on the relation of the mitochondria and golgi apparatus to...

On the relation of the Mitochondria and GolgiApparatus to yolk-formation in the Egg-cellsof the common Earthworm, Lumbricust e r r e s t r i s .

By

Leslie A. Harvey, A.H.C.S., B.Sc, D.I.C.,Beit Scientific Research Fellow, Imperial College of Science.

With Plates 18 and 19 and 1 Text-figure.

CONTENTS.PAGE

1 . I N T R O D U C T I O N . . . . . . . . . 2 9 1

2 . T E C H N I Q U E . . . . . . . . . . 2 9 2

3 . M I T O C H O N D R I A A N D Y O L K - N U C L E U S . . . . . 2 9 4

4 . G O L G I B O D I E S . . . . . . . . . 2 9 7

5 . Y O L K 2 9 9

6 . N U C L E O L U S . . . . . . . . . . 3 0 1

7 . D I S C U S S I O N 3 0 2

8 . S U M M A R Y 3 1 2

1. INTRODUCTION.

THE following piece of work has been carried out as a pre-liminary to a general investigation into the problem of thefunction of the Golgi bodies, mitochondria, and yolk-nucleusin the growing egg-cell, more especially with regard to the forma-tion of yolk.

A great deal of general work has been carried out on thecytoplasmic inclusions of the germ-cells, but most workershave confined themselves almost entirely to the male germ-cells.However, a certain amount of work has been done on yolk-formation in the egg-cells of various animals, and this work hasbeen summarized by Gatenby (31). A glance at this paper willshow that really very little is known about the formation of

U2

292 LESLIE A. HARVEY

yolk. Various authors have supposed yolk to be derived fromquite different cytoplasmic inclusions in the egg. But in veryfew cases indeed have any conclusions with regard to thisquestion been based on actual observations on the living egg.The static pictures given by fixed and stained preparationsdo not seem to the author to be sufficient evidence for makingpositive statements on such an essentially dynamic questionas that involved in yolk-formation. To state that yolk isformed from the mitochondria, or from Golgi elements, becausemitochondria or Golgi elements are seen lying on the surfaceof yolk-droplets in fixed preparations is making rather asweeping statement on very insufficient grounds. Until avery much more efficient i n t r a v i t a m technique is developedwhereby mitochondria, fat, yolk, and Golgi bodies can bedemonstrated and at the same time differentiated in the livingcells, problems such as the present one are likely to be thesubject of much controversy and little explanation.

It is a pleasure to express my thanks to Professor E. W.MacBride, in whose laboratories at the Imperial College ofScience this work has been carried out, and also to Mr. H.Graham Cannon for reading through the manuscript and formuch extremely useful help and advice.

2. TECHNIQUE.

The material used in this research was, where possible,obtained from worms directly they were brought into thelaboratory, as the action of many if not all fixatives seems todepend to a very large extent on the physiological conditionof the animal. Not only does the condition of the animalaffect the fixation with regard to shrinkage and the generalrelations between the various cell elements, but also with regardto the preservation of the actual cytoplasmic inclusions them-selves.

For the mitochondria the best fixation was obtained by themethod of Kolatschev somewhat modified. The full techniqueof the fixation is given by Dr. Nassonov in a recent paper (35).This method gives excellent results for the mitochondria, and

YOLK-FORMATION IN LUMBRICUS 293

requires no mordanting in order to use Altmann's anilin fuchsinand methyl green or thionin as stains. The Golgi apparatusis also fixed by this method, but, as it was used chiefly for themitochondria, the osmication has been carried out usuallyat room temperature for from three to six days instead of atthe higher temperature advocated by Kolatschev. Thiseliminates most of the risk of over-impregnation and brittle-ness, and the Golgi elements can be easily made out. UsingEegaud's haematoxylin instead of Heidenhain's after thistechnique, the mitochondria are a rather palish blue and theGolgi apparatus deep blue, nearly black.

Memming without acetic acid was also used as a well triedand extremely good mitochondrial fixative. Occasionally themethod of Champy-Kull gave very good results, but thistechnique is rather uncertain and consequently not verysatisfactory as a routine method.

For the Golgi apparatus undoubtedly the best fixation wasobtained by means of Da Fano's silver impregnation method,slightly modified, by increasing the formalin from 15 c.c. to25 c.c. per 100 c.c. of water and adding 1 gm. of cobalt nitrate.Mann-Kopsch and similar methods involving long treatmentwith osmic acid are open to the same objection as the originalKolatschev technique, that is that the eggs are apt to becomebrittle and over-impregnated. Besides this, as will be seenbelow, the fixation obtained by these methods is open tosuspicion, and consequently they were avoided except fora few specimens which Avere not very satisfactory. Otherimperfect fixations of the Golgi apparatus were obtained withF.w.a. (Flemming without acetic acid), Kolatschev, andoccasionally Champy-Kull.

Minor techniques used include Bouin, alcoholic Flemming,Mann's corrosive osmic fluid, Carnoy, and various i n t r av i t a rn and fat techniques.

The stains used include Altmann's anilin fuchsin followed bythionin and aurantia, and also by methyl green, Heidenhain'siron haematoxylin, and also Regaud's haematoxylin. (This,used as replacing the Heidenhain's haematoxylin in that


method, gave excellent results.) Benda's crystal violet alsomade an excellent mitochondrial stain. Best's carmine wasused for glycogen after Carnoy fixation, and minor stains includemethylene blue and eosin and also Ehrlich's haematoxylin.

3. THE MITOCHONDRIA AND YOLK-NUCLEUS.

In the youngest oogonia there can be detected no certainsigns of the mitochondria, but a slight granular appearance inthe cytoplasm after certain techniques may represent them.The nucleus is large and occupies practically the whole of thecell at this stage, there being only a thin skin of superficialcytoplasm. The cell is oval in section, the nucleus beingexcentrically placed. In the freer mass of cytoplasm thefaint granulations may be detected.

In P.w.a. and Kolatschev preparations usually the cyto-plasm contains a few large yolk-droplets besides the mito-chondria (PL 18, fig. 1), and occasionally Golgi bodies are fixedas well, but this will be dealt with later.

Very early on, a definite mitochondrial cap may be detectedfitting closely over the nucleus on the side nearest the centreof the cell (PI. 18, fig. 2). This cap consists of a faintly staining,nebulous cloud with more definite, darker-staining patches init (PI. 18, fig. 2). It can be demonstrated by F.w.a., Kolatschev,Champy, and other mitochondrial fixatives. From this earlystage the cap grows in size, getting thicker and more cup-shaped, and at the same time denser and more deeply staining,but it still fits closely over the nucleus (PI. 18, figs. 2-4).Whether the growth is due to division of the original mito-chondrial elements, or to the origin of fresh ones from thecytoplasm, it is impossible to say. Although the original capis very faint and indistinct, indications of a fibrous structurecan be made out, and this becomes more apparent as thecap grows.

In the very young oocyte the mitochondria occur, still asa compact cap over the nucleus, but now the cap is movingoutwards towards the peripheral regions of the cell. Thismovement takes place as a whole, but at the same time the

YOLK-FQRMATION IN LUMBEICUS 295

inner margin of the cap becomes jagged and irregular (PI. IS,fig. 5), as if portions of the cap were not moving outwards asrapidly as others. This process continues during the growthof the oocyte until, when the latter is quite large, the mito-chondria form a deep cup-shaped mass in the peripheral regionsof the cytoplasm (PL 18, fig. 6).

The mitochondrial cap is evidently the structure that Calkins(10) has described as the yolk-nucleus. He stated that it arosefrom the chromatin of the nucleus. As growth proceeded itbroke away from the chromatin and moved towards theperiphery of the cell. Here it broke up into smaller masses ofyolk-nuclear material. These then became homogeneous andwere called by Calkins the ' yolk-plates '. In his sections theyolk-nucleus appeared granular, but this can only be ascribedto the imperfect technique of the day, for actually it is distinctlyfibrous in structure. In the present investigation the ' yolk-plates ' have been found, but the yolk-nuclear masses giverise, primarily at any rate, to the mitochondrial threads whichare scattered over the cytoplasm of the cell as the egg ripens(PL 18, fig. 9, and PL 19, figs. 12 and 13).

In the fairly late oocyte stage described above and figured inPL 18, fig. 6, the yolk-nucleus stains intensely black in ironhaematoxylin after F.w.a. and Kolatschev, &c. A closeexamination of this mass reveals it as composed entirely ofvery closely intertwined, rather stout threads.

The next process is simply one of distribution. The cap firstseparates into smaller masses of threads, and these thenspread throughout the whole of the cytoplasm of the cell(PL 18, figs. 7 and 8). Calkins has described the breaking upof the yolk-nucleus into smaller masses ; he found no threads,but described the whole mass as granular. The smaller masses,according to his description, become homogeneous and formthe yolk-plates. This will be discussed later. After the mito-chondrial cap has broken up into smaller masses, each littlemass itself breaks up and scatters its component mitochondrialthreads throughout the cytoplasm of the cell (PL 18, fig. 9).During this process the threads seem to become much thinner


in cross-section and perhaps longer, but no estimate of their 'length could be made with any accuracy, at any time, becauseof the degree of intertwining exhibited. In the riper oocytesoccasionally a thread can be seen running for some distanceacross a section Avhen its length is seen to be considerable(PI. 19, figs. 12 and 13).

On the completion of this spreading process the evolution ofthe mitochondria in the ovarian egg is completed. The threadsremain distributed in the cytoplasm as long as the egg remainsin the ovary, and after this stage no further observationshave been made.

It should be mentioned here that the stage in which theyolk-nucleus is found as a compact, deeply staining cloud inthe peripheral cytoplasm of the half-grown oocyte is not oftenobserved in ovaries from mature worms, that is in wormswith a well-developed clitellum. In these worms the spread-ing of the mitochondria over the cell seems to take placeearly in the growth of the oocyte, as mitochondria maybe observed distributed over the cell before the oocyte ishalf grown. But in ovaries from worms which have not yetreached maturity, as indicated by their small size and thelack of a clitellum, the mitochondria remain clumped togetherfor a rather longer period during the growth of the oocyte.This seems to indicate a greater activity on the part of themitochondria, at least during maturity.

In the nearly ripe oocytes at the free end of the ovary maybe seen large patches of some homogeneous material scatteredthrough the cytoplasm irregularly (PL 18, fig. 9, and PL 19,figs. 12 and 13). These bodies, which are somewhat indefinitein outline in many cases, have been described by Calkins, asmentioned above, under the name of ' yolk-plates '. Theycan, however, be traced through two or three sections, each ofHfi. thickness, and this, combined with the diameter of theircross-section, points to their being solid masses rather thanplates. In iron haematoxylin preparations, after fixation inE.w.a. or Kolatschev, they stain blue-grey, slightly darkerthan does the surrounding cytoplasm, and in Altmann's anilin


^fuchsin, after Champy's fluid, they stain rather deep orange.In the living cell they occur as transparent bodies of a slightlyhigher refractive index than the cytoplasm. Figs. 12 and 13,PL 19, give a very good idea of their distribution in the cell.As a rule they are free from any mitochondrial threads, yolk-droplets, or Golgi platelets, but occasionally there are yolk-droplets inside them, and cells have been found in which theyolk-droplets are practically confined to them. Undoubtedlythese plates are not yolk, for their staining reactions, asdescribed, are not those of yolk. Besides this, true yolk-droplets are present as spheres showing the usual micro-chemical reactions of yolk, that is, they do not go red in Champy-Kull or Bensley-Cowdry, they are yellowish or brownish inmost chrome-osmium preparations, are easily decolorized byturpentine after Mann-Kopsch, do not disappear after Carnoyfixation, &c.

Foot (19), working on the eggs of the allied form E i s e n i a( A l l o l o b o p h o r a ) f oe t i d a, obtained these same largebodies, but regarded them as the products of the degenerationof the eggs, as she could only find them in eggs from ovariesof worms past the breeding period, as indicated by there beingno definite clitellar region. In Lumbricus, however, theseplates were found in practically all preparations, and the onlyworms used without a clitellum were individuals which had notyet developed one owing to immaturity.

4. THE GOLGI BODIES.

Unlike the mitochondria, the Golgi apparatus is definitelypresent in the youngest oogonia (PL 18, figs. 10 and 11). Inthese cells the number of Golgi bodies present is very small,usually not more than four or five, and often only two. No stagehas been found in which the apparatus is collected round onecentral mass of archoplasm, as has been described for thespermatozoa and also for some eggs.

In the oogonia by Da Pano's method the Golgi elementsappear as platelets of a lightly impregnating material sur-rounded by a heavily impregnating rim outside. In chrome-


osmium preparations the inner plate is often destroyed, and therim is broken up into three or four distinct rodlets. AfterDa Pano's fixation the inner plate may be hardly impregnatedat all, but it is distinctly not composed of the ordinary cyto-plasm of the cell, for it appears homogeneous and may bewrinkled on its surface.

These platelets lie for the most part on the surface of thenucleus in the young oogonia, usually flattened closely on thenuclear membrane. However, it must be remembered thatthe nucleus is excentrically placed in an oval cell. Conse-quently there is only a thin skin of superficial cytoplasm overone side of the nucleus, and here the Golgi bodies have noroom except to lie closely flattened on the nuclear membrane.At the other side of the cell the Golgi bodies lie free in thecytoplasm, not necessarily near the nucleus, and with noorientation with regard to the nucleus or to any other cell-body(PL 18, figs. 9 and 10).

The only change undergone by the Golgi bodies duringoogenesis is the increase in numbers of the platelets present ineach egg-cell. Little increase in number is shown during theoogonial stages, but in later stages there are one or two moreplatelets present (PI. 19, fig. 14). But as the oocyte grows, theplatelets commence to increase rapidly, and before it is halfgrown the oocyte contains large numbers of Golgi elements(PI. 19, fig. 15). Whether this increase is due to new platesarising from the cytoplasm, or to the division of theoriginal plates, cannot be stated definitely. From the fact

.that aggregates of plates lying closely together, and chainsof three or four plates may be found (PI. 19, figs. 15and 16), it seems probable that they arise by division ofthe original Golgi bodies, or are formed by the divisionof a central mass of some substance arising from the cyto-plasm. On the other hand, the bodies vary very greatlyin size, and only the larger ones are seen closely apposedtogether in threes or fours. Very small elements are scatteredsingly throughout the cytoplasm in quite large numbers, and,since these bodies are present in the older oocytes when the

YOLK-FOEMATION IN LUMBBICUS 299

numbers of the Golgi bodies are still increasing, they cannotbe postulated as the remnants of elements, once large, butnow degenerating. They are also present in the youngeroocytes in quite large numbers (PI. 19, fig. 15), which seemsto point to the origin of new Golgi bodies from thecytoplasm.

In the nearly ripe oocytes at the free end of the ovary thenumber of Golgi elements is very considerable indeed (PI. 19,fig. 16), and they form a very conspicuous cell-organ in prepara-tions made by Da Fano's method.

Throughout the whole of oogenesis the Golgi platelets arearranged quite at haphazard in the cytoplasm. There is neverany attempt at fusion or concentration in large masses, noris the Golgi apparatus composed of one mass in the earlystages of oogenesis. There is no orientation observable,either with regard to the nucleus or to any other body inthe cell.

In PI. 19, fig. 15, there is figured a very deeply impregnatedregion above the nucleus of the right-hand oocyte. Similarmasses occur in many of the younger stages, fixed by DaFano's method. They correspond in appearance and positionwith the yolk-nucleus as fixed by a chrome-osmium method,but no definite thread-like structure is observable. It issimply a cloud of deeply impregnated matter, showing nostructure at all. As the yolk-nucleus may be fixed by Da Fanotechnique under certain circumstances, this cloud is probablythe remains of that body, which has been rather badly fixed.

5. YOLK.

The following reactions have been taken as distinguishingyolk from fat :

Method.Fat vacuoles.Yolk-droplets.

Chrome-osmium.Black.Yellowish grey.

Chcmvpy-KuU.Black.Yellowish or unstained.

Benda.Black.Yellowish.

A fair amount of yolk is present in the developing eggs ofLumbricus as quite large droplets. Although not universally


present in the oogonial and young oocyte stages, yet thesedroplets are to be found in all the stages, and it is significantthat they are present in the young oogonium before themitochondrial cap can be detected with certainty (PI. 18,fig. 1).

•No spatial relations could be found existing between theyolk-droplets and the other cytoplasmic inclusions or thecell-body. In the oogonia only a few large drops of yolk arepresent, distributed with no relation to the yolk-nucleus(PI. 18, figs. 1 and 4). There are more droplets present in theoocyte, just scattered in the cytoplasm (PI. 18, figs. 6 and 7),some near the yolk-nucleus, others on the opposite side of thecell. In only a few cases could any association between yolk-droplets and Golgi elements be detected. In figs. 6 and 7of PI. 18 the Golgi plates in a few cases are lying closely apposedto a yolk-droplet, but this association does not appear to beof any significance.

In the nearly ripe oocytes, Avhere the mitochondria arescattered over the entire cytoplasm of the cell, the yolk-droplets can be seen, each with a circle of intensely staining' granules ' surrounding it (PI. 18, fig. 9, and PI. 19, figs. 12and 13). On closer examination it is seen that these ' granules 'are really the optical sections of mitochondrial threads, andthese threads may be traced running over the surface of thedrop. This is probably due to close packing in the cell. Themitochondrial threads occur all over the cell in large numbers,and the yolk-droplets are also present in quite large numbers,so that the yolk-droplets must lie surrounded by mitochondria.In such a case it is extremely likely that the mitochondria willcome into contact with the liquid surfaces of the drops andadhere, so giving the appearance observed in the sections.Once the threads had come into contact with the yolk-droplets, probably the surface-tension of the yolk-fluid wouldbe strong enough to hold the mitochondria on the surface,and probably this phenomenon occurs in cells in which thereare only a few mitochondria and yolk-droplets, but as thechances of the two elements coming into contact are very


much less than in the eggs in question, the phenomenon isnot noticeable.

In the riper oocytes at the free end of the ovary no case atall of the association of Golgi elements with yolk-dropletshas been observed.

6. THE NUCLEOLUS.

In the oogonia there is a small nucleolus, apparently solid, orsemi-solid, for, although it is more or less spherical or circularin shape, its actual outline is irregular. Very fine strands con-nect it with the nuclear reticulum (PI. 18, figs. 3, 4, 5, and 10).

The microchemical reactions of this nucleolus seem to be asfollows: after chrome-osmium fixation and staining in ironhaematoxylin, black ; after Champy-Kull technique, red : afterDa Fano, very faint purple, hardly impregnated at all; afterBenda, purple; with Bouin and Ehrlich's haematoxylin,faint purple ; finally with Mann, Mann-Kopsch, osmic vapour,Bouin, &c, and staining in methylene blue and eosin, blue.These reactions, in the main, point to a basophil com-position, and consequently this nucleolus is probably akaryosome.

In the later oogonia there appears a second larger body in thenucleus. This is probably liquid in consistency, for it isspherical in shape and has a regular outline (PI. 18, figs. 6 and 7,and PI. 19, fig. 15). This nucleolus stains as follows : aftermost chrome-osmium fixations and staining in iron haema-toxylin, black ; after Benda, brownish purple ; after alcoholicFlemming and iron haematoxylin, brownish yellow with largepale droplets inside and darker brown mottling due to yellowish,rather irregular droplets of some substance having a veryhigh refractive index; with Champy-Kull technique it isorange red to yellowish green ; Da Fano fixation leaves ita fairly dark purple with a mottling of very dark, nearly blackgranules ; fixation in Bouin followed by staining in Ehrlich'shaematoxylin shows the nucleolus faint purple with deep,nearly black mottling and with several large pale dropletsinside. Staining with methylene blue and eosin after this


fixation gives a uniform brick-red-stained nucleolus, andthis same appearance is seen after fixing in Mann, Mann-Kopsch, osrnic vapour, &c, and staining in methylene blueand eosin.

These reactions, on the whole, point to an oxyphil composi-tion of the second nucleolus, which may therefore be calleda plasmosome. The droplets inside it appear to be of twokinds : (1) large droplets which do not take up the stainsused very strongly ; sometimes these may be half as largeas the nucleolus itself; (2) smaller droplets which also do nottake up the stains readily but are oily looking, have a veryhigh refractive index, and occur in quite large numbers, eachindividual droplet being quite small. These droplets are veryobvious in preparations made by Bouin, osmic vapour, Mann,Mann-Kopsch, often Champy-Kull, and rarely Plemming andP.w.a. techniques. In Da Fano preparations they seem to becollapsed, leaving a granular, argentophil substance in thenucleolus.

In the later oocytes the first appearing nucleolus, or karyo-some, has disappeared, leaving only the plasmosome. Thisplasmosome remains in the nucleus while the eggs remain inthe ovary. In the nuclei of these later, nearly ripe oocytesthere may occur little droplets of material, similar in reactionsto the plasmosome. They may be lying on the surface of thisbody, or may be entirely separate. Occasionally they are seenon the nuclear membrane, but never outside it. This maypoint to extrusion of nucleolar fragments, but only on a verysmall scale and never into the cytoplasm.

7. DISCUSSION.

Before entering into a discussion of the results detailed above,it will be as Avell to examine the value of the type of evidencethat is obtained by modern cytological methods.

The appearance of yolk in the region where certain cyto-plasmic inclusions occur does not necessarily indicate thatthose bodies metamorphose directly into yolk ; further evidence


is necessary before anything definite can be stated. If theyolk-droplets appear as a transformation series, from theinclusion at one end, to the yolk at the other, then one canstate with confidence that the inclusion is metamorphosinginto yolk, or the yolk into the inclusion ; the direction in whichthe change is going will be indicated by the relative increaseor decrease of the yolk or of the inclusion in the cell. That is,if yolk is increasing in amount as the cell grows, and theinclusions are decreasing, then the inclusion may be statedto be metamorphosing into yolk. On the other hand, if theinclusions are increasing and the yolk decreasing, then theyolk may be stated to be metamorphosing into the inclusion.

If the yolk-droplets appear where the cytoplasmic inclusionsoccur, and there are no transformation stages between theinclusions and the yolk, but if, at the same time, yolk does notappear elsewhere in the cell, then it may be inferred that theyolk may arise from the cytoplasm through the action of theinclusions on the cytoplasm.

If, again, yolk appears where the cytoplasmic inclusions donot occur, and there is no transformation series from theinclusion to yolk, then that is evidence that the inclusion isprobably not connected with yolk-formation at all.

The possibilities indicated above are not, however, the onlyones. In the case of yolk-droplets appearing where the cyto-plasmic inclusions do not occur, it must be borne in mind thata body, such as a nucleolus, to take an example, may disin-tegrate, and its products may diffuse in certain directionsaccording to physiological gradients, diffusion currents, &c,in the cell. In this condition these products may be unstainableand consequently not to be demonstrated by ordinary cyto-logical methods. Then, on reaching certain regions of the cell,or certain types of cytoplasm, they may be precipitated assolid masses that take up stains. This would be as much aprocess of nucleolar extrusion as when pieces of the nucleolarmaterial migrate through the nuclear membrane as solid (whenfixed), demonstrable pieces, and yet this process could not beestablished by the inspection of normal cytological preparations.


This same reasoning may be applied to the smaller and morenumerous bodies in the cytoplasm, such as the mitochondriaand Golgi apparatus. They may transform into yolk by somesuch solution-precipitation process, but, as stated above, sucha process cannot be demonstrated by ordinary cytologicaltechniques now in vogue.

Also, if yolk is formed in this manner from some definite,demonstrable cytoplasmic inclusion, then even i n t r a v i t a mmethods will not be of much use in investigating the process.For, obviously, if the metamorphoses of a body are to bedemonstrated by an i n t r a v i t a m stain, then that bodymust necessarily retain its identity as a discrete particle, andmust not go into solution. Hence we see that even i n t r av i t a m methods may prove useless in studying such a questionas yolk-formation. But apart from this, the fact that the eggof Lumbricus is full of highly refractive granules and globulesof yolk, fat, &c, which obscure any signs of the fine, thread-like mitochondria, makes the study of the inclusions in theliving cell impossible.

Before attacking the major problem, it will be as well firstto clear up some minor points. First, to refer to the yolk-nucleus : Calkins (10) summarizes his work as follows : ' Theyolk-nucleus is chromatin in the form of a mass of granules ;this granular mass disintegrates and the parts form the yolk-plates of the egg, after undergoing change in their chemicalcomposition.' He also states in his paper that this yolk-nucleus is in direct continuity with the chromatin of thenucleus, and that it loses this connexion as it moves awayfrom the nuclear membrane. It is evident, however, from theresults of more accurate modern technique, that firstly theyolk-nucleus has no connexion with the nuclear chromatin atany time. Nor does the yolk-nucleus consist of granules, butof a mass of closely intertwined threads. Secondly, with regardto the origin of the ' yolk-plates ', it is certain that, at any rate,they are not the primary products of the yolk-nucleus. Theyolk-nucleus breaks up into its component mitochondrialthreads, which remain in the cytoplasm of the cell while

YOLK-FORMATION IN LUMBBICUS 305

the egg remains in the ovary. The ' yolk-plates ' appearin the oocytes after the yolk-nucleus has spread throughoutthe cell, but as to where they come from nothing can besaid.

It has been stated above, that in the growing oocyte themitochondrial cap migrates as a whole from lying closely overthe nucleus into the peripheral cytoplasm of the egg, butthat the inner edge of the cap becomes jagged, as if some ofthe component mitochondrial threads were moving lessrapidly than others. This movement may be due to one ofthree alternatives : (1) active movement of the mitochondrialthreads themselves ; (2) passive reaction of the mitochondriato a field of force created in the cell as it grows into the oocyte ;or (3) disintegration of mitochondria near the nuclear mem-brane and origin of fresh ones towards the periphery of thecell, due to some gradient arising in the cell.

The second two alternatives do not explain the spreadingof the mitochondria throughout the cytoplasm after the generalmigration outwards, but this may be a second operation afterthe finish of the first. On the whole, however, the first alterna-tive would seem to be more probable, especially as mitochondriahave been observed exhibiting amoeboid movement. Unfor-tunately, in this piece of work i n t r a v i t a m techniques,as stated above, are useless for studying mitochondria living,and consequently the matter cannot be entered into morefully.

In such an investigation as the present one it is most impor-tant that the various cell elements should be easily identifiable,and that no confusion should arise between any two inclusions,and here again cytological technique is far from perfect.With regard to yolk, the tests used were those given by BollesLee in his ' Microtomist's Vade Mecum ', 1921, and by Gatenbyin his paper on yolk-formation (31). A glance at either of thesesources of reference will show that the two elements, fat andyolk, can quite easily be confused ; for most of the reactionsof either body have an alternative, which renders it difficultto distinguish between these two bodies. In order to emphasize

NO. 274 X

306 LESLIE A. HAKVBY

this statement, the following extract from Gatenby's paperwill serve. The spaced words are mine.

Method.

Yolkgranules.

F a tvacuoles.

Cajal.

Eitherwill notshow,greyish orgoldenbrown.

Do notshow.

Kopschseries.

Yellowish,or black,easily de-colorizedin turps .

Black,easily de-colorizedin turps .

Chrome-osmlum andiron haem.

(or Altmann).

May ormay not goblack (orunstainedvery rarelyred).

Black inunstainedprepara-tions (orblack).

Bourn andcorrosiveacetic andEhrlich's

haem.N o tstained oryellowish.

No tstained,washed away.

ChampKvU.

Yellow-ish orblack

Black

The only critical difference between the two inclusions isthat after Carnoy, Bouin and corrosive acetic, Petrunkewitsch,&c, the yolk-droplets remain, usually unstained, whereas thefat vacuoles are washed away. But the fixation of a yolkyegg after such techniques is usually such as to render themuseless. In consequence the worker has to rely on the reactionsof the two bodies not overlapping, in order to be able to distin-guish between them in the same section. As stated above, thereactions exhibited by the yolk of Lumbricus are as follows :

-.* ., j \ Qhronie-osrnvinn and ' ~ 7 „ 7I -n j

Method. | iron haem. | Ohampy-Ktdl. Benda.Yolk. Yellowish grev. i Yellow or unstained. Yellowish.Fat. Black. " ] Black. Black.

Prom the above table it will be seen that the yolk can bedistinguished from fat, but even this distinction is not critical,for the blackening of the fat vacuoles is liable to fade veryrapidly in many cases, and an examination of slides sometime after their preparation would lead to some confusionwithout a previous knowledge of the appearance of the sections.

But this is not the only difficulty, for yolk may quite con-ceivably be confused with portions of the Golgi apparatus.


Here a divergence is necessary with regard to the relative valuesof the fixatives used for demonstrating the Golgi apparatus.Da Pano's silver impregnation method gives the most completepicture of the Golgi platelets. By this method the- elementsappear as little plates, with a heavily impregnated rim anda lightly impregnated centre (Text-fig. 1, a). This may hejust a collapsed sphere, but on this supposition it would bedifficult to account for the heavily impregnated outer rim.The probability is that the body is a spheroid, very muchresembling a blood corpuscle in shape. Occasionally by themethod of Kolatschev, and more rarely after F.w.a. andChampy-Kiill, Golgi platelets are found with the inner platefixed and staining a rather darker grey than the surroundingcytoplasm of the cell, after iron haematoxylin. The outerrim is also present, and either is broken into two or threerodlets (Text-fig. 1, c) or else consists of tM7o or three thickregions connected by thin strands of the same darkly stainedmaterial, so as to complete the rim (Text-fig. 1, b). Very oftenthe whole plate is distorted, as indeed the plates may be afterDa Fano fixation. An extremely common form is found inKolatschev preparations and quite frequently after F.w.a.This is just a thick, heavily staining rodlet, which may be bentinto a V shape (Text-fig. 1, d and/), or may be straight (Text-fig. 1, h) or there may be .two rodlets lying parallel or approach-ing one another at one end (Text-fig. 1, e and g). These rodletslie at haphazard in the cytoplasm and are of varying lengths,from the short stumpy rodlets of figure g to the long straightrod of figure h or the horse-shoe-shaped rodlet of figure d. Thisseries seems to point to the fact that chrome-osmium fixationis inferior to that of Da Pano's cobalt nitrate-formalin, in theproblem under discussion at any rate. It might be arguedthat the Golgi elements consist of little rodlets lying on thesurface of plates or spheres of some substance having lessaffinity for osmium tetroxide than the rodlets, and that inDa Fano fixation the rods had broken down and run together.But if such is the case it remains to be explained why the innerplate is so rarely fixed by such rapid fixatives as Kolatscbev

X 2


and F.w.a., and why, when the inner plate is preserved at all,the rods tend to fuse to form one deeply staining rim (PI. 19,fig. 17). In PL 19, fig. 17, is drawn a fairly young oocyte fixed byP.w.a. and stained in iron haematoxylin. In this cell the Golgiplatelets have been fixed quite well, and here several intermediatestages between the plates of a Da Fano preparation and therods of a Kolatschev may be seen. It would seem that thechrome-osmium method and also plain osmic techniquesdemonstrate that the rim of a Golgi platelet is not of homo-

TEXT-FIG. 1.

Series showing action of fixatives on woigi JJI»Io, Da Fano ; b-h, chrome-osmium techniques. See text.

geneous structure all round, but that there are certain regionswhich are less resistant to these fixatives than other regions.

Eeturning to the question under discussion, it was stated thatyolk may conceivably be confused with portions of the Golgiplatelets. This statement has been made as a suggestedexplanation of the statements of various authors, e. g. Gatenby(31) and Ludford (32) especially, that Golgi bodies have beenobserved on the surface of yolk-droplets. The various appear-ances of yolk have been indicated above, and it is extremelypossible that-the inner plate of a Golgi element, fixed by anosmication method, might be mistaken for yolk, and thusform the ground for the statements of the above-mentionedauthors, that they have observed Golgi rodlets on the surfaceof yolk-droplets. It is true that the figures given by Ludfordwould seem to mitigate against any such confusion arising, but,


on the other hand, he does not seem to regard the Da Panofixation of the Golgi apparatus as at all significant.

That the Golgi element is a plate, sphere or spheroid, andnot a rod, seems very probable, especially as there can be norodlets observed in i n t r a v i t a m examination of the cells.Even were the refractive index of the rods the same as that ofthe cytoplasm, they would still be visible i n t r a v i t a m ,their presence being revealed by reflection froni their surfaceor by their possessing a different dispersive index from that ofthe cytoplasm. On the other hand, were the Golgi bodiesspheres without a distinctive i n t r a v i t a m staining, itwould be very difficult to distinguish them from yolk or fatspheres which occur in abundance in the eggs.

A perusal of Gatenby's paper on yolk-formation (31) willshow that the origin of yolk is still a very much disputedquestion, and that various workers have claimed its originfrom the ground cytoplasm, from the mitochondria, the Golgiapparatus, and from the nucleolus. In Lumbricus no evidencehas been discovered in support of the hypothesis that themitochondria and Golgi platelets metamorphoses into yolk.There are yolk-droplets present in the young oogonia beforea definite rnitochondrial cap can be detected, and when theyolk-nucleus is in the form of a compact cloud or cap of threads,the yolk-droplets may be seen scattered throughout the cell-cytoplasm. They are rarely associated with the rnitochondrialthreads. The fact that the two elements are closely associatedtogether in the nearly ripe oocyte is merely due to the largenumbers of mitochondria occupying the whole of the cytoplasmof the cell, and, in consequence, surrounding the yolk-dropletsand probably adhering to the liquid surfaces of the drops.The close association of the two bodies in the late oocytesupports the idea that the mitochondria may affect the cyto-plasm in such a way as to cause the formation of yolk-droplets.But, on the other hand, the almost complete dissociation of thetwo elements in the younger oocytes is stronger evidence againstit, unless one accepts the idea that extrusions from the mito-chondria are carried in an unstainable condition through the


cytoplasm, until they are precipitated on coming into adifferent region of the cytoplasm, and there form yolk-dropletsas was discussed above. There is no evidence for direct meta-morphosis of mitochondria into yolk-droplets; on the otherhand, the absence of intermediate stages showing this meta-morphosis in progress is direct evidence against this view.

Although Golgi platelets are present in the youngest oogonia,yet in very few cases are they associated with yolk-droplets.Ludford, on the oogenesis of Patella, has used both Da Fanoand osmic techniques, but, owing to inability to obtain goodfixation with the Da Fano method, has not placed muchimportance on the results obtained by this method. However,in the one figure of this technique that he publishes (32, PI. i,fig. 2) the Golgi elements are spheres or spheroids, whereasby osmic techniques they seem to consist of one or two rodletssurrounding a central plate of archoplasm. It is unfortunatethat he does not state whether this central archoplasm stainsblack after an osmic technique in the very early stages ofoogenesis, comparable to those figured. He is not at all clearon this point, as will be seen from the following quotation :' In fig. 4 is shown a cell with the Golgi batonettes just com-mencing to disperse. This is drawn from a Mann-Kopschpreparation which has been treated with turpentine to removethe blackened fat. The archoplasm is therefore colourless.'From this statement one naturally infers that fat is alreadypresent in the archoplasm of the Golgi platelets, or ratherbatonettes. But he goes on to state : ' In fig. 8, however,which has not been treated with turpentine, the whole of thecompact part of the Golgi apparatus appears black, showingthat a fatty substance has already been formed at this stage.'Fig. 8 is obviously a much later stage than that of fig. 4, yetthe inference from Ludford's second statement is that this isthe first stage at which the fatty substance has been observedin the Golgi platelets. It is also unfortunate that he does notstate whether the Da Fano fixation of the Golgi elementsremains the same all through the oogenesis. If it is the sameall through, then the central archoplasm of the Golgi bodies


cannot be converted into fat or yolk, for these bodies areconverted by the formalin into substances soluble in alcohol,and consequently the inner plate of the Golgi body •would notappear in a finished Da Fano preparation.

With regard to the oogenesis of Limnaea, Gatenby hasstated that yolk is formed by direct metamorphosis of the Golgielements (31). The only real evidence brought forward is thatGolgi bodies and yolk-droplets occur together, the Golgibodies lying on the surface of the yolk-droplets in some cases.He figures an oocyte in which there are large numbers of smallyolk-droplets with no trace of any Golgi rodlets on theirsurface, whereas the majority of the large yolk-droplets haveGolgi rodlets on their surface. If, then, there are many verysmall yolk-droplets formed by complete metamorphosis of theGolgi bodies, then one must either assume that varying amountsof yolk are formed from Golgi elements which are all more orless of the same size, or else that the Golgi bodies vary in sizeto a considerable extent. The first supposition is extremelyunlikely, and such diversity of the Golgi elements as is sug-gested by the second of the assumptions, would be strikingenough to have called for comment, of which there is nonein the paper quoted. The other alternative is that there maybe another source of yolk in the egg.

With regard to the nucleolus, there is no evidence for nucleolarextrusion into the cytoplasm, but, on the other hand, thesuggestion made above, of soluble extrusions, may apply here.The small globules of plasmosome-like material seen in thenucleus of the later oocyte may be a stage in this process, thebodies losing their staining power later, before becomingsoluble and passing into the cytoplasm.

Before concluding, another point arising out of the appear-ance of the Golgi platelets after an osmication fixation may bediscussed. Gatenby has based his description of the divisionof the Golgi bodies on this rod-like appearance of the Golgielements after an osmic technique (24 and 31). The rod-likeappearance after certain techniques may conceivably give anindication of the method of division of the platelets. The


weak places in the rims may indicate regions where it isseparating into two or three parts, preparatory to the divisionof the whole platelet. This is supported by the fact thatGolgi bodies may occasionally be found lying in a line or in theform of a triangle in Da Fano preparations, with the sides ofthe elements closely apposed (PI. 19, fig. 16). But one is loathto draw conclusions upon such an essentially dynamic pheno-menon as this, from the static pictures offered by fixed andstained preparations, especially when one considers that inosmic preparations of the eggs of Lumbricus the Golgi bodiesconsist almost entirely of these rod-like bodies in all stagesof the oogenesis. Consequently one would necessarily be forcedto conclude that the Golgi elements are dividing continuouslythroughout the oogenesis and even in the nearly ripe eggs atthe free end of the ovary. If such were the case, the cells wouldvery rapidly become choked with masses of Golgi platelets,unless there were a compensating disintegration of the olderelements, as might be indicated by the presence in Da Fanopreparations of smaller, heavily impregnating granules than theGolgi platelets (PI. 19, figs. 15 and 16). But, on the whole,it seems more probable that the rod-like fixation has nosignificance with regard to the division of the Golgi platelets,or to the growth of the Golgi apparatus as a whole. The moreprobable explanation of the heavily impregnating granulesobserved in Da Fano preparations is that they are new Golgielements arising from the cytoplasm.

An analysis of the facts with regard to the behaviour andstructure of the cytoplasmic inclusions in the oogenesis ofL u m b r i c u s t e r r e s t r i s , leads one to the conclusion thatthe yolk is not derived from either mitochondria, Golgiapparatus, or nucleolus, but that it is probably derived fromthe cytoplasm of the cell.

8. SUMMARY.

1. The yolk-nucleus is merely a mass of mitochondria.2. The mitochondria arise as a cap of threads over the

nucleus, and this cap grows in size and density, migrates away

YOLK-FORMATION IN LUMBRIOUS 313

from the nuclear membrane and breaks up into its componentmitochondrial threads. These threads become evenly spreadthroughout the cytoplasm of the cell.

3. The mitochondria are not clearly denned in the veryyoung oogonia.

4. The Golgi apparatus consists of numbers of Golgi elementslying separate in the cytoplasm. There is never any attemptat concentration of these elements round one central mass.

5. The Golgi elements are probably little platelets or spheroidssomewhat resembling blood corpuscles in shape. They are notrods. As fixed by Da Fano technique, each element is a littleplate with a very lightly impregnating centre and a very heavilyimpregnating rim.

6. The Golgi elements may probably arise from the cyto-plasm.

7. The nucleus contains two nucleoli; an early arisingkaryosome, homogeneous and solid in structure, and a plasmo-some arising later. This plasmosome is liquid in consistencyand contains an argentophil core. The karyosome disappearsbefore the oocyte is half grown, but the plasmosome remainsin the nucleus while the egg remains in the ovary.

8. No visible nucleolar extrusions into the cytoplasm wereobserved.

9. Yolk probably arises from the cytoplasm ; no directmetamorphosis of either mitochondria, Golgi apparatus, ornucleolus into yolk was observed.

L I T E R A T U R E .

1. Beckwith, C. J.—"The Genesis of Plasma Structures in the Egg ofHydractinia ", ' Journ. Morph.', xxv, 1914.

2. R. H. Bowen.—Studies on Insect Spermatogenesis. " I. The Historyof the Cytoplasmic Components of the Sperm in Hemiptera",' Biol. Bull.', xxxix, 1920.

3. " II. The Components of the Spermatid and their Role in theFormation of the Sperm in Hemiptera ", ' Journ. Morph.', xxxvii,1922.

4. " III. On the Structure of the Nebenkern in the Insect Spermatidand the Origin of Nebenkern Patterns ", ' Biol. Bull.', xlii, 1922.


5. R. H. Bowen.— " IV. The Phenomenon of Polymegaly in the SpermCells of the Family Pentatomidae ", ' Proc. Amer. Acad. Arts andSciences ', lvii, 1922.

6. "V. Sperm Formation in the Lepidoptera", 'Quart. Journ.Micr. Sci.', lxvi, 1922.

7. " On certain Features of Spermatogenesis in Amphibia andInsects ", ' Amer. Journ. Anat.', xxx, 1922. •

8. ——•" The Occurrence of Abnormal Mitoses in Spermatogenesis ",' Biol. Bull.', 1922.

9. " On the Idiosome, Golgi Apparatus, and Acrosome in the MaleGerm Cells ", ' Anat. Rec.', xxiv, 1922.

10. Calkins, G. N.—" Observations on the Yolk Nucleus in the Egg-cellsof Lumbricus ", ' Trans. N.Y. Acad. Sci.', 1895.

11. Chubb, G. C—"The Growth of the Oocyte in Antedon", 'Phil.Trans.', B. 198, 1906.

12. Cowdry, E. V.—" The Mitochondria! Constituents of the Protoplasm ",Coht. to ' Embryology', nos. 24-6, 1918.

13. Cowdry, N. H.—" Comparison of the Mitochondria in Plant andAnimal Cells ", ' Biol. Bull.', xxxiii, 1917.

14. Da Fano, C—" Method for the Demonstration of the Golgi Apparatusin Nervous and other Tissues ", ' Journ. Roy. Micr. Soc.', 1920.

15. Dendy, A.—" The Gametogenesis of Grantia cornpressa ", ' Quart.Journ. Micr. Sci.', 1920.

16. Doncaster, L., and Cannon, H. G.—" On the Spermatogenesis of theLouse ", ibid., lxiv, 1920.

17. Drew, A. H.—"Preliminary Tests on the Homologue of the GolgiApparatus in Plants ", ' Journ. Roy. Micr. Soc.', 1920.

18. Duesberg, J.—" Nouvelles recherches sur l'appareil mitochondrial descellules seminales ", ' Arch. f. Zellf.', vi, 1910.

19. Foot, K.—" Yolk Nucleus and Polar Rings ", ' Journ. Morph.', xii,1896.

20. Gatenby, J. B.—" The Cytoplasmic Inclusions of the Germ Cells.I. Lepidoptera ", ' Quart. Journ. Micr. Sci.', lxii, 1917.

21. • " II. Helix aspersa ", ibid., lxii, 1917.22. " III. Other Pulmonates ", ibid., lxiii, 1918.23. "IV. Paludina and Testacella", ibid., lxiii, 1918.24. " V. Limnaea ", ibid., lxiii, 1919.25. "VI. Apanteles ", ibid., lxiv, 1919.26. " VII. Modern Cytological Technique ", ibid., lxiv, 1920.27. " VIII. Grantia compressa ", ' Journ. Linn. Soc.', 1920.28. Gatenby and Woodger.—" The Origin of the Golgi Apparatus in the

Middle Piece of the Ripe Sperm of Cavia, and the Development ofthe Acrosome ", ' Quart. Journ. Micr. Sci.', 1921.

29. Gatenby, J. B.—" X. The Gametogenesis of Saccocirrus ", ibid., 1922.


30. Gatenby and King, S.—" Golgi Bodies in a Coccidian ", ibid., 1923.31. Gatenby, J. B.—" On the Relationships between the Formation of

Yolk and the Mitochondria and Golgi Apparatus during Oogenesis ",' Journ. Roy. Mior. Soc.', 1920.

32. Ludford, L. J.—" Contributions to the Study of the Oogenesis ofPatella ", ibid., 1921.

33. " The Behaviour of the Nucleolus during Oogenesis, with SpecialReference to the Mollusc Patella ", ibid., 1921.

34. Munson, J. S.—" A Comparative Study of the Structure and Originof the Yolk Nucleus ", ' Arch. f. Zellf.', viii, 1912.

35. Nassonov, D.—" Das Golgische Binnennetz und seine Beziehungenzu der Sekretion ", ' Arch. f. mikr. Anat. u. Entwick.', 1924.

DESOEIPTION OP PLATES 18 AND 19.

LETTERING.

OP, Golgi platelets; M, mitochondria; N, nucleus ; Nli, nucleolusfirst appearing, i. e. karyosome; Nlii, nucleolus appearing later, i. e.plasmosome ; YD, yolk-droplets ; YP, ' yolk-plates '.

PLATE 18.

Pig. 1.—Very young oogonium showing yolk-droplets but no inito-ohondrial cap. x 1,450. F.w.a.

Kg. 1 A.—Portion of the cytoplasm of a nearly ripe oocyte, very muchenlarged, to show mitochondria, yolk-droplets, Golgi elements.

Kg. 2.—Oogonium with mitocliondrial cap just formed. x 1,450.F.w.a.

Fig. 3.—Oogonium with cap a little larger, x 1,450. F.w.a.Fig. 4.—Oogonium showing cap of mitochondria and also yolk-droplets

and Golgi platelets, x 1,450. Kolatschev.Fig. 5.—Oogonium with cap beginning to move away from the nuclear

wall, x 1,450. Kolatschev.Fig. 6.—Two young oocytes showing yolk-nucleus or mitochondria

forming a cloud in the peripheral region of the cell; a few yolk-dropletsare present and also Golgi bodies, x 930. F.w.a.

Fig. 7.—Similar cell to those of fig. 6, but with more yolk-droplets andwith the yolk-nucleus commencing to break up. x 930. F.w.a.

The last two figures were taken from cells in ovaries of worms not yetmature, as indicated by their small size and the lack of a clitellum.

Fig. 8.—Slightly older oocyte in which the yolk-nucleus is breaking upand spreading over the entire cytoplasm of the cell, x 930. F.w.a.

Fig. 9.—Nearly mature oocyte in which the mitochondria have spread


over the entire cytoplasm of the cell, showing yolk-droplets lying sur-rounded by the mitochondria and also showing the ' yolk-plates' ofCalkins, x 1,450. Champy.

Figs. 10-11.—Very young oogonia showing Golgi plates lying on thesurface of the nucleus; note also the faintly impregnated nucleolus.x 1,450. Da Fano, slightly modified.

PLATE 19.

Figs. 12-13.—Ripe oocytes showing mitochondria distributed over thewhole of the cytoplasm of the cell and surrounding the yolk-dropleta.The darkly stained bodies are Golgi platelets. Note also the ' yolk-plates '.x 1,450. Kolatschev.

Fig. 14.—Late oogonium showing the Golgi bodies lying in the cytoplasm.X 1,450. Da Fano, slightly modified.

Fig. 15.—Two fairly young oocytes with large number of Golgi platelets;the dark cloud round the nucleus in the cell on the right is probably ofmitochondrial origin. The second, larger nucleolus has now appeared,x 930. Da Fano, slightly modified.

Fig. 16.—Ripe oocyte showing the Golgi bodies still spread over theentire cytoplasm of the cell. Only the second nucleolus is now present.x 930. Da Fano, slightly modified.

Fig. 17.—Fairly young oocyte showing inferior fixation of the Golgiplatelets by F.w.a. fixation, x 1,450. F.w.a.

>//r/.- Sri. Vol. 69, N.S., PI. 18-

L.A.narvey del.

Q*iart. Jovrn. Micr. Sci. Vol. 69. NX PL 19M

< v .• ; • . - • • • • )

. A.Harvey del.

on the relation of the mitochondria and golgi apparatus to...

Documents