124 Cytologia 26

Morphological and Histochemical Studies on the Uropygial Glands of Pigeon and Domestic Fowl

Kuldip Chand Kanwar1

Department of Zoology, Panjab University, Hoshiarpur, India

Received June 28, 1960

Introduction

Although quite a good deal of cytological work has been done in recent years on glands secreting triglycerides (e. g., sebaceous glands, mammary glands and Harderian glands), uropygial glands or more popularly known as oil glands of birds have not received closer attention of the workers. A few papers available (Altmann 1894, Bowen 1926, Bhatia 1943, Das and Ghosh 1959) dealing with the morphological and histochemical studies of these glands supply insufficient data as to how exactly the secretion is manufactured inside these holocrine glands and more particularly, as to what is the role of the mitochondria during the secretory genesis. Altmann (1894) and Bowen (1926) employed, almost exclusively, techniques involving prolonged osmication which, as Nath (1944) and Baker (1953) have also observed, have little morphological and absolutely no histochemical validity. Similarly Bhatia (1943) also depended mainly on Kolatchev and F. W. A./ preparations. Recently Das and Ghosh (1959), who worked out the histochemistry of the oil gland cells of the pigeon, have published only a brief note. As early as 1894, Altmann observed that the peripheral envelope of each secretion droplet differs chemically from the central part. He based his observations on the fact that in various osmium preparations it is only the periphery of the secretion droplet which gets impregnated whereas the se cretion droplet proper remains untinged. Bowen (1926), who worked on a variety of holocrine cells, contrary to the findings of Altmann (1894), established that there is no peripheral im pregnation of the secretion droplets and whatever is impregnated in various osmium preparations is not a part of the secretion droplets but the remnant of the 'Golgi apparatus'. Bhatia (1943) recorded that the secretion is always formed within the 'Golgi interna' surrounded with highly o smiophil cortices ('Golgi externa') . Quite recently, Baker (1954, 1957) and Nath (1957, 1959) have argued that the use of the terms like 'Golgi material', 'Golgi apparatus', 'Golgi

1 Present address: Department of Zoology, Panjab University, Chandigarh, India. 1961 Morphological and Histochemical Studies on the Uropygial Glands 125 bodies' etc. should be dropped , as these terms do not refer to structures which can be held homologous in different types of cells . These renowned cytologists further deny the existence of the classical 'Golgi apparatus' , from all types of cells, to which Bowen (1926) attributes a crucial role in the synthesis of the secretion. These observations of Baker and Nath call for fresh investigations by employing the rational morphological and various other cytochemical techniques. The purpose of this communication is to correlate the present findings with those of other authors and, if possible, to bring about unanimity in the varied descriptions available upto this date.

Material and techniques Only the male pigeons and fowls were used for the present investiga tions. The animals were dissected alive and the uropygial glands were taken out. These were further cut into small pieces which in turn were transferred to the physiological solution. The material was then dropped into various fixatives and processed accordingly. The various techniques which have been employed include formaldehyde osmium (Baker 1944) followed by gelatin as well as paraffin embedding, Kolatchev, formaldehyde calcium (Baker 1946) -both with and without postchroming , Regaud, Lewitsky saline (Baker 1956a), Zenker and Carnoy. The paraffin sections of the last four fixatives were usually stained with 0.5% iron haematoxylin. Regaud fixed material was also coloured with Sudan black B (SBB) and also treated with acid for the demonstration of the mitochondria. Carnoy fixed material was mainly used for with mercuric bromophenol blue (Hg-BPB) for the detection of proteins (Mazia et al. 1953). The various employed for colouring the include ethanolic Sudan black B (Baker 1949), nile blue sulphate (Cain 1947), Sudan III and IV (Kay and Whitehead 1941) and Fettrot (Pearse 1954). These have been mainly used on gelatin sections. Extractions with like acetone and were tried on formaldehyde calcium (FCa) unpostchromed gelatin sections. In addition to all this, acid haematein (AH) technique for the identification of phospholipids (Baker 1946) along with its pyridine extraction control, Performic acid-Schiff (Pearse 1951) for unsaturated bonds and Fischler's test for fatty acids (Pearse 1954) have also been employed. Schultz's test for cholesterols (Gomori 1952) was also performed.

Periodic acid-Schiff (PAS) technique (Hotchkiss 1948) for the detection of general (1•F2 glycol group) coupled with its acetylation control and KOH reversal treatment (Gersh 1949) was used both on paraffin and gelatin sections. For the cytochemical detection of RNA (cytoplasmic basiphilia), paraffin sections of Zenker (chilled) fixed material were stained with pyronin G/methyl 126 K. C. Kanwar Cytologia 26

green (Jordan and Baker 1955). Control sections, prior to their staining with pyronin G/methyl green, were treated with trichloroacetic acid (Schneider 1945) and perchloric acid (Erickson et al. 1949), both of which dissolve out the RNA completely.

Observations The uropygial glands of the pigeon and the fowl are multiple acinar holocrine glands. The whole gland, in both the animals, consists essentially of a sac with a central lumen. Since the new cells are regularly being added to replace those which are constantly being destroyed along with the dis charge of the secretion products (holocrine), all stages of secretory cycle can be observed even in a small portion of the gland. The glandular acini consist of small undifferentiated peripheral cells and large 'vacuolated' cells. Progressive maturity of the cells from the periphery towards the centre is indicated by the increasing quantity of lipid (sudanophil) material in the cytoplasm-the cells just near the lumen being fully packed with lipid droplets (Fig. 22). The gland cells form a stratified epithelium arranged in the form of long tubes. The centre of each tube is pierced by a narrow lumen through which the secretion products are discharged into the common duct. The earliest undifferentiated cells or 'indifferent cells' of Das and Ghosh (1959) are small, peripheral in position and these rest on an acinar basement membrane. There is a gradual increase in size of these cells from the peri phery towards the lumen. These earliest cells-farthest from the lumen, contain comparatively large, more or less centrally placed nuclei (with one or sometimes more nucleoli each). The cytoplasm of these cells is more or less homogeneously basiphil. Since the basiphilia is labile in salivary RNase, perchloric acid and trich loroacetic acid, it is evidently due to the presence of RNA. The ground cytoplasm of these cells is stained appreciably with mercuric bromophenol blue and also gives homogeneous diffuse pink coloration after periodic acid -Schiff . There is observed a gradual decrease in the cytoplasmic basiphilia and cytoplasmic affinity for mercuric bromophenol blue and periodic acid -Schiff from periphery towards lumen, so much so that the cells adjoining the lumen are almost completely devoid of these various colorations. Mitochondria. The cytoplasm of the early undifferentiated cells reveals uniformly distributed mitochondria which are in the form of rods or small stout filaments, each usually with one granule (Figs. 1-10). These mitochondria appear sufficiently impregnated in Kolatchev and formaldehyde osmium preparations (Fig. 1). The surface or end granules in such preparations appear jet black. The mitochondria are very well preserved in all the fixatives employed. In FCa/SBB (Fig. 4) and Lewitsky saline/SBB (Fig. 6) preparations, the mitochondria are stained distinctly with intense 1961 Morphological and Histochemical St udies on the Uropygial Glands 127

Figs. 1-22. Abbreviations. LG, lipid granule. M, mitochondria. N, nucleus. N, nucleolus . P, peripheral investment. PR, peripheral remnants. S, secretion granule or droplet . SG, surface granule. SV, secretion vacuoles. All the figures have been drawn with the help of camera lucida. 1, fowl. Kolatchev. 2, 3, fowl. Formaldehyde calcium/acid haematein- Sudan III and IV. 4, pigeon. Formaldehyde calcium/Sudan black B. 5, pigeon. Formaldehyde calcium/nile blue. 6, pigeon. Lewitsky saline/Sudan black B. 7, fowl. Carnoy/mercuric bromophenol blue. 8, pigeon. Formaldehyde calcium/acid haematein- Sudan III and IV . 9, fowl. Formaldehyde calcium (unpostchromed)-acetone extraction/Sudan black B . 10, fowl. Formaldehyde calcium (unpostchromed) ethanol extraction Sudan black B. 11, pigeon. Formaldehyde calcium/nile blue. 12, pigeon. Formaldehyde osmium. 13, fowl. Kolatchev . 14, fowl. Formaldehyde calcium/acid haematein-Sudan III and IV. 15, pigeon. Formaldehyde calcium/acid haematein-Sudan III and IV. 16, fowl. Formaldehyde calcium/nile blue . 17, fowl. Lewitsky saline,/Sudan black B. 18, pigeon. Lewitsky saline/Sudan black B. 19, fowl. Carnoy/mercuric bromophenol blue. 20, fowl. Formaldehyde calcium (unpostchromed) -acetone extraction/Sudan black B. 21, pigeon. Formaldehyde calcium (unpostchromed)- ethanol extraction/Sudan black B. 22, fowl. Formaldehyde calcium/Sudan black B. 128 K. C. Kanwar Cytologia 26 blue black granules on their surfaces. Similarly these mitochondria are clearly stained in Lewitsky saline/haematoxylin, acid haematein (even after pyridine extraction) (Figs. 2, 3, 8), Carnoy/haematoxylin, and Hg-BPB (Fig. 7), and Regaud/haematoxylin and acid fuchsine preparations. These mito chondria are also appreciably coloured with SBB, even when the FCa (un postchromed) sections, prior to their colouring with SBB, are extracted in cold acetone and ethanol for 24 hours (Figs. 9, 10). All this indicates that the mitochondria in the present material have the usual lipoprotein nature, the lipids in all probability being phospholipids. The mitochondria along with associated granules are distinctly stained blue in FCa/nile blue preparations (Fig. 5). In later stages, when the cells are engaged in active neutral lipid synthesis, the mitochondria are not easily detected owing the over crowding of the lipid droplets. In the fowl, however, the filamentous mitochondria have been observed in the thin strands of the cytoplasm, left in between the growing secretion droplets (vacuoles in this case) during pretty late stages of the secretory synthesis in even Carnoy/haematoxylin, and also in Kolatchev preparations. Lipids. In these undifferentiated cells, as well as in slightly later stages, the cytoplasm reveals sufficient number of uniformly distributed granules of varying sizes in addition to the mitochondria (Figs. 3-7). These granules are also lipoproteinous in nature and can be seen in all the preparations men tioned above. In the oil gland cells of the pigeon, sometimes, certain pro tein bodies (Hg-BPB positive) with incomplete lipid (sudanophil) ensheathments have also been observed during these stages. Upto these stages all the cell organelles seem to resist paraffin embedding since no difference is observed between paraffin and gelatin preparations. There now starts active lipid synthesis in the cells which, owing to the increasing accumulation of the lipids in the cytoplasm, go on increasing in size considerably, though gradually, along with the advancement in the se cretory phase. Now the lipid granules which are still very small, start revealing duplex nature in formaldehyde osmium (Fig. 12), Aoyama and Lewitsky saline/SBB (paraffin) preparations with distinctly osmiophil, ar gentophil and sudanophil cortices and osmiophobe, argentophobe and sudan ophobe interna respectively. In all these paraffin preparations these duplex bodies rarely reveal uniform cortices. Instead the outer coat is usually reduced to thin crescents or rodlets and, in later stages (vide infra), even to a very thin membrane which binds the internum completely, and on this binding membrane are seen studded one, two or even three lipoproteinous granules which too are osmiophil, argentophil and sudanophil. The interna of these duplex bodies in osmium preparations appear yellowish (Fig. 12). In slightly later stages the outer lipid cortices along with the associated granules come out as blue structures in acid haematein preparations and when such prepara tions are counterstained with a mixture of Sudan III and IV-which colours 1961 Morphological and Histochemical Studies on the Uropygial Glands 129 the interna of these bodies pink, a good contrast is achieved (Fig. 8). The Lewitsky saline/SBB (paraffin) preparations reveal distinctly the sudanophil cortices whereas the interna (hitherto sudanophobe), in which now has started the accumulation of the neutral lipids (triglycerides) , though as yet in smaller quantity, catch this with lesser intensity; thus pointing towards the fact that neutral lipids are not suitably preserved in this fixative (vide, infra) and are partly dissolved out during the course of paraffin treatment . This view is further strengthened when the corresponding stages are studied from FCa/nile blue preparations. This dye colours the peripheries of these bodies blue and the interna either as violet or slightly pinkish (Fig. 5). In these stages, even the osmium impregnation in the interna is comparatively intense as compared with the impregnation of more matured neutral lipid droplets encountered in still later stages. In FCa/SBB preparations, however, these bodies appear as intensely coloured homogeneous structures (Fig. 4). Now coupled with the further increase in the size of the cells there is a rapid synthesis of the neutral lipids. Cells undergoing this phase of active lipid synthesis reveal, in the unstained FCa preparations, growing secretion droplets as clear greyish shining spheroids in contrast to the dull yellow cyto plasmic background. In these 'intermediate cells' too, the secretory droplets in osmium preparations reveal sufficiently impregnated peripheral investments of variable thickness (Fig. 12). In slightly later stages in osmium prepara tions, the peripheral investments are seen as broken rods closely associated with the secretion droplets (Fig. 13). These stages are best studied in acid haematein counterstained with Sudan III and IV (Figs. 8, 14, 15), and nile blue preparations (Figs. 11, 16). In both these preparations a good contrast between the secretion droplets (which appear pink) and their ensheathments along with associated granules (which stain blue) is achieved. In such pre parations it becomes perfectly clear that every secretion (neutral fat) droplet is invariably bound by an outer variably thick membrane, studded quite often with granules-both having the same lipoproteinous nature. These investing membranes, and their associated granules resist cold acetone (Figs. 9, 20), cold ethanol (Figs. 10, 21) and hot pyridine extractions. These investments are very clearly seen in Lewitsky saline/haematoxylin preparations and are also stained with acid fuchsine and Hg-BPB when applied on FCa, Zenker and Carnoy (Fig. 19) sections. All these reactions confirm their lipoprotein ous nature. When the cells with considerable amount of neutral lipids in the cyto plasm are studied from the paraffin preparations, these present a much dis torted appearance. Such cells in Lewitsky saline/SBB and haematoxylin preparations reveal clear vacuoles (formed obviously after the dissolution of neutral lipids) with scanty cytoplasm (interspaces of vacuoles) which itself is sufficiently coloured with SBB as well as with haematoxylin. In such paraffin 130 K. C. Kanwar Cytologia 26 preparations there is not even suitable preservation of the investing membranes of the individual secretion droplets. In Lewitsky saline/SBB preparations, it has been observed that a thick peripheral band of the cytoplasm (just inner to the cell membrane) is more intensely coloured with this colorant. Similarly this peripheral cytoplasmic band is also appreciably stained with Hg-BPB when applied on Carnoy and FCa fixed materials (not shown in the figures). Some of other interesting observations which were made during the pre sent study are enumerated below:- 1. Lewitsky saline does not fix the neutral lipids very satisfactorily. It has been observed, both in the pigeon and the fowl, that during the inter mediate and still later stages there are seen in the cells, homogeneously coloured sudanophil droplets and duplex bodies of the same size with sudan ophil cortices and either completely sudanophobe or lightly coloured interna (Figs. 17, 18). When corresponding stages are studied from Lewitsky nile blue sulphate preparations the duplex bodies give exactly the same appearance. Since such duplex structures are not seen in the corresponding stages in FCa/SBB preparations (vide supra) these must be regarded as fixation arti facts. 2. Secretion droplets (neutral lipids) do not resist paraffin treatment, even after postchroming the material for about 8 days as in Regaud technique, indicating thereby their highly saturated nature. This is further strengthened by performic acid-Schiff technique which constantly gave negative results. These secretion droplets seem to resist paraffin treatment when the is postosmicated for 5 to 6 days as in Kolatchev preparations. Neutral lipid globules in such preparations appear as slightly greyish spheres.

3. Periodic acid-Schiff test was tried to detect the presence of carbo hydrates (1•F2 glycol group). After acetylation control and KOH reversal treatment it was ascertained that the younger cells reveal diffuse pink colora tion in the ground cytoplasm. The Schiff's coloration goes on diminishing in the more matured cells till, after the intermediate stages, there is practi cally left no such coloration in the cells. 4. Cytoplasmic basiphilia (RNA) has been studied with the help of pyronin G/methyl green technique. The distribution of the cytoplasmic RNA is exactly the same as has been noticed in case of carbohydrates. 5. In the lumen, in addition to the big neutral lipid globules appear ing pink in nile blue preparations, there are seen clear vacuoles. Such clear vacuoles are also seen in FCa/SBB preparations. When Hg-HPB and periodic acid-Schiff preparation are studied, it is established that there is a good amount of material in the lumen which is positive to both these reagents, revealing thereby the presence of proteins and carbohydrates in the lumina. It is probable that these Hg-BPB and PAS positive masses represent the same bodies which appear vacuolar in various SBB preparations. Similarly acid 1961 Morphological and Histochemical Studies on the Uropygial Glands 131 haematein preparations (both FCa and pyridine extracted sections) reveal a considerable amount of positive material in the lumen. 6. Schultz test for cholesterols was also tried several times and it gave constantly negative results. 7. Feyrter's 'enclosure' method for the detection of metachromasia was also employed. In none of the cells at any stage does the metachro matic coloration appear. However, in certain lumina of the pigeon there were seen metachromatic masses. This too, however, was not a constant feature. 8. Fischler test for fatty acids was also performed. This too yielded negative results.

Discussion Although the mitochondria are easily discernible in the oil gland cells, especially in the early stages, yet very little information is available concern ing their morphology, chemical nature and their role in the secretory genesis. Even the recent histochemical studies carried out by Das and Ghosh (1959) on the uropygial gland of the pigeon are silent with regard to the mito chondoria. Whatever, in detail, is known about the morphology of the mitochondria, their chemical nature and role in the secretary genesis in the holocrine cells, is based, almost exclusively, on observations collected from other tissues similarly engaged in synthesizing neutral lipids, e. g. sebaceous glands, Harderian glands, inguinal glands etc. etc. The mitochondria during the course of present investigations were found to be in the form of either rods or rodlets or stout filaments, each with one, two or sometimes more surface granules. These come up very well in Lewitsky saline/Sudan black B, acid fuchsine, haematoxylin, Carnoy/haema toxylin and Hg-BPB, formaldehyde calcium/SBB, Hg-BPB and nile blue (blue) and acid haematein preparations. These mitochondria along with their sur face granules resist cold acetone and ethanol extractions done for 24 hours or even more and when such extracted sections are treated with SBB these organelles are appreciably coloured. These also come up very well in pyri dine extracted material treated afterwards for acid haematein. All this towards the conclusion that the mitochondria, as usual, in the present material too are composed of lipoprotein complex, lipids most probably being phos pholipids (Baker 1956b and Brachet 1957). The mitochondria are also stained with (supervitam). In early cells, wherein secretion synthesis (neutral lipids) has not yet actually begun, these mitochondria are found in comparatively large number and are uniformly distributed throughout the cytoplasm. With the initiation of rapid lipid synthesis, their visibility is hindered (see also Kanwar 1959a and b) probably due to the over-crowding of the lipid droplets. In case of fowl, however, the filamentous mitochondria have been observed compressed in the thin strands of the scanty cytoplasm-left in between the growing 132 K. C. Kanwar Cytologia 26

secretion droplets (vacuoles in this case), even during the last stages of secretory synthesis in even Carnoy/haematoxylin preparations. This indicates that even when these cytoplasmic organelles are not easily observed in most of the preparations, they are present in the cytoplasm. The present findings broadly coincide with those of Montagna (1955) so far as the presence of the mitochondria in late secretory stages is concerned. It is, however, difficult to state that the mitochondria do not vary in number in various phases of secretory cycle, as Montagna (1955) has observed. The findings of Nicolas and others (1914) and Ludford (1925), who observed that the mitochondria increase in number at the beginning of the secretion synthesis and afterwards become fewer, seem to be nearer to the present observations. Various earlier workers attributed an interesting role of lipid synthesis (neutral lipids) to these mitochondria. Perhaps Nicolas and others (1914) were the first to believe that the lipid droplets first appear within the mitochon drial filaments. Murray (1916) claims to have observed the formation of lipid globules within the mitochandria in the cells of transplantable sarcoma of the guinea pig. Similarly Kater and Smith (1932) are also the ardent supporters of such a direct origin of lipid bodies from the mitochondria. What appears to the author is that the surface or end granules of the mitochondria get detached from the rods or filaments, grow in size, become vacuolated and finally serve as the sites of condensation for the secretory products. For detailed discussion about the origin of the lipid bodies ('Golgi bodies') from the mitochondria, reference is made to Lever (1955, 1956), Rishi (1956, 1957), Nath and Gupta (1956) and Kanwar (1958, 1959a and b). Contrary to the opinions expressed by Nicolas and others (1914) and Kater and Smith (1932), neither Ludford (1925) nor Montagna (1955) recorded any such direct transformation of the mitochondria into secretion droplets. The present observations broadly support the views of Ludford and Montagna in not establishing any direct transformation of the mitochondria into the secretion globules. ' Golgi apparatus'. There exists nothing which could even remotely be compared with the classical 'apparato reticolare interno' of Golgi (1898). Even Bowen (1926), who has described the definite existence of a reticular ' Golgi apparatus' in various other vertebrate exocrine glands, denies the existence of a typical net-like 'Golgi apparatus' in the oil gland cells. He states, ".... the dispersion of the Golgi material in the form of discrete pieces is accomplished before secretion actually begins and at no stage is there any reticular development ." In the final stages, Bowen (1926) observed that 'no Golgi material is impregnated except small crescentic rods or caps which are fitted around the individual granules.' Bowen further adds that 'the entire apparatus becomes distributed to the granules' in the final stages. 1961 Morphological and Histochemical Studies on the U ropygial Glands 133

What appears is that Bowen considered the outer lipoproteinous sheaths (of variable thickness) along with their associated granules as the 'reminiscent' of the 'Golgi apparatus' . These ensheathments in younger stages are com paratively thick and appear as 'rodlets' or 'caps' and are sufficiently impreg nated by osminum tetroxide . In later stages when these lipoproteinous sheaths become very thin and are seen only with difficulty , the identity of granules studded on them becomes comparatively conspicuous . Probably this led Bowen to conclude that the 'granules' are the remnants of the 'Golgi apparatus' in these stages. Observations regarding the 'Golgi element' in the various holocrine glands were also made by various other workers. Ludford (1925), Melczer and Deme (1943) observed that as the lipid (neutral fat) globules increase in size, the osmiophilic bodies or their 'Golgi element' (peripheral ensheath ments of the present investigations) become reduced to curved rods or 'shells' around them. Gibbs (1956) while working on the buccal glands of the lamprey states, "The division of th e lipid droplets into phospholipid-rich shells and triglyceride -containing centers is reminiscent of the so-called Golgi externa and interna of Hirsch (1939)." The above findings of Gibbs stand in strict conformity with the present observations. Exactly similar findings with regard to the 'Golgi apparatus' have been made by Quay (1954) who worked on the holocrine skin glands of Kangroo rat. Recently Baker (1953, 1954, 1957) and Nath (1957, 1959) have also denied the existence of the 'Golgi apparatus' from all types of animal cells. Lipids. Altmann (1894), who worked on a variety of holocrine glands, for the first time observed that the peripheral envelope of each droplet differs chemically from the central part. In all his preparations he observed that only the peripheral envelope is impregnated while the secretion droplet proper remains untinged. This is exactly what the author has observed during the course of current investigations. Here too the lipoproteinous peripheries (around the secretion droplets) have only been observed to be impregnated and the secretion droplets proper, which are composed of neutral lipids (triglycerides), remain totally unimpregnated by osmium tetroxide. Bowen (1926), while working on various holocrine glands, contrary to the views of Altmann (1894) established that there is no peripheral impregna tion of the secretion droplets and whatever is impregnated is the 'Golgi apparatus.' Leave aside Bowen (1926), who probably misinterpreted the outer en sheathments as 'reminiscent' of the 'Golgi apparatus' (vide supra), though he observed such structures, Das and Ghosh (1959), while working on the uropygial gland of the pigeon after employing various histochemical techniques, sur prisingly failed to observe any coverings or limiting membranes around

Cytologia 26, 1961 10 134 K. C. Kanwar Cytologia 26 individual secretion droplets. Since these investigators employed mainly Sudan III and IV for the study of the lipids, they do not seem to have detected peripheral ensheathments which in such preparations do not give contrasting colour as compared to the secretion droplets. The above observations find support from the works of Montagna and Noback (1947), and Quay (1954). Montagna and Noback (1947) likewise observed that phospholipids are present in the form of granules and rodlets closely associated with the secretion globules. Quay (1954), while working on the holocrine skin glands of Kangaroo rat, similarly observed that granules which lie on the surface of neutral droplets (secretion globules) are embedded in acidic caps (since these are stained blue in nile blue). Quay (1954) further observes that the 'granules' and the 'black lining of vesicle walls' remain after 15 hours ether extraction and also after pyridine extraction (since these are lipoproteinous in nature). Above all, the present findings are fully supported by Worley (1946), Baker (1949), Hirsch (1955), Rishi (1956, 1957) and Kanwar (1959a and b), all of whom hold the view that the secretion droplets never appear 'loose' in the cytoplasm. The 'Golgi bodies' or the lipid vesicles serve as the sites of condensation for the secretion products. Cytoplasmic Basiphilia. The early cells or 'undifferentiated cells' are more basiphil than the intermediate ones. The cells which have almost completed the secretory cycle and are about to be disintegrated reveal hardly any cytoplasmic RNA. These observations find support from the latest com munication of Brachet (1960). Brachet also is of the view that the cells, which are not engaged in the synthesis of proteins and yet are metabolically quite active otherwise, are weakly basiphil.

Summary The present communication records observations on the cytoplasmic inclusions of the uropygial glands of the pigeon and the domestic duck. During the course of present investigations, classical 'Golgi techniques ,' morphological techniques of known chemical reactions and other current histo chemical techniques have been employed. The mitochondria, which are of usual lipoproteinous nature, are in the form of rods or filaments, each having one or more surface granules . These surface granules detach themselves from their respective mitochondrion, grow in size, get vacuolated and serve as sites for the condensation of neutral lipids (secretion). Every secretion droplet is invariably ensheathed by thin lipoproteinous membrane with which are sometimes seen associated granules of similar chemical nature . There has been observed a gradual decrease in the cytoplasmic basiphilia (RNA) and cytoplasmic affinity for Hg-BPB and PAS colourations from periphery to wards lumen, whereas reverse is the case with sudanophilia . In the lumina, in addition to the secretory products (neutral lipids), the presence of proteins 1961 Morphological and Histochemical Studies on the Uropygial Glands 135 and carbohydrates has also been detected. There has been observed nothing which could even remotely be com pared with the much publicized 'apparato reticolare interno' of Golgi.

Acknowledgements The author is deeply indebted to Dr. Vishwa Nath, Sc. D. (Cantab.), F. N. I., formerly Professor and Head of the Panjab University Zoology Department and now Emeritus Professor of Zoology, under whose direct supervision the present investigations were carried out. Help rendered by Mr. Sohan Singh Jande is sincerely acknowledged. Grateful thanks are also due to Dr. G. P. Sharma, Ph. D. (Pb. and Edin), Professor and Head of Panjab University, Zoology Department, for providing research facilities.

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