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Histochemistry of Lipids in Oogenesis
Histochemistry of Lipids in Oogenesis VISHWA N A T H Department of Zoology, Punjab University, Hoshiarpur, Chndigarh, Punjab, India I. Introduction ......................................................... 11. Technique ........................................................... 111. Results .............................................................. A. Insects .......................................................... 1. Panoistic Type .............................................. 2. Polytrophic Type ............................................. 3. Telotrophic Type ............................................. B. Spiders ......................................................... C. Earthworm ...................................................... D. Fishes .......................................................... E. Frogs and Toads ................................................ F. Reptiles, Birds, Mammals ....................................... 1. Reptiles ..................................................... 2. Birds ........................................................ 3. Mammals .................................................... IV. Conclusions .......................................................... Acknowledgment ..................................................... References ...........................................................
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I. Introduction Until very recently work on animal oogenesis has been carried out by what may be described as the orthodox classical techniques of fixation and staining, such as chrome-osmium, long osmication, and silver impregnation. At best these techniques gave us only a rough idea of the morphology and chemical composition of the cytoplasmic inclusions such as “Golgi bodies,” mitochondria, vacuome, nuclear extrusions, and yolk, fatty and protein, met with in animal oocytes. It was concluded that the Golgi bodies and the mitochondria contained lipids and lipoproteins respectively. Gatenby and Woodger (1920) showed that the “Golgi dictyosomes” secrete the fatty yolk in the oogenesis of Patella vulgaris. This claim was subsequently confirmed by Ludford (1921) and Brambell (1924) ; but it is important to note that Ludford clearly mentioned that some of the “Golgi elements” are directly metamorphosed into such yolk, as claimed by Hirschler (1917) for the ascidian egg. Speaking of centrifuged eggs of Saccocirrus, Gatenby (1922) wrote that “the upper cap is formed of delicate granules which, I think, are fatty yolk and probably of the Golgi elements ; these granules will go yellow-green after prolonged osmication.” Since 1928 the author has been laying great stress on the importance of the study of fresh oocytes for determining the form and functions of the Golgi bodies, mitochondria, and vacuome in oogenesis (see Nath, 1957, 305
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for references). Nath (1931) and Nath and Nangia (1931) showed for the first time in oogenesis that the neutral red-staining watery vacuoles of Parat were independent of the classic lipoidal “Golgi apparatus” and the mitochondria in the frog and fish respectively. But, as pointed out above, all these techniques give at best only a rough idea of the chemical composition of these cytoplasmic inclusions. During the last few years studies on oogenesis of some insects, spiders, fishes, frogs, reptiles, birds, and mammals have been carried out in this laboratory by the modern techniques of histochemistry mentioned below. These investigations have very clearly brought out that lipids may exist not only in the Golgi bodies and the mitochondria, but also, in many forms of oogenesis, in what is usually named as the “albuminous” or “protein” yolk. 11. Technique The histochemical methods for the study of lipids in animal cells have been excellently reviewed by Cain (1950) and more recently by Deane (1958). Further details of these methods can be found in many books, e.g., Pearse (1954), Lison ( 1953), Lillie ( 1954). Of all the various fixatives it has been found that formaldehyde-calcium followed by postchroming, as recommended by Baker (1946, 1956), with gelatin embedding, is undoubtedly the best for the preservation of lipids in oocytes. The presence of calcium ions in the fixative and the postchroming treatment seem to be indispensable for the proper preservation of lipids in oogenesis. Moreover, almost all the tests for lipids, with the possible exception of performic acid-Schiff for the unsaturated bonds (Pearse, 1954), can be tried on these sections. I n many cases the Lewitsky-saline technique of Baker (1956) may also be employed, particularly if the arrangement of cells is also to be studied, as the cell membranes are fixed much better in this fixative. Sudan black B used as a saturated ethanolic solution (Baker, 1949) has been found to be the most successful lipid colorant. This colorant may also be used in propylene glycol (Chiffelle and Putt, 1951), since in many cases it reveals much more of lipids than the alcoholic solution. Sudan I11 and IV (Kay and Whitehead, 1941 ; Govan, 1944) or Fettrot 7B (Pearse, 1954), generally recommended as the colorants for the neutral fats (triglycerides), are not at all specific; they can at best be considered as general lipid colorants but much less vigorous than Sudan black B. They can be employed most usefully, however, as counterstains for the acid-hematein (Baker, 1946) preparations. Such counterstained preparations are extremely useful in oogenesis since they present the various types of lipid bodies in different colors in the same preparation (see Nath et al., 1 9 5 9 ~ ) .
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It has often been found that Sudan black B does not color the larger lipid bodies in the oocytes homogeneously, owing to the presence of either solid or masked lipids (Gupta, 1958a). It is advisable, therefore, to try this colorant at 60” also. If even this technique fails, one or all of the various techniques for “unmasking” the lipids (Gupta, 1958a, 1959; Clayton, 1958; Berenbaum, 1958) may be tried before deciding finally that a lipid body really possesses a nonlipid core, as such “duplex” bodies do not seem to occur in the animal oocytes (Gupta, 1958b). It is unfortunate that no specific histochemical technique is available for the triglycerides, occurring widely in oogenesis. The most useful method available is the Nile blue sulfate method of Cain (1947, 1948) used with Keilig-type extractions as control. Thus a body which gives a negative reaction to the tests for other lipids, which appears pink in Nile blue sulfate, and which is soluble in cold acetone may be considered to be composed of triglycerides. The presence of unsaturated bonds is generally revealed by the performic acid-Schiff technique (Pearse, 1951 ; Lillie, 1952). However, the degree of saturation of the various lipids is best revealed by the graded postchroming technique of Ciaccio followed by paraffin embedding (Lison, 1953; Bradbury, 1956). Fresh cover slip preparations treated with 2% Os04 solution may also yield useful information in this respect (Wigglesworth, 1957 ; Nath, 1957). For cholesterols and their esters the modification of Schultz technique by Weber et al. (1956) has been found to give trouble-free results. Keilig-type extractions with the various lipid solvents (Pearse, 1954) have proved very useful in determining the detailed chemical nature of the composite lipid bodies (L, and L).Such extractions when tried on the fresh oocytes do not generally yield good results. More useful information may be gained by extracting gelatin sections of the material fixed in formaldehyde-calcium for 6 hours, either with or without postchroming. For further details reference may be made to Nath et al. (1958a). For comparison and additional information, mercuric-bromophenol blue (Mazia et al., 1953) for proteins and the periodic acid-Schiff test for carbohydrates (Hotchkiss, 1948 ; Pearse, 1954) are the most useful techniques.
111. Results
A. INSECTS Wigglesworth ( 1950) has described three chief types of insect ovaries, viz., ( 1 ) the panoistic type, in which there are no special nurse cells, (2) the polytrophic type, in which every oocyte has its own nurse cell or cells,
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and ( 3 ) the telotrophic type, in which every ovariole has a nutritive chamber or trophic core in its germarium.
1. Panoistic T y p e T o the best of my knowledge Nath, Gupta, and La1 (1958a), and Nath, Gupta, and Aggarwal (1959a) are the only workers who have studied the histochemistry of lipids in the oogenesis of Periplaneta amen’ccuna, and Chrotogonus trachypterus and Gryllodes sigillatus, respectively. a. Cockroach. Nath et d.(1958a) have described three kinds of lipid bodies in the cockroach: (1) L1 bodies, present in the earliest oocyte, which persist until the oocyte measures approximately 0.5 mm. and which contain phospholipids only, possibily having more lecithins than cephalins ; ( 2 ) Lz bodies, which first arise in the oocyte measuring 0.4 mm. and have a complete or incomplete sheath of phospholipids surrounding a medulla of triglycerides, and which are rather highly saturated; and ( 3 ) La bodies, which are the only types of lipids in the oocytes measuring more than 0.65 mm. and consist of rather highly saturated triglycerides. Some of the larger La bodies have been described as giving a “ringed” or “crescentic” appearance in Sudan black when used at room temperatures (12°C. to 40°C.) but appearing mostly homogeneous when the coloring agent is used at 60°C. The L1 bodies of these authors correspond to the “Golgi bodies” of Nath and Mohan (1929), but they do not have the duplex structure attributed to them. The duplex structure of the Golgi bodies of this cell appears to be due to incomplete reduction of osmium tetroxide. The Lz bodies, which directly arise from L1 bodies, have undoubtedly a duplex structure and correspond to the duplex Golgi vesicles of Nath and Mohan (1929). The LB bodies, which arise directly from L2 bodies, correspond to the “fatty yolk” of these authors. Nath et al. (1958a) studied the rodlets called “bacterioid forms” by Blochmann (1884, 1887), and Nath and Mohan (1929). These are situated just below the follicular epithelium, and they contain free fatty acids and phospholipids. Nath et al. (1958a) conclude that the bacterioid forms participate in lipid synthesis in the cockroach oocyte, as suggested by the fact that all the lipid bodies grow in size and number near the layer of bacterioid forms. This conclusion is further supported by the fact that when the synthesis of lipids is over the bacterioid forms are no longer positive to any lipid test. b. Chrotogonus and Gryllodes. Nath et d. (1959a) have studied the lipids in the oocytes of Chrotogonus trachypterus and Gryllodes sigillatus. The pattern of lipid synthesis in these two orthopteran forms is similar to the cockroach except that (1) L1 bodies in both the forms consist of
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lipoproteins, (2) both the L1 and Lz bodies in Chrotogonus contain “masked” lipids, and ( 3 ) there are no bacterioid forms. Gupta (195%) has made a valuable contribution in demonstrating the presence of some “masked” lipids in the oocytes of Chrotogonus and their “unmasking” by Sudan black B and phenol technique. This author found that a “ringed” or “crescent” appearance, produced by a sudanophil sheath surrounding a non-sundanophil core, was given by a great many of the lipid spheres. This occurred even in material fixed by formaldehydecalcium postchromined and stained in Sudan black B in propylene glycol at 60” for 1 hour. Various tests failed to demonstrate either cholesterol or its esters in these bodies. These results demonstrated that there must be a masking of the lipids in the core if they are present at all. Gupta (1958a) “unmasked” these lipids of Chrotogonus oocytes by fixing the material in formaldehyde-calcium for 6 hours and then treating it with 1% phenol solution at 37” for 24 hours. Sudan black B stained gelatin sections failed to show any “ringed” or “crescentic” structures ; instead the spheres were stained a uniform blue-black color. This demonstrates quite clearly that the cores of the lipid spheres in Chrotogonus oocytes were “masked” presumably by proteins as evidenced by the positive reaction they give with mercuric-bromophenol blue. Finally, it may be mentioned that Nath et al. (1958a) have described in the cockroach and Nath et al. (195%) have recorded in Chrotogonus and Gryllodes the presence of large quantities of lipid bodies, only of L1 and L2 types, in the follicular epithelial cells ; these latter, in the absence of special nurse cells, are the only source of raw materials to the oocyte.
2. Polytrophic T y p e In the polytrophic type of ovary each oocyte may have either only one nurse cell associated with it as in two species of earwigs (Nath et al., 1959b), or a number of them as in Culex fatigans (Nath et al., 1 9 5 8 ~ ) . As far as the author is aware, there is no other work on the lipids in this type of ovary. a. Culex fatiguns. Nath et al. ( 1 9 5 8 ~ )have demonstrated three types of lipid bodies again in the oocyte of Culex fatiguns, viz., L1, L,and L3. The L1 bodies, consisting of phospholipids and lipoproteins, give rise to the L2 bodies. The L2 bodies originally consist of phospholipids, lipoproteins, and triglycerides, but later they lose their protein constituents and then have triglycerides in their core and phospholipids in the cortex. A gradual attenuation of the phospholipid rim ultimately transforms the L2 bodies into L3 bodies ; the L2bodies, however, do not grow to form the Idsbodies as in many other forms.
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The lipid bodies of the nurse cells and the follicular epithelial cells are of L1 type only ; but the lipids are very scarce in the nurse cells. Nath et al. ( 1 9 5 8 ~ )have pointed out that most of the supply of the raw material to the oocyte in Culex comes through the follicular epithelial cells directly. The nurse cells seem only to provide their protein component to the growing oocyte, and they do not contribute to the oocyte any carbohydrate or lipid substances which could be detected histochemically. b. Earwigs, Labidura ripark, and L . bengalensis. Nath et al. (1959b) have again demonstrated three types of lipid bodies in the earwig, viz., LI, Lz, and Ls. The L1 bodies contain phospholipids only and directly give rise to LZ bodies, which contain triglycerides in their core and phospholipids in the cortex. In Labidura bengalensis the Lz bodies appear very late in the oocyte and are fewer in number as compared with L . riparia; they also contain some masked lipids, which were unmasked by phenol treatment. The L2 bodies in both species of earwigs directly give rise to larger Ls bodies, which contain triglycerides only. It has already been pointed out that in earwigs each oocyte carries with it its own nurse cell, from which it is cut off by a septum containing a central pore. Nath, Gupta, and Aggarwal have shown that in both the species of earwigs the associated nurse cell forms the chief source of the supply of lipids to the oocyte, in contrast with Culex, in which the nurse cells do not make any lipid contribution. These authors have seen a stream of mitochondria and L1 bodies passing from the nurse cell to the oocyte through the central pore in the septum; they also describe infiltration of lipid bodies through the septum itself.
3. Telotrophic Type The only paper on lipids of the telotrophic ovary is by Bonhag (1955). This author, working on Oncopeltus farciatus, states (1) that it is almost certain that sudanophil lipids are contributed to the oocytes by the apical trophic tissue ; (2) that there is circumstantial evidence that sudanophil lipids (of a non-phospholipid type) and phospholipids (or their precursors) are contributed to the oocytes by the follicular epithelium; ( 3 ) that in oocytes having a complete or nearly complete endowment of yolk, most of the sudanophil lipid occurs in distinctive bodies called the “coarse sudanophilic bodies” ; (4) that these bodies appear to be formed first in the oocytes from precursor bodies which utilize lipids contributed by way of the nutritive cords from the apical trophic tissue; ( 5 ) that in late oocytes, however, these “coarse sudanophilic bodies” seem to be formed from precursor bodies, which utilize lipids obtained from the follicular epithelium ; (6) that these “coarse sudanophilic bodies” are uniformly dispersed throughout the oocyte cytoplasm between the larger
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protein-carbohydrate yolk granules ; and (7) that there is a progressive decrease of phospholipids in the course of oogenesis. Nath et al. (1959d) in the telotrophic ovary of Laccotrephes maculatus and L . ruber have found three types of lipid bodies, viz., L1 bodies containing phospholipids and perhaps also phosphatidic acid, Lz bodies containing phospholipids and triglycerides, and L3 bodies containing triglycerides only. It is obvious that the LBbodies, and LIand L2 bodies of these authors correspond to the “coarse sudanophilic bodies” and to the “phospholipids” of Bonhag respectively. These authors also agree with Bonhag that the apical trophic tissue and the follicular epithelium are the only sources of lipid supply to the oocytes. B. SPIDERS Krishna (1953), working on the oocytes of the Indian water spider, Lycosa birmanica, states that the fine bodies resembling Golgi bodies are granular and contain “phospholipins, lipo-protein, and protein,” but no triglyceride. In a subsequent communication Krishna ( 195S), describing very briefly the chemical composition of the “yolk-nucleus” of Lycosa birmunica, states that this structure is free from triglycerides, and its concentric lamellae show a thick coating of “phospholipins” covering “lipo-proteins of varying consistency.” Nath et al. ( 1 9 5 9 ~ )have worked out the details of the lipids in the oogenesis of the spider, Plexippus paykulli, with special reference to the “yolk-nucleus.” They have shown that there is in this spider a very prominent yolk-nucleus consisting of four zones, viz., an innermost zone of diffused lipids, a clear zone, a zone of circularly arranged and closely packed mitochondria1 fibers, and an outermost zone of diffused lipids. The yolk-nucleus is the dynamic seat of lipid synthesis ; all the lipid bodies in the cell take their origin in the yolk-nucleus, and there are at least four distinct cycles of lipid synthesis in the yolk-nucleus. These authors have further described three categories of lipid bodies in this material, viz., L1 bodies consisting of phospholipids only, Lz bodies having a phospholipid core and a triglyceride sheath, and L3 bodies having triglycerides only. These authors have shown that the L1bodies are restricted to the innermost zone of the yolk-nucleus where they take their origin. They pass to the outer zones of the yolk-nucleus, and during this outward migration they develop a thick triglyceride sheath and thus form La bodies. These last accumulate in the outermost zone of the yolk-nucleus where they gradually lose their phospholipid content to form Ls bodies, which are released into the cytoplasm from the yolk-nucleus at the close
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of every cycle, in the form of a large mass. They gradually disperse in the cytoplasm. It is interesting to note that the L3 bodies do not grow, but remain granular throughout oogenesis. Nath, Gupta, and Manocha have also described large quantities of phospholipids and triglycerides in the yolk globules of Plexippus paykulli in addition to the protein-carbohydrate complex. Nath and associates (1958e) have shown that there is no true yolknucleus in the spider Crossopriza lyoni, the so-called yolk-nucleus of other authors (see Nath, 1957) being a mere aggregation of mitochondria and Golgi bodies. These authors have described in this spider also three types of lipid bodies, viz., L1, b,and Ls ; but the L1 and L2 bodies differ from the similar bodies in Plexippus inasmuch as they contain proteins also in addition to phospholipids, and to phospholipids and triglycerides, respectively. The L3 bodies of Crossopriza, which consist of pure triglycerides as usual, remain granular throughout oogenesis as in Plexippus. These authors have described five kinds of yolk globules, Y1 to YE. Of these, the Y1 globules consist of proteins and carbohydrates, blended with phospholipids and triglycerides, while the Y2 and Y3 globules contain mainly proteins and carbohydrates with small quantities of triglycerides. The Y d and YE globules do not contain any lipids.
C. EARTHWORM Nath et al. (1958b) in Pheretimu posthum have shown that the earthworm oocyte contains lipids of L1 and L2 categories only, the LBtype of lipid bodies being entirely absent. The L1 bodies are smaller, appear as homogeneous dark granules under the phase-contrast microscope, and have a protein-phospholipid core surrounded by a thick sheath of phospholipids only. The bodies, which arise as a result of growth and chemical change in L1 bodies, have a pure phospholipid core surrounded by a thick triglyceride sheath. They give a ringed appearance under the phase-contrast microscope ;but under the interference microscope this ringed appearance is shown to be an optical artifact (see also Gupta, 1958b). The lipid spheres present in the follicular epithelium contain phospholipids only. These authors have further shown that their L1 and L2 bodies correspond very closely to. the “Golgi granules” and Sudan IV-coloring spherules respectively of Nath and Bhatia ( 1944).
D. FISHES Chopra in this laboratory at Panjab University has carried out morphological and histochemical studies on the oogenesis of eleven species of fish with particular reference to lipids.
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I n his first paper (Chopra, 19%) this author has studied the cytoplasmic inclusions in the growing oocytes of the fish Ophiocephalus punctatus. The nonyolky oocytes of early stages show cytoplasmic granules of two types, namely, the mitochondria and the lipid granules of the first category (LI). The latter are bigger than the mitochondria, consist of phospholipids only, and correspond to the “Golgi bodies” of Nath and Nangia (1931). Chopra describes a second category of lipid bodies (L2) , consisting of phospholipids and triglycerides to begin with, but only triglycerides in the mature eggs. This author does not describe L3 bodies as such; however, it is obvious from his description that he has in his material two kinds of L2 bodies: L2 bodies proper consisting of phospholipids and triglycerides, and L3 bodies consisting of triglycerides only, as in most forms of oogenesis described here. Nath and Nangia (1931) had described two kinds of yolk globules only in Ophiocephalus, viz., “fatty yolk,” which corresponds to the triglyceride spheres of Chopra, and “albuminous yolk,” which develops in vacuoles. Chopra (1958a) has described this “albuminous yolk” of Nath and Nangia as a “vacuolar yolk” and has shown it to consist of carbohydrates and proteins. But this author has also described a third category of yolk globules containing proteins and lipoproteins, which do not develop in vacuoles. I t is this lipoprotein yolk which was completely missed by Nath and Nangia (1931) and Nath et al. (1944). I n his second publication on Barbus ticto Chopra (1958b) has described lipid granules consisting of phospholipids only and corresponding to the “Golgi bodies’’ of earlier workers. The author has also described in this material: (1) triglyceride yolk globules, corresponding to his L2 bodies in Ophiocephalus and the “fatty yolk” of earlier workers; (2) Y2 yolk globules, which are rich in proteins with some traces of triglycerides and which correspond to the “albuminous yolk” or “protein yolk” of earlier workers ; and ( 3 ) the “vacuolar yolk,” which develops in vacuoles and is very rich in carbohydrates with some traces of proteins. Chopra (19.58~)has studied the lipid bodies and yolk in nine more species of fish and has found, more or less, the same pattern of vitellogenesis as in Ophiocephalus and Barbus.
E. FROGS AND TOADS Nath et al. (1958d) have made histochemical studies of the cytoplasmic inclusions in the oocytes of Ram tigrina, with particular reference to lipids. There are three kinds of lipid bodies in the frog, viz., pure phospholipid L1 bodies, phospholipid-triglyceride L2 bodies, and pure triglyceride La bodies. Nath (1931, 1932) had worked earlier on the oogenesis of Rana tigrina
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and Rana cyanophlyctis. Nath (1931) had described certain dark grayish refractile bodies in the fresh young oocytes, which grow into tiny vesicles with a dark grayish cortex and a less refractile medulla in later stages. H e labeled these bodies as the “Golgi bodies.” Nath (1932) showed that in the breeding season some of the “Golgi bodies” grow into fatty yolk spheres. Nath and Malhotra (1954, 1955) later confirmed the findings of Nath that “Golgi vesicles” directly give rise to the fatty yolk spheres. Nath and Malhotra (1955) state that in the toad, the treatment of young oocytes with Sudan I V (Kay and Whitehead, 1941) demonstrates no positive elements but by the time the oocytes have reached a diameter of 0.35 mm. a reaction is given by the Golgi bodies. Subsequent growth of the oocytes is accompanied by an increase in the size of Golgi bodies and Nath and Malhotra believe that ultimately they form the fatty yolk. A positive reaction is given by the Golgi bodies with Sudan I V at all these stages and at the later period the yolk and the Golgi bodies cannot be distinguished by this reagent since they both stain positively with it. It is clear that the “Golgi bodies” of Nath and Malhotra not colored with Sudan I V are homologous with the L1 bodies of Nath et al. (1958d) ; “Golgi vesicles,” which color with Sudan I V in later stages, with the L2 bodies ;and the “fatty yolk spherules” with the La bodies.
F. REPTILES,BIRDS,MAMMALS Guraya in this laboratory has been engaged now for some years in the study of lipids in the oogenesis of reptiles, birds, and mammals. The histochemical investigations of this author, and of Gupta et al. (1959), have shown that the lipid synthesis in these forms is much more complicated, and there exist more than three categories of lipid bodies.
1. Reptiles Guraya (1959a) has described three types of lipid bodies (L1, Lz, L3) of different chemical composition and morphology in two species of lizards (Calotes versicolor, Uromastix hardwickii), two species of snakes (Lycodon aulicus aulicus, Boiga trigonuta) , and one species of fresh-water turtle (Lissemys punctata punctata) . The L1 bodies of Guraya (1959a) are in the form of granules and spheres consisting of phospholipids and triglycerides of an unsaturated nature. They correSpond to the so-called “Golgi bodies” of earlier workers on the oogenesis of reptiles and .react like them with osmium tetroxide and silver nitrate. The L3 bodies arise in the oocytes, when still young, in the form of
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comparatively large spheres, consisting of unsaturated triglycerides only. They seem to develop from L1 bodies by some chemical transformation, and they correspond to the “fatty yolk” of earlier workers. They are usually used up during the growth of the oocyte before the appearance of yolk globules. The L2 bodies of Guraya (19%) are a new category of lipid bodies not present in insects, spiders, earthworm, fish, and frog; they arise de novo in the early oocytes in the form of granules lying in groups, after the appearance of L1 and L3 bodies; and they consist of a peculiar type of phospholipids, which are fully preserved only after fixation in formaldehyde-calcium and postchroming as recommended by Baker ( 1946, 1956). The L2 granules grow into duplex vesicles, spheroids, plates, and various other forms having sudanophobe areas which react negatively to all the tests used; they develop simultaneously a small amount of triglycerides, which, however, are absent in the Lz bodies of Lissemys and Lycodon. bodies have either been missed by Guraya (1959a) states that his the earlier workers, or their incompletely fixed forms were also identified as Golgi bodies. Further, the L1 and L2 bodies are also present in the follicular epithelial cells with the exception of Lissemys, in which only L1 bodies are found. Guraya (1959a) has also observed the infiltration of L1 bodies from the follicular epithelium into the oocyte through the zona radiata. The yolk of ripe ova consists of two types of bodies: (1) triglyceride yolk spheres composed of unsaturated triglycerides and having no connection whatsoever with L3 bodies, and ( 2 ) compound yolk, corresponding to the albuminous or proteid yolk of earlier authors and consisting mainly of carbohydrates and proteins with some triglycerides and lipoproteins. Guraya (1958) had arrived at similar conclusions in the lizard Hemidactylus flawiviridis.
2. Birds Guraya (1959b) has investigated the oogenesis of three species of birds, viz., one of the fowl, Gallus domesticus, and two of the dove, Streptopelia senigalensis cambaiensis and S. decaocto decaocto. In these birds this author has described the same pattern of lipid bodies as in reptiles. In these species of birds also the L1 bodies consist of unsaturated phospholipids and triglycerides, corresponding to the so-called Golgi bodies of earlier workers on bird oogenesis; the L3 bodies, which seem to develop from L1 bodies as in reptiles, consist of unsaturated tri-
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glycerides and cholesterol and its esters, and correspond to the “fat globules” or “fatty yolk” of earlier workers. The L3 bodies disappear in the course of oogenesis as in reptiles. The L2 bodies correspond exactly to the Lz bodies of reptiles. They arise de novo at the periphery of the oocytes, have no connection with L1 and L3 bodies, and consist of some specific phospholipids with some amount of unsaturated triglycerides, which can be fully preserved only in formaldehyde-calcium followed by postchroming, as in reptiles. Again the La bodies appear under different forms as in reptiles. Guraya (1959b) has described three kinds of yolk globules in birds, all of which contain lipids. They are (1) triglyceride spheres of highly unsaturated nature, (2) homogeneous compound yolk spheres, consisting of carbohydrates, proteins, and lipoproteins, and (3) heterogeneous compound yolk spheres, with a matrix consisting of carbohydrates, proteins, and unsaturated triglycerides, in which are embedded spheres of homogeneous compound yolk. In his first publication Guraya (1957) has described exactly the same types of lipid inclusions in the oogenesis of the pigeon, Columba livia, but unfortunately has used a different terminology.
3. Mammals Deane and Barker (1952) and Deane (1952) have studied the distribution of lipids in the ovary of rat and sow, and of albino rat respectively during the estrous cycle. Their main object was to study the sites of steroid activity in the ovary, and for this reason they did not work out the details of the lipids in the normal oocyte. Gupta et al. (1959) have described four distinct categories of lipid bodies in the ovary of rat, viz., L1, Lz, L3, and fenestrated lipid bodies ( F N L ) . The L1 bodies consist of phospholipids, the L2 of phospholipids and neutral lipids, the L3 of neutral lipids, and the F N L bodies of phospholipids and traces of triglycerides. The normal oocyte of the rat, according to these authors, contains the L1 bodies only, beside the mitochondria and the yolk granules. The granulosa of the normal follicles contains L1, Lz, and F N L bodies. It may be noted carefully that the LZ bodies of the granulosa cells of the normal follicles are composed of phospholipid and triglycerides, but with the start of atresia the L2 bodies of the granulosa cells show the presence of steroids. It may be noted further that the presence of fenestrated lipid bodies ( F N L ) is a characteristic feature of the granulosa of the normal follicle; they start disappearing with the onset of atresia. The phospholipids of the F N L bodies can be preserved only after fixation in formaldehyde-calcium followed by postchroming in dichromate-
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calcium as recommended by Baker (1946, 1956) ; in this respect they are strictly homologous with the Lz bodies of Guraya described in reptiles and birds in this article. Deane and Barker (1952) and D a n e (1952) seem to have missed these lipid bodies completely, because they employed pure formaldehyde as a fixative and did no postchroming in dichromatecalcium. According to Gupta et al. (1959) the theca cells of the normal follicle also contain L1 and L2 bodies, but the latter contain some quantities of steroids in addition to phospholipids and triglycerides. On the contrary, the atretic follicles, both primary and vesicular, contain large quantities of L3 bodies in addition to the L1 and L2. Further, the Lz and L3 bodies of these follicles contain appreciable quantities of steroids in addition to the triglycerides. Gupta et d. (1959) have also described traces of phospholipids in the yolk bodies found in the normal oocytes of the rat. Guraya ( 1 9 5 9 ~ )described in the rabbit and the hare also the fenestrated type of lipid bodies, containing phospholipid and traces of triglycerides, in the normal follicles, having the same specific nature as described by Gupta et d. (1959) for the rat. Further, these bodies are homologous with the Lz bodies of Guraya described in the oocytes of reptiles and birds. Guraya ( 1959c) describes phospholipids and triglycerides in the yolk spheres also in the oocytes of rabbit and hare.
IV. Conclusions It is certainly possible now to make some general statements with regard to the scheme of lipid synthesis in animal oogenesis. A very important conclusion which has emerged from the investigations of Nath and his co-workers is that the synthesis of triglyceride globules in the oocytes occurs in three stages. The earliest lipid granules in oogenesis may consist of phospholipids only (as in cockroach, earwigs, water scorpions, Plexippus, fish, frog, and rat), or of phospholipids and proteins (as in mosquito, earthworm, and Crossopriza) , or of lipoproteins only (as in Gryllodes and Chrotogorcus) . With the growth of the oocyte these earliest lipid granules grow and increase in number greatly and develop triglycerides either in their cores (as in cockroach, Gryllodes, Chrotogonus, Culex, earwigs, and water scorpions), or in their cortex (as in spiders and earthworm). But in all vertebrate species reviewed here the phospholipids and triglycerides are mixed. I n the late stages of oogenesis, as a rule, the lipid spheres are large and consist of neutral lipids (generally triglycerides) only; but in Culex and
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in spiders the triglyceride bodies are small and granular. I n the alecithal eggs of the earthworm, however, pure triglyceride bodies do not develop at all, while in the normal oocytes of the rat the lipid bodies consist of phospholipids only, there being no triglyceride synthesis. Another very important conclusion which we have arrived at is the presence of an altogether new type of lipid bodies, occurring as “fenestrated” masses in the oocytes of the Amniota only; they consist of some specific phospholipids which can be preserved only in formaldehydecalcium followed by postchroming in dichromate-calcium. Again these bodies seem to be independent of the normal graded lipid bodies described above. In mammals these fenestrated bodies occur in the granulosa of the normal follicles only; they disappear as soon as these follicles start developing atresia. The author since 1928 has been developing and advocating the view that the “fatty yolk” in eggs is directly derived from the “Golgi vesicles” by the deposition of fat and gradual attenuation of the rim of the vesicles. For example, Nath and Mohan (1929) in reference to the cockroach wrote: “From the time the circumnuclear ring of Golgi vesicles breaks away from the nuclear membrane, the majority of the vesicles continue to grow in size and become more and more fatty till in the most advanced oocyte (about 4 mm. long) they assume huge dimensions. . . . Pari passu with this enlargement the membrane of the Golgi vesicles becomes more and more attenuated. These enlarged Golgi vesicles are the fatty yolk.” Bonhag (1958), reviewing the work of Ries and van Wee1 ( 1934), states “that ‘lipochondria’ enlarge and multiply during oocyte growth, gradually accumulate fatty material, and transform into the lipid yolk of Pediculus. These authors considered the lipochondria to be comparable to the Golgi vesicles of Nath and his co-workers, but did not think that they could be homologized with the typical Golgi apparatus of vertebrate cells.” Nath (1958) has fully confirmed this view. The earlier conclusions of the author (see Nath, 1957), therefore, have received full support by the histochemical work under review, as the lipid bodies of the first two categories (L, and Ls of Nath and others) are homologous with the “Golgi vesicles” of Nath, and the third category of lipid bodies (L3 bodies of Nath and others) with the “fatty yolk” of Nath. Finally, the investigations of Nath and collaborators quoted here strongly point toward the conclusion that the lipid granules first arise either from or under the direct influence of the mitochondria. This view is strongly supported by the recent studies of Lever (1955) on adrenal cortex with the electron microscope.
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Acknowledgment I should like to thank Mr. B. L. Gupta for much technical assistance in the preparation of this review.
REFERENCES Baker, J. R. (1946) Quart. J. Microscop. Sci. 87, 441. Baker, J. R. (1949) Quart. J. Microscop. Sci. 90, 293. Baker, J. R. (1956) Quart. J. Microscop. Sci.97, 621. Berenbaum, M. C. (1958) Quart. J. Microscop. Sci. 99, 231. Blochmann, F. (1884,1887) Quoted by E. B. Wilson In “The Cell in Development and Heredity.” Macmillan, New York. Bonhag, P. F. (1955) J. Morphol. 97, 283. Bonhag, P. F. (1958) Ann. Rev. Entomol. 3, 137. Bradbury, S. (1956) Quart. J. Microscop. Sci. 97, 499. Brambell, F.W. R. (1924) Brit. 1.Exptl. Biol. 1, 501. Cain, A. J. (1947) Quart. J. Microscop. Sci.88, 383. Cain, A. J. (1948) Quurt. J. Microscop. Sci. 89, 429. Cain, A. J. (1950) Biol. Revs. Cambridge Phil. SOC.26, 73. Chiffelle, T. L., and Putt, F. A. (1951) Stain Technol. !28, 51. Chopra, H. C. (1958a) Quart. J. Microscop. Sci.99,149. Chopra, H. C. (1958b) Research Bull. Punjab Univ. 162, 211. Chopra, H. C. (1958~)Unpublished. Clayton, B. P. (1958) Quart. J. Microscop. Sci. 99,453. Deane, H. W. (1952) A m . J. Anat. 91, 363. Deane, H. W. (1958) In “Frontiers in Cytology” (S. L. Palay, ed.), p. 227. Yale Univ. Press, New Haven, Connecticut. Deane, H. W., and Barker, W. L. (1952) In “Studies on Testis and Ovary, Eggs and Sperm” (E. T. Engle, ed.), p. 176. C. C Thomas, Springfield, Illinois. Gatenby, J. B. (1922) Quart. J. Microscop. Sci.66,1. Gatenby, J. B., and Woodger, J. H. (1920) J. Roy. Microscop. SOC.40, 129. Govan, A. D. T. (1944) I. Pathol. Bacteriol. 66,262. Gupta, B. L. (1958a) Nature 181, 555. Gupta, B. L. (1958b) Nature 182, 537. Gupta, B. L. (1959) Experientia 16, 223. Gupta, B. L., Kaur, R., and Nath, V. (1959) Research Bull. Punjab Univ. rN.S.1 10, 390.
Guraya, S. S. (1957) Quart. J . Microscop. Sci.98,407. Guraya, S. S. (1958) Research Bull. Punjab Univ. 166, 245. Guraya, S. S. (195%) Sci. 6.Culture Calcutta 24,390. Guraya, S. S. (1959b) Research Bull. Punjab Univ. [N.S.] 10, 119. Guraya, S. S. (1959~) Research Bull. Punjab Univ. [N.S.] 10, 81. Hirschler, J. (1917) Arch. mikroskop. Anat. u. Entwicklungsmech. Abt. 11, 69, 1. Hotchkiss, R. D. (1948) Arch. Biochm. 16, 131. Kay, W. W., and Whitehead, R. (1941) J . Pathol. Bacteriol. 63, 279. Krishna, D. (1953) Quart. J. Microscop. Sci. 94,314. Krishna, D. (1958) Sci. 6.Culture Calcutta a3, 651. Lever, J. D. (1955) Am. J. Anat. 97, 409. Lillie, R. D. (1952) Stain Technol. 27, 37. Lillie, R. D. (1954) “Histopathologic Technic and Practical Histochemistry.” Blakiston Div., McGraw-Hill, New York.
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Lison, L. (1953) “Histochimie et Cytochimie Animales.” Gauthier-Villars, Paris. Ludford, R. J. (1921) J . Roy. Microscop. SOC.U , 1. Mazia, D., Brewer, P. A., and Alfert, M. (1953) Biol. Bull. 104, 57. Nath, V. (1931) Z . Zellforsch. u. mikroskop. Anat. 13, 82. Nath, V. (1932) Nature 190,204. Nath, V. (1957) Research Bull. Punjab Univ. B8, 145. Nath, V. (1958) 15th Intern. Congr. Zool. London p. 721. Nath, V., and Bhatia, C. L. (1944) Proc. Natl. Inst. Sci. India B10, 231. Nath, V., and Malhotra, S. K. (1954) Research Bull. Punjab Univ. 69, 149. Nath, V., and Malhotra, S. K. (1955) Research Bull. Punjab Univ. 68, 39. Nath, V., and Mohan, P. (1929) J. Morphol. and Physiol. 48, 253. Nath, V., and Nangia, M. D. (1931) J. Morphol. 62, 277. Nath, V., Singh, B., and Bakr, A. (1944) Proc. Natl. Inst. Sci. India B10, 247. Nath, V., Gupta, B. L., and Lal, B. (19%) Quart. J. Microscop. Sci. 99, 315. Nath. V., Gupta, B. L., and Manocha, S. L. (1958b) Quart. J. Microscop. Sci. 99, 475. Nath, V., Gupta, B. L., and Bains, G. S. (1958~) Research Bull. Punjab Univ. 148, 135. Nath, V., Gupta, B. L., and Kaur, R. (1958d) Research Bull. Punjab Univ. 159,223. Nath, V., Gupta, B. L., and Rani, V. (1958e) Research Bull. Punjab Univ. 1w,235. Nath, V., Gupta, B. L., and Aggarwal, D. K. (195%) Cellule 60, 79. Nath, V., Gupta, B. L., and Aggarwal, S. K. (1959b) Research Bull. Punjab Univ. [N.S.] 10, 315. Nath, V., Gupta, B. L., and Manocha, S. L. (1959~) Cellule 69, 385. Nath, V., Gupta, B. L., and Lal, B. (1959d) Research Bull. Punjab Univ. [N.S.] 10, 375. Sci. O ~92, . 4. Pearse, A. G. E. (1951) Quart. J. M ~ C T O S C Pearse, A. G. E. (1954) “Histochemistry.” Churchill, London. Ries, E., and van Weel, P. (1934) Z . Zellforsch u. mikroskop. Anat. 20, 565. Weber, A. F., Phillip, M. G., and Bell, T. T. (1956) J . Histochem. and Cytochem. 4, 308. Wigglesworth, V. B. (1950) “The Principles of Insect Physiology.” Methuens, London. Wigglesworth, V. B. (1957) Proc. Roy. SOC.B147, 185.
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I. Introduction Until very recently work on animal oogenesis has been carried out by what may be described as the orthodox classical techniques of fixation and staining, such as chrome-osmium, long osmication, and silver impregnation. At best these techniques gave us only a rough idea of the morphology and chemical composition of the cytoplasmic inclusions such as “Golgi bodies,” mitochondria, vacuome, nuclear extrusions, and yolk, fatty and protein, met with in animal oocytes. It was concluded that the Golgi bodies and the mitochondria contained lipids and lipoproteins respectively. Gatenby and Woodger (1920) showed that the “Golgi dictyosomes” secrete the fatty yolk in the oogenesis of Patella vulgaris. This claim was subsequently confirmed by Ludford (1921) and Brambell (1924) ; but it is important to note that Ludford clearly mentioned that some of the “Golgi elements” are directly metamorphosed into such yolk, as claimed by Hirschler (1917) for the ascidian egg. Speaking of centrifuged eggs of Saccocirrus, Gatenby (1922) wrote that “the upper cap is formed of delicate granules which, I think, are fatty yolk and probably of the Golgi elements ; these granules will go yellow-green after prolonged osmication.” Since 1928 the author has been laying great stress on the importance of the study of fresh oocytes for determining the form and functions of the Golgi bodies, mitochondria, and vacuome in oogenesis (see Nath, 1957, 305
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for references). Nath (1931) and Nath and Nangia (1931) showed for the first time in oogenesis that the neutral red-staining watery vacuoles of Parat were independent of the classic lipoidal “Golgi apparatus” and the mitochondria in the frog and fish respectively. But, as pointed out above, all these techniques give at best only a rough idea of the chemical composition of these cytoplasmic inclusions. During the last few years studies on oogenesis of some insects, spiders, fishes, frogs, reptiles, birds, and mammals have been carried out in this laboratory by the modern techniques of histochemistry mentioned below. These investigations have very clearly brought out that lipids may exist not only in the Golgi bodies and the mitochondria, but also, in many forms of oogenesis, in what is usually named as the “albuminous” or “protein” yolk. 11. Technique The histochemical methods for the study of lipids in animal cells have been excellently reviewed by Cain (1950) and more recently by Deane (1958). Further details of these methods can be found in many books, e.g., Pearse (1954), Lison ( 1953), Lillie ( 1954). Of all the various fixatives it has been found that formaldehyde-calcium followed by postchroming, as recommended by Baker (1946, 1956), with gelatin embedding, is undoubtedly the best for the preservation of lipids in oocytes. The presence of calcium ions in the fixative and the postchroming treatment seem to be indispensable for the proper preservation of lipids in oogenesis. Moreover, almost all the tests for lipids, with the possible exception of performic acid-Schiff for the unsaturated bonds (Pearse, 1954), can be tried on these sections. I n many cases the Lewitsky-saline technique of Baker (1956) may also be employed, particularly if the arrangement of cells is also to be studied, as the cell membranes are fixed much better in this fixative. Sudan black B used as a saturated ethanolic solution (Baker, 1949) has been found to be the most successful lipid colorant. This colorant may also be used in propylene glycol (Chiffelle and Putt, 1951), since in many cases it reveals much more of lipids than the alcoholic solution. Sudan I11 and IV (Kay and Whitehead, 1941 ; Govan, 1944) or Fettrot 7B (Pearse, 1954), generally recommended as the colorants for the neutral fats (triglycerides), are not at all specific; they can at best be considered as general lipid colorants but much less vigorous than Sudan black B. They can be employed most usefully, however, as counterstains for the acid-hematein (Baker, 1946) preparations. Such counterstained preparations are extremely useful in oogenesis since they present the various types of lipid bodies in different colors in the same preparation (see Nath et al., 1 9 5 9 ~ ) .
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It has often been found that Sudan black B does not color the larger lipid bodies in the oocytes homogeneously, owing to the presence of either solid or masked lipids (Gupta, 1958a). It is advisable, therefore, to try this colorant at 60” also. If even this technique fails, one or all of the various techniques for “unmasking” the lipids (Gupta, 1958a, 1959; Clayton, 1958; Berenbaum, 1958) may be tried before deciding finally that a lipid body really possesses a nonlipid core, as such “duplex” bodies do not seem to occur in the animal oocytes (Gupta, 1958b). It is unfortunate that no specific histochemical technique is available for the triglycerides, occurring widely in oogenesis. The most useful method available is the Nile blue sulfate method of Cain (1947, 1948) used with Keilig-type extractions as control. Thus a body which gives a negative reaction to the tests for other lipids, which appears pink in Nile blue sulfate, and which is soluble in cold acetone may be considered to be composed of triglycerides. The presence of unsaturated bonds is generally revealed by the performic acid-Schiff technique (Pearse, 1951 ; Lillie, 1952). However, the degree of saturation of the various lipids is best revealed by the graded postchroming technique of Ciaccio followed by paraffin embedding (Lison, 1953; Bradbury, 1956). Fresh cover slip preparations treated with 2% Os04 solution may also yield useful information in this respect (Wigglesworth, 1957 ; Nath, 1957). For cholesterols and their esters the modification of Schultz technique by Weber et al. (1956) has been found to give trouble-free results. Keilig-type extractions with the various lipid solvents (Pearse, 1954) have proved very useful in determining the detailed chemical nature of the composite lipid bodies (L, and L).Such extractions when tried on the fresh oocytes do not generally yield good results. More useful information may be gained by extracting gelatin sections of the material fixed in formaldehyde-calcium for 6 hours, either with or without postchroming. For further details reference may be made to Nath et al. (1958a). For comparison and additional information, mercuric-bromophenol blue (Mazia et al., 1953) for proteins and the periodic acid-Schiff test for carbohydrates (Hotchkiss, 1948 ; Pearse, 1954) are the most useful techniques.
111. Results
A. INSECTS Wigglesworth ( 1950) has described three chief types of insect ovaries, viz., ( 1 ) the panoistic type, in which there are no special nurse cells, (2) the polytrophic type, in which every oocyte has its own nurse cell or cells,
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and ( 3 ) the telotrophic type, in which every ovariole has a nutritive chamber or trophic core in its germarium.
1. Panoistic T y p e T o the best of my knowledge Nath, Gupta, and La1 (1958a), and Nath, Gupta, and Aggarwal (1959a) are the only workers who have studied the histochemistry of lipids in the oogenesis of Periplaneta amen’ccuna, and Chrotogonus trachypterus and Gryllodes sigillatus, respectively. a. Cockroach. Nath et d.(1958a) have described three kinds of lipid bodies in the cockroach: (1) L1 bodies, present in the earliest oocyte, which persist until the oocyte measures approximately 0.5 mm. and which contain phospholipids only, possibily having more lecithins than cephalins ; ( 2 ) Lz bodies, which first arise in the oocyte measuring 0.4 mm. and have a complete or incomplete sheath of phospholipids surrounding a medulla of triglycerides, and which are rather highly saturated; and ( 3 ) La bodies, which are the only types of lipids in the oocytes measuring more than 0.65 mm. and consist of rather highly saturated triglycerides. Some of the larger La bodies have been described as giving a “ringed” or “crescentic” appearance in Sudan black when used at room temperatures (12°C. to 40°C.) but appearing mostly homogeneous when the coloring agent is used at 60°C. The L1 bodies of these authors correspond to the “Golgi bodies” of Nath and Mohan (1929), but they do not have the duplex structure attributed to them. The duplex structure of the Golgi bodies of this cell appears to be due to incomplete reduction of osmium tetroxide. The Lz bodies, which directly arise from L1 bodies, have undoubtedly a duplex structure and correspond to the duplex Golgi vesicles of Nath and Mohan (1929). The LB bodies, which arise directly from L2 bodies, correspond to the “fatty yolk” of these authors. Nath et al. (1958a) studied the rodlets called “bacterioid forms” by Blochmann (1884, 1887), and Nath and Mohan (1929). These are situated just below the follicular epithelium, and they contain free fatty acids and phospholipids. Nath et al. (1958a) conclude that the bacterioid forms participate in lipid synthesis in the cockroach oocyte, as suggested by the fact that all the lipid bodies grow in size and number near the layer of bacterioid forms. This conclusion is further supported by the fact that when the synthesis of lipids is over the bacterioid forms are no longer positive to any lipid test. b. Chrotogonus and Gryllodes. Nath et d. (1959a) have studied the lipids in the oocytes of Chrotogonus trachypterus and Gryllodes sigillatus. The pattern of lipid synthesis in these two orthopteran forms is similar to the cockroach except that (1) L1 bodies in both the forms consist of
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lipoproteins, (2) both the L1 and Lz bodies in Chrotogonus contain “masked” lipids, and ( 3 ) there are no bacterioid forms. Gupta (195%) has made a valuable contribution in demonstrating the presence of some “masked” lipids in the oocytes of Chrotogonus and their “unmasking” by Sudan black B and phenol technique. This author found that a “ringed” or “crescent” appearance, produced by a sudanophil sheath surrounding a non-sundanophil core, was given by a great many of the lipid spheres. This occurred even in material fixed by formaldehydecalcium postchromined and stained in Sudan black B in propylene glycol at 60” for 1 hour. Various tests failed to demonstrate either cholesterol or its esters in these bodies. These results demonstrated that there must be a masking of the lipids in the core if they are present at all. Gupta (1958a) “unmasked” these lipids of Chrotogonus oocytes by fixing the material in formaldehyde-calcium for 6 hours and then treating it with 1% phenol solution at 37” for 24 hours. Sudan black B stained gelatin sections failed to show any “ringed” or “crescentic” structures ; instead the spheres were stained a uniform blue-black color. This demonstrates quite clearly that the cores of the lipid spheres in Chrotogonus oocytes were “masked” presumably by proteins as evidenced by the positive reaction they give with mercuric-bromophenol blue. Finally, it may be mentioned that Nath et al. (1958a) have described in the cockroach and Nath et al. (195%) have recorded in Chrotogonus and Gryllodes the presence of large quantities of lipid bodies, only of L1 and L2 types, in the follicular epithelial cells ; these latter, in the absence of special nurse cells, are the only source of raw materials to the oocyte.
2. Polytrophic T y p e In the polytrophic type of ovary each oocyte may have either only one nurse cell associated with it as in two species of earwigs (Nath et al., 1959b), or a number of them as in Culex fatigans (Nath et al., 1 9 5 8 ~ ) . As far as the author is aware, there is no other work on the lipids in this type of ovary. a. Culex fatiguns. Nath et al. ( 1 9 5 8 ~ )have demonstrated three types of lipid bodies again in the oocyte of Culex fatiguns, viz., L1, L,and L3. The L1 bodies, consisting of phospholipids and lipoproteins, give rise to the L2 bodies. The L2 bodies originally consist of phospholipids, lipoproteins, and triglycerides, but later they lose their protein constituents and then have triglycerides in their core and phospholipids in the cortex. A gradual attenuation of the phospholipid rim ultimately transforms the L2 bodies into L3 bodies ; the L2bodies, however, do not grow to form the Idsbodies as in many other forms.
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The lipid bodies of the nurse cells and the follicular epithelial cells are of L1 type only ; but the lipids are very scarce in the nurse cells. Nath et al. ( 1 9 5 8 ~ )have pointed out that most of the supply of the raw material to the oocyte in Culex comes through the follicular epithelial cells directly. The nurse cells seem only to provide their protein component to the growing oocyte, and they do not contribute to the oocyte any carbohydrate or lipid substances which could be detected histochemically. b. Earwigs, Labidura ripark, and L . bengalensis. Nath et al. (1959b) have again demonstrated three types of lipid bodies in the earwig, viz., LI, Lz, and Ls. The L1 bodies contain phospholipids only and directly give rise to LZ bodies, which contain triglycerides in their core and phospholipids in the cortex. In Labidura bengalensis the Lz bodies appear very late in the oocyte and are fewer in number as compared with L . riparia; they also contain some masked lipids, which were unmasked by phenol treatment. The L2 bodies in both species of earwigs directly give rise to larger Ls bodies, which contain triglycerides only. It has already been pointed out that in earwigs each oocyte carries with it its own nurse cell, from which it is cut off by a septum containing a central pore. Nath, Gupta, and Aggarwal have shown that in both the species of earwigs the associated nurse cell forms the chief source of the supply of lipids to the oocyte, in contrast with Culex, in which the nurse cells do not make any lipid contribution. These authors have seen a stream of mitochondria and L1 bodies passing from the nurse cell to the oocyte through the central pore in the septum; they also describe infiltration of lipid bodies through the septum itself.
3. Telotrophic Type The only paper on lipids of the telotrophic ovary is by Bonhag (1955). This author, working on Oncopeltus farciatus, states (1) that it is almost certain that sudanophil lipids are contributed to the oocytes by the apical trophic tissue ; (2) that there is circumstantial evidence that sudanophil lipids (of a non-phospholipid type) and phospholipids (or their precursors) are contributed to the oocytes by the follicular epithelium; ( 3 ) that in oocytes having a complete or nearly complete endowment of yolk, most of the sudanophil lipid occurs in distinctive bodies called the “coarse sudanophilic bodies” ; (4) that these bodies appear to be formed first in the oocytes from precursor bodies which utilize lipids contributed by way of the nutritive cords from the apical trophic tissue; ( 5 ) that in late oocytes, however, these “coarse sudanophilic bodies” seem to be formed from precursor bodies, which utilize lipids obtained from the follicular epithelium ; (6) that these “coarse sudanophilic bodies” are uniformly dispersed throughout the oocyte cytoplasm between the larger
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protein-carbohydrate yolk granules ; and (7) that there is a progressive decrease of phospholipids in the course of oogenesis. Nath et al. (1959d) in the telotrophic ovary of Laccotrephes maculatus and L . ruber have found three types of lipid bodies, viz., L1 bodies containing phospholipids and perhaps also phosphatidic acid, Lz bodies containing phospholipids and triglycerides, and L3 bodies containing triglycerides only. It is obvious that the LBbodies, and LIand L2 bodies of these authors correspond to the “coarse sudanophilic bodies” and to the “phospholipids” of Bonhag respectively. These authors also agree with Bonhag that the apical trophic tissue and the follicular epithelium are the only sources of lipid supply to the oocytes. B. SPIDERS Krishna (1953), working on the oocytes of the Indian water spider, Lycosa birmanica, states that the fine bodies resembling Golgi bodies are granular and contain “phospholipins, lipo-protein, and protein,” but no triglyceride. In a subsequent communication Krishna ( 195S), describing very briefly the chemical composition of the “yolk-nucleus” of Lycosa birmunica, states that this structure is free from triglycerides, and its concentric lamellae show a thick coating of “phospholipins” covering “lipo-proteins of varying consistency.” Nath et al. ( 1 9 5 9 ~ )have worked out the details of the lipids in the oogenesis of the spider, Plexippus paykulli, with special reference to the “yolk-nucleus.” They have shown that there is in this spider a very prominent yolk-nucleus consisting of four zones, viz., an innermost zone of diffused lipids, a clear zone, a zone of circularly arranged and closely packed mitochondria1 fibers, and an outermost zone of diffused lipids. The yolk-nucleus is the dynamic seat of lipid synthesis ; all the lipid bodies in the cell take their origin in the yolk-nucleus, and there are at least four distinct cycles of lipid synthesis in the yolk-nucleus. These authors have further described three categories of lipid bodies in this material, viz., L1 bodies consisting of phospholipids only, Lz bodies having a phospholipid core and a triglyceride sheath, and L3 bodies having triglycerides only. These authors have shown that the L1bodies are restricted to the innermost zone of the yolk-nucleus where they take their origin. They pass to the outer zones of the yolk-nucleus, and during this outward migration they develop a thick triglyceride sheath and thus form La bodies. These last accumulate in the outermost zone of the yolk-nucleus where they gradually lose their phospholipid content to form Ls bodies, which are released into the cytoplasm from the yolk-nucleus at the close
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of every cycle, in the form of a large mass. They gradually disperse in the cytoplasm. It is interesting to note that the L3 bodies do not grow, but remain granular throughout oogenesis. Nath, Gupta, and Manocha have also described large quantities of phospholipids and triglycerides in the yolk globules of Plexippus paykulli in addition to the protein-carbohydrate complex. Nath and associates (1958e) have shown that there is no true yolknucleus in the spider Crossopriza lyoni, the so-called yolk-nucleus of other authors (see Nath, 1957) being a mere aggregation of mitochondria and Golgi bodies. These authors have described in this spider also three types of lipid bodies, viz., L1, b,and Ls ; but the L1 and L2 bodies differ from the similar bodies in Plexippus inasmuch as they contain proteins also in addition to phospholipids, and to phospholipids and triglycerides, respectively. The L3 bodies of Crossopriza, which consist of pure triglycerides as usual, remain granular throughout oogenesis as in Plexippus. These authors have described five kinds of yolk globules, Y1 to YE. Of these, the Y1 globules consist of proteins and carbohydrates, blended with phospholipids and triglycerides, while the Y2 and Y3 globules contain mainly proteins and carbohydrates with small quantities of triglycerides. The Y d and YE globules do not contain any lipids.
C. EARTHWORM Nath et al. (1958b) in Pheretimu posthum have shown that the earthworm oocyte contains lipids of L1 and L2 categories only, the LBtype of lipid bodies being entirely absent. The L1 bodies are smaller, appear as homogeneous dark granules under the phase-contrast microscope, and have a protein-phospholipid core surrounded by a thick sheath of phospholipids only. The bodies, which arise as a result of growth and chemical change in L1 bodies, have a pure phospholipid core surrounded by a thick triglyceride sheath. They give a ringed appearance under the phase-contrast microscope ;but under the interference microscope this ringed appearance is shown to be an optical artifact (see also Gupta, 1958b). The lipid spheres present in the follicular epithelium contain phospholipids only. These authors have further shown that their L1 and L2 bodies correspond very closely to. the “Golgi granules” and Sudan IV-coloring spherules respectively of Nath and Bhatia ( 1944).
D. FISHES Chopra in this laboratory at Panjab University has carried out morphological and histochemical studies on the oogenesis of eleven species of fish with particular reference to lipids.
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I n his first paper (Chopra, 19%) this author has studied the cytoplasmic inclusions in the growing oocytes of the fish Ophiocephalus punctatus. The nonyolky oocytes of early stages show cytoplasmic granules of two types, namely, the mitochondria and the lipid granules of the first category (LI). The latter are bigger than the mitochondria, consist of phospholipids only, and correspond to the “Golgi bodies” of Nath and Nangia (1931). Chopra describes a second category of lipid bodies (L2) , consisting of phospholipids and triglycerides to begin with, but only triglycerides in the mature eggs. This author does not describe L3 bodies as such; however, it is obvious from his description that he has in his material two kinds of L2 bodies: L2 bodies proper consisting of phospholipids and triglycerides, and L3 bodies consisting of triglycerides only, as in most forms of oogenesis described here. Nath and Nangia (1931) had described two kinds of yolk globules only in Ophiocephalus, viz., “fatty yolk,” which corresponds to the triglyceride spheres of Chopra, and “albuminous yolk,” which develops in vacuoles. Chopra (1958a) has described this “albuminous yolk” of Nath and Nangia as a “vacuolar yolk” and has shown it to consist of carbohydrates and proteins. But this author has also described a third category of yolk globules containing proteins and lipoproteins, which do not develop in vacuoles. I t is this lipoprotein yolk which was completely missed by Nath and Nangia (1931) and Nath et al. (1944). I n his second publication on Barbus ticto Chopra (1958b) has described lipid granules consisting of phospholipids only and corresponding to the “Golgi bodies’’ of earlier workers. The author has also described in this material: (1) triglyceride yolk globules, corresponding to his L2 bodies in Ophiocephalus and the “fatty yolk” of earlier workers; (2) Y2 yolk globules, which are rich in proteins with some traces of triglycerides and which correspond to the “albuminous yolk” or “protein yolk” of earlier workers ; and ( 3 ) the “vacuolar yolk,” which develops in vacuoles and is very rich in carbohydrates with some traces of proteins. Chopra (19.58~)has studied the lipid bodies and yolk in nine more species of fish and has found, more or less, the same pattern of vitellogenesis as in Ophiocephalus and Barbus.
E. FROGS AND TOADS Nath et al. (1958d) have made histochemical studies of the cytoplasmic inclusions in the oocytes of Ram tigrina, with particular reference to lipids. There are three kinds of lipid bodies in the frog, viz., pure phospholipid L1 bodies, phospholipid-triglyceride L2 bodies, and pure triglyceride La bodies. Nath (1931, 1932) had worked earlier on the oogenesis of Rana tigrina
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and Rana cyanophlyctis. Nath (1931) had described certain dark grayish refractile bodies in the fresh young oocytes, which grow into tiny vesicles with a dark grayish cortex and a less refractile medulla in later stages. H e labeled these bodies as the “Golgi bodies.” Nath (1932) showed that in the breeding season some of the “Golgi bodies” grow into fatty yolk spheres. Nath and Malhotra (1954, 1955) later confirmed the findings of Nath that “Golgi vesicles” directly give rise to the fatty yolk spheres. Nath and Malhotra (1955) state that in the toad, the treatment of young oocytes with Sudan I V (Kay and Whitehead, 1941) demonstrates no positive elements but by the time the oocytes have reached a diameter of 0.35 mm. a reaction is given by the Golgi bodies. Subsequent growth of the oocytes is accompanied by an increase in the size of Golgi bodies and Nath and Malhotra believe that ultimately they form the fatty yolk. A positive reaction is given by the Golgi bodies with Sudan I V at all these stages and at the later period the yolk and the Golgi bodies cannot be distinguished by this reagent since they both stain positively with it. It is clear that the “Golgi bodies” of Nath and Malhotra not colored with Sudan I V are homologous with the L1 bodies of Nath et al. (1958d) ; “Golgi vesicles,” which color with Sudan I V in later stages, with the L2 bodies ;and the “fatty yolk spherules” with the La bodies.
F. REPTILES,BIRDS,MAMMALS Guraya in this laboratory has been engaged now for some years in the study of lipids in the oogenesis of reptiles, birds, and mammals. The histochemical investigations of this author, and of Gupta et al. (1959), have shown that the lipid synthesis in these forms is much more complicated, and there exist more than three categories of lipid bodies.
1. Reptiles Guraya (1959a) has described three types of lipid bodies (L1, Lz, L3) of different chemical composition and morphology in two species of lizards (Calotes versicolor, Uromastix hardwickii), two species of snakes (Lycodon aulicus aulicus, Boiga trigonuta) , and one species of fresh-water turtle (Lissemys punctata punctata) . The L1 bodies of Guraya (1959a) are in the form of granules and spheres consisting of phospholipids and triglycerides of an unsaturated nature. They correSpond to the so-called “Golgi bodies” of earlier workers on the oogenesis of reptiles and .react like them with osmium tetroxide and silver nitrate. The L3 bodies arise in the oocytes, when still young, in the form of
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comparatively large spheres, consisting of unsaturated triglycerides only. They seem to develop from L1 bodies by some chemical transformation, and they correspond to the “fatty yolk” of earlier workers. They are usually used up during the growth of the oocyte before the appearance of yolk globules. The L2 bodies of Guraya (19%) are a new category of lipid bodies not present in insects, spiders, earthworm, fish, and frog; they arise de novo in the early oocytes in the form of granules lying in groups, after the appearance of L1 and L3 bodies; and they consist of a peculiar type of phospholipids, which are fully preserved only after fixation in formaldehyde-calcium and postchroming as recommended by Baker ( 1946, 1956). The L2 granules grow into duplex vesicles, spheroids, plates, and various other forms having sudanophobe areas which react negatively to all the tests used; they develop simultaneously a small amount of triglycerides, which, however, are absent in the Lz bodies of Lissemys and Lycodon. bodies have either been missed by Guraya (1959a) states that his the earlier workers, or their incompletely fixed forms were also identified as Golgi bodies. Further, the L1 and L2 bodies are also present in the follicular epithelial cells with the exception of Lissemys, in which only L1 bodies are found. Guraya (1959a) has also observed the infiltration of L1 bodies from the follicular epithelium into the oocyte through the zona radiata. The yolk of ripe ova consists of two types of bodies: (1) triglyceride yolk spheres composed of unsaturated triglycerides and having no connection whatsoever with L3 bodies, and ( 2 ) compound yolk, corresponding to the albuminous or proteid yolk of earlier authors and consisting mainly of carbohydrates and proteins with some triglycerides and lipoproteins. Guraya (1958) had arrived at similar conclusions in the lizard Hemidactylus flawiviridis.
2. Birds Guraya (1959b) has investigated the oogenesis of three species of birds, viz., one of the fowl, Gallus domesticus, and two of the dove, Streptopelia senigalensis cambaiensis and S. decaocto decaocto. In these birds this author has described the same pattern of lipid bodies as in reptiles. In these species of birds also the L1 bodies consist of unsaturated phospholipids and triglycerides, corresponding to the so-called Golgi bodies of earlier workers on bird oogenesis; the L3 bodies, which seem to develop from L1 bodies as in reptiles, consist of unsaturated tri-
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glycerides and cholesterol and its esters, and correspond to the “fat globules” or “fatty yolk” of earlier workers. The L3 bodies disappear in the course of oogenesis as in reptiles. The L2 bodies correspond exactly to the Lz bodies of reptiles. They arise de novo at the periphery of the oocytes, have no connection with L1 and L3 bodies, and consist of some specific phospholipids with some amount of unsaturated triglycerides, which can be fully preserved only in formaldehyde-calcium followed by postchroming, as in reptiles. Again the La bodies appear under different forms as in reptiles. Guraya (1959b) has described three kinds of yolk globules in birds, all of which contain lipids. They are (1) triglyceride spheres of highly unsaturated nature, (2) homogeneous compound yolk spheres, consisting of carbohydrates, proteins, and lipoproteins, and (3) heterogeneous compound yolk spheres, with a matrix consisting of carbohydrates, proteins, and unsaturated triglycerides, in which are embedded spheres of homogeneous compound yolk. In his first publication Guraya (1957) has described exactly the same types of lipid inclusions in the oogenesis of the pigeon, Columba livia, but unfortunately has used a different terminology.
3. Mammals Deane and Barker (1952) and Deane (1952) have studied the distribution of lipids in the ovary of rat and sow, and of albino rat respectively during the estrous cycle. Their main object was to study the sites of steroid activity in the ovary, and for this reason they did not work out the details of the lipids in the normal oocyte. Gupta et al. (1959) have described four distinct categories of lipid bodies in the ovary of rat, viz., L1, Lz, L3, and fenestrated lipid bodies ( F N L ) . The L1 bodies consist of phospholipids, the L2 of phospholipids and neutral lipids, the L3 of neutral lipids, and the F N L bodies of phospholipids and traces of triglycerides. The normal oocyte of the rat, according to these authors, contains the L1 bodies only, beside the mitochondria and the yolk granules. The granulosa of the normal follicles contains L1, Lz, and F N L bodies. It may be noted carefully that the LZ bodies of the granulosa cells of the normal follicles are composed of phospholipid and triglycerides, but with the start of atresia the L2 bodies of the granulosa cells show the presence of steroids. It may be noted further that the presence of fenestrated lipid bodies ( F N L ) is a characteristic feature of the granulosa of the normal follicle; they start disappearing with the onset of atresia. The phospholipids of the F N L bodies can be preserved only after fixation in formaldehyde-calcium followed by postchroming in dichromate-
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calcium as recommended by Baker (1946, 1956) ; in this respect they are strictly homologous with the Lz bodies of Guraya described in reptiles and birds in this article. Deane and Barker (1952) and D a n e (1952) seem to have missed these lipid bodies completely, because they employed pure formaldehyde as a fixative and did no postchroming in dichromatecalcium. According to Gupta et al. (1959) the theca cells of the normal follicle also contain L1 and L2 bodies, but the latter contain some quantities of steroids in addition to phospholipids and triglycerides. On the contrary, the atretic follicles, both primary and vesicular, contain large quantities of L3 bodies in addition to the L1 and L2. Further, the Lz and L3 bodies of these follicles contain appreciable quantities of steroids in addition to the triglycerides. Gupta et d. (1959) have also described traces of phospholipids in the yolk bodies found in the normal oocytes of the rat. Guraya ( 1 9 5 9 ~ )described in the rabbit and the hare also the fenestrated type of lipid bodies, containing phospholipid and traces of triglycerides, in the normal follicles, having the same specific nature as described by Gupta et d. (1959) for the rat. Further, these bodies are homologous with the Lz bodies of Guraya described in the oocytes of reptiles and birds. Guraya ( 1959c) describes phospholipids and triglycerides in the yolk spheres also in the oocytes of rabbit and hare.
IV. Conclusions It is certainly possible now to make some general statements with regard to the scheme of lipid synthesis in animal oogenesis. A very important conclusion which has emerged from the investigations of Nath and his co-workers is that the synthesis of triglyceride globules in the oocytes occurs in three stages. The earliest lipid granules in oogenesis may consist of phospholipids only (as in cockroach, earwigs, water scorpions, Plexippus, fish, frog, and rat), or of phospholipids and proteins (as in mosquito, earthworm, and Crossopriza) , or of lipoproteins only (as in Gryllodes and Chrotogorcus) . With the growth of the oocyte these earliest lipid granules grow and increase in number greatly and develop triglycerides either in their cores (as in cockroach, Gryllodes, Chrotogonus, Culex, earwigs, and water scorpions), or in their cortex (as in spiders and earthworm). But in all vertebrate species reviewed here the phospholipids and triglycerides are mixed. I n the late stages of oogenesis, as a rule, the lipid spheres are large and consist of neutral lipids (generally triglycerides) only; but in Culex and
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in spiders the triglyceride bodies are small and granular. I n the alecithal eggs of the earthworm, however, pure triglyceride bodies do not develop at all, while in the normal oocytes of the rat the lipid bodies consist of phospholipids only, there being no triglyceride synthesis. Another very important conclusion which we have arrived at is the presence of an altogether new type of lipid bodies, occurring as “fenestrated” masses in the oocytes of the Amniota only; they consist of some specific phospholipids which can be preserved only in formaldehydecalcium followed by postchroming in dichromate-calcium. Again these bodies seem to be independent of the normal graded lipid bodies described above. In mammals these fenestrated bodies occur in the granulosa of the normal follicles only; they disappear as soon as these follicles start developing atresia. The author since 1928 has been developing and advocating the view that the “fatty yolk” in eggs is directly derived from the “Golgi vesicles” by the deposition of fat and gradual attenuation of the rim of the vesicles. For example, Nath and Mohan (1929) in reference to the cockroach wrote: “From the time the circumnuclear ring of Golgi vesicles breaks away from the nuclear membrane, the majority of the vesicles continue to grow in size and become more and more fatty till in the most advanced oocyte (about 4 mm. long) they assume huge dimensions. . . . Pari passu with this enlargement the membrane of the Golgi vesicles becomes more and more attenuated. These enlarged Golgi vesicles are the fatty yolk.” Bonhag (1958), reviewing the work of Ries and van Wee1 ( 1934), states “that ‘lipochondria’ enlarge and multiply during oocyte growth, gradually accumulate fatty material, and transform into the lipid yolk of Pediculus. These authors considered the lipochondria to be comparable to the Golgi vesicles of Nath and his co-workers, but did not think that they could be homologized with the typical Golgi apparatus of vertebrate cells.” Nath (1958) has fully confirmed this view. The earlier conclusions of the author (see Nath, 1957), therefore, have received full support by the histochemical work under review, as the lipid bodies of the first two categories (L, and Ls of Nath and others) are homologous with the “Golgi vesicles” of Nath, and the third category of lipid bodies (L3 bodies of Nath and others) with the “fatty yolk” of Nath. Finally, the investigations of Nath and collaborators quoted here strongly point toward the conclusion that the lipid granules first arise either from or under the direct influence of the mitochondria. This view is strongly supported by the recent studies of Lever (1955) on adrenal cortex with the electron microscope.
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Acknowledgment I should like to thank Mr. B. L. Gupta for much technical assistance in the preparation of this review.
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