.

Centrifugal non-granular layer shown.

PLATE 35.

Photomicrographs. Fig. 11 from Aoyama preparation. Fig. 12 frompreparation fixed in formol and stained in Sudan IV. All other figuresfrom osmic preparations.

Figs. 9 and 10.—Centrifuged mature oocytes. Nuclei not shown.Fig. 11.—Uncentrifuged oocyte at approximately the same stage as

eggs in figs. 9 and 10.Fig. 12.—Uncentrifuged atretic follicle. Egg has undergone fragmenta-

tion. Fat-globules shown.Fig. 13.—Centrifuged mature oocyte. Nucleus very irregular in outline;

nueleolus at centrifugal pole.Fig. 14.—Centrifuged mature ooeyte. Nucleus dumb-bell shaped;

nucleolus at centrifugal pole.