Ultrastructural observations on the granular leucocytes of the tuatara Sphenodon punctatus (gray)

TISSUE & CELL 1979 11 (4) 7033715 Published by Longman Group Ltd. Printed in Great Brtiain

SHERWIN

S. DESSER and IRIS WELLER

ULTRASTRUCTURAL OBSERVATIONS GRANULAR LEUCOCYTES OF THE SPHENODON PUNCTATUS (GRAY)

ON THE TUATARA

ABSTRACT. The granular leucocytes of an active, mature female tuatara, Sphenodon prrncfatus (Gray) were examined in the electron microscope. Eosinophils contained a lobulated nucleus, homogeneous, dense, irregularly shaped granules, assorted smaller granular inclusions, mitochondria and p-glycogen. Endoplasmic reticulum, Golgi complexes and ribosomes were scanty. Immature neutrophils (myelocytes) were regular in outline and contained a compact nucleus. In the adjacent centrosomal region were paired centrioles with connected microtubules, and Golgi complexes. Ovoid electrondense granules, mitochondria, lipid droplets and numerous microfilaments arranged randomly or in bundles. lay in the cytoplasm. Mature neutrophils were often highly irregular in outline, had a segmented nucleus and contained possibly a second type of granular inclusion. The basophils were regular in outline with a compact nucleus. Numerous ovoid homogeneous, electron-dense granules, mitochondria, fl-glycogen particles and some microfilaments were seen in the cytoplasm. The granules in many basophils appeared ‘altered’ or degenerate and most of these contained microtubules The cytology of the granulocytes of the tuatara is compared with that in other vertebrates.

Introduction THE tuatara, Sphenodon punctatus, the only surviving member of the reptilian order Rhyncocephalia, is confined to about 20 small offshore islands of New Zealand (Dawbin, 1962). This reptile is a relict species which has remained virtually unchanged for up to 200 million years (Robb, 1973), and is frequently referred to as a ‘living fossil’ or ‘dinosaur’. As such it has attracted the attention of many zoologists and during the past century several studies of its anatomy, histology and physiology have appeared. Recent haematological studies on an adult female tuatara from Stephens Island included morphological, cytochemical and biochemical data (Desser, 1978, 1979). In the present study the ultrastructure of the granular leucocytes, including the eosinoDepartment of Microbiology and Parasitology, Faculty of Medicine, University ofToronto, Toronto, Ontario, Canada M5S IAI. Received 16 January 1979. Revised 19 July 1979.

Phil, neutrophil and basophil is described and compared with that of similar cells of amphibians, other reptiles and of higher vertebrates. Materials and Methods A sexually mature female tuatara was captured on Stephens Island on 31 October 1977 and brought to the Zoology Department, University of Canterbury, Christchurch, New Zealand, where it was kept outdoors in a special cage (see Desser, 1978, for details of housing, diet and general care). On three occasions during the summer months (November to January), when the animal was active and feeding regularly, blood was taken and fixed for electron microscopy. Blood from a clipped digit was introduced directly into a solution containing glutaraldehyde and osmium tetroxide buffered with sodium cacodylate and fixed at room temperature for 1 hr according to the method of Franke et al. (1969). The blood was then collected in microhaematocrit tubes and spun at 11,000

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rev/min for 5 min. The buffy layer was removed from the tubes and post-fixed in osmic acid. Subsequently the tissue was dehydrated in a graded alcohol series followed by propylene oxide and embedded in a mixture of Epon-Araldite. Thin sections were stained in a saturated solution of uranyl acetate in methanol followed by lead citrate. For detection of glycogen other sections were collected on gold grids and stained by the periodic acid thiosemicarbazide silver proteinate (PA-TSC-AG protein) method of Thiery (1967) using 30 min oxidation in 1% (w/v) periodic acid, 30 min in 1 % (w/v) thiosemicarbazide and 30 min in a 1% (w/v) solution of silver proteinate (Protargol from Roboz Surgical Instrument Co., Washington, D.C.). Control sample were processed as above omitting the periodic acid oxidation. The sections were examined in a Zeiss EM 9A electron microscope. Light photomicrographs of Giemsa-stained blood films were taken on Kodak Panatomic X film with a Zeiss photomicroscope. Results Eosinophil

In Giemsa-stained films, eosinophils measured about 17pm and were the smallest granular leucocyte. The nucleus was usually bi- or trilobed and the cytoplasm contained numerous closely spaced eosinophilic granules. One or more pseudopodia were often seen around the periphery of these cells (Fig. I).

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It was observed ultrastructurally that the chromatin in the lobulated nuclei of mature eosinophils was rather condensed and marginated (Fig. 2). Elements of granular endoplasmic reticulum (ER) and Golgi were sparse and a few small mitochondria were evident. Free ribosomes were not seen (Fig. 3). Numerous large (0.74.8 pm), irregularly shaped, homogeneous, dense granules occupied much of the cytoplasm (Figs. 2, 4, 5). Some of the large homogeneous granules appeared disrupted in many eosinophils. In some cases the core of these granules appeared empty with some residual electron-dense material around the periphery (Fig. 2); in others membranous structures were seen within the altered granules (Figs. 2, 3). Pronounced indentations were seen at the periphery of several large granules (Fig. 2). Among the large granules were smaller circular ones of varying electron density and small dense, dumb-bell-shaped inclusions (Figs. 4, 5). Microfilaments were seen between the granules (Fig. 4) and were especially concentrated at the juctions between the granulebearing cytoplasm and pseudopodia (Fig. 5). The PA-TSC-Ag protein technique revealed the presence of numerous dense figlycogen-like, particles in the cytoplasm (Fig. 6). These particles were concentrated more heavily in the pseudopodia than in the granule-bearing cytoplasm. Control specimens contained no stained particles (Fig. 7).

Abbreviations Ce, centrioles Er, endoplasmic reticulum Go, Golgi complex Li, lipid

used in figures Mf, microfilaments Mi, mitochondrion Nu, nucleus Ps, pseudopod

Fig. I. Photomicrograph of Giemsa-stained eosinophil with a bilobed nucleus, eosinophilic granules, and several peripherally arranged pseudopodia. x 1040. Fig. 2. Electron micrograph of eosinophil with two nuclear lobes (Nu) containing condensed chromatin. The cytoplasm contains numerous large homogeneous, electrondense granules, some of which are irregularly shaped with peripheral indentations (arrows). Other large granules (asterisks) appear ‘empty’ with some of the dense matrix material around their periphery. Numerous smaller granules including dense spherical ones lie in the cytoplasm. Note the large pseudopod (Ps) devoid of granular inclusions. x 17,680.

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Neutrophil In Giemsa-stained films, neutrophils measured about 23p.m and were the largest granulocyte. They were roughly circular and the nucleus of mature cells appeared segmented, often containing three to five lobes. The cytoplasm was stained faintly and appeared reticulate. Granules were not clearly discernible in the cytoplasm (Fig. 8). In the electron microscope many immature neutrophils (myelocytes) were observed. The nucleus of the myelocytes appeared relatively homogeneous with a slight condensation of chromatin around the nuclear margins. Nucleoli were not seen at this stage. Adjacent to the nucleus was an obvious centrosomal area containing scattered Golgi membranes and many small vesicles (Fig. 9). Within the centrosome was a pair of centrioles and the associated microtubules. At higher magnification the perpendicularly arranged centrioles could be resolved more clearly. Microtubules were attached to the satellite of one of the centrioles and numerous small vesicles and granules lay in the adjacent cytoplasm (Fig. 10). Granular reticulum, several mitochondria, lipid inclusions and microfilaments were seen in the cytoplasm (Fig. 9). At this stage of maturation the cytoplasmic granules appeared to consist primarily of electrondense, spheroidal bodies measuring 0.4-0.6 pm. Mature neutrophils were more irregular in outline than the myelocytes and their nuclear chromatin was considerably more

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condensed. The cytoplasm of the mature cells contained at least two morphologically distinguishable spheroidal granules; some which were electron dense as those in the myelocytes, and others which were similar in size but with a lighter, more granular matrix (Fig. 12). Smaller granules, presumably immature stages, with light cores or with dense inclusions were also seen (Figs. 10, 12). Granular reticulum, microtubules and scattered microfilaments occurred in the cytoplasm of mature neutrophils. The microfilaments were occasionally arranged into discrete bundles which coursed through the cytoplasm (Fig. 11). Basophil In Giemsa-stained films the basophils were roughly circular and were intermediate in size between the eosinophils and neutrophils measuring about 21 pm in diameter. They were characterized by numerous large, spherical, dark-staining granules which partially obscured a compact nucleus (Fig. 13). The chromatin of the nucleus of mature basophils was quite condensed and nucleoli were not seen (Fig. 14). Large, spherical, uniformly electron-dense granules which measured 0.8-l .Oprn were abundant in the cytoplasm in which granular reticulum and ribosomes were scarce. Some mitochondria were seen and microfilaments were scattered in the granular The granules

altered.

cytoplasmic

matrix.

in many basophils appeared Such granules assumed a variety of

Fig. 3. An ‘altered’ eosinophilic granule with membranous matrix (arrow). adjacent intact homogeneous granules and mitochondrion (Mi). x 38,000.

Note

Fig. 4. Cytoplasm of eosinophil illustrating in addition to the large homogeneous granules, smaller spherical and dumb-bell shaped forms. Note microfilaments in the cytoplasmic matrix. x 42,000. Fig. 5. Junction between granule-bearing cytoplasm and pseudopod of an eosinophil. Note various types of granules and the discrete band of microfilaments (Mf) which lies between the granule-bearing cytoplasm and pseudopod. x 18,000. Fig. 6. PA-TSC-Ag-protein-stained particles in the intergranular cytoplasm.

eosinophil x 35,000.

Fig. 7. PA-TSC-Ag-protein unoxidized control glycogen-like particles are not stained. x 38,000.

with specimen

numerous

j3-glycogen-like

of an eosinophil

in which

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Fig. 8. Photomicrograph of a Giemsa-stained mented nucleus. The cytoplasm is faintly stained apparent. x 1040.

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neutrophil with characteristx segand reticulate and granules are not

Fig. 9. Electron micrograph of a neutrophil myelocytc with typical compact nucleus, adjacent to which lie paired centrioles (Ce) with associated microtubules. In the centrosomal region are many small electron-lucent vesicles and some Golgi membranes (Go). Several ovoid, uniformly electron-dense granules (arrows), mitochondria (Mi) and lipid droplets (Li) lie in the cytoplasm which contains also numerous small vesicles and microfilaments x 17,600.

appearances; some retained much of their dense contents, while others appeared less dense and contained myelin-like membranous whorls; many granules contained microtubules (Figs. 15-17). In slightly altered granules the microtubules were arranged in parallel arrays while in more advanced stages of ‘degranulation’, the microtubules were more randomly oriented (Fig. 16).

Centrioles with their associated microtubules were seen occasionally adjacent to the nucleus and were most apparent in basophils with altered granules (Fig. 17). The diameter of the intragranular microtubules (0.024~m) was similar to those associated with the centrioles. In basophils stained by the PA-TX-Ag protein method, numerous /3-glycogen-like

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particles were scattered among in the cytoplasm (Fig. 18).

the granules

Discussion Each type of granulocyte of the tuatara will be discussed separately and later their morphological and cytochemical features compared collectively with those of other vertebrates. Eosinophils The basic ultrastructural features of the tuatara eosinophil, including the segmented nucleus with its condensed and marginated chromatin, absence of nucleolus and reduced cytoplasmic reticulum, ribosomes and Golgi, are similar to those of turtles, lizards and amphibians (Kel&nyi and Nemeth, 1969; Taylor et al., 1963) and also of mammals (Bessis, 1973; Cline, 1975). The prominent pseudopodia seen in tuatara eosinophils fixed in either methanol or glutaraldehyde are a feature which has not been recorded from these cells in other vertebrates. The large, homogeneous granules of the tuatara eosinophil resemble those seen in these cells in frogs, turtles, lizards and birds (Dhingra et al., 1969; KelCnyi and Nemeth, 1969; Taylor et al., 1963). Crystalloid cores like those in the eosinophilic granules of most mammals, were not observed in the tuatara or in most other lower vertebrates with the possible exception of certain teleost fishes (Smith et [I[., 1970; Weinreb, 1963) and some birds. In a recent fine structural study of the eosinophils of 39 species of aquatic and terrestrial birds, Maxwell (1978) noted that their eosinophilic granules fell into three groups, those with crystalline interna, those with non-crystalline interna and the largest group, with homogeneous granules and no interna (like those of the tuatara). Maxwell was unable to attribute any phylogenetic importance to the granular morphology of avian eosjnophils. The positive benzidine peroxidase reaction of the eosinophilic granules of the tuatara (Desser, 1978) and of other lower vertebrates (Kelenyi and Nemeth, 1969) suggests that crystalloid inclusions are not necessarily related to the peroxidase activity and acidophilia commonly associated with these granules. Other features shared by the eosinophils of the tuatara and other verte44

brates include a positive PAS reaction (probably due in part to numerous glycogen inclusions) and acid phosphatase activity in the eosinophilic granules (Desser, 1978). Acid phosphatase activity in mammalian eosinophils is apparently associated with the small dense, spherical granules and not the large crystalloid bearing ones (Cline, 1975). Ultrastructural disorganization of the eosinophilic granules like that observed in the tuatara, has also been described in turtles and amphibians and according to KelCnyi and NCmeth (1969) may be due to ‘certain conditions (fixation, solvent effect, cellular breakdown, enhanced functional activity) which may lead to granular, vesicular or fibrillar matrix formation’. Peripheral indentations in the eosinophilic granules of birds and reptiles are similar to those observed in the tuatara and were thought by Keltnyi and N&meth (1969) to represent sites of interaction between granules and cytoplasm. Centrioles which were commonly seen in neutrophils and basophils in this study were not observed in eosinophils, and to our knowledge have not been recorded from mature eosinophils in other vertebrates. Ntw f rvphils The neutrophils of the tuatara share many common features with those of other vertebrates. In the myelocyte stage for example, the nucleus is compact with diffuse chromatin and without a nucleolus. The cytoplasmic reticulum and ribosomes are scanty and the Golgi complexes form a small spherical zone in the cell centre (centrosome), containing two centrioles from which several microtubules eminate. The neutrophil myelocyte of the tuafara contains primarily one type of spheroidal, electron-dense granule in contrast to the two types seen in mature cells. The distinction between the two ‘types’ is uncertain however, as they are of similar size and shape and differ only in the density of their matrix material. With progressive maturation, neutrophils in several mammalian species exhibit an increase in the variety and number of neutrophilic granules (Bessis, 1973). The morphology of the neutrophilic granules of the tuatara differs from that described for certain other reptiles and birds, in which rounded granules in immature cells become increasingly elongated and electron-

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dense. It is perhaps noteworthy that in some respects tuatara neutrophils resemble those of amphibians more than other reptiles. For example the neutrophilic granules of the frog (Rana esculenta) and the salamanders (Triturus cristatus and T. vulgaris) are small, beyond the resolution of the light microscope. Mixed populations of granules including smaller electron-dense and larger ovoid forms were seen in the electron microscope (Kelenyi and Nemeth, 1969). The neutrophilic granules of the tuatara and other reptiles, unlike the mammals, do not contain crystalloid cores, These cores are not, however, restricted to the higher vertebrates, as they have been recorded in amphibians (Kelenyi and Nemeth, 1969) and teleost fishes (Lester and Desser, 1975). While there is relatively little comparative data on the ultrastructural and chemical features of the neutrophilic granules of lower vertebrates, those of mammals have been studied extensively and are generally included in three groups which differ greatly, depending upon the host species and the method of fixation (Be&s, 1973). Unlike the neutrophilic granules of most other lower vertebrates, those of the tuatara react positively to peroxidase, acid phosphatase and esterase (Desser, 1978). Information on the specific localization of enzymes in

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granulocytes of mammals is much more advanced. Bainton and Farquhar (1968a, b) showed, for example, that peroxidase, acid phosphatase and esterase activities were localized in the azurophilic granules of neutrophils of rabbit and man and not in the neutrophilic (specific) granules, which did, however, react positively for alkaline phosphatase. Basophils

Unlike the neutrophils, the majority of basophils in the peripheral blood of the tuatara were mature. These basophils share many features with those of mammals including prominent electron-dense granules, scanty granular reticulum and ribosomes, a small centrosomal region with a relatively inactive Golgi, which surrounds a typical pair of centrioles, several mitochondria and glycogen particles (Cline, 1975). The nucleus of mature basophils of the tuatara contains condensed chromatin and does not have a nucleolus; it differs from the mammalian basophil nucleus in being compact and rarely segmented. The altered appearance of many of the granules in the basophils of the tuatara may have resulted from normal degranulation or possibly from the extraction of their electrondense matrix material during fixation and

Fig. 10. Paired centrioles are typically aligned perpendicular to each other in the centrosomal region of the neutrophil. The attachment of microtubules to a satellite of one of the centrioles is illustrated (arrow). Note the adjacent small vesicles and dense granules which commonly occur in this region. x 43,000. Fig. Il. Cytoplasm of mature neutrophil with prominent microfilament bundle (Mf). A large vacuole (arrow), dense granules and randomly arranged microfilaments can also be seen in the adjacent cytoplasm. x 23.500. Fig. 12. The cytoplasmic inclusions of mature neutrophils include spherical electrondense and less dense granules. Scattered in the matrix along with smaller granules and vesicles are ER cisternae (Er), mitochondria and many microfilaments. Mature neutrophils are irregular in outline (arrow). x 22,000. Fig. 13. Photomicrograph of a Giemsa-stained basophil prominent, spherical, dark staining granules. x 1040.

with a compact

Fig. 14. Electron micrograph of mature basophil with a compact condensed chromatin and large, spherical electron-dense cytoplasmic Fig. 15. Basophil with ‘altered granules’. Microtubular granules; a myelin figure is apparent (arrow), x 10,100.

nucleus and

nucleus containing granules. x 18,000.

profiles can be seen in some

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Fig. 16. Highly magnified port~n of cytoplawn of an ‘altered’ basophil. The mmotubules in the granule on the right appear to be oriented in parallel, in contrast to the granule on the left in which the tubules appear to be oriented mow randomly. x 55.000. Fig. I?. A centriole hamphil. x 36,000.

and associated

microtubules

further processing for electron microscopy as has been described for the basophilic granules of mammals (Cline, 1975). Intact granules appeared uniformly electron-dense with RO apparent substructure in contrast to the lamehar or crystalline matrix observed in the basophilic granules of man and certain other mammals (Cline, 1969; 1975 ; Murata, Zucker-Franklin, 1967).

adjacent

to the nucleus of an altered

In the altered granules oftuatara basophils in which the electronidense matrix material had been partially or wholly extracted, microtubular cores were evident These tubules, were similar in appearance and dimension to the microtubules associated with the centrioles and to our knowledge are a unique feature in the basophils of vertebrates.

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Unfortunately comparative information on the ultrastructure and cytochemistry of basophils of other lower vertebrates is scanty. It is of interest that granulocytic leucocytes which resembled basophils were observed by Lester and Desser (1975) in the blood of the teleost, Catostomus commersoni. These cells in the fish were fixed in the same manner as the tuatara blood and the matrix of their basophilic granules contained no discernible substructure. Certain fixation ‘artifacts’ in the granules of tuatara basophils, such as membranous compartmentalization and myelin figures, resemble those seen in human basophils (Murata, 1969; Zucker-Franklin, 1967). Cytochemical observations on the basophils of the tuatara revealed a positive PAS reaction and no reactivity for acid and alkaline phosphatase, esterase or peroxidase (Desser, 1978). These cytochemical features are also exhibited by mammalian basophils (Cline, 1975). It would be of interest to ascertain whether basophilic granules of Sphenodon are rich in heparin like those of mammals. Actin-like filaments and fibre bundles were a constant feature in the three granulocytes of the tuatara and occur commonly in these cells in other vertebrates. While in certain cases such as the formation of the sperm acrosomal process, contractile proteins may play a purely cytoskeletal role, strong circumstantial evidence suggests that actin and myosin-containing filaments and fibre bundles are involved in cell motility (Hitchcock, 1977). The clear distinction between the three

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types of granular leucocytes as seen in the primitive reptile S. punctutus beomes somewhat hazy among the teleost fishes where differentiation between the neutrophils and eosinophils has been controversial (Fey, 1966; Lester and Daniels, 1975). In a recent ultrastructural study of the blood of the primitive cyclostome Myxine glutinosa, only a single type of granulocyte could be distinguished (Mattisson and Flnge, 1977). The granules in these cells were peroxidase negative, unlike in the majority of higher vertebrates where the specific granules are thought to be ‘lysosomal’ in function. The above authors suggested that a ‘different system’ effective against microorganisms probably exists in the primitive hagfishes. From the available evidence it is apparent that the morphological and certain cytochemical features of the tuatara granulocytes are more similar to those of the higher vertebrates than to those of lower forms. It is likely that in many respects the eosinophils, neutrophils and basophils of the tuatara are also functionally homoiogous to those in the higher vertebrates, including mammals. Acknowledgements

We are indebted to Bertha ‘Frances’ Allison for her enthusiastic assistance in the field and for financial support from the University of Canterbury Research Committee and the Natural Sciences and Engineering Research Council of Canada, Grant no. 6956. We are grateful also to the New Zealand Wildlife Service, Department of Internal Affairs, for permission to visit Stephens Island and to retain a tuatara for this study.

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