The Formation of the Secretory Granules in the Liver-cells of the Slug, Anadenus altivagus

In a previous publication on the liver-cells of the slug, Anadenus altivagus (Rishi, 1956), I have described the morphological aspect of the Golgi bodies and their transformation into the secretory granules as seen under the phase-contrast microscope. In that paper the probable origin of the Golgi granules of dark contrast (Praesubstanz of Hirsch, 1939) from the tip-granules of the fibrillar mitochondria was described. These Golgi granules were shown to form the Golgi spheroids, which ultimately were transformed into the secretory granules.

In the present communication I have examined the same phenomenon in the liver-cells of the same species of slug, in fixed material. Special care has been taken to employ the techniques of known reactions. Techniques in-volving excessive precipitation of metallic silver or osmium have been carefully avoided.

The material for the present study of the liver-cells of the slug, Anadenus altivagus Theobald, was fixed during the months of August and September 1955 at Simla, Panjab, India. Small pieces of the liver were removed from the living animals and placed directly in various fixatives. The fixatives employed were Helly, Regaud, Aoyama, formaldehyde-saline, Altmann, and Champy. The material was also postchromed in a saturated solution of potassium dichromate at 37° C for 48 h after Helly, Aoyama, and formaldehyde-saline. The paraffin method of embedding was employed. Sections were cut at 5 μ.

The sections of the postchromed material fixed in Helly, Regaud, Aoyama, and formaldehyde-saline were coloured with Sudan black B according to the technique described by Thomas (1948). The best results were obtained from the Regaud-fixed material.

The material fixed in Altmann and Champy was post-osmicated in a 2% solution of osmium tetroxide at 37° C for 48 h. Unstained preparations of Altmann material were also studied. The silver nitrate technique of Aoyama was also tried, but it introduced serious artifacts in the tissue.

Some sections of the Regaud-fixed material were also stained with 0-5% iron haematoxylin.

Some of the slides fixed in Regaud were first coloured with Sudan black, and the position of particular cells noted. The Sudan black was then extracted in 70% alcohol and the slides were stained with 0-5% iron haematoxylin to elucidate the relationship between the sudanophil granules (Golgi granules) and the mitochondria.

The cells of the liver of Anadenus, in the earliest secretory phase, show a large number of sudanophil granules filling up the whole of the nuclear end of the cell, which is on the side opposite to the lumen of the alveolus. These granules are very closely packed in this region. It also appears that some diffuse sudanophil material is present between the granules. These sudanophil granules correspond to the ‘Golgi’ granules of the author (Rishi, 1956). Besides these there is no indication of the presence of any secretory granules in such cells, although a few duplex spheroids (appearing as sudanophil rings or crescents in optical section), with a sudanophil rim and a sudanophobe sphere, may be seen towards the lumen end of the cell (fig. 1, A).

If such cells are stained with iron haematoxylin after the extraction of the Sudan black, one can make out a large number of fine mitochondrial fibrillae, each fibrilla bearing one deeply staining granule at each tip (fig. 1, B). Some of the mitochondrial fibrillae, however, do not show these granules. In addition to the tip-granules of the mitochondria there are other small, deeply-staining granules present in this region. These are comparable to the Golgi pre-substance of the author (Rishi, 1956). The mass of the sudanophil granules does not take up haematoxylin at all. The cytoplasm of this region, however, stains slightly more deeply with haematoxylin than the cytoplasm of the lumen end.

With further progress in the secretory activity of the cell, the number of sudanophil granules increases in the basal end of the cell (fig. 1, c). It also appears that these granules now start spreading towards the lumen end of the cell. In addition to these solid sudanophil granules, one can now make out a few spheroids of varying sizes, appearing as crescents or rings in optical sections, in the cytoplasm of the lumen end of the cell. Among the spheroids can also be seen some solid sudanophil bodies, representing the intermediate stages between the spheroids and minute granules.

With advancement in the process of secretion the sudanophil granules shift further towards the lumen end of the cell and become transformed into the spheroids of duplex structure, with a completely sudanophobe ‘internum’ or medulla surrounded by a complete or incomplete sudanophil sheath (fig. i, D). The number of spheroids seems to be inversely proportional to that of the sudanophil granules. The latter seem to be continuously drifting towards the lumen end of the cell and becoming differentiated into the spheroids.

The duplex spheroids in the lumen end of the cell become larger and larger and their sudanophobe medulla seems to grow at the expense of the sudanophil cortex, which appears to become attenuated progressively under the pressure of the medullary growth, till ultimately a completely sudanophobe secretory granule is formed. The cell in this stage still exhibits a marked polarity in the distribution of its contents; the small sudanophil granules occupying one end of the cell, while the secretory granules and the growing spheroids occupy the other end, which is invariably the lumen end (fig. 1, E). The amount of secretory granules continues to increase in the cell with a corresponding decrease in the amount of the sudanophil material. Ultimately the cell becomes filled up with the secretory granules, among which some sudanophil granules may be dispersed (fig. 1, F).

Neither the sudanophil granules, nor the duplex spheroids, nor again the secretory granules take up any haematoxylin after Regaud fixation.

Osmium tetroxide after Altmann blackens the sudanophil granules and the rim of the spheroids but leaves the secretory granules untouched. An exactly similar picture is presented by Altmann preparations that have not been postosmicated.

The fully formed granules, and even the larger immature secretory ones, generally appear to be surrounded by a clear space, giving the appearance of a granule within a vacuole. But since I have never seen such a space encircling the secretory granule in the living material studied under the phase-contrast microscope (Rishi, 1956), these spaces are presumably artifacts produced by shrinkage on fixation. .

The present study not only confirms my earlier observations (Rishi, 1956), but also elucidates the following points.

1. The secretory granules, as well as the grey medulla of the duplex spheroids, are non-lipid in nature as they do not take up Sudan black in postchromed Helly, Regaud, or formaldehyde-saline material. The secretory granules are also completely osmiophobe and chromophobe in stained and unstained chromeosmium preparations.

2. The dark sheaths of the duplex spheroids as well as the solid granules are lipid in nature, since they take up Sudan black intensely and are also osmiophil in unstained Altmann preparations. It seems that the sudanophil and osmiophil sheaths of the duplex spheroids undergo some chemical change and become transformed into the sudanophobe and osmiophobe secretory material.

An almost similar process in the formation of a secretory product has been described by Bhatia (1945) in the oil-gland of the common Indian duck.

Many other authors also have described the elaboration of the secretory granules in the medulla of the ‘Golgi bodies’. Hirsch (1939) has shown that the secretion is differentiated in the internum of the Golgi system (duplex spheroids). Similarly Hsu (1947, 1948) has shown that the secretory droplets in the mid-gut epithelium and salivary glands of the larvae of Drosophila melanogaster are elaborated in the ‘Golgi bodies’. According to this author, as the individual ‘Golgi granule’ increases in size, a light area (secretion droplet) appears in it, forming what he calls the ‘Golgi-material- and-secretion complex’. Duthie (1934), working on the Harderian gland of the rat, showed that the secretory granules arise in the vacuole-like medulla of the ‘Golgi apparatus’, which is surrounded by an osmiophil crescentic cortex. The granule later on fills the medulla completely.

Worley and Worley (1943) also describe a similar origin of the fat and the protein in the developing veliger larva of the tectibranch mollusc, Navanax inermis.

Thomas (1948) showed that the smallest secretory granules in the sympathetic neurones appear to be formed within the lipoidal pellicle of a single ‘Golgi system’, for it can be shown that they are at first completely covered with a sudanophil sheath. He homologizes the smallest osmiophil and sudano-phil bodies to the Praesubstanz of Hirsch (1939).

Similarly, Nath in a number of publications since 1924 has been advocating the direct origin of fatty yolk from the ‘Golgi vesicles’ of duplex structure in many forms of oogenesis. For a full bibliography of the subject reference may be made to Nath (1957).

Some authors believe that chromophil or dark sheath surrounding the developing secretory granule breaks itself loose from the fully formed secretory granule, assumes the form of the ‘Golgi granules’, and restarts the process of secretion. This view does not seem to be applicable in the present case, because, as has been pointed out earlier, the lipoidal sheath of the duplex spheroids seems to disappear during the formation of the secretory granules, exactly as described by Nath (1957). I myself have already described how the ‘Golgi granules’ differentiate from the granular ‘Golgi pre-substance’, which in turn seems to arise as tip-granules of the fibrillar mitochondria (Rishi, 1956). A similar view on the origin of the Golgi bodies (lipoidal bodies) has been expressed by Hirsch (1939).

In Sudan black preparations of these cells it is not possible to differentiate any mitochondrial material or pre-substance granules. This is due to the fact that the lipid granules are so closely packed in the basal region of the cell that it is not possible to make out any underlying structure. But if the Sudan black of such preparations is extracted and the slides restained with iron haematoxylin, one can clearly make out fibrillar mitochondria in the region of the cell where the densely packed sudanophil material was lying. Most of them have a deeply stained granule at each end. One can also see a few deeply-staining granules lying separately amongst the mitochondrial fibres, whereas some of the mitochondrial fibres are devoid of the tip-granules. It appears to the author that these granules, which seem to correspond to the Praesubstanz of Hirsch (1939) and the pre-substance of the author (Rishi, 1956), arise as tip-granules of the mitochondria, from which they later disassociate. These pre-substance granules seem to act as nuclei for the lipid synthesis, and later become ‘Golgi’ granules (lipid granules) by the accumulation of lipid round them. Such a view has also been advocated by Bourne (1951).

It is interesting that the mitochondrial origin of lipid granules and the subsequent conversion of the latter directly into secretory granules has been described by Fujimura (1921) in the human placenta and decidua.

In none of the fixed preparations has the author been able to make out any 240 Secretory Granules in the Liver-cells of Anadenus structure that could be homologized with the ‘canaliculi’ described by Lacy (1954) in the cells of the pancreas.

In conclusion it may be stated that in the liver-cells of Anadenus there are no structures that can possibly be compared with the networks of Golgi.

I wish to thank Prof. Vishwa Nath for providing facilities for the work, and for his ever-ready help and encouragement throughout the study. I also thank Mr. B. L. Gupta, Technician, Panjab University Department of Zoology, for a great deal of skilful practical assistance.