Cytologia 39: 619-631, 1974

Cytochemical Studies on the Origin and Composition of Yolk in stollii ()

G. P. Verma1 and A. K. Basiston

Post-Graduate Department of Zoology, Berhampur University Orissa,

Received November 28, 1972

Broadly speaking two types of yolks (fatty and compound) have been found to be synthesized in the oocyte during vitellogenesis of different . However, these yolks vary from to insect in their origin and chemical composition. Whileorigin of fatty yolk has been attributed to mitochondria (Hsu 1953), to the dense bodies that arise within the mitochondrial aggregation (King 1960) and to nucleolarextrusions (Machida 1941), most of the workers agree with the fact that the Golgi elements get directly transformed into fatty yolk. The compound yolks have been recorded to originate from the nucleolar ex trusions (Nath and Mehta 1927, 1929, Nath and Mohan 1929, Bhandari and Nath 1930,Gresson 1931, Aggarwal 1964) on one hand, and possibly from mitochondria (King1960, Nath et al. 1958e, Sareen 1965) and from yolk precursors in the follicular epithelium(Nath et al. 1959d, Bonhag 1955, Aggarwal 1964) on the other hand. Similarlythe compound yolks also vary in their chemical nature in different insects. Thus, protein-carbohydrate nature of the yolk has been recorded by Nath et al. (1958a,c,1959 a,b,d), Gupta (1968), Verma and Das (1974); protein-phospholipid nature by King (1960); protein-acid mucopolysaccharide nature by Nath et al. (1959a, b), Aggarwal (1960, 1964) and King (1960). In order to ascertain the origin and chemical composition of different yolks in a bug , the present work was undertaken using cytochemical techniques.

Material and methods

Female specimens of Chrysocoris stollii were collected from the local field and rearedon the branches of their host plant Croton sparsiflorous in normal laboratory condition. The ovaries, which are of telotrophic type, were dissected out and fixed in differentfixatives: Carnoy, Bouin, 10% neutral formalin, Zenker, Helly, formol calcium,Aoyama and Champy. The materials fixed in formol-calcium were post chromedand embedded in gelatin for frozen sections, and those in other fixatives wereprocessed as usual for paraffin sections. Feulgennucleal test (Feulgen and Rossenbeck 1924) alongwith its trichloroacetic acidcontrol and methyl green-pyronin (Brachet 1942, 1953) before and after treatment with RNAase (as control) were adopted for the detection of nucleic acids. For

1 Present address: P. G. Department of Zoology., Bihar University, L. S. College, Muzaffar pur, India. 620 G. P. Verma and A. K. Basiston Cytologia 39 carbohydrates, periodic acid-Schiff (PAS) specific for 1:2 glycol group was attempted in the conventionall-manner before and after malt diastase (Pearse 1968) and also after acetylation and KOH reversal (McManus and Cason 1950). For the study of glycogen sections were stained with Best's carmine (Best 1906) before and after malt diastase. Acid mucopolysaccharides were detected by the alcian blue method of Steedman (1950) and dialysed iron method of Hale (1946). Mercuric bromophenol blue (Mazia, Brewer and Alfert 1953) for basic proteins and coupled tetrazonium with its performic acid, dinitrofluorobenzene, and benzoylation controls for the protein bound amino acids were performed as suggested by Danielli (1947). Frozen sections were stained in sudan black B (Baker 1946) using 70% ethanol and propylene glycol as media (Chiffelle and Putt 1951) for lipids. Sudan III and IV were used for the study of neutral fats (Kay and Whitehead 1941), while for the study of phospholipids acid haematin test of Baker (1946) was done. To differentiate phospholipids and neutral fats, nile blue sulphate (Cain 1947) was tried. Saturated and unsaturated nature of the lipids were detected by performic acid-Schiff of Lillie (1952) with bromination control (Lillie 1954). Golgi bodies were located with Da Fano's (1921) silver nitrate-gold chloride method. For mitochondria Kull's (1913) acid fuchsin-toluidine blue-aurantia method was adopted. The presence of mitochondria was further ascertained by Nadi-reaction (Gurr 1958, page 189) and succinodehydro genase (Gurr 1958, page 204) activity.

Observations Golgi bodies and lipids Golgi bodies of the early oocyte of Chrysocoris stollii appear as minute granules in the perinuclear zone (Fig. 1). As the growth proceeds the minute granules spread over the whole ooplasm (Fig.2), enlarge in size and start assuming duplex structure (Fig.3) in the cortical zone. During vitellogenesis these duplex bodies migrate to the central ooplasm. The perinuclear Golgi bodies in the early stage give positive reaction to per formic acid-Schiff (PFAS) indicating that they contain unsaturated lipids. When treated with acid-haematin (AH) of Baker, these Golgi elements gave positive reac tion for phospholipids. These unsaturated phospholipid content of Golgi bodies has been designated as Ll bodies. They appear as bluish granules in ethanolic propylene glycolic sudan black B (SBB) and intense blue in nile blue sulphate (NBS) preparations. During previtellogenesis the Li bodies spread up uniformly throughout the ooplasm, grow in size and become duplex in appearance near the periphery. The duplex structure appears due to the development of masked lipids in the Ll bodies of the cortical ooplasm. Such structures (duplex) have been called as L2 bodies. In lipid colourants (sudan) each of the L2 bodies has a sudanophilic "externum" and sudanophobic "internum" (Fig.4). They, however, give uniform colouration (with no duplex appearance) when treated with 1% phenol (Gupta 1958)for 48 hours prior to staining with SBB. This confirms the development of masked lipids in the zone of internum. These L2 bodies give an intense blue colouration in the externum after AH while the internum remains gray. In sudan III and IV preparation the externum stains pink whereas the internum orange. When treated with PFAS, 1974 Cytochemical Studies on the Origin and Composition of Yolk in Chrysocoris stollii 621 the L2 bodies give negative result in internum and positive in externum. Thus these indicate that the L2 bodies consist of unsaturated phospholipid sheath and a tri glyceride core of saturated masked lipids. During vitellogenesis the La bodies move towards the central ooplasm, where they are converted to L3 bodies by enlarging and assuming round sphere after loosing duplex appearance (Fig. 5). The L3 bodies stain uniform ly pink with NBS, orange with sudanIII and IV, and negatively with AH showing the presence of triglyceridesand absence of phos pholipids in them. Besides, negative reaction with PFAS reveals their saturated nature. The L3 bodies are considered to be the fatty yolks, which are scattered here and there in bet ween the compound yolk glo bules(Fig. 5). Compoundyolk The compound yolk bodies formed during oogenesis of Chrysocorisstollii can be divided intothree categories according to their source of development and different chemical constituents. These yolks develop from three different sources: 1) nucleolar extrusions,2) mitochondria, and 3) yolk precursors in the fol licular epithelium. Figs. 1-3. Camera lucida diagrams of oocytes (Da Yolksoriginating from the nucleo Fano •~430) with follicular epithelium (FE). 1, show lar extrusions ing perinuclear Golgi bodies (GB). 2, showing dispers ed Golgi bodies. 3, showing duplex appearance of Golgi At the time of differentiating bodies. oogonia the nucleoli are clearly identifiedand contain RNA (Fig. 6), proteins and little carbohydrate. The ooplasm, however,does not take the stain of RNA or carbohydrate, though a weak reaction withHg-bromophenol blue indicates the presence of little protein in it. In several oogonialnuclei the nucleolus is seen dividing into few nucleoli-the nucleolar ex trusions,at this stage. When the growth period starts and the ova measure 0.084mm in diameterthe minute nucleolar extrusions fill up the nucleoplasm (Fig. 7) and ulti matelystart diffusing in the ooplasm. These extrusions now fuse together to form severallarger granules. These granular extrusions give strong positive reactions to PAS(Fig. 8), Best's carmine (negative after malt diastase), Hg-bromophenol blue and pyronin(negative after RNAase) indicating the presence of polysaccharides (1:2 622 G. P. Verma and A. K. Basiston Cytologia 39 glycol group), glycogen, basic proteins and RNA respectively. During further growth when the ova measure 0.140mm in diameter these nucleolar extrusions migrate to the

Figs. 4-8. 4, photomicrograph showing duplex L2 bodies (sudan III and IV). •~430. 5,

photomicrograph showing L2 bodies (arrow), L3 bodies (FY) and compound yolk (CY) (sudan III and IV)•~1000. 6, photomicrograph showing differentiating oogonia with pyrophil nucleoli

(visible as clear spaces) in the methyl green-positive nucleoplasm (visible as dark bodies) (methyl green/pyronin). •~430. 7, photomicrograph showing PAS-positive minute nucleolar extrusions in the oocyte nucleus. •~1000. 8, photomicrograph showing dispersing PAS-positive nucleolar extrusions in the ooplasm (unfortunately tangential section passing through little

portion of the oocyte nucleus).•~430. 1974 Cytochemical Studies on the Origin and Composition of Yolk in Chrysocoris stollii 623 peripheryof the oocyte (Fig. 9), where they enlarge to form round yolk globules, which in due course disperse throughout the ooplasm (Fig. 10). The chemical constituents of the nucleolar extrusions persist in these yolk bodies. The maximum sizeof these yolks has been recorded to be 0.006mm in diameter.

Figs. 9-13. 9, photomicrograph showing PAS-positive materials in the peripheral ooplasm. •~430. 10, photomicrograph showing PAS-positive yolk globules dispersing throughout the ooplasm. •~

430. 11, photomicrograph showing mitochondria (arrow) and developing fuchsinophil (dark) and aurantiaphilic yolk bodies (light) (after Kull). •~430. 12, photomicrograph showing ap

pearance of yolk (arrow) in between the mitochondria (after Kull). •~430. 13, photomicrograph showing bicoloured yolk (after Kull). •~1000. 624 G. P. Verma and A. K. Basiston Cytologia 39

Yolks originating from mitochondria The terminal filament (TF) of the ovary is devoid of any mitochondria easily resolvable. When the oocyte starts developing the mitochondrial granules appear in the perinuclear zone (Fig. 14) as red granules in Kull's acid fuchsin-toluidine blue - aurantia preparation. Later on, as the oocyte grows and measures 0.075mm in dia meter they become more or less uniformly distributed in the ooplasm (Fig. 15). The mitochondrial granules in due course (when the ova are 0.195 mm in diameter) start concentrat ing in the cortical ooplasm (Fig. 16) and enlarging in size. These mitochondrial granules give blue colouration in Nadi and deep red colouration in succinodehydro -genase reactions indicating the activities of cytochrome oxidase and succinic dehydrogenase re spectively in them. Besides,they have also been observed react positively to acid haematin test, Hg-bromophenol blue and pyro nin (negative after RNAase) showing phospholipids, proteins and RNA respectively as their constituents. During previtel logenesis the mitochondrial gra nules start decreasing in number and yolk bodies appear in between them (Figs. 17 and 12). These

Figs. 14-17. Camera lucida diagrams (after Kull). •~ yolk bodies have been referred to 430. 14, showing perinuclear mitochondria. 15, as mitochondrial yolks. When the showing dispersed mitochondria. 16, showing con oocyte is 0.210mm in diameter centration of mitochondria in peripheral ooplasm. these yolk bodies migrate towards 17, showing appearance of yolk (Y) in between the the centre to choke up the whole mitochondria (M). ooplasm. When treated with Champy-acid fuchsin-toluidine blue-aurantia (after Kull 1913), the mitochondria and the developing yolk bodies of oocyte measuring 0.406mm in diameter are conspicuously marked (Fig. 11). Thus while mitochondria and certain yolk bodies are stained red with acid fuchsin some of the yolk bodies are bicoloured (Fig. 13) with both acid fuchsin and aurantia, and others with only aurantia. At this stage no yolk bodies are stained with toluidine blue. This indicates that the mitochondria first swell up to form fuchsinophil yolk bodies, which in turn start converting into aurantiaphilic yolks. The yolk bodies having both stains (acid fuchsin and aurantia) are the intermediate stage between the acid fuchsin-and aurantia-positive yolks. The different histochemical tests reveal that these yolk bodies are composed of 1974 Cytochemical Studies on the Origin and Composition of Yolk in Chrysocoris stollii 625 proteins,phospholipids and RNA (Fig. 18). Such yolk bodies attain the maximum sizeof 0.039mm in diameter. Yolksoriginating from yolk precursors in the follicular epithelium During vitellogenesis, when the oocyte measures 0.700mm in diameter, the

Figs. 18-21. 18, photomicrograph showing RNA-positive yolk bodies (methyl green/pyronin). •~ 430. 19, photomicrograph showing yolk precursors in follicular epithelium (arrow) and acid mucopolysaccharide-positive yolks (alcian blue). •~430. 20, photomicrograph showing bicoloured (arrow), toluidine blue-positive (dark) and aurantiaphilic (light) yolk bodies (after Kull). •~430. 21, photomicrograph showing frothy yolk bodies (after Kull). •~1000. 626 G. P. Verma and A. K. Basiston Cytologia 39 minute granular yolk precursors are seen in the follicular epithelium (Fig. 19). These yolk precursors are histochemically composed of acid mucopolysaccharides, proteins and RNA; migrate to the peripheral ooplasm where they enlarge and form yolk bodies. In Champy-acid fuchsin -toluidine blue-aurantia prepara tion these yolk bodies are toluid ine blue-positive. Some of them are, however, stained with both toluidine blue and aurantia (Fig. 20), and others with only auran tia. This indicates the transfor mation of toluidine blue-positive yolks into aurantiaphilic ones and the bicoloured yolk globules are the intermediate stage. None of the yolk bodies, however, takes the stain of acid fuchsin. These yolk bodies measure 0.045mm in diameter and contain acid muco polysaccharide (Fig. 19), protein

Fig. 22. Schematic diagram of an ovariole showing yolk bodies' cycle during oogenesis of Chrysocoris stol lii (for details see text). CY1-com pound yolk originates from nucleolar extrusions and is composed of 1:2 glycol group, glycogen, protein and RNA; CY2-compound yolk origin ates from mitochondria and is com posed of phospholipids, proteins and RNA; CY3-compound yolk origin ates from the yolk precursors in fol licular epithelium and is composed of acid mucopolysaccharide, protein and RNA; FY-fatty yolk originates from Golgi bodies and is composed of triglycerides; FrY-frothy yolk; BY1-Bicoloured yolk both acid fuch sin and aurantia positive; BY2-bico loured yolk both toluidine blue and aurantia positive.

and RNA, since they react positively with alcian blue and dialysed iron, Hg-bro mophenol blue and pyronin (negative after RNAase). When the egg becomes mature and ready to be laid, some of the compound yolks present a very interesting picture. They appear frothy, showing a number of round vacuoles (Fig. 21) in their interior. This frothy appearance is conspicuous in Bouin's 1974 Cytochemical Studies on the Origin and Composition of Yolk in Chrysocoris stollii 627

fixedpyridine extraction technique, Carnoy's fixed material and in Champy-acid fuchsin-toluidineblue-aurantia preparations. However, the frothy appearance is not seen after formol-calcium/post chroming technique. Thus it seems that the vacuolationsof the yolk bodies in the mature egg is due to the loss of some alcohol or pyridine-soluble material, which of course, is not lipid, since they are sudan ophobic. The whole cycle of these yolk bodies during oogenesis of Chrysocoris stollii can be surveyed in Fig. 22.

Discussion While Gresson (1930) and Payne (1932) are of the opinion that the Golgi ele ments take no part in the formation of fatty yolk, most of the workers, who have studied the yolk formation, agree with the fact that the Golgi elements get directly transformed into or at least take some part in the yolk formation. A number of cytologistswith the help of histochemical tests have found three types of lipid bodies (L1,L2 and L3) in the Golgi zone during oogenesis of different including insects. Nath and others (1958a) in Periplaneta have homologized these L1, L2 and L3 bodies with the 'Golgi bodies', 'Golgi vesicles' and 'fatty yolk' respectively of Nath and Mohan (1929), Gresson (1931) and Ranade (1933). These lipid bodies (L1,L2 and L3) have been observed in the present study, similar to those described by Nath and others (1958a,b, 1959a,b) and Aggarwal (1962, 1964). The specific histo chemicaltests have revealed that the L1 bodies are homogeneous granules of un saturatedphospholipid nature. The L2 bodies are duplex in appearance and each with a unsaturated phospholipid sheath and a saturated triglyceride core. The L3 bodiesare triglyceride spheres of saturated nature . The transformation of the Ll into L2 and L3 bodies during oogenesis is very similar to that described in different insectsby Nath and others (1959a,b) and Aggarwal (1962, 1964). Machida (1941) has observed two types of fatty yolks during oogenesis of silk worm: a) fatty yolk arising from the Golgi elements of the oocyte, trophocytes and the follicular epithelium and b) fatty yolk arising from the nucleolar extrusions of the trophocytes. In the same insect Aggarwal (1962) has pointed out that the fattyyolk (L3 bodies) is derived from duplex L2 bodies, which have originated from L1bodies. Machida observed synchronization of the appearance of the nucleolar extrusionsand the fatty yolk b, on the basis of which he concluded the derivation of latter from the former . Aggarwal with the help of his histochemical studies finds that the fatty yolk a and b of Machida correspond his L1 and L2, and L3 bodies res pectively. He further argues that mere synchronization of the appearance of the nucleolarextrusions and the fatty yolk does not mean that one is derived from the other. The gradual variation of size is bound to be there when the fatty yolk b (L3 -triglyceridebodies) is derived from the fatty yolk a (L1 and L2 bodies) as revealed fromhis studies. In the present study also no evidence has been found as to the originof fatty yolk from the nucleolar extrusions . There are different opinions regarding the participation of nucleolar extrusions in the synthesis of compound yolk. While Nath and Mehta (1927, 1929), Nath and Mohan (1929), Bhandari and Nath (1930), Gresson (1931) and Aggarwal (1964) 628 G. P. Verma and A. K. Basiston Cytologia 39 believe that nucleolar extrusions are directly involved in the formation of proteid yolk in different insects, Bonhag (1959),Aggarwal (1960)and Anderson (1964)feel that the nucleolar extrusions do not directly give rise to yolk in certain insects. Though some of the authors believe the yolk bodies originated from the nucleolar extrusions are composed of protein-carbohydratecomplex, they are of the opinion that these extrusions contribute only the protein part of the yolk (and hencethe proteid yolk) while the carbohydrate moiety is being supplied by the follicularepi thelium (Nath et al. 1958c, Anderson 1964). Vincet (1955),Brown and Ris (1959), Sareen (1961, 1962, 1965) have observed some carbohydrate in the nucleolusof different 's oocytes but they deny any contribution of carbohydratemoiety of the proteid yolk by the nucleolar extrusions. Nucleolus and nucleolarextrusions of germinal vesicle during early period of oogenesis in Chrysocorisstollii contain protein, RNA and carbohydrate. During further growth these extrusionsmigrate into the ooplasm, where they enlarge and are transformed into yolks, which also contain RNA and protein in addition to carbohydrate. Since these extrusionsin the ooplasm react positivelywith Hg-bromophenol blue and PAS it is evidentthat both the protein and carbohydrate contents of the yolks are contributedby them. Besides,no PAS-positivematerial has either been seen in or coming out of the folli cular epithelium in the present case. Such yolk spheres of Chrysocoris,however, resemble broadly speaking, in their histochemicalnature, the yolk spheres of cock roach (Nath et al. 1959d), Culex (Nath et al. 1958a), Chrotogonus,Gryllodes and Labidura(Nath et al. 1959a,b),Laccotrephes (Nath et al. 1959d),Oncopeltus (Bonhag 1955),Locusta (Gupta 1968) and Oxya (Verma and Das 1974) inasmuchas they are protein-carbohydratein nature. Nath et al. (1958a,c; 1959a,b,d) and Aggarwal (1962) have studied the dis tribution and chemical composition of mitochondria during oogenesis of several insects. But in no case the involvement of mitochondria in the formation of yolk has been recorded. Unlike to these authors, however, King (1960) in Drosophila, has found alpha yolk spheres, which possibly originate from the mitochondria and are composed of phospholipids and proteins. Similar to his findings the present authors have also noted the remarkable indulgement of mitochondria during ooge nesis of Chrysocoris in the synthesis of yolks, which have been designated by them as mitochondrial yolks. At first the mitochondrial granules are perinuclear in posi tion, then disperse uniformly throughout the ooplasm, in the cortical zone of which they concentrate, enlarge and convert to aurantiaphilic yolk bodies. These yolks are composed of phospholipids, proteins and RNA. Among arachnids also, Nath et al. (1958e) and Sareen (1965) have found the synthesis of some of the yolk glo bules under the influence of mitochondria. Yolk precursors in the follicular epithelium have been observed by several workers (Nath et al. 1959d, Bonhag 1955, Aggarwal 1964) in different insects. Simi lar to these, in the case of Chrysocoris during vitellogenesis the yolk precursors are noted in the follicular epithelium. These come out to the ooplasm where they grow to form yolk bodies, which are composed of acid mucopolysaccharide, protein and RNA. Histochemically these yolk spheres of Chrysocoris resemble the yolk spheres of Chrotogonus (Nath et al. 1959b), Labidura (Nath et al. 1959a), Perip- 1974 Cytochemical Studies on the Origin and Composition of Yolk in Chrysocoris stollii 629

planeta(Aggarwal 1960) and Tenebrio (Aggarwal 1964). King (1960) has also observ ed such yolks (beta spheres), which are composed of acid mucopolysaccharide and phospholipidsin the case of Drosophila. From the above account it seems that histochemically four types of yolks are synthesizedfrom different sources during oogenesis of Chrysocoris stollii. Thus the yolks of fatty nature from Golgi bodies, yolks of carbohydrate/protein/RNA nature from nucleolar extrusions, yolks of phospholipid/protein/RNA nature from mitochondriaand yolks of acid mucopolysaccharide/protein/RNA nature from the yolkprecursors in the follicular epithelium. During maturation period, when the eggs are ready to be laid, some of the yolk spheres of Chrysocoris are becoming frothy in appearance due to the develop mentof vacuoles in their interior. Such frothy yolks have also been observed in the caseof cockroach (Nath et al. 1958c), Chrotogonus and Gryllodes (Nath et al. 1959b) and Culex (Nath et al. 1958a). The frothy globules were erroneously described as vacuolatednucleolar extrusions by earlier workers like Hogben (1920), Nath and Mohan (1929) and Gresson (1931). The possibility of joining of smaller yolk globulesto form larger frothy globules has been suggested by Bonhag (1956) and Nath et al. (1959b). When studied with histochemical techniques, it was found by Nath and his collaborators that the yolk globules present a frothy appearance both in PAS and Hg-bromophenol blue only after certain technique, viz. pyridine extrac tion method, hot ethanol extraction, chloroform/methanol extraction and Carnoy's. And they give a perfectly homogeneous appearance after the other fixatives like formaldehyde-calcium,Orth's, etc. This led them to the conclusion that the frothy appearancemight be due to the removal of some alcohol or pyridine-soluble material by the various solvents. Similar conclusion has also been drawn by the present authors in the case of Chrysocoris, since the frothy appearance of the yolk is lost in formol-calcium/post-chroming technique.

Summary

Four types of yolk bodies develop during the oogenesis of Chrysocoris stollii: 1. Yolks, which originate from the Golgi bodies and are of fatty (triglycer ides)nature. 2. Yolks, which originate from the nucleolar extrusions and are of carbo hydrate-protein-RNA nature. 3. Yolks, which originate from the mitochondria and are of phospholipid -protein-RNA nature. 4. Yolks, which originate from the yolk precursors in the follicular epithelium and are of acid mucopolysaccharide-protein-RNA nature. The involvement of different ooplasmic organelles and follicular epithelium in thesynthesis of these yolks has been discussed.

Acknowledgements

The authors are highly thankful to Dr. C. C. Das, Professor and Head of the Post-Graduate Department of Zoology , Berhampur University for his supervision 630 G. P. Verma and A. K. Basiston Cytologia 39 and encouragements throughout the course of present work. Thanks are also due to University Grants Commission, India for the award of Junior Research Fellow ship to one of us (A.K. B.), during the tenure of which this work could be done.

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