Cytochemical Studies on the Origin and Composition of Yolk in Chrysocoris Stollii (Hemiptera)
Total Page：16
File Type：pdf, Size：1020Kb
Cytologia 39: 619-631, 1974 Cytochemical Studies on the Origin and Composition of Yolk in Chrysocoris stollii (Hemiptera) G. P. Verma1 and A. K. Basiston Post-Graduate Department of Zoology, Berhampur University Orissa, India 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 insects. However, these yolks vary from insect 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 Chrysocoris stollii, 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