Studies on the Origin of Yolk. II. Oogenesis of the Scolopendra, Otostigmus Feae (Pocock)

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Studies on the Origin of Yolk. II. Oogenesis of the Scolopendra, Otostigmus Feae (Pocock). By Yishwa Nath, M.Sc, Ph.D. (Cantab.) and Mian Tasdique Husain, M.Sc. Department of Zoology, Government College, University of the Punjab, Lahore.

With 11 Text-figures.

IN a previous paper (1924) on the egg of the centipede, Lithobius f orficatus, one of us (V. N.) described two kinds of yolk, albuminous and fatty. The albuminous yolk is pre- ceded by nucleolar extrusions of a remarkable type, and its origin seems to be associated with them, although no evidence could be adduced that they are directly transformed into the yolk. It was further shown that the juxta-nuclear Golgi apparatus fragments into small granules and small crescent- shaped Golgi elements. The former grow in size and give rise to the fatty yolk. Miss S. D. King (1924) confirms the above account of the association of the albuminous yolk with the nucleolar extrusions, but in her opinion this type of yolk arises directly from these extrusions. With regard to the Golgi apparatus she admits that it fragments into small granules. Furthermore she describes fatty yolk :—' the origin of this fatty yolk is doubtful, but it may possibly be connected with the Golgi apparatus, although no evidence in support of this theory has been discovered '. In our opinion Miss King is doubtful of the origin of fatty yolk from the Golgi apparatus, because ' both Mann-Kopsch and Da Fano material was studied, but the latter gave such favourable results that it was used almost exclusively ' (spaced words ours). Now it is well NO. 287 D d 404 VISHWA NATH AND MIAN TASDIQUE HUSAIN known that the Da Fano method fails to show fat in the finished slides, while the Golgi apparatus is preserved. On the other hand the Mann-Kopsch method preserves both Golgi apparatus and fat, and thus enables one to study the trans- formation of the former into the latter. Furthermore, Flem- ming-without-acetic, which Miss King has used, shows fat, but not the Golgi apparatus, at least in the egg ofLithobius. In a later paper (1926), however, Miss King has shown that ' the formation of fatty yolk from the Golgi elements in Oniscus is strictly comparable to the process described by Nath in Lithobius. . . .' In view of the above conflicting evidence it was thought desir- able to work out the origin of yolk in the eggs of the centipede Scolopendra. Qur observations, both on fixed and fresh material, unmistakably show the origin of the fatty yolk from the Golgi elements and furnish a strong confirmation of the same process in Lithobius. The nucleolar extrusions of Scolopendra are few and they disappear at a particular stage ; and between their disappearance and the origin of albuminous yolk there is a long pause which eliminates all possibility of the direct origin of this type of yolk from these extrusions. The routine laboratory technique employed is mentioned in the text and in the explanatory notes on the figures. One of us (V. N.) has also studied fresh material with the help of neutral red and janus B. All diagrams have been drawn by Vishwa Nath. It is a very great pleasure to us to thank Colonel S. E. Christophers, F.E.S., Director of the Central Eesearch Institute, Kasauli, who gave us all facilities for this work in his institute in the summer of 1926. Our thanks are also due to the authorities of the Indian Museum for identifying the Scolopendra and for supplying us with the necessary literature. OOGENESIS OF SCOLOPENDKA 405

OBSERVATIONS. Staining with vital dyes gave good results. The ovary is kept in neutral red or janus green B for about fifteen minutes. It is then mounted on a slide in a drop of the stain, covered with a cover-slip, and studied under the high power of the micro- scope. Text-fig. 9 represents a fairly young oocyte. The whole oocyte is full of very clear colourless vacuoles of similar size distributed in a uniform manner throughout the cytoplasm ; these are the Golgi vacuoles. In the spaces between the vacuoles lie the granular mitochondria also distributed uniformly. Both the cytoplasm and the mitochondria are stained red or green according to the dye used. The mitochondria, however, stain deeper than the cytoplasm and appear more distinctly with janus green B than with neutral red. With the growth of the oocyte both the Golgi vacuoles and the mitochondria increase in number (Text-fig. 10). The mitochondria remain granular, but some of the Golgi vacuoles increase in size and give rise to the fatty yolk. Both the Golgi vacuoles and the fatty yolk- vacuoles appear as colourless vacuoles, but their contents are chemically different, as will soon appear. Excellent results are obtained when a most highly developed egg is stained with neutral red. Such an egg can be studied only under the lower power as the big size of the egg does not allow the use of the cover-slip. In Text-fig, lla portion of such an egg stained with neutral red is represented. It is studied under the low power and is magnified only forty times. Nevertheless appearances are brilliant. At A.Y. is the albuminous yolk which appears as colourless, solid, and homogeneous spheres. At F.Y. are the fatty yolk-vacuoles which appear brilliantly red as the stain has penetrated inside them. The smaller Golgi vacuoles, however, remain colourless. The granular mitochondria cannot be seen with this magnification. If now the egg is broken with a needle the solid albuminous yolk-spheres are seen scattered on the slide, but the vacuoles, both Golgi and fatty yolk, being delicate are ruptured and do not appear. In some cases, however, a small portion of the cytoplasm of the ruptured egg D d 2 406 VISHWA NATH AND MIAN TASDIQUB HUSAIN remains intact and the vacuoles can be seen, as if held in position by the solid albuminous yolk-spheres. On account of the paucity of material oocytes younger than that represented in Text-fig. 9 could not be studied with the vital dyes. Text-fig. 1 represents an unstained Mann-Kopsch preparation

G V

Youngest oocyte showing Golgi vacuoles and mitochondrial ring. Mann-Kopsch unstained, x 2,100.

EXPLANATION or LETTERING OF TEXT-FIGURES. AY, albuminous yolk; 0, cytoplasm; FY, fatty yolk; OV, Golgi vacuole; OV, Golgi vacuole looking solid on account of excessive impregnation; G V", Golgi' crescent' with ' idiosome'; 0 V", Golgi ' crescent' without ' idiosome'; M, mitochondria; N, nucleus; N', nucleolus ; N", secondary nucleoli; NE, nucleolar extrusions. of the youngest ooeyte that we have been able to obtain. The mitochondria appear granular and brownish and form a complete ring round the nucleus. At G.V. are the Golgi vacuoles, each with a sharp black chromophilic rim and a central chromo- phobic substance. The contents of each vacuole are watery. At G.V.' the Golgi vacuoles appear solid on account of the precipitation of the metallic osmium inside their interior. With the growth of the oocyte the mitochondrial ring spreads out at its OOGENBSIS OF SCOLOPENDRA 407 periphery and the Golgi vacuoles increase in number, as shown in Text-fig. 2 which represents a Mann-Kopsch preparation stained with acid fuchsin. In Text-fig. 3, which is a Mann- Kopsch unstained preparation, the rnitochondrial ring has

Slightly older oocyte showing proliferation of the Golgi vacuoles and the spreading out of the initochondrial ring. Mann-Kopsch stained. X 1,800. spread out and is touching the egg-membrane at the two poles. The Golgi vacuoles have further increased in number and most of them look solid for the reason given above. With the further growth of the oocyte a change takes place in the contents of the Golgi vacuoles. Text-fig. 4 represents a Champy-Kull preparation stained with acid fuchsin. At G.V. are the Golgi vacuoles which are stained with acid fuchsin. Each vacuole shows a red 408 VISHWA NATH AND MIAN TASDIQUB HUSAIN

rim and a clear central substance. The contents of these vacuoles are undoubtedly watery and non-fatty. At F.Y. are shown vacuoles whose contents have become fatty. In unstained Champy-Kull preparations these vacuoles appear solid and dull black on account of the coagulation and osmication of the fatty contents by osmic acid. But when the slide is heated with the

TEXT-FIG. 3. GV

Older oocyte showing the same phenomenon. Mann-Kopsch unstained. X 1,680. acid fuchsin stain floating on it, these solid and dull black bodies appear as clear vacuoles on account of their decolorization by acid fuchsin aided probably by heat. How acid fuchsin which is dissolved in anilin-oil water decolorizes osmicated bodies we cannot explain. It is, however, certain that acid fuchsin tends to decolorize osmicated bodies in much the same way as turpen- tine and xylol. It will be noticed that in Text-fig. 4 the number OOGENESIS OF SCOLOPENDRA 409 of Golgi vacuoles is much smaller than in Text-fig. 3, although the latter represents a younger oocyte. This, however, is due to the Champy-Kull method which is certainly inferior to the Mann-Kopsch method so far as the fixation of the Golgi vacuoles with Avatery contents is concerned. Text-fig. 5 represents an oocyte of a later stage fixed with Mann-Kopsch and stained with acid fuchsin. This is one of the best preparations in our posses- sion as it comes very near to fresh eggs studied with vital dyes. The whole oocyte is full of Golgi vacuoles, each having a sharp

A young oooyte showing Golgi vacuoles, fatty yolk-vacuoles, mitochondria, and nucleolar budding. Champy-Kull stained with acid fuchsin. x 920. black rim and a central clear substance. The uniform arrange- ment of these vacuoles that we find in fresh eggs (Text-fig. 10) is disturbed undoubtedly by the action of the fixative. At F.Y. are the fatty yolk-vacuoles which are bigger than the Golgi vacuoles. In unstained preparations they appear solid and dull black on account of the coagulation and osmication by osmic acid, but when the slide is stained with acid fuchsin they appear as colourless vacuoles without a sharp black chromophilic rim 410 VISHWA NATH AND MIAN TASDIQUE HUSAIN

TEXT-FIG. 5.

G,V

An oocyte showing Golgi vacuoles, fatty yolk, mitochondria, and two nucleolar extrusions. Mann-Kopsoh stained, x 300. OOGBNBSIS OF SOOLOPENDRA 411 of the Golgi vacuole. The mitochondria remain granular and henceforth are uniformly distributed throughout the cytoplasm.

Older oocyte showing the failure of 0s04 to impregnate all the vacuoles. Mitochondria and nucleolar extrusions are also shown. Mann-Kopsch stained, x 550.

Text-fig. 6, which is a stained Mann-Kopsch preparation, represents an oocyte of a later stage than that represented in Text- fig. 5. This figure is drawn to show that osmic acid sometimes 412 VISHWA NATH AND MIAN TASDIQUE HUSAIN gives unsatisfactory results without any apparent reason. When compared with Text-fig. 5 it will be seen that many Golgi vacuoles have missed impregnation, as is shown by a smaller number of these elements in this diagram, although the fresh eggs of this stage are full of these vacuoles. In addition to this failure of OsO4 to impregnate the Golgi vacuoles, the acid may

TEXT-FIG. 7.

GV"

GVl

Older oocyte showing the failure of OsO4 to impregnate the Golgi vacuoles completely. Hence result ' crescents ' with or without idiosome. Fatty yolk solid and dull black. Nucleolar extrusions degenerating. Mann-Kopsch stained, x 270. cause certain artefacts. The artificial solid appearance of these vacuoles on account of excessive impregnation has already been mentioned. But osmic acid may incompletely impregnate a Golgi vacuole. In Text-fig. 7, which is a stained Mann-Kopsch preparation, certain crescent-shaped Golgi elements are shown. OOGENESIS OF SCOLOPBNDRA 413 Some of these crescents at G.V." have a clear substance attached to them which has been described as the idiosome or the archo- plasm by various workers. Other crescents, however, may have no clear substance attached to them, e.g. at G.V." It is certain that these appearances are artefacts due to the osmication of a portion only of the ' wall ' of the vacuole. We do not see these

TEXT-FIG. 8.

GV1

Very old oooyte showing a few Golgi vacuoles and a number of fatty yolk-vacuoles. Albuminous yolk putting in its appearance. Mann-Kopsch stained, x 700. crescents in Text-fig. 5 which we consider to be an ideal preparation, and what is more important they are entirely absent from fresh eggs. We are not prepared to make the general statement that crescent-shaped Golgi elements are never met with in any type of cell, especially when such competent workers as Bowen and Gatenby have consistently described them in the sperm- forming cells of insects, &c. We shall, however, at present content ourselves with saying that the solid granular type of Golgi element is an artefact wherever it has been described, due 414 VISHWA NATH AND MIAN TASDIQUE HUSAIN to the excessive precipitation of metallic silver or osmium, and that crescents of the egg of Scolopendra at least are also artefacts due to incomplete impregnation. On the other hand, the crescents may represent only the optical sections of osmicated Golgi vacuoles. In the same figure at F.Y. are the fatty yolk-bodies which appear solid and dull black in spite of the fact that the slide has been stained with acid fuchsin. The inferiority of this preparation is made evident not only by the

TEXT-FIG. 9.

A young oocyte stained with neutral red showing Golgi vacuoles of uniform size and granular mitochondria. Nucleu3 out of focus. x210. artefacts described above but also by the presence of fewer Golgi vacuoles and fatty yolk-vacuoles as compared with Text-fig. 5, although the latter represents a younger oocyte. The origin of the albuminous yolk is very interesting. Long before the mitochondria and the Golgi elements get uniformly dispersed in the cytoplasm the nucleolus shows signs of budding (Text-fig. 4). The number of these nucleolar nucleoli quickly increases and some of them pass into the cytoplasm and grow OOGENESIS OF SCOLOPENDHA 415 in size (Text-fig. 6). Although there are numerous nucleoli, nucleolar extrusion are few. At first sight it appeared to us that these extrusions give rise to the albuminous yolk, since histo- chemically they are very much like the latter and also because they precede them. Study of later stages, however, soon showed

TEXT-FIG. 10.

An older oocyte stained with neutral red showing the growth of some of the Golgi vacuoles to give rise to the fatty yolk-vacuoles. Mito- chondria granular. Nucleus out of focus, x 190. that these extrusions disappear long before the albuminous yolk puts in its appearance. The process of their disappearance may thus be described. At first the extrusions swell up probably by absorbing some liquid from the cytoplasm, and then each one of them undergoes disintegration (Text-fig. 7, N.E.). The pieces that result from this disintegration are irregular in form and show obvious signs of degeneration. Gradually they disappear. The albuminous yolk arises de novo for the first time 416 VISHWA NATH AND MIAN TASDIQUE HUSAIN when the egg has reached the dimensions represented by Text- fig. 8. The oocytes between the stages represented by Text- figs. 7 and 8 are devoid of any nucleolar extrusions, so that a direct origin of the albuminous yolks from the extrusions is out of the question. The possibility remains, however, that when the extrusions disappear their material might contribute towards

TEXT-FIG. 11.

GV A portion of the most highly developed egg stained with neutral red showing albuminous yolk, Golgi and fatty yolk-vacuoles. Mito- chondria cannot be seen with this magnification, x 40. the formation of albuminous yolk, but there is no way of ascer- taining this. New albuminous yolk-spheres arise rapidly till we get the most highly developed ovarian egg (Text-fig. 11).

DISCUSSION. In support of our claim that the Golgi elements give rise to the fatty yolk we have shown that the latter puts in its appearance after the Golgi elements begin to increase in number and spread out in the cytoplasm, and that between the small Golgi vacuoles and the big fatty yolk-vacuoles there are vacuoles of intermediate dimensions. But above all we emphasize the fundamental morphological similarity between the Golgi elements and the fatty yolk, inasmuch as both are vacuoles. OOGBNESIS OF SCOLOPBNDRA 417

This has been ascertained not only by good Mann-Kopsch preparations but also by the study of fresh eggs. It will thus appear that as in spiders (Nath, in press) and Lithobius (Nath, 1924) the Golgi vacuole, whose contents are watery and non-fatty, gives rise to the fatty yolk-vacuole by a process of growth and deposition in its interior of a colloid in the form of free fat not miscible with the general cytoplasm. Although nucleolar extrusions clearly give rise to the albuminous yolk in various eggs, e.g. Saccocirrus (Gatenby, 1922), the Cockroach (Hogben, 1920), Lithobius (King, 1924, and Nath, 1924), and Luciola (Nath and Mehta, in press), &c, this is not the case in Scolopendra. The direct origin of this type of yolk from the nucleolar extrusions is impossible, as there is a long pause between the disappearance of the extrusions and the first appearance of the yolk; besides, nucleolar extrusions are very few.

SUMMARY. 1. The oogenesis of Scolopendra has been worked out with vital dyes and the routine laboratory technique. 2. The Golgi elements exist in the form of vacuoles with watery and non-fatty contents. 3. The solid Golgi elements are artefacts due to the excessive precipitation of metallic osmium inside the vacuoles. 4. The Golgi crescents are either artefacts due to incomplete blackening of a vacuole, or they are simply the optical sections of an osmicated vacuole. 5." The Golgi vacuoles give rise to fatty yolk-vacuoles by a process of growth and deposition inside them of free fat not miscible with the general cytoplasm. 6. The mitochondrial granules form a complete ring round the nucleus of the youngest oocyte. Later they are uniformly distributed throughout the cytoplasm. 7. There are a few nucleolar extrusions but they disappear long before the albuminous yolk puts in its appearance de novo in the cytoplasm. 418 VISHWA NATH AND MIAN TASDIQUE HUSAIN

BIBLIOGRAPHY. 1. Gatenby, J. Bronte (1922).—' Quart. Journ. Micr. Sci.', vol. 66. 2. Hogben, L. (1920).—' Proc. Roy. Soc. London ', Series B, vol. xci. 3. King, S. D. (1924).—' Scient. Proc. Roy. Dub. Soc.', vol. 18. 4. (1926).—' Proc. Roy. Soc. London ', Series B, vol. c. 5. Nath, V. (1924).—' Proc. Phil. Soc. Camb. Biol. Sciences ', vol. i.