20 e. s. duthie. - royal...

20 E. S. Duthie.

Hill and Parkes (1932). ‘ Proc. Roy. Soc.,’ B, vol. 112, p. 138.Moller-Christensen (1933). “ Acta Path, et Microbiol. Scand.,” vol. 10, p. 136.Smith (1930). ‘ Amer. J. Anat.,’ vol. 45, p. 205.Thompson (1932). ‘ Endocrinol,’ vol. 16, p. 257.White (1933). ‘ Proc. Roy. Soc.,’ B, vol. 114, p. 64.

DESCRIPTION OF PLATE.

F ig. 7. Reproductive organs of the adult male hedgehog during the breeding season. Killed May 19. X J.

Fro. 8.—Reproductive organs of the hypophysectomized male hedgehog during the breeding season. Hypophysectomized February 24. Killed May 13. x f .

612.343

Studies in the Secretion of the Pancreas and Salivary Glands.

By E. S. D uthie, M.B., M.Sc., Rockefeller Foundation Fellow in ExperimentalCytology.

(From the Zoological Department, Trinity College, Dublin, Ireland.)

(Communicated by J. A. Murray, F.R.S.—Received May 10, 1933.)

In recent years considerable attention has been paid by various workers to the cytological constituents of various gland cells. In invertebrates following the lead of Parat and Painleve (1924), the so-called salivary glands of Chiro- nomous have been the favourite objects of study (Beams and Goldschmidt, 1930 ; Gatenby, 1932), while in vertebrates the pancreas has been more usually chosen (Parat, 1928 ; Coveil, 1928 ; Ludford, 1930 ; Beams, 1930 ; Gatenby, 1931).

The chief object of these researches has been to discover what relationship, if any, exists between the Golgi apparatus and the cell bodies revealed by intra- vitam staining with neutral red (so-called vacuome of Parat), but the conclusions reached have been in every case conflicting. In consideration of the great diversity of opinion existing between these workers it was hoped in the present instance that a reinvestigation of the cytology of the pancreas together with the salivary glands might throw light on the subject, since the problem in each must be fundamentally the same. The objects of the present work were then :—

(a) A complete morphological study of the Golgi apparatus in the cells of these glands.

on May 24, 2018http://rspb.royalsocietypublishing.org/Downloaded from

http://rspb.royalsocietypublishing.org/

Secretion of the Pancreas and Salivary Glands. 21

(c) A determination of the relationship between these structures and theknown cell constituents.

(d) A study of the various secretory stages in the cell activity.

The first portion of the paper deals with the pancreas, the salivary glands being treated in the second part. The author here takes the opportunity of thanking Professor J. Bronte Gatenby, under whose inspiration the work was carried out, for his helpful criticism and advice. The animal experimentation was carried out at 33, Upper Fitzwilliam Street, Dublin.

Part I .—Pancreas.

Material and Methods.

The mouse and frog were chosen for this study, the former being most suitable for the experimental studies, while the large secretory and fat granules in the latter were very helpful in fixed preparations. Fixation for mitochondria was carried out by Zenker formol or by Regaud’s fixative, the latter being injected through a blood vessel following saline perfusion. For the Golgi apparatus the best results were obtained by following Nassonow’s technique (1924). Fixation in Champy’s fixative for 12-16 hours was followed by 12 hours washing in running water and incubation for 8 hours in 2% osmic acid at 40° C. followed by 3-6 days at 37° C. Fixation with Mann-Kopsch was also used, but had no advantages. Fat granules were shown by fixation in Champy alone or by prefixation in a formol containing fixative such as Regaud’s or Hellv’s fluid, followed by 1 day at 30° C. in 1% osmic acid. Using Regaud’s fixative this allowed mitochondrial demonstration as well. Intra-vitam staining with neutral red w'as carried out with various doses, depending on the degree of staining required. In general in the mouse two doses of 0*5 c.c. of 0*5% solution of neutral red administered intra-peritoneally at intervals of half an hour gave excellent results, the animal being killed 1 hour after the last dose. The neutral red used was supplied by the National Anilin Company of New York. Staining supra-vitally with neutral red was carried out with a solution 1 : 30,000 neutral red (light red colour) at 37° C. and usually occurred in about 5 minutes. Similar results were obtained by perfusion in the rat. In order to ensure good results, it is advisable to use several strengths of solution either stronger or weaker than this. I t is extremely important that the neutral red solution should be quite fresh as stock solutions deteriorate. Mitochondria were stained by Hoechst Janus green, 1 : 100,000.



22 E. S. Duthie.

Observations on the Living Cell.

The method of Ilirsch (1932, 6) was adopted and was found to be perfectly satisfactory. Cibalgin 0*6 c.c. of 1/10 solution was used as an anaesthetic. Exhaustion of the pancreas was carried out by four injections of 0*25 c.c. of 0*1% solution of pilocarpine administered at half-hour intervals. Neutral red was then administered 1 hour later by two intra-peritoneal injections of 0*5 c.c. of 0-5% solution of neutral red in Ringer at 37° C., the operation being commenced some 20 minutes later.

Operation.

Having clipped the hairs on the left side over the splenic region, a short cut is made in the skin parallel to the lower border of the ribs and across the j unction of the back and lower surface of the animal. The skin having been retracted the underlying muscle is cut, and the peritoneal cavity opened by a short incision over the lower border of the spleen which can usually be seen at this stage. The lower pole of the spleen is then gripped with a suitably padded forceps, and is withdrawn through the wound bringing with it the attached mesentry and tail of the pancreas. The animal is now strapped to a suitable stage attached to the mechanical stage of the microscope, and the spleen manipulated gently with cotton swabs soaked in warm saline so that the pancreas tail comes to rest on a glass slide. Saline is added to the preparation, and a very thin coverslip, specially blown, is dropped over the end. If the operation has been successfully performed, it is possible to view the cells where the preparation is not more than two or three cells thick with the oil immersion lens using strong illumination. Very little, if any bleeding will occur in a successful experiment. Trouble may sometimes occur owing to the pancreas tail being too short, or through its being tightly bound to the spleen by thick mesentry. Starvation of the animals for at least 24 hours is always essential, since an empty stomach facilitates drawing out of the pancreas. The reader is referred to the work of O’Leary (1930) and Hirsch (1932, a, b) for further details.

In animals not previously stained, staining may be carried out during the experiment by the addition of 1 : 30,000 neutral red to the preparation, mitochondria being stained in the same manner by 1 : 100,000 Janus green in Ringer at 37° C. I t is, of course, important that the preparation should be kept moist and warm, mainly through the constant addition of Ringer at 37° C. The dosage of neutral red employed by the author is greatly in excess




F igs. 1-5.—Pancreas cells of mouse and rat, fixed and fresh preparations. A, newly formed secretory granules; GA, Golgi apparatus; M, mitochondria; F, fa t; S, secretion. Fig 1.—Cell in stage of restitution hours after 0-0005 gm. pilocarpine showing young zymogen granules, A, and fat spaces, F, at cell periphery (Zenker formol iron alum haematoxylin). Fig. 2.—Cell of rat in stage similar to fig. 1 showing mitochondria, M, and their relation to the fat droplets, F (Regaud’s fixation with post osmication stained acid fuchsin methyl green). Fig. 3.—Cell of mouse in restitution stage 3 J hours after pilocarpine 0-0005 gm. showing relation of Golgi apparatus, GA, to zymogen granules. Champy fixation after Nassonow. F ig. 4.—Cell of mouse in restitution stage 4 hours after pilocarpine showing prozymogen granules, A, at periphery of zymogen, S, and similar stained prozymogen granules, B, outside Golgi zone. Protocol of Experiment—Four injections of 0-25 c.c. of 6-1% solution pilocarpine at half-hourly intervals. Fig. 5.—Resting cell of mouse pancreas following massive dose of neutral red showing segregation of dye in large vacuoles at the periphery of the secretion. Toxic condition of Parat, fresh preparation. Protocol of Experiment—Two doses of 0*5 c.c. saturated solution of neutral red at 1 hour intervals. Animal killed 2 hours after last dose.



24 E. S. Duthie,

of that used by Hirsch, which was found inadequate to give a permanent staining to the granules. I t is also to be noted that the amount of pilocarpine was considerably greater. In Hirsch’s experiments 20 c.mm. of 0-2% solution were administered, which is 1 /25th of that used by the present author.

Observations on the Pancreas.

Fig. 1 shows a pancreas cell stained with iron alum haematoxylin after Zenker formol fixation, and illustrates well the plan of the cell. At the apex of the cell lies the secretion mass stained black, while through the cytoplasm towards the cell base are smaller stained granules, marked A, similar in appearance to the secretion granules. Fat spaces indicated by the stippled rings, F, are to be seen at the base of the cell, and occasionally through the cytoplasm.

Fig. 2 is a drawing of a similar cell fixed in Regaud’s fluid post-osmicated and stained with acid fuschsin methyl green. In addition to themitochondria, marked M, the fat droplets, F, in one corner will be noted. Spaces are also present at the cell periphery and are probably due to non-osmicated fat droplets. The mitochondria are here typically filamentous bodies, as described by Morelle, and lie either in the long axis of the cell or at the periphery at right angles to this. The Golgi apparatus was found in all cases to lie completely on the lumen side of the nucleus, and appears as a network embracing the zymogen granules. In fig. 3 it was observed to become more compact, and to retain its position relative to the zymogen granules following pilocarpinization. The exact relationship of the Golgi material is difficult to determine, but it seems probable that it actually embraces the zymogen granules at the periphery.

Vital Staining.

In the pancreas cell it is possible to distinguish various stages in staining with vital dyes. Using a very small dose of neutral red, two injections of 0 • 5 c.c. of 0• 5% solution at an interval of one hour, or after supra-vital staining in 1/30,000 of neutral red in Ringer at 37° C. for about 3 minutes one obtains an appearance corresponding to the minimal degree of staining of the cell. Fig. 4 shows a partially exhausted cell stained in this manner during the stage of refilling. A group of small neutral red stained granules, A, is lying at the edge of the secretory mass or in the clear area, B, of the cell. The granules are of various sizes, being usually about the size of the largest secretory granules, and are rarely to be found in cells full of secretion. These granules may also




be stained by Janus green, and very frequently the periphery of the granule is the only portion staining, it being possible to focus the unstained granule surrounded by a red staining cortex. These may be identified immediately with the granules described bv Michaelis (1900), Bensley (1911), Coveil (1928), Beams (1930), and Gatenby (1931). The number of these granules appears to be increased following pilocarpine stimulation, and there is little doubt that the granules stained are the newly formed zymogen granules. From the fact that a colourless granule is often seen to be surrounded by a red stained cortex, one concludes that the dye collects in some form of vacuole surrounding these maturing granules. By longer staining with neutral red supra-vitally, or by using a heavier dose in intra-vitam staining such as 0 • 5 c.c. of a saturated solution, the appearance shown in fig. 5 may be found. The neutral red collects into larger or smaller masses outside the zymogen granules, and occasionally through the free cytoplasm. These masses are generally rounded or elliptical, and are of a dull red colour. If squeezed out of the cell they rapidly lose their colouring matter, and become lightly stained vesicles which do not break up for some time. The addition of N/10 NaOH causes the zymogen granules to disappear, and the large masses to lose their shape, being merely crenated vacuoles. Very often these vacuoles are separate structures lying in the clear area of the cell, and appear to be quite discrete. They may be preserved by various fixatives, especially Mann’s and Champy’s, and appear in osmicated specimens as blackened masses lying usually in contact with the Golgi substance. This appearance is undoubtedly that described by Parat (1928) as being a toxic condition, and later by Ludford (1930), who identified it with Chlopin’s Krinom (1927). By observing the progress of the staining in supra-vitam preparations it is possible to note that these large Krinom masses are derived directly from the red stained structures identified in fig. 4 with the prozymogen granules. I t is possible in all case3 to note, especially in the larger granules, a swelling of the surrounding vesicle, which later loses its round appearance, and becomes more irregular. In addition smaller granules shown in fig. 5 appear, but they are probably not derived in the same manner.

Observations on the Living Pancreas Cell.

In the main it has been possible to confirm the findings of Hirsch (1932, a, b) regarding the genesis of the secretory granules in the pancreas cell. In suitably exhausted cells, such as may be found 2^ hours after pilocarpine, it has been possible to note the origin of the small secretory granules at the cell base, and



26 E. S. Duthie.

to trace their passage inward to join the main secretion mass. Fig. 6 illustrates the passage of one of these granules inwards in a cell studied over a period of 3 hours, the diagrams being chosen from a series made about every o minutes. The cell was stained with neutral red and Janus green. At 9.25 p.m. in addition to the mass of colourless secretion granules next to the

Fro. 6.—Successive stages in passage inward to Golgi zone of a newly formed secretory granule marked X in a living pancreas cell observed over 3 hours. A, young zymogen granules stained with neutral red; B, young zymogen granule formed in relation to mitochondrion, M, and X, particular granule observed over whole period. Protocol of Experiment—Mouse unfed for 36 hours, 0 • 25 c.c. of 0 • 5% pilocarpine 4.30 p.m., 0 • 5 c.c. of 0*25% neutral red at 5 p.m., and at 5.30 p.m., Cibalgin 0-6 c.c. of 1/10 solution at 6.30 p.m. Operation at 7.15 p.m.

lumen, there are a number of neutral red stained granules, A, at the periphery of the mass, while at the cell base there are three neutral red stained granules, one of which marked B had been noted to appear in connection with the adjacent mitochondrion. The granule marked X which was slightly larger than the



others was chosen, and was watched over the whole period until it reached the Golgi zone, the various stages in the movement being noted. The mitochondria which were freshly stained at the beginning of the observation had lost their staining after a short time, and are not shown in the later figures. The appearance and disappearance of neutral red granules in the figures is a constant feature in these cells, and is due to the granules going out of focus. As will be noted the passage inward is marked by a slight increase in size. The cell shown in this diagram is a very typical example, such as is described by Hirsch who has observed the whole process extremely carefully. He finds that the granules arise in connection with the mitochondria, and remain in contact with them for 10-17 minutes. This is followed by an irregular movement of the granule in the basal region of the cell lasting 60-90 minutes followed by a movement towards the Golgi field lasting for the same period. The average speed of the granule is 1 jx in 7-13 minutes, and he states that the small granules move more quickly. In the present author’s experience, however, this is not always so. Very often, especially with smaller granules, the passage from the cell periphery inwards is extremely quick, lasting about 5-7 minutes. This is usually accompanied by a good deal of oscillatory movement, the granules advancing a distance of 2 or 3 u in about 3 minutes (exact figures are difficult to obtain) and then just as quickly retreating to their previous position, the path in such movements being usually fairly straight. This is especially true in the neighbourhood of the Golgi field, where a granule by a sudden movement may travel half the nucleus length into the Golgi zone only to retreat again. One sometimes gains the impression that the Golgi apparatus itself, which is invisible, presents a temporary barrier to its further progress. This type of movement is seen usually in cells which have not been studied for too long, or have not been stained with Janus green. I t is possible that the slow but steady progress depicted in fig. 6 and described by Hirsch is not the normal type of movement, but occurs only through injury or both types may be present in the same cell. The cause of such a forward movement of the granules to the Golgi zone is unknown, but from the present author’s investigations it would seem unlikely that there is any general movement of the protoplasm. Hirsch states that he could find no such movement in the cell when examined with dark ground illumination, but believes that it may be due to a localized movement. In the present author’s experience this, too, is unlikely, since cells have been observed in which of two granules lying together about 2 [x from the Golgi field one has moved forward the intervening distance in about 2 minutes, the other being left behind and following some 10 minutes later.




28 E. S. Duthie.

like diplococci, such as is described by Hirsch (1932, a, b). This sometimes gives rise to the impression that it is due to division of a granule, but in the author’s experience this is probably not so, since a double granule by a rotary movement may simulate a single granule. Careful watching of such granules, however, usually reveals a reappearance of the double condition. The actual transformation of these neutral red staining granules into unstained secretory granules has not been observed, though the author has no doubt that this eventually occurs. There is little doubt that these neutral red granules are secretory granules and not new formations, since (a) the whole process of their origin, growth and movement inwards may be followed though not so easily in the unstained cell, and (6) by the addition of 1/30,000 neutral red, the preparation may be temporarily stained so that the gradual reversion of these stained granules to the unstained state may be clearly observed. I t is to be noted that these granules stain equally well with Janus green as observed by Michaelis (1900) and by Hirsch (1932, a, b), so that caution is necessary in adding the former dye. The Janus green has a tendency to fade from the cell after some time, and a second addition is very apt to stain the prozymogen granules both at the periphery and in the Golgi field, leaving the mitochondria unstained. For this reason it is better to employ Janus green as little as possible. The appearance shown by Hirsch of a red stained granule with peripheral green ring is often obtainable. The relationship of the newly formed granules to the mitochondria is not always as clear as one might expect. There is no doubt that some of the mitochondria may remain unstained by Janus green, so that the appearance of a granule in one area of the cell not occupied by visible mitochondria is no proof that such a relationship does not exist. There is no doubt, however, that the granules arise if not in actual contact with the mitochondria at least a little distance from them, so that there is no question of either bleb formation or fragmentation occurring as described by previous authors, Saguchi (1920), Morelle (1925), Horning (1925).

A remarkable phenomenon in these cells is the appearance of fat granules at the periphery, and occasionally in the Golgi zone following cell activity. These fat droplets appear to be normal constituents of the pancreas cell, though they are greatly increased by excitation. Heidenhain (1880) seems to have been the first to describe them, and showed that they might be easily differentiated from the zymogen granules by the addition of caustic potash or acetic acid, which dissolved the latter, leaving the former alone. Since then they have been described by numerous workers, Mathews (1899), von Laguesse



(1900), Champy (1911), Mislawsky (1913), Maximow (1916), Saguchi (1920), Ma (1924), and Hirsch (1932, a, b). In the living cell they are more retractile, and are usually slightly larger than the zymogen granules, fig. 4, and can be fairly easily distinguished from them in the unstained cell. They are apparently the granules described by Coveil (1928), though while he describes their passage from the cell into the intercellular space, no such movement has been noted by the present author. The formation of these fat granules is usually associated with a decrease in the mitochondrial substance visible in that area, many of the longer mitochondria breaking up and being replaced by short rods associated with groups of the granules. The appearance of new fat granules in such groups has been noted, and it is believed that direct transformation between the two occurs especially since many of the fat granules become visibly tinted with Janus green when this is added. If Regaud-fixed tissues be blackened with osmic acid as described, the granules can be demonstrated side by side with the mitochondria as in fig. 2, the acid fuchsin stained substance sometimes giving rise to the crescent shaped appearance on one side of the fat granule described by Ma (1924). The granules themselves are well shown in any osmic fixative such as Champy or by osmication following prefixation in a formalin containing fixative. Osmication alone produces a greying or browning of the globules. In the living cell a slow oscillation of these granules about their point of origin has been noted, and they have a tendency to pass towards the Golgi zone, though this has not been followed. A similar oscillatory movement is to be noted in the mitochondria, and is probably due to local changes in surface tension brought about by the absorption of fluid from the outside.

Part II .—Salivary Glands.

As far as can be ascertained there is no complete study of the salivary glands of the rat in the literature. Most investigators have contented themselves with a description of one particular gland, or with a very brief description of these glands in the course of a comparative study. Before dealing with the various histological characteristics these references will be briefly mentioned. Ranvier (1886) included a description of the submaxillary glands of the rat in a review of these glands in various animals. He noted that there were two glands present, a submaxillary and retrolingual, and that they lay in the same capsule. The submaxillary he described as a serous gland, the retrolingual being mucous, a few serous cells being included at the base of the acini.

In a more recent paper by Honda (1927) four types of cell are mentioned in connection with the submaxillary gland structures, (a) in the epithelium of the




30 E. S. Duthie.

duct, (b) the serozymogen cell characterized by the presence of large granules stainable with acid fuchsin, (c) serous or true acinar cell, (d) the mucous cell which is apparently confined to the retrolingual gland.

Stormont (1928) in reviewing the literature dealing with the submaxillary glands in general, does much to clarify the various distinctions which have arisen, and in this paper the classification adopted by him will be used for the sake of simplicity. In addition to mucous and serous cells, the latter are further divided into chromophil and chromophobe, depending on the presence or absence of chromophil (basophil) material in the gland cell as described first by Heidenhain (1868). This substance has been described in the acinar cells of the pancreas, chief cells of the stomach and in the parotid cells of most animals investigated. Stormont then restricts the term serozymogen to a gland cell characterized by the presence of zymogen granules plus chromophil material. Other cells not containing chromophil material, including all those found in the submaxillary of the rat, he includes under the term special serous cells. In the rat as in a number of other mammals the submaxillary gland proper contains two types of cell, neither of which contains chromophil material, and which are therefore not serozymogen cells but special serous cells.

Cohoe (1907) working on the submaxillary gland of the rabbit found that the two types of cell found in the submaxillary had different properties, when stained with muci-carmine, muci-hsematin and thionin after formol-bichromate fixation, the secretion of the clear acinar cells staining metachromatically in every case in contrast to that of the ducts. Bensley (1908) termed this character trophochromatism, the clear metachromatically staining cells being named trophochrome in contrast to the duct cells which are homochrome. This distinction is noted in the present paper, though the author feels that more information regarding the comparative histology of the glands is required, before the criteria may be accepted as being definite.

Cytological studies have been confined to descriptions of the Golgi apparatus, mitochondria, and secretion in the salivary glands of various animals.

The Golgi apparatus was first described in the salivary glands of the dog by von Bergen (1904), using the silver impregnation technique. He described it as a series of granules, rods, twisted threads, and networks. Cajal (1915) described the Golgi apparatus in the salivary glands of the rabbit, cat and guinea pig, as a network or horseshoe shaped group of concentric bands, between the nucleus and the gland lumen. Fragmentation was found to occur after pilocarpine. Bowen (1926) included the salivary glands of the cat in his masterly survey of the Golgi apparatus in various gland cells. He found that




it underwent considerable hypertrophy during the various stages of the secretory cycle, but while in the serous cells of the parotid during refilling the apparatus tended to extend through the whole mass of secretory granules, in mucous cells it retained a more compact and peripheral relationship.

Investigation of the secretion formation is confined mainly to older investigators. Langley (1879) described the changes which occur in the fresh gland cell, following various forms of stimulation. In the serous cells of the rabbit’s parotid, the effects of stimulation were followed both in living cells and in those examined immediately after death. The granules which were easily visible were found, in the resting state, to fill the cell, and after stimulation were greatly diminished in number and came to line the sides and free end of the cell. From his description one gathers that the morphological appearance of these glands, parotid, submaxillary, and retrolingual is very similar to that in the rat.

Noll (1902) examined the formation of the secretion in various salivary gland cells. In mucous cells he described the appearance of very small granules in the region of the nucleus following stimulation. These granules were deeply fuchsinopliil when stained by Altmann’s method, and occurred mainly at the periphery of the mucous cells, though often scattered through the vacuoles left after the extrusion of the mucous. These granules he believed were visible in the fresh condition, and from the fact that they were not present in the fully filled cell he concluded that they were newly formed secretory granules. Regaud and Mawas (1909, a, b) studied the secretory process in the human submaxillary gland. They described the appearance of swellings in connection with the mitochondria, which they considered to be the matrix of the future secretory granules. Further development they believed to occur after separation. Honda (1927) studied the mitochondria in the salivary gland of the rat. In the duct cells he believed that he detected a transformation of the mitochondria into secretory granules. This he was unable to confirm in either the serous or mucous cells.

Material and Methods.The mouse and rat were used in this study. As far as could be determined

there was little distinction between the salivary glands of either animal except that of size. As before a comparison was made whenever possible between the structures observable in the fresh condition and those found in fixed preparations. Vital staining with neutral red was carried out either by a perfusion of the whole animal with 1/15,000 solution of neutral red in Ringer at 37° C., or by intra-vitam staining. In the latter case rats were given a total of 2 c.c.



32 E. S. Duthie.

of a saturated solution of neutral red in two doses with 1-| hours interval, the rat being killed 2 hours after the last injection. In mice 0*5 c.c. of 0*5% solution was given twice at hour intervals. All injections in this experiment were given subcutaneously in the region of the neck. The neutral red apparently travels in the fascial planes, and it is difficult to achieve any degree of uniform staining. Janus green staining for mitochondria was carried out by perfusion.

Fixation for mitochondria and Golgi body was carried out exactly as for the pancreas ; in the latter it is often difficult to secure successful impregnation. For ordinary histological examination formol, Zenker formol, and Podwyssozki’s fluid were used. Mucin was stained by Mayer’s muci-carmine prepared according to the directions in Carlton’s “ Histological Technique,” and also by thionin.

The glands dealt with are shown in fig. 7. Of these the parotid, A, lies at the side of the head. The submaxillary, D, and retrolingual, C, lie on either side

F ig. 7.—Dissection of submaxillary region in rat showing salivary glands. A, parotid;B, sublingual; C, retrolingual; D, submaxillary.

of the mid-line enclosed in a common caspule, while the fourth gland, B, which the author believes to be the sublingual is a diffuse whitish mass lying lateral to and outside the common caspule of these, and opens by a separate duct into the floor of the mouth.

In sections of the submaxillary gland it is easily seen that it contains two types of secretory cell. One of these, the sero-acinar cell is the clear cell forming the gland tubules, while the other which is filled with large, coarse, deeply stained granules occupies the terminal portion of the intra-lobular




ducts, and will be referred to here as the duct cell. These correspond respectively to the acinar and sero-zymogen cells of Honda, Or to the clear and dark cells of earlier authors, Mueller (1896), Illirig (1904). When examined fresh in the resting condition the sero-acinar cell is completely filled with a large mass of secretory granules, which are almost invisible owing to their low refractive index, fig. 8. Should the cell become injured in any way, especially if it is kept for some time in cold Ringer, the secretion granules become more

F ig. 8.—Resting acinar cell of submaxillary gland of mouse intra-vitaliy stained with neutral red showing neutral red granules, A, at the periphery of cell, fat-granules, F. and zymogen granules, S. Fresh preparation. Protocol of Experiment—Two injections of 0 • 5 c.c. of 0 • 5% neutral red at 1 hour intervals killed in 1 hour. F ig. 9.—Sero- acinar cell of submaxillary gland of mouse in restitution stage following pilocarpine. Note absence of formed secretory elements and great increase in number of neutral red staining granules. A few fully formed zymogen granules could be distinguished, but are not shown in the figure. Fresh preparation. Protocol of Experiment—Four injections of 0-25 c.c. of 0*1% solution pilocarpine. I hour later two injections of 0-5 c.c. of 0-5% neutral red in Ringer. Killed in 1 hour. F ig. 10.—Sero-acinar cell of rat submaxillary gland showing mitochondria. Rat fed about 6 hours previously. Fixation Zenker formol. Iron alum hsematoxylin. F ig. 11.—Golgi apparatus in sero- acinar cell of rat in restitution stage 1£ hours after 0*001 gm. pilocarpine. Champy- Kull method after Nassonow.

VOL. CXIV.— B. D



34 E. S. Duthie.

highly refractile, and appear homogeneous in size filling the cell completely. In addition there are a varying number of smaller and more highly refractile granules scattered through the cell. The majority of these are insoluble in alkali (unlike the secretory granules), and may be blackened in osmic following formol fixation. I t is concluded that they are of a fatty nature. After feeding or pilocarpinization, however* the picture changes, and following the discharge of the main secretion mass, the number of neutral red granules is greatly increased, fig. 9. Their bright red colour is very similar to that found in the prozymogen granules of the pancreas and stages showing increase in size and loss of staining capacity may be found so that it is not difficult to trace their conversion into the unstained secretory granules. Like those found in the pancreas they stain equally well in Janus green. There is no special region for their occurrence as in the pancreas, though as the cell fills they tend to be more numerous in the region of the nucleus, fig. 9. Like the secretory granules and in contrast to the fat granules, they are soluble in alkali. The increase in number of the neutral red granules following stimulation, and the various sizes found make it certain that they are the prozymogen granules. An examination of the mitochondria in these cells shows that they consist of irregular filamentous bodies of all shapes and sizes scattered through the cell. Fig. 10 shows their appearance in a section fixed in Zenker formol and stained with iron alum haematoxylin. In addition to the filamentous bodies, small round deeply stained bodies are shown through the cytoplasm. I t may be that these granular bodies are young secretory granules, since similar mitochondria are not found in the fresh condition. Secretory granules are often shown in this method especially if the destaining is not carried too far, and the author is inclined to regard them as mature or young prozymogen granules, which they resemble in number and size. Staining of Regaud-fixed sections of this gland with muci-carmine or thionin reveals a pronounced metachromatic reaction in these cells, but not in the duct cells. They are accordingly trophochrome on Bensley’s classification.

Fig. 11 shows the type of Golgi apparatus found in pilocarpinized animals. Figures of the Golgi apparatus in the resting cell are not easy to obtain. Many attempts have been made, but even in the half-filled cells shown, some idea of its extraordinary complexity is obtained, since it spreads through the whole cell embracing the nucleus. This extremely open network is possibly to be related to the scattered arrangement of the prozymogen granules. In this case there is no differentiation of the cell into clear and secretogenous areas as in the pancreas. Staining with basic dyes reveals no chromophil substance,




and doubtless the formation and maturation of young zymogen granules occurs all through the cell. Bowen (1926) has described what may be termed a very similar condition in the parotid gland of the cat. Here, according to Bowen, the Golgi apparatus displays a distinct tendency to spread through the cell, so as to embrace the zymogen granules where formed. This Bowen contrasts with the mucous cells which more nearly resemble those of the pancreas, pointing out that in the parotid, the spreading of the Golgi apparatus is necessitated by the fact that all the granules undergo a uniform and simultaneous maturation. While the present author has been unable to confirm this description in the parotid, he feels that the sero-acinar cell is a cell of the type which Bowen has described.

Duct Cells.

When the submaxillary is examined in the fresh condition, these cells are extremely conspicuous by reason of their being filled with large highly refractile secretory granules, fig. 12. The granules at the lumenal end are in general much larger than those on the outside and varying numbers of small granules are to be seen lying in the clear area outside the nucleus. Some of these peripheral granules stain with neutral red as in fig. 12, and very often the secretory granules next the nucleus are slightly tinted. In general it is extremely difficult to stain these cells, and the condition shown in fig. 12 is largely the result of overstaining intra-vitally, when the dye droplets tend to collect in groups at the periphery of the formed secretion or as smaller droplets lying among the secretory granules. I t is, however, probable that we are again dealing with a staining of the young granules, this being borne out by the fact that the neutral red staining granules are more numerous following secretion when intermediate stages may be found. Usually they lie at the periphery among the mitochondrial threads which are now easily visible, and the process may also be followed in fixed preparations as in fig. 13. One cannot regard all these granules seen in fresh preparations as being secretory in character, since some in contrast to the secretory granules are insoluble in dilute alkali. These latter are usually to be seen at the periphery in the living cell. Fig. 13 shows a section of one of these ducts stained with iron alum hsematoxylin following pilocarpinization. Many of the cells are still filled with secretion, and do not contain any visible mitochondria, the latter being prominent only in the discharged cells. The mitochondria are long filamentous bodies lying in the long axis of the cell, and being especially numerous in the basal region. Amongst them are to be seen the newly formed



36 E. S. Duthie.

secretion granules, A. Fig. 14 shows preparations of the Golgi bodies in these cells. I t appears to be distinctly polarized, and is a small compact network, lying between the nucleus and the main secretion mass. In over-osmicated specimens the secretion granules themselves are but poorly preserved, and here small blackened particles are to be seen both in the secretion mass and at the

F ig. 12.—Duet cell of submaxillary gland of rat stained supra-vitally. Note neutral red is collected in groups of granules (NR) at periphery of zymogen granules, and is distributed in smaller globules (C) through them. Also newly forming granules, A. F ig. 13.—Duct cells of submaxillary gland of rat after pilocarpinization. Note in exhausted cells young zymogen granules, A, and mitochondria, M. Fixation Zenker formol stained iron alum hematoxylin. F ig. 14.—Duct cell of mouse showing Golgi body and its relation to the zymogen granules. Champy-Kull after Nassonow.



periphery. These the author regards as being blackened mitochondria, since they have no connection with the main Golgi body-mass, and there is no evidence that this ever expands into a widely flung network to embrace the individual secretion granules. The poor preservation of the granules makes it often difficult to say whether a particular cell is in the active or resting condition. In pilocarpinized animals, however, no appreciable change in the form of the network is visible, and one must conclude that it undergoes very little if any alteration. No penetration of the nucleus by the mitochondria as described by Honda (1927) been seen.


Retrolingual ( Mucous Salivary Gland).

As first described by Langley (1889) the mucous cell in the living condition is filled when resting with large numbers of granules, which are scarcely visible owing to their low refractive index. The nucleus is pushed to the periphery and assumes a flattened shape. In the region of the nucleus a few smaller and more highly refractile granules are to be seen, some of which stain with neutral red, fig. 15. Discharge and refilling brings about an immense increase in the number of the neutral red staining granules, which come to lie in the neighbourhood of the nucleus, or may be scattered through the cell. These granules appear first at the basal region being very small in size, and are later found in the region of the nucleus. Here they undergo increase in size with loss of colourability with neutral red and Janus green, finally turning into the larger but less refractive secretory granules. Other non-vitally staining granules of various sizes are visible, but are not like the secretory or neutral red granules soluble in alkali and give positive fat tests.

In specimens fixed in Regaud’s fluid and stained with acid fuchsin methyl green, there are to be seen in the region of the nucleus small groups of brightly stained fuchsinophil granules, fig. 17. These granules extend down to the region of the mucous secretion, but do not stain with specific mucin stains. They may be demonstrated rather faintly with iron alum hsematoxylin. They are not to be regarded as mitochondria which are shown both in fixed and fresh preparations, fig. 16, as long filamentous bodies, but correspond in position and occurrence to the vital staining granules found in the fresh cell. In active cells they are extremely numerous, but with the refilling of the cell they tend to disappear and are clearly the young secretory granules. As will be seen from figs. 19 and 20 the Golgi body in these cells is a network lying at the periphery of the mucous mass. In the resting cell, fig. 17, it is very much



38 E. S. Duthie.

flattened and lies around the nucleus, but with retreat of the secretion it apparently increases in substance, and advances towards the free end of the cell, fig. 18. In the Golgi preparations it is unfortunately impossible to demonstrate the fuchsinophil granules too, but a comparison of figs. 16 and 18 will make it evident that the boundary between the fuchsinophil granules and the mucous secretion is the Golgi zone, where presumably the transformation of the former into mucin is carried out. These fuchsinophil granules are those described by Noll (1902), who correctly regarded them as young secretory

F ig. 15.—Resting cell of retrolingual gland of mouse, stained intra-vitally with neutral red. Note neutral red stained granules, A, at periphery around the nucleus. Protocol of Experiment—Two injections of 0*5 c.c. of 0*5% solution of neutral red. F ig. 16.—Cell of retrolingual gland of rat killed 8 hours after feeding showing relationship of fuchsinophil granules of Noll R to nucleus and fully formed mucus. Note fat spaces, F , and mitochondria, M. F ig. 17.—Cell of the retrolingual gland of rat in resting stage showing Golgi body. F ig. 18.—Cells of the retrolingual gland of mouse half an hour after pilocarpine showing relation of Golgi material, GA, to mucus.




granules. Krause (1895) noted that in the retrolingual gland of the hedgehog the transformation of the antecedents into stainable mucin took place first in the region of the nucleus, and this was later confirmed by Maximow (1901). The fact that both in the living and fixed preparation, they may be found at the extreme periphery of the cell, makes it fairly certain that they do not arise in the Golgi area.

Sublingual Gland.

When a portion of the gland is examined in warm Ringer, the cells are seen to be filled with large numbers of highly refractile granules leaving a clear area at the periphery. The nucleus is easily visible, and in this area are a variable number of smaller granules usually stainable with neutral red or Janus green though other granules may be present which do not so stain. During simulation vacuoles are formed at the free end of the cell, and the secretion is discharged. Refilling is brought about by the appearance of large numbers of small neutral red staining granules in the cell, fig. 19, some of which may be observed at the extreme periphery. Transitions between these small neutral red stained granules and the larger unstained granules may be found. If alkali is added the neutral red and fully formed secretory granules dissolve, while many fat granules previously unstained remain. The mitochondria as shown by acid fuchsin appear as short or long rods and dots disposed parallel to the long axis of the cell, and are extremely numerous. Fresh preparations show a similar appearance. The secretory granules while preserved in Zenker formol are not easy to demonstrate since they blacken only very slightly with iron alum haematoxylin. They may, however, be fixed in Podwyssotzky’s fluid, when they display a certain degree of selective staining with acid fuchsin. Staining with haematoxylin reveals a basic staining of the outer portions of the cell as in the pancreas, no metachromatism is apparent with muci-carmine or with thionin so that it is to be regarded as a sero-zymogen gland.

Preparations of the Golgi body, figs. 20 and 21, show that it is a network lying at the border of the secretory granules in the resting gland, fig. 20, and that following the extrusion of the secretory granules it advances towards the free end of the cell, fig. 21. The condition of over-staining with neutral red shown in fig. 22 readily occurs in this gland, owing to its lack of capsule and extreme diffuseness. As in the pancreas neutral red collects in large masses of all shapes and sizes at the periphery of the secretion.



40 E. S. Duthie. .

Parotid.

In the fresh resting gland the cells are filled with large granules of a low refractive index. Pilocarpine brings about a dissolution of the granules into vacuoles, the remainder consisting of a small group at the free end of the cell. At the same time large groups of very small granules make their appearance at the cell base, fig. 23, and some of these, as indicated, are stainable in neutral red and Janus green. The neutral red granules like the secretory granules are

P ig. 19.—Cell of sublingual gland of rat 6 hours after feeding, and stained intra-vitally with 2 c.c. of saturated neutral red subcutaneously. Note neutral red staining granules, A, also unstained granules. P ig. 20.—Cell of sublingual gland of mouse 8 hours after feeding showing relation of Golgi apparatus, GA, to secretory granules. Champy-Kull technique after Nassonow. Killed under Cibalgin. P ig. 21.—Cell of sublingual gland of mouse 1J hours after 0*001 gm. Pilocarpine showing Golgi apparatus. (Champy-Kull after Nassonow.) P ig. 22.—Cell of sublingual gland of mouse in resting stage showing massive staining with neutral red. Note discrete nature of vacuoles. Protocol—Mouse unfed 36 hours, two doses of 0 • 5 c.c. of 1 % neutral red subcutaneously. Killed 2 hours after last dose. Presh preparation.




soluble in alkali, while the non-stainable granules are not. The re-formation of the zymogen granules appears to be brought about by a passage of these neutral red granules inwards to the Golgi area with increase in size and progressive decolourization, the stages of which can easily be followed. Fig. 24 shows an iron alum haematoxylin preparation of such a cell from a mouse parotid. In this animal the granules display a selective staining with iron alum hsematoxylin, which is not so apparent in the rat, and the various stages in the passage of the zymogen granules inwards can be followed. The large groups of smaller non-neutral red staining granules are not to be seen in fixed preparations. Fat spaces do appear however, and it is possible that all these

Fig. 23.—Parotid cell of mouse 8 hours after feeding showing fully formed secretory granules, S, neutral red stained granules, A, and mass of unstained granules at periphery of cell which is probably fat. Fresh preparation. Protocol of Experiment—Neutral red in two doses of 0*5 c.c. of 0*5% solution hours before killing. F ig. 24.—Parotid of mouse 8 hours after feeding. Fixed preparation showing zymogen granules, S, at cell apex and at periphery, A. Also fat granules, F. (Zenker, formol fixation. Iron alum hsematoxylin.) .

non-staining granules are of a fatty character. The mitochondria when stained with Janus green are disposed either in the long axis of the cell or parallel to the periphery, fig. 25. They are mainly straight, or short curved rods, and bear no special relationship to the non-staining granules at the periphery of fresh preparations. Their peculiar arrangement parallel to the side walls of the cell is to be noted. The Golgi body as shown in fig. 26 is seen to be an extended network lying along the border of the secretion mass. Overstaining causes some blackening among the secretory granules, but at no time does one get the impression of there being a definite network extending among



42 E. S. Duthie.

the secretory granules. Bowen (1926), however, has described such an appearance in the parotid of the cat, in which he pictures the Golgi network of the resting cell as lying dispersed throughout the whole secretion mass, so that the Golgi threads embraced the individual zymogen granules. The author considers this concept inadmissable, since in the parotid of neither the rat, mouse nor cat has he been able to obtain any clear cut evidence of the distribution described by Bowen. Bowen believed that the peculiar extension of the Golgi network was necessitated by a simultaneous ripening of the zymogen

F ig. 25.—Parotid cell of rat during restitution showing mitochondria, M, stained with Janus green. Fully formed zymogen granules, S, and mass of smaller granules at periphery. Note especially the distribution of mitochondria along lateral walls of cel]. (Fresh preparation.) F ig. 26.—Parotid cell of mouse 8 hours after feeding showing Golgi apparatus and its relation to the secretory granules. (Champy-Kull after Nassonow and killed under Cibalgin.)

granules in contrast with that occurring in mucous glands. In the present author’s opinion, however, the appearance described by Bowen is due to a blackening of the secretory granules themselves. I t is clear from the observation on the fresh cell that the full maturation of the granules which implies (a) loss of staining power with neutral red, (b) increase in size, (c) decrease in refractive index, occurs in the Golgi zone, basally to the fully formed secretion.

Discussion.The origin of the secretory granules in various gland cells is a subject which

has occupied the attention of an increasing number of cytologists in the last




fifty years. Earlier workers such as Altmann (1894), Bourn (1905), Regaud and Mawas (1909), Hoven (1910, 1912), Champy (1911), Arnold (1912) and Maximow (1916) believed that they were derived directly or indirectly from the mitochondria. Michaelis (1900) used Janus green and neutral red in combination for the vital staining of the pancreas and salivary glands of various species. He noted that in addition to the mitochondrial filaments stained green and zymogen granules stained red, there were in the basal portions of the pancreas cell minute granules which stained partly in Janus green and partly in neutral red, which he believed to be young zymogen granules. From this he concluded that the zymogen granules arose in connection with the filaments, and so partly anticipated the later work of Hirsch (1932, a, b).

Saguchi (1920) sought to reconcile this view of the mitochondrial origin of the secretory granules with the fact that the new granules were in his experience only to be found in the area between the nucleus and the secretion mass, which he termed the secretogenous area. I t was the breakdown of the mitochondria in this region alone, which gave rise to the future secretory granules.

Following on the work of Gatenby (1921) and others on the formation of the acrosome by the Golgi body in insect spermatogenesis, Nassonow (1923) postulated his theory of the “ bound secretion.” In this and in subsequent papers (1924) it was shown convincingly that the secretion in a number of gland cells studied, arose in the meshes of the Golgi network, so that the latter came to be accepted as the source of secretion. This theory was later developed by Bowen (1926), who in a study of the secretory stages in various gland cells showed in a very convincing manner that the Golgi apparatus underwent very striking changes in the active cell, “ a very close topographical relationship being found between the Golgi network and the developing secretory granules.” He confirmed the previous work of Nassonow in that he described the granules as appearing first in the reticulum and undergoing maturation there. In 1925 Morelle published a very careful survey of the pancreas secretion in a large number of animals. In this publication he endeavoured to reconcile the views of Saguchi with those of Nassonow and his supporters. The secretory granules were formed by a disintegration of those mitochondria in the Golgi area (secretogenous area of Saguchi), and their further growth and maturation occurred within the network. Morelle’s views have for many reasons failed to meet with wide acceptance. Ludford (1925) accepted the theory of Nassonow, and has since produced a very considerable body of evidence bearing on the importance of the Golgi body in the various stages of the secretory cycle. In the thyroid it has been shown by Cramer and Ludford (1928), that during secretion both



44 E. S. Duthie.

the Golgi body and mitochondria are enlarged. This they explained by saying that while the Golgi apparatus is actively engaged in secretion, the mitochondrial variation produces corresponding variations in surface energy within the cytoplasm and so effects a redistribution of the fats. In the liver and kidney, however, the concentration of dye droplets bore no relationship to the mitochondria, and occurred in the region of the Golgi apparatus. Ludford, however, is unwilling to consider the mitochondria as being completely inactive members of the cell economy. He takes the view of Cowdry (1926), that at the mitochondrial cytoplasmic surface synthesis by enzymes occurs. The resulting products continually diffuse into the cytoplasm, and at the surface of the Golgi apparatus the elaborated products are concentrated into droplets prior to their elimination. Of later workers the most recent is Florey (1932, a, b), who in dealing with the mucous secretory cells of the vagina and the crypts of Lieberkuehn has shown clearly that in both areas the newly formed mucin arises as small droplets in the Golgi area, the author accepting the views of Nassonow (1923). The question of the origin of the secretory granules in the pancreas has, however, been decided, by the study of the changes occurring in the living cell by Hirsch (1932). This method in the present author’s experience shows clearly that for the pancreas the early secretory elements are formed at the cell base, in fairly close topographical relationship to the mitochondria. How far then is this applicable to other glandular systems ? In all five types of salivary gland cell studied, it has been possible to identify with some certainty the young secretory granules with those staining with neutral red or Janus green, and in three of these cells the submaxillary duct-cell, parotid and mucous cells it has also been possible to identify them both in fixed and stained preparations. In all these cases the young secretory granules have been found in the basal region of the cell away from the Golgi area, so that their origin there in connection with the mitochondria seems to be very probable. I t is to be emphasized that the total number of yoimg secretory granules lying outside the Golgi network at any one time is never very large. One must remember that in the pancreas the time taken to travel into the Golgi zone is only 2 to 3 hours, and that during the earlier portion of this time the granules are so small that it is difficult to locate them in osmicated preparations. This is in all probability the reason why Nassonow and subsequent workers failed to notice them.

Plan of Secretion Formation.Appearance at the cell periphery of a small vitally staining granule in relation

to mitochondria. Duration 5-10 minutes (pancreas).



Passage inwards to the Golgi zone with gradual increase in size. Time 15 minutes to 3 hours (pancreas).

Loss of staining capacity with vital dyes (Janus green and neutral red) and decrease in size leading to formation of mature secretory granule. Time 8 hours to 9 hours (Hirsch, 1932) (pancreas).

Nature of the Vacuome.

The vacuome of the pancreas cell was described by Parat (1928) in the cells of Scyllium catulus. From Parat’s description we learn that it consists of a series of deeply stained vacuoles in the Golgi zone, i x ., between the secretion granules and the nucleus. He describes two kinds, in one the “ rhagocrine,” the vacuome is not completely stained, but contains inside a small unstained secretory droplet; while in the other “ plasmocrine 55 this does not occur. In addition he describes a light staining of the periphery of the prozymogen granules grouped around it. Parat believes the secretory droplets to be formed in the vacuome, so that by a gradual increase in size of the granule at the expense of the surrounding vacuolar substance, there appeared the condition in which the prozymogen granules have lightly stained peripheries. His figures show a grouping of the mitochondria in this area around the vacuole, and he believes the Golgi body to be formed by a blackening of these structures.

I t is clear from the present work and that of Hirsch (1932, a, b) that there is no necessary connection between the vacuome and the Golgi body, since the former first appears in the basal region of the cell, away from the Golgi area. On the other hand, one must recognize the truth of Parat’s observations in that the secretory granules are first formed inside vacuoles in the cell, these vacuoles being also the spaces inside which neutral red and Janus greeen are concentrated. Parat in addition recognized the neutral red formations shown in fig. 5, and obtained by heavier and more prolonged staining, but believed them to be toxic effects. On this point it seems that a misconception may have arisen in the minds of various authors. I t is clear from the papers of Coveil (1928), Beams (1930) that the structures they describe as being the vacuome are the prozymogen granules, correctly described as such by Gatenby (1931). Ludford (1930), on the other hand, was dealing with much larger structures similar to those shown in fig. 5. In the present author’s opinion these are segregation vacuoles in the cell formed in the great majority of cells by an enlargement of the space in which the prozymogen granule is forming. They are therefore similar to the “ Krinom ” structures of Chlopin, and as shown by him in other cells, they may be fixed by various reagents, and exhibit an

Secretion of the Pancreas and Salivary Glands. 45 on May 24, 2018http://rspb.royalsocietypublishing.org/Downloaded from


46 E. S. Duthie.

affinity for basic dyes. When squeezed out of the cell they are thin walled sacs similar to those described by Young in the nerve cells of Cephalopods, and may readily be crenated by mechanical means, such as hypertonic solutions. The exact point of transition from prozymogen vesicle to Krinom is impossible to determine. In unstained preparations a clear space may usually be distinguished around the granules at the edge of the secretion mass. I t is this space which takes up vital dyes, but the transition from a light staining of this space to one of slight enlargement with deposition of protein matter around the dye, so that it can be fixed and stained to constitute a Krinom, is too gradual to be observable.

Conclusions.(1) Observations on the living pancreas cell have been carried out, which

support the observations of Hirsch (1932) regarding the origin of the zymogen granules in relation to the mitochondria. The passage of these granules inwards to the Golgi zone, and their increase in size has been observed.

(2) These early zymogen granules were found to be equally stainable with neutral red and Janus green, thus bearing out the work of Michaelis (1900), and later of Bensley (1911) and Gatenby (1931).

(3) The vacuome of the pancreas cell described by Parat (1928) is the vesicle in which the prozymogen granules are formed. Enlargement of these vesicles occurs through overstaining, giving rise to new formations in the cell. Neither the vacuome nor these new formations bear any necessary relation to the Golgi body.

(4) In the cells of the salivary glands of the rat and mouse the young secretory granules have been shown to originate in the periphery of the cell, outside the Golgi zone. Movement is believed to take place into this latter area where maturation occurs. In all types of cell examined these young secretory granules, as in the pancreas, stained equally well in neutral red and Janus green.

(5) Fat granules have been found to occur in all these cells following stimulation. I t is believed that they arise through the disintegration of the mitochondria.

REFERENCES.

Altmann, R. (1894). “ Die Elementarorganismen ” 2nd Ed., Leipzig.Arnold, G. (1912). 6 Arch. Zellforsch.,’ vol. 8, p. 251.Beams, H. W., and Goldschmidt, J. B. (1931). 4 J. Morph.,’ vol. 50, p. 4.Beams, H. W. (1920). 6 Anat. Rec.,’ vol. 45, p. 137.Bensley, R. R. (1908). 4 Anat. Rec.,’ vol. 2, p. 105.------(1911). c Amer. J. Anat.,’ vol. 12, p. 297.Bergen, F. von (1904). 4 Arch. mikr. Anat.,’ vol. 64, p. 498.Bouin, P. (1905). 4 C.R. Soc. Biol. Paris,’ vol. 58, p. 916.



Secretion o f the Pancreas and Salivary Glands.

Bowen, R. H. (1926). ‘ Quart. J. Micr. Sci.,’ vol. 70, p. 75.Cajal, Ramon J. (1915). 4 Trab. Lab. Invest, biol. Univ. Madrid,’ vol. 12, p. 127.Champy, C. (1911). 4 Arch. anat. micr.,’ vol. 13, p. 55.Chlopin, N. G. (1927). 4 Arch. exp. Zellforsch.,’ vol. 4, p. 462.Cohoe, B. H. (1907). 4 Amer. J. Anat.,’ vol. 6, p. 167.Coveil, W. P. (1928). 4 Anat. Rec.,’ vol. 40, p. 213.Cowdry, E. V. (1926). 4 Amer. Nat.,’ vol. 60, p. 157.Cramer, W., and Ludford, R. J. (1928). 4 Proc. Roy. Soc.,’ B, vol. 104, p. 28.Florey, H. (1932, a). 4 Brit. J. Exp. Path.,’ vol. 12, p. 323.------(1932, b). Ibid., vol. 13, p. 349.Gatenby, J. B. (1921). 4 Quart. J. Microsc. Sc.,’ vol. 65, p. 265.------(1931). 4 Amer. J. Anat.,’ vol. 48, p. 421.------(1932). Ibid., vol. 51, p. 253.Heidenhain, R. (1868). 44 Studien Physiol. Inst. Breslau,” p. 488.------(1880). 4 Handbuch der Physiol, von L. Hermann,’ vol. 5.Hirsch, G. C. (1932, a). 4 Z. Zellforsch.,’ vol. 14, p. 517.------(1932, b). Ibid., vol. 15, p. 37.Honda, R. (1927). 4 Anat. Rec.,’ vol. 34, p. 301.Hoven, H. (1910). 4 Anat. Anz.,’ vol. 37, p. 343.------(1912). 4 Arch. Zellforsch.,’ vol. 8, p. 553.Horning, E. S. (1925). 4 Austral. J. Exp. Biol. & Med.,’ vol. 2, p. 135.Tiling, G. (1904). 4 Anat. Hefte,’ vol. 26, p. 385.Krause, R. (1895). 4 Arch. mikr. Anat.,’ vol. 45, p. 93.Laguesse, E. von (1900). Ibid., vol. 52, p. 706.Langley, J. N. (1879). 4 J. Physiol.,’ vol. 2, p. 261.------(1889). 4 J. Physiol.,’ vol. 10, p. 433.Ludford, R. J. (1925). 4 Proc. Roy. Soc.,’ B, vol. 98, p. 354.------(1930). Ibid., vol. 107, p. 101.Ma, W. C. (1924). 4 Anat. Rec.,’ vol. 27, p. 47.Mathews, A. (1899). 4 J. Morph.,’ vol. 15, Suppl., p. 171.Maximow, A. (1901). 4 Arch. mikr. Anat.,’ vol. 58, p. 1.------(1916). 4 C.R. Soc. Biol. Paris,’ vol. 79, p. 462.Michaelis, L (1900). 4 Arch. mikr. Anat.,’ vol. 55, p. 558.Mislawsky, A. N. (1913). 4 Arch. mikr. Anat.,’ vol. 81, p. 394.Morelle, J. (1925). 44 La Cellule ” volume Jubilaire V. Gregoire, vol. 37, p. 77.Mueller, E. (1896). 4 Arch. Anat. Physiol., Anat. Abt.,’ p. 305.Nassonow, D. (1923). 4 Arch. mikr. Anat. Abt.,’ vol. 97, p. 136.------(1924). Ibid., vol. 100, p. 433.Noll, A. (1902). 4 Arch. Anat. Physiol. Leipzig, Physiol. Abt.,’ p. 166.O’Leary, J. L. (1930). 4 Anat. Rec.,’ vol. 45, p. 27.Parat, M., and Painlev6, J. (1924). 4 C.R. Acad. Sci. Paris,’ vol. 179, p. 543.Parat, M. (1928). 4 Arch. Anat. micr.,’ vol. 24, p. 73.Ranvier, L. A. (1886). 4 Arch. Physiol, norm, et path.,’ vol. 8, p. 223.Regaud, C , and Mawas, J. (1909, a). 4 C.R. Soc. Biol. Paris,’ vol. 66, p. 97.------(1909, b). Ibid., vol. 66, p. 461.Saguchi, S. (1920). 4 Amer. J. Anat.,’ vol. 20, p. 347.Scott, W. J. M. (1916). 4 Amer. J. Anat.,’ vol. 20, p. 237.Stormont, D. L. (1928). 44 Special Cytology ” edited by E. V. Cowdry, vol. 1.Young, J. Z. (1932). 4 Quart. J. Micr. Sci.,’ vol. 75, p. 1.



20 e. s. duthie. - royal...

Documents