Studies in the Origin of Yolk.

III. Oogenesis of the Firefly,Luciola gorhami.

By

Vishwa Nath, M.Sc, Ph.D. (Cantab.),

and

Dev Raj Mehta, M.Sc.

Department of Zoology, Government College, University of thePunjab, Lahore.

With 16 Text-figures.

IN the first two papers of this series1 one of us has shown,by studying both fixed and fresh preparations stained withvital dyes, that the Golgi elements in the youngest oocytesof the spider and S c o l o p e n d r a consist of vacuoles which,after treatment with various fixatives, do not show any coagu-lum inside their interior, indicating that their contents arewatery and non-fatty. When the oocyte begins to enlarge, themajority of these vacuoles, by a process of growth and depositioninside their interior of colloids in the form of free fat not misciblewith the general cytoplasm, give rise to fatty yolk-vacuoles,which henceforward show a distinct black coagulum inside theirinterior after treatment Avith osmic acid. By this process therefractive index of the Golgi vacuoles is considerably raised, andit becomes increasingly easier to study them in fresh prepara-tions. In the case of the firefly, L u c i o l a g o r h a m i , however,which forms the subject of the present paper, we observed theremarkable thing that the Golgi vacuoles of the female primor-dial germ-cells contain free fat as proved by their blackening in2 per cent, osmic acid in ten minutes. When the oocyte is

1 'Quart. Jour. Micr. Sci.', vol. 72, Parts II and III.

8 VISHWA NATH AND DEV RAJ MEHTA

differentiated and begins to enlarge many of these vacuoles growin size and give rise to fatty yolk-vacuoles. L u c i o l a , there-fore, is a very valuable material for the demonstration of theorigin of the fatty yolk-vacuoles from the Golgi vacuoles, inas-much as the latter are fatty from the very beginning.

The technique used is mentioned in the explanatory notes on

TEXT-FIG. 1.

NYoungest oocyte. The surrounding follicle cells have not been shown.

Mann-Kopsch stained with acid fuchsin. X 1,120.

EXPLANATION OF LETTERING.

A.Y., albuminous yolk; C, cytoplasm ; F.C., follicle cells ; F.E.,follieular epithelium ; O.V., Golgi vacuole ; O.V, Golgi vacuolelooking solid on account of osmication; M., mitochondria;M', egg membrane ; N., nucleus ; N', nuoleolus ; N", secondarynucleoli; N.F., nucleus of the follicle cell; 0., oocyte.

the figures and also in the text. In addition to this routinetechnique one of us (V. N.) has used the vital dyes neutral redand Janus green B, as also the technique described below. Whilethese dyes brought out most satisfactorily the Golgi vacuolesand the mitochondria in the primordial germ-cells, they totallyfailed to show these inclusions in the oocytes for the followingreason. When the oocyte differentiates it is surrounded on allsides by a large number of follicle cells which do not allow asufficient amount of light to pass through the follicle to enableone to see the inclusions in the oocyte as stained with neutralred or Janus green B. By the time most of the follicle cells areused up by the oocyte for nourishment and the remaining onesform a thin follieular epithelium round it, albuminous yolk

OOGENBSIS OF LUCIOLA

N

A little more advanced oocytes. Only the nuclei of the follicle cellshave been shown as the cell boundaries did not come up well. TheGolgi vacuoles of the follicle cells have not been drawn. Mann-Kopscb. stained. Both are magnified 520 times.

10 VISHWA NATH AND DEV EAJ MEHTA

appears in the oocyte and makes it opaque. Another technique,therefore, had to be used. The ovary is kept in 2 per cent, osmicfor about ten minutes and is then studied in a drop of waterunder the microscope. Both the Golgi vacuoles and the fatty

TEXT-FIG. 4.

N"

N

Ay

GV'

Text-figs. 4 and 5. Still more advanced oocytes. The follicle cellsare now arranged in a single-layered epithelium whose details havebeen omitted. Albuminous yolk is beginning to appear. Mann-Kopsoh stained. Text-fig. 4 magnified 420 times, and Text-fig. 5magnified 360 times.

yolk-vacuoles are blackened by the OsO4, but not so much asto make them look solid as they do after prolonged treatmentwith this acid. During this short period the vacuoles becomeslightly black, with the result that each vacuole shows a blackchromophilic rim and a central clear substance. This methodproved highly satisfactory, and brought out the vacuoles in allstages of oogenesis. As was expected it failed to show the mito-chondria. This technique cannot be strictly called ' vital', but

OOGENBSIS OF LUCIOLA

TEXT-FIG. 5.

11

it comes very near it as the amount of the coagulation of theprotoplasm is very small, and there is hardly any possibility ofan artifact being introduced. Experience has shown us thatosmic acid used for a few minutes is the best fixative known.All the diagrams have been drawn by Vishwa Nath.

12 VISHWA NATH AND DBV RAJ MEHTA

It is a great pleasure to us to thank Colonel S. E. Christophers,F.E.S., Director of the Central Eesearch Institute, Kasauli, forgiving us all facilities for this work in his laboratory in the sum-mer of 1926, and to the authorities of the Indian Museum forsupplying us with the necessary literature.

M

A portion of the most highly advanced ovarian oocyte. The follicularepithelium has disappeared and the egg membrane has appeared.Champy-Kull unstained. X 500.

OBSERVATIONS.

The ovary is kept in 2 per cent, osmic acid for about tenminutes, mounted in a drop of water on a slide, covered witha cover-slip, and studied under the & objective. Text-figs. 12-16are drawn from such a material. Text-fig. 16 represents thegreater portion of the sac containing the primordial germ-cells.The nuclei of these germ-cells appear distinctly, but the cellboundaries cannot be made out. In the spaces between the

OOGBNESIS OF LUCIOLA 13

nuclei one can see distinct vacuoles with a rim gone black due toosmication and a clear central substance. These are the Golgivacuoles. If, however, the ovary is kept in 2 per cent, osmicacid for more than half an hour, these vacuoles are blackened

FE

\J

A portion of an oocyte younger than that represented in Text-fig. 6. Champy-KuU stained with acid fuchsin. X 500.

considerably and then appear solid. Such quick blackening ofthese vacuoles in osmic acid unmistakably shows that theircontents are fatty. The number of these vacuoles is about threeor four in each cell, as can be determined more accurately by thestudy of fixed preparations, and they are not closely aggregatedin each cell. In chrome-osmium and Mann-Kopsch preparations

14 VISHWA NATH AND DEV RAJ MEHTA

they appear solid and black, but they can be easily decolorizedwith xylol or turpentine, after which they appear as clearvacuoles. With Janus green B or neutral red, in which theovary is kept for about fifteen minutes, they appear as clear

TEXT-FIG. 8.

N"

A centrifuged oooyte. Mann-Kopsch stained with acid fuchsin. X 160.

vacuoles, and the mitochondria are stained green or red accord-ing to the vital dye used but they are much more distinct aftertreatment with Janus green B. For reasons already explainedthese vital dyes fail to show either the mitochondria or the Golgivacuoles in the oocytes. When the oocyte is differentiated fromthe follicle cells 2 per cent, osmic is the only reagent which showsthe Golgi vacuoles in fresh preparations.

OOGENESIS OF LUCIOLA 15

In Text-fig. 12 the oocyte has differentiated from the sur-rounding follicle cells. The Golgi vacuoles are found at the peri-phery of the cytoplasm of the oocyte. Why these vacuoles arepresent only at the periphery we are unable to explain. We are,however, certain that this peripheral distribution is a constantfeature of the oocytes of this age. In Text-fig. 13 the oocyte hasgrown in size, and the Golgi vacuoles have increased and areuniformly distributed throughout the cytoplasm. The nucleolihave become prominent and are shown at N". They appearsolid and colourless or very slightly brownish. Although thenuclear membrane cannot be made out the size of the nucleuscan be more or less determined by the clear space round thegroup of nucleoli. Text-fig. 14 represents an older oocyte. Thealbuminous yolk has begun to be deposited and appears ascolourless or very slightly brownish, solid, and homogeneousdiscs. The Golgi vacuoles have increased in number and aredistributed uniformly throughout the cytoplasm. They show avery distinct black rim and a clear interior, and are thus veryeasily distinguished from the albuminous yolk. The nucleoli areincreasing in number and the nucleus is represented by a clearspace round them. After the stage represented in Text-fig. 14the oocyte is quickly packed with albuminous yolk, which makesit very opaque and unfit for study in whole mounts. When suchan oocyte is treated with 2 per cent, osmic for a short time, itbecomes dark and unfit for study unless sections are cut. If,however, it is ruptured by a needle its contents which flow outcan be studied with remarkable ease. Text-fig. 15 represents afragment of an osmicated and highly developed oocyte whichhas been ruptured with a needle. The albuminous yolk appearsas colourless, solid, and homogeneous discs, while the Golgivacuoles of different sizes are blackened with 0s04 and occupythe spaces between the yolk-discs.

Text-figs. 1-5 are stained Mann-Kopsch preparations. Text-fig. 1 represents the youngest oocyte that we have been able toobtain. At G.V are the Golgi vacuoles that look solid on accountof osmication. In Text-figs. 2 and 3 the vacuoles have increasedin number and have been decolorized by xylol. In Text-fig. 4,

16 VISHWA NATH AND DEV RAJ MEHTA

TEXT-FIGS. 9, 10, AND 11.

, G VIN

M

GV

Ciro. 11.

Young oooytes showing Golgi vacuoles and mitochondria. Champy-Kulland iron haematoxyhn. Text-figs. 9 and 10 x 500, 11 x 420.

which is a more advanced oocyte than that represented in Text-fig. 3, most of the Golgi elements look solid on account of osmica-tion, but some of them shown at G.V. are decolorized by xyloland appear as clear vacuoles. In Text-fig. 5, which is a still more

OOGBNESIS OF LUOIOLA 17

advanced oocyte, many Golgi elements have swollen up. Mostof them look solid and black on account of osmication. Never-theless, xylol has decolorized some, e.g. at G.V. Experiencewith a large number of different eggs shows that xylol or tur-pentine does not decolorize all the osmicated fatty bodies at thesame rate. Probably this is connected with the amount of freefat present inside these vacuoles, and the degree of coagulationthat it undergoes in osmic acid. In all these preparations(Text-figs. 1-5) the mitochondria cannot be seen because it isvery difficult to stain them after keeping the ovary in 2 per cent,osmic acid for from fourteen days to three weeks. Text-figs. 9-11are chrome-osmium preparations stained with iron-alum haerna-toxylin. In all these preparations the osmicated Golgi elementshave been decolorized by xylol and appear as clear vacuoles.Naturally enough, xylol will decolorize much more rapidly theosmicated Golgi vacuoles of oocytes fixed in chrome-osmiumthan the vacuoles of those oocytes fixed in Mann-Kopsch. Inthe former technique the tissue is exposed to osmic acid forabout twenty-four hours, whereas in the latter the tissue is keptin the acid for from fourteen days to three weeks. In thesepreparations the mitochondria appear as small granules verysharply stained with haematoxylin, whereas the general cyto-plasm is of a much lighter colour. Text-fig. 7 represents a por-tion of an advanced oocyte fixed according to the Champy-Kullmethod and stained with acid fuchsin. The osmicated Golgielements have been decolorized by xylol and appear as clearvacuoles of different sizes. The mitochondria are strongly fuchsi-nophil and are uniformly distributed throughout the cytoplasm.Text-fig. 6 represents a portion of a most highly developedunstained ovarian oocyte prepared according to the Champy-Kull technique. The Golgi vacuoles are osmicated and are notdecolorized by xylol as the slide is passed quickly throughit, whereas the albuminous yolk-discs are yellowish in colour.

In the above account we have deliberately avoided the wordsfatty yolk for obvious reasons. In the eggs of spiders andSco lopend ra the Golgi vacuoles of the youngest oocyte con-tain a watery non-fatty substance. By a process of growth and

NO. 289 C

18 VISHWA NATH AND DEV RAJ MBHTA

deposition inside them of free fat they give rise to the fatty yolk-vacuoles. As soon as free fat appears in the Golgi vacuoles theyare called fatty yolk-vacuoles. But in the case of L u c i o l a ,free fat is present in the Golgi vacuoles of even the undif-ferentiated germ-cells, so that it is impossible to determine at

TEXT-FIGS. 12-16. These are drawn from fresh material kept in2 per cent, osmic for about ten minutes.

TEXT-ITG. 12.

o

cv ovA young oocyte showing Golgi vacuoles at the periphery. The

follicle cells also show these vacuoles. x 330.

what stage of swelling a Golgi vacuole may be called a fattyyolk-vacuole. To avoid this confusion we have called thesevacuoles the Golgi vacuoles irrespective of their size. It is highlyprobable that the big vacuoles will be used as nourishmentduring embryogeny; but it is impossible to be certain about thisunless we study the segmentation of the fertilized egg.

The behaviour of the oocyte nucleolus is remarkable. In theprimordial germ-cells the nucleolus is a small basophil body; butas soon as the oocyte is differentiated from the follicle cells itbreaks up into a number of smaller basophil pieces which lieclosely aggregated (Text-figs. 1 and 9). Indeed, this is the first

OOGENESIS OF LUCIOLA 19

sign of the differentiation of the future oocyte. Gradually thesebasophil nucleolar pieces move apart, and they are then seento be embedded in an acidophil ground-substance of plastin(Text-figs. 2 and 10). The whole nucleolus, therefore, is am-phophil at this stage with an acidophil ground-substance em-bedded in which are round basophil bodies of different sizes.

ovOV

A more advanced oocyte showing the increase and the uniform dis-tribution of the Golgi vacuoles. Nucleoli are also shown with aclear space round them which represents the nucleus, x 290.

Some of these bigger basophil bodies again become amphophilin exactly the same way as the original nucleolus did (Text-figs. 3, 4, and 7). The plastin ground-substance of the nucleolusgoes on growing (Text-figs. 2, 8,4, 5,10, and 11) till it completelyoccupies the whole space within the nuclear membrane (Text-fig. 7). At the stage represented in Text-fig. 4 the basophil roundnucleolar bodies begin to migrate into the cytoplasm of theoocyte and directly give rise to the albuminous yolk. In Text-fig. 5 this process has proceeded a little farther, and in Text-fig. 7 it has reached an advanced stage. There seems to be little

c2

20

CV

VISHWA NATH AND DEV RAJ MBHTA

TEXT-FIG. 14.

CV

PO

toi

I'd

\Ok

fQ?

NF

A still more advanced oooyte showing albuminous yolk and theincrease in the number of the Golgi vacuoles. x 250.

doubt that the nucleolar extrusions pierce the nuclear mem-brane as whole bodies. In the cytoplasm the nucleolar extru-sions do not divide further but simply grow in size. The amountof albuminous yolk in a fully developed oocyte is very large(Text-fig. 6), but the nucleolar bodies are also numerous

OOGBNBSIS OF LUCIOLA 21

(Text-figs. 5 and 7) and there can be no doubt that they giverise to this type of yolk. Besides the nucleolar activity syn-chronizes with the appearance of yolk, and the histochemicalreactions of both types of bodies are exactly similar. Further,it is noteworthy that the migration of the nucleoli into the cyto-plasm does not stop with the appearance of albuminous yolkas it does in L i t h o b u s (Nath, 1924) and B u t h u s and

TEXT-FIG. 15.

.GV

A fragment of a highly advanced oocyte showing albuminous yolkand Golgi vacuoles.

E u s c o r p i u s (Nath, 1925), but continues till the oocyte isfully grown.

We have sometimes observed a phenomenon in fixed prepara-tions, namely, that at about the stage represented in Text-fig.14 the albuminous yolk is arranged mostly at the periphery ofthe oocyte. At first we thought that this migration towards theperiphery is real, and is undertaken with a view to derivenourishment for growth from the follicle cells. But an extensivestudy of fresh oocytes has convinced one of us that this peri-pheral position of the yolk-discs is really an artifact. Howexactly this artifact comes about we cannot definitely explain.Possibly when the ovaries are placed in the fixatives most of theyolk-discs are carried towards the peripheral cytoplasm alongwith the diffusion currents that might be caused by the fixatives.

22 VISHWA NATH AND DEV RAJ MEHTA

Experiments with the centrifuge gave us very satisfactoryresults. Text-fig. 8 represents an advanced centrifuged ooeytefixed with Mann-Kopsch and stained with acid fuchsin. At the

TEXT-FIG. 16.

. •'&' °o • . .

Greater part of the sac containing the primordial germ-cells. Nucleiand Golgi vacuoles are shown but the cell boundaries cannot beseen.

lower pole are the heavy albuminous yolk-discs that are stronglyfuchsinophil and at the opposite pole are the osmicated Golgivaeuoles of all sizes. The central area consists of granular mito-chondria that did not stain very strongly, and also containsthe nucleus which is always slightly pushed into the pole of the

OOGBNBSIS OF LUCIOLA 23

Golgi vacuoles-. It will be observed that a few osmicated Golgivacuoles are present in the pole of the albuminous yolk, butthis is due to mechanical causes inasmuch as they are caughtbetween the big yolk-discs.

DISCUSSION.

With regard to the origin of fatty yolk-vacuoles from theGolgi vacuoles we do not wish to say more except emphasizethe very important fact that in Luc io l a the Golgi vacuoles ofeven the uridifferentiated germ-cells contain a colloid in theform of free fat inside them, whereas the Golgi vacuoles of theyoungest oocytes of the spider and S c o l o p e n d r a contain awatery non-fatty substance. In the latter cases these vacuolesby a process of growth and deposition of free fat inside themgive rise to fatty yolk-vacuoles. In L u c i o l a , however, theGolgi vacuoles simply grow in size and give rise to the fatty yolk-vacuoles. It is likely that in this process of growth more andmore free fat is deposited in their interior.

The process of the origin of albuminous yolk from nucleolarextrusions is reminiscent of what has been described in thecockroach by Hogben (1920) and in S a c c o c i r r u s by Gatenby(1922). Nucleolar extrusions preceding the appearance ofalbuminous yolk have been described by one of us in L i t h o -b ius (1924) and B u t h u s and E u s c o r p i u s (1925), and byvarious other writers both in vertebrates and invertebrates.But in Luc io l a it is noteworthy that the process of nucleolarbudding lasts practically throughout oogenesis, and the processof the growth of nucleolar extrusions into the albuminous yolk-spheres can be studied with diagrammatic clearness.

SUMMARY.

1. Both fresh and fixed eggs have been studied.2. A remarkable phenomenon has been discovered in

Luc io l a : that the Golgi elements which are in the form ofvacuoles contain free fat even in the undifferentiated germ-cells.

3. The Golgi vacuoles containing free fat swell up in theoocytes and give rise to fatty yolk-vacuoles.

24 VISHWA NATH AND DEV RAJ MEHTA

4. In the youngest oocyte the nucleolus is amphophil, con-sisting of deeply basophil round bodies, the nucleoli, embeddedin an acidophil ground-substance.

5. The plastin ground-substance of the nucleolus grows andentirely fills up the whole space within the nuclear membrane.

6. The nucleoli multiply rapidly, migrate into the cytoplasm,and directly give rise to albuminous yolk.

7. Nucleolar budding lasts throughout oogenesis.8. The mitochondria are granular and remain so throughout

oogenesis.

BIBLIOGRAPHY.

1. Gatenby, J. Bronte (1922).—' Quart. Journ. Micr. Sci.', vol. 66.2. Hogben, L. (1920).—' Proc. Eoy. Soo. Lond.', Series B, vol. 91.3. Nath, V. (1924).—' Proc Phil. Soc. Camb. Biol. Sciences ', vol. i.4. (1925).—' Proc. Roy. Soc. Lond.', Series B, vol. 98.