The stratification of various cell organelles and of important chemical substances have been studied in the eggs of the slug, after moderate centrifugation.

As in other molluscs the egg contents stratify typically into three well-defined zones - lipid, hyaline and yolky zones - but a distinct equatorial band of inclusions consisting mainly of phagosomes and associated lysosomes was detected in the most centrifugal region of the hyaline zone.

The sub-stratification of various cell inclusions in their respective zones was determined in some detail.

The role of the cell membrane and egg cortex in the redistribution of these inclusions and the nature of the ergastoplasm are discussed in the light of electron-microscope studies of eggs of this slug and of the sea urchin.

The centrifuge has been an extremely valuable tool in cytological and cytochemical research, especially on eggs. Unlike differential centrifugation and fractionation procedures used to isolate cellular components in artificial media, by this method the cell inclusions could be concentrated within their cell membrane and studied in their natural environment.

Such studies have been made of eggs of various other molluscs, including Limnaea (see Raven, 1958). Recently, centrifuged eggs of the sea urchin have been examined with an electron microscope (Mercer & Wolpert, 1962). The main aim of this paper is to determine in some detail the stratification of various cell organelles and of certain important chemical substances in a molluscan egg.

Entire egg capsules with ova, 2-cell and 4-cell stages, were subjected to a centrifugal force of 950 g (at 3750 rev/min) for 6 min using a Cambridge electric centrifuge. The embryos were then quickly decapsulated in tap-water, rinsed in isotonic saline (0·28 % NaCl) and examined by vital methods or fixed, sectioned and studied by cytological and cytochemical methods.

Vital studies included examination of embryos by phase-contrast microscopy and after vital staining and incubating them alive for certain enzymes - ‘vital enzymology’ (Sathananthan, 1968). For phase-contrast work the embryos were squashed in isotonic saline and examined under oil immersion with a Wild M 20 phase-microscope. Vital staining was carried out according to Baker (1958). Embryos were stained in Grubler’s neutral red, Nile blue, toluidine blue and methylene blue for 5–40 min using a 0·01 % solution of the dye in isotonic saline, rinsed, squashed and examined with an apochromatic oil-immersion objective. Most of the enzyme tests were carried out successfully by incubating alive whole stratified embryos in various media and examining them in much the same way as in the vital staining procedure. Cytochrome oxidase, succinic dehydrogenase, acid phosphatase, benzidine peroxidase and thiamine pyrophosphatase were the enzymes studied in this manner (Table 1).

Table 1.

Summary of important staining and cytochemical reactions of centrifuged embryos

Summary of important staining and cytochemical reactions of centrifuged embryos
Summary of important staining and cytochemical reactions of centrifuged embryos

Centrifuged embryos were also fixed in a wide variety of fixatives, including Altmann, Champy, Mann, Helly, Zenker, Ciaccio, Perenyi, Bouin, Carnoy and acetone/ethanol, impregnated with celloidin and rapidly embedded in ceresin/wax (56 °C) according to Peterfi’s method (Pantin, 1960). Serial sections (4 μm thick) of each embryo were cut with a Cambridge rocking microtome and the distribution of mitochondria, lysosomes, Golgi, yolk and important cytochemical substances such as lipids, nucleic acids, polysaccharides, muco-substances, vitamin C, iron, calcium, pigment, thiamine pyrophosphatase and alkaline phosphatase were determined using appropriate methods (Table 1). Some embryos were fixed in formaldehyde/saline (10% neutral formalin in 0·28 % NaCl), embedded in 15 % gelatin (Pearse, 1961) and frozen sections (6 μm thick) were cut in a cryostat. These were used to study lipids and acid phosphatase activity. All colour reactions were visualized with a Zeiss apochromatic objective and photomicrographs were taken with a Zeiss photomicroscope using Ilford Pan-F film.

Fig. 1.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 1. General distribution of cell organelles in lipid, hyaline and yolky zones.

Fig. 1.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 1. General distribution of cell organelles in lipid, hyaline and yolky zones.

Fairly good stratification of various cell inclusions and very sharp demarcations between three well-defined zones (fat cap, hyaline and yolky zones) were obtained immediately after centrifugation (Figs. 1, 6). Broadly speaking the relative amounts of lipid andprotein present in an inclusion seems to determine its stratification. Centrifuged eggs are oval but on standing they round up in a few minutes and redispersion of certain inclusions begins almost immediately, especially between the lipid and hyaline zones. The demarcation between the hyaline and yolky zones, however, persists for about 1 h or more. Prolonged centrifugation tends to constrict the embryo at the equator and sever it into two segments. If centrifuged eggs are left to develop in saline or within the capsule cleavage continues apparently normally though slightly retarded. The cleavage furrows, when established, prevent further redistribution of visible inclusions.

As in other molluscs (Raven, 1958) the egg contents stratify typically into three distinct major zones (Fig. 1) according to their specific gravity: (a) centripetal oil cap zone, which is lightest and consists of lipid globules; (b) intermediate clear hyaline zone with most of the ergastoplasm, mitochondria, lysosomes, phagosomes, nuclear inclusions and asters; (c) centrifugal yolky zone, which is heaviest and consists mostly of yolk spherules and Golgi bodies. The most remarkable feature in this embryo is that an equatorial band of inclusions consisting mainly of albumen vesicles (phagosomes) and lysosomes was detected in the most centrifugal region of the hyaline zone.

The axis of stratification is usually more or less parallel or is oblique to the animal-vegetal axis of the embryo, and the polar bodies are usually attached to its lighter half. Exceptionally they may be found attached to the yolky half.

Substratification of cell organelles

The distribution of various cell organelles in their respective zones will now be dealt with. Most of the important cell constituents stratify in the hyaline zone (Fig. 1).

Ergastoplasm is the chief component of this zone. It consists of ground cytoplasm (hyaloplasm), ribosomes and elements of the endoplasmic reticulum (ER). These observations are supported by cytochemical evidence and also ultrastructural studies of normal eggs (Sathananthan, 1966). The presence of ribosomes is deduced by the stratification RNA in this zone (Fig. 12). Electron micrographs of unstratified eggs show that there is an abundance of free ribosomes in ova and that the ER is sparsely developed and consists of vesicular elements (microsomes) and a few elongate cisternae, sometimes associated with mitochondria and yolk spherules. Further, strands of ER were also seen after staining with pyronin and Sudan black and some of these may remain in the yolky zone on account of their association with yolk spherules.

Mitochondria are easily stainable with acid fuchsin and show cytochrome oxidase and succinic dehydrogenase activities. They stratify chiefly in the hyaline zone, forming a very broad band above the equator (Fig. 10). They are more abundant towards the equator and decrease gradually in number towards the oil cap zone. There is invariably an almost clear region just below the fat cap with few visible inclusions, which presumably abounds with ribosomes and perhaps microsomes. A few mitochondria are always seen in the yolky zone evenly distributed between the yolk spherules or form rows or chains around the latter.

Lysosomes stratify among the mitochondria in the hyaline zone and could be best demonstrated by the acid-phosphatase test and the periodic acid/Schiff’s (PAS) reaction. They also show vital metachromasy with toluidine blue and are neutral red-positive and contain peroxidase. They are, however, more abundant towards the equator and together with larger bodies called phagosomes, with which they are associated as ‘satellites’, they form a distinct supra-equatorial band (Figs. 1, 7–9). The phagosomes are, in fact, tiny albumen vesicles which stratify above the equator in the most centrifugal region of the hyaline zone. Albumen is ingested at the egg surface from the very onset of development (Sathananthan, 1968). Perhaps this is the first time that such a layer has been detected in a molluscan egg.

Nuclear inclusions, etc. Nuclei with nucleoli, maturation and mitotic figures and associated chromosomes stratify in the hyaline zone. The nuclei are sometimes displaced centripetally towards the oil cap (Fig. 6). The second maturation figure and chromosomes are often seen on a side associated with the first polar body (Fig. 7) and are not displaced from their normal site at the animal pole. The mitotic figures lie freely in the hyaloplasm and usually occupy a more central position in the hyaline zone (Fig. 9).

The heaviest inclusions, chiefly consisting of yolk and Golgi bodies, stratify in the yolky zone (Figs. 1, 9, 11).

Golgi bodies exist in a variety of forms and range from tiny spheres (dictyoles), duplex vesicles, crescents, bean-shaped bodies to rodlets, as revealed in Mann-Kopsch preparations, where they are heavily impregnated with osmium (Fig. 1). They also show thiamine pyrophosphatase activity and are easily stainable with the vital dyes used. While the dictyoles and vesicles are independent objects the other dictyosomes are associated with yolk spherules and are inseparable on centrifugation. The dictyoles and vesicles which have yolky interna usually stratify in the most centripetal region of this zone just below the equator and a few may occasionally invade the clear hyaline zone.

Yolk spherules could be best demonstrated by tests for thiamine pyrophosphatase and alkaline phosphatase and by the PAS reaction. They are also stained by vital dyes but less intensely than Golgi bodies. Each spherule is duplex in structure, with a membranous externum and a yolky internum. They vary in size and are sometimes arranged in their zone along a gradient, the smaller ones being more centripetal while the larger mature ones are displaced centrifugally. Yolk is chemically very complex, being glyco-lipo-protein in nature, with proteins predominating.

Stratification of chemical substances

These substances are either found freely or are bound to some cell organelle, in which case their stratification will conform to that of the latter. The cytochemical methods used to identify these substances are given in Table 1.

Lipids

Free lipids stratify mainly in the oil cap zone (Figs. 1,7). The oil cap stains very intensely with Fettrot and Sudan black B and consists of numerous minute globules and also some diffuse fat. Certain lipid inclusions remain in the yolky zone (Fig. 7) and appear as blobs or flecks in association with a few mitochondria. All these lipid inclusions are composed of both neutral fats and phospholipids. There are also discrete membrane-bound lipid bodies (lipo-chondria) which stratify close to the oil cap zone or are associated with asters or are found in polar bodies. They are probably overgrown lysosomes impregnated with lipid (Sathananthan, 1968). They have a strong affinity for Sudan black and are stained by vital dyes such as neutral red and Nile blue and hence seem to be predominantly phospholipid. Sudan black also stains the membranebound lipids of various cell organelles (Fig. 7). Thus mitochondria, lysosomes, Golgi, yolk membranes, plasma and vitelline membranes, and even strands of the ER appear in these preparations. These protein-bound or masked lipids are essentially phospholipid in nature. The phagosomes that stratify at the equator, chromosomes and nucleoli too have appreciable amounts of lipid.

Oxidative enzymes

When centrifuged eggs are incubated for cytochrome oxidase and succinic dehydrogenase, the sites of enzyme activity closely correspond to the distribution of mitochondria (Fig. 2). The hyaline zone gives an intense positive reaction, the region above the equator being most intense. On closer examination, the mitochondria are the chief sites of enzyme activity. Those in the yolky zone and the Golgi bodies there are also intensely positive. The results obtained on incubation for succinic dehydrogenase, however, are less striking than those obtained for cytochrome oxidase (see Sathananthan, 1970).

Fig. 2.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 2. Cytochrome oxidase-and succinic dehydrogenase-positive granules (mitochondria and Golgi).

Fig. 2.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 2. Cytochrome oxidase-and succinic dehydrogenase-positive granules (mitochondria and Golgi).

Acid-phosphatase activity

Lysosomes, phagosomes and Golgi bodies are strongly acid phosphatase-positive (Fig. 3). When incubated by the azo dye method the hyaline zone is intensely stained and there appears the supra-equatorial band which consists of phagosomes and associated lysosomes. The Golgi bodies are found distributed among the yolk spherules, which are acid-phosphatase-negative. Acid-phosphatase-rich granules, chiefly lysosomes and a few lipochondria, are also seen around nuclei and asters in the hyaline zone and in polar bodies.

Fig. 3.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 3. Acid phosphatase-positive granules (lysosomes, phagosomes and Golgi).

Fig. 3.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 3. Acid phosphatase-positive granules (lysosomes, phagosomes and Golgi).

Benzidine peroxidase activity

A remarkable reaction is seen when centrifuged eggs are tested for this enzyme (Fig. 4). A sharply defined, intense blue, supra-equatorial band appears in about 20 min of incubation, similar to that observed in Limnaea (Raven, 1958). The picture resembles very much the reaction seen in embryos incubated for acid phosphatase. When examined closely the blue band is seen to consist of fine irregular granules (lysosomes) and larger rounded bodies (phagosomes). A few of these inclusions are also found in the hyaloplasm, especially around nuclei and asters. A diffuse cytoplasmic reaction in the band region is also evident and needle-like spicules are sometimes seen among the inclusions, which is probably an artifact. The identification of benzidine peroxidase as an intrinsic component of lysosomes and phagosomes, perhaps for the first time, is rather significant (see Sathananthan, 1968).

Fig. 4.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 4. Benzidine peroxidase activity (pronuclei are copulating).

Fig. 4.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 4. Benzidine peroxidase activity (pronuclei are copulating).

Thiamine pyrophosphatase activity is almost exclusively limited to the yolky zone (Fig. 11). The Golgi bodies and yolk membranes (externa of yolk spherules) are intensely positive while the yolk interna also show some activity. Golgi are probably involved in vitellogenesis and the yolk membranes are very likely derived from Golgi membranes (Sathananthan, 1966). Nucleoli in the hyaline zone are also positive.

Alkaline-phosphatase activity

Results are exactly similar to those obtained for thiamine pyrophosphatase activity.

Nucleic acids

Free RNA stratifies in the hyaline zone (Fig. 12) and presumably corresponds to the distribution of ribosomes in the hyaloplasm. A few strands of ER that remain in the yolky zone are RNA-positive and these seem to be associated with yolk spherules. Bound RNA is mainly found in the yolk membranes. The mitochondria in the hyaline zone are also feebly positive while the nuclear RNA is also stainable. The presence of RNA was confirmed with ribonuclease. DNA is found in the nuclei that stratify in the hyaline zone but is rather difficult to demonstrate in ova. The chromosomes, however, are readily DNA-positive and could be easily stained with acid fuchsin in mitochondrial preparations (Fig. 10).

Polysaccharides and mucosubstances

Glycogen appears finely granular and stratifies in the hyaline zone (Fig. 5). It could be stained with Best’s carmine or PAS reagent and removed with saliva. Traces of galactogen could also be demonstrated in the phagosomes by the same methods, by predigesting with pectinase.

Fig. 5.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 5. Glycogen (fine stippling) and vitamin C (coarse granules).

Fig. 5.

Figs. 1–5. Illustrations showing distributions of various cell inclusions in centrifuged ova.

Fig. 5. Glycogen (fine stippling) and vitamin C (coarse granules).

The PAS reaction also reveals larger polymorphic granules (lysosomes) in the hyaline zone and a fairly distinct equatorial band consisting of phagosomes and associated lysosomes (Figs. 8, 9) corresponding to that seen after incubation for acid phosphatase and benzidine peroxidase. Some of these inclusions are also seen, as usual, around nuclei and asters and in polar bodies. In the yolky zone, the yolk membranes and Golgi are also intensely PAS-positive, while the flecks of lipids give a weak reaction. Various mucosubstances and glycolipids are known to answer the PAS test.

Fig. 6.

v.s. mature ovum (Helly/acid fuchsin).

Fig. 6.

v.s. mature ovum (Helly/acid fuchsin).

Fig. 7.

v.s. ovum at second maturation (Helly/Sudan black B). Note equatorial band and flecks of lipid in yolky zone.

Fig. 7.

v.s. ovum at second maturation (Helly/Sudan black B). Note equatorial band and flecks of lipid in yolky zone.

Fig. 8.

v.s. ovum at second maturation (Helly/PAS). Note equatorial band.

Fig. 8.

v.s. ovum at second maturation (Helly/PAS). Note equatorial band.

Fig. 9.

v.s. 2-cell stage (Helly/PAS). Note phagosomes and lysosomes at the equator.

Fig. 9.

v.s. 2-cell stage (Helly/PAS). Note phagosomes and lysosomes at the equator.

Fig. 10.

v.s. ovum at second maturation showing mitochondria in hyaline zone (Helly/acid fuchsin). Note redispersion of lipid has begun.

Fig. 10.

v.s. ovum at second maturation showing mitochondria in hyaline zone (Helly/acid fuchsin). Note redispersion of lipid has begun.

Fig. 11.

v.s. ovum fixed in acetone/ethanol and incubated for thiamine pyrophosphatase.

Fig. 11.

v.s. ovum fixed in acetone/ethanol and incubated for thiamine pyrophosphatase.

Fig. 12.

v.s. ovum showing distribution of RNA (Kelly/pyr on in Y). Note diffuse staining in hyaline zone and pyroninophily of yolk granules.

Fig. 12.

v.s. ovum showing distribution of RNA (Kelly/pyr on in Y). Note diffuse staining in hyaline zone and pyroninophily of yolk granules.

The distribution of acid mucopolysaccharides (AMP) in the hyaline zone also conforms to that of the phagosomes and lysosomes but the results are not as striking as those obtained with the PAS reaction due to a weaker reaction and a diffuse staining of the hyaloplasm. Aldehyde fuchsin, Alcian blue and Hale’s reagent were used to demonstrate AMP. More striking results were obtained using vital metachromasy. Lysosomes and phagosomes stain purple with toluidine blue, methylene blue and sometimes with Nile blue. The hyaline zone as a whole, however, stains diffuse purple while a more intense meta-chromatic band is seen above the equator corresponding to that seen earlier. The diffuse staining could be due to some free AMP and also RNA that stratifies here, which is feebly metachromatic. AMP in bound form is also present in the Golgi bodies, yolk membranes (externa), nucleoli, plasma and vitelline membranes. All results indicate that most inclusions have a higher content of sulphated AMP, the exceptions being the yolky interna and vitelline membrane which have more non-sulphated AMP.

Vitamin C exists freely as sharply defined coarse granules (1–2 μm in diameter), strongly resembling Golgi bodies, and stratifies in the yolky zone (Fig. 5). The substance, however, is not bound to Golgi or any other inclusion.

Iron

Ferric iron is found distributed diffusely as fine irregular particles (0·3–1 μm in diameter), which stratify in the yolky zone. It seems to be also bound to Golgi bodies and to a lesser extent to nucleoli.

Calcium is bound to Golgi bodies and yolk spherules that stratify in the yolky zone. The yolk membranes are more intensely positive than their interna. Nucleoli are also calcium-positive. Golgi seem to be involved in the metabolism of calcium.

Pigment

Golgi bodies and yolk spherules have a yellowish-brown pigment which has been identified as melanin. Its distribution is similar to that of calcium.

The fat cap is also pigmented brown, but this pigment has not been confirmed as melanin.

The results show that by moderate centrifugation one could obtain a fairly sharp stratification of cell inclusions. This undoubtedly has been most valuable in subcellular studies of this egg. Although stratification is sharp, it is by no means complete. A few mitochondria and elements of the ergastoplasm are always seen in the yolky zone. Likewise some lipid inclusions are retained in the yolky half or may invade the hyaloplasm. Exceptionally a few Golgi bodies or yolk spherules may be found in the hyaline zone. This confirms the view that complete stratification cannot be achieved by the moderate centrifugation commonly used in embryological studies.

The centrifugal force causes an inevitable stretching of the cell membrane but it is capable of regaining its original form when the force ceases to act. This indicates that the membrane is fairly elastic in nature. The tension developed in it probably causes the egg to round up soon after centrifugation. In Aplysia the elastic recovery of the spherical shape by the elongated egg after centrifugation is thought to play an important part in the redistribution of its cytoplasmic contents (see Raven, 1958). This seems to be the case in Arion.

It has been also postulated that in certain molluscs the egg cortex may play a part in the redistribution of inclusions (ooplasmic re-segregation) after centrifugation, and that it is the seat of polarity of the egg (Raven, 1958). It is believed that this cortex is a gel that cannot be displaced by moderate centrifugation. This is also the commonly accepted view in the case of the sea urchin. There is no structural evidence of a cortical gel layer in the egg of this slug. Electron micrographs of normal eggs (Sathananthan, 1966) show a well-defined ‘unit’ cell membrane and a zone usually devoid of larger granules (about 1 μm thick) below it, corresponding to a clear agranular cortical region seen with the light microscope. This zone has an abundance of ribosomes and also vesicular elements of the ER and those of micropinocytotic origin. Occasionally larger inclusions like mitochondria and lysosomes invade it and are found very close to the cell membrane, indicating the absence of a cortical gel layer. Further, ultrastructural studies of both normal and centrifuged eggs of the sea urchin (Mercer & Wolpert, 1962) have failed to detect a gel-like cortex, beneath the cell membrane. These authors have, however, shown that the small particulate cytoplasmic fraction (probably ergastoplasm) appears to be relatively unaffected by centrifugation and forms a continuous ‘ground substance’ in which are found the larger mobile inclusions, such as mitochondria and yolk granules. This, indeed, is the impression gained by the present study. The incomplete stratification of the ER and undoubtedly of the hyaloplasm which suspends the granules even in the yolky zone is evidence that the ergastoplasm is relatively unaffected. A similar electron-microscope investigation of centrifuged eggs, however, needs to be carried out for further confirmation of these views.

I am most grateful to Professor Alastair Graham and Dr Vera Fretter for providing the facilities for my work at the University of Reading, U.K., and for their supervision and encouragement.

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  • EB

    equatorial band

  •  
  • ER

    endoplasmic reticulum

  •  
  • G

    Golgi

  •  
  • HZ

    hyaline zone

  •  
  • L

    lysosome

  •  
  • LG

    lipid globule

  •  
  • LZ

    lipid zone

  •  
  • M

    mitochondrion

  •  
  • m

    microsome

  •  
  • N

    nucleus

  •  
  • n

    nucleolus

  •  
  • P

    phagosome

  •  
  • PB

    polar body

  •  
  • R

    ribosome

  •  
  • Y

    yolk

  •  
  • YZ

    yolky zone