Serous cutaneous glands in Phyllomedusa hypochondrialis (Anura, Hylidae): secretory patterns during ontogenesis

Tissue & Cell, 1998 30 (1) 30-40 © 1998 Harcourt Brace & Co. Ltd

Serous cutaneous glands in

Phyllomedusa hypochondrialis (Anura, Hylidae): secretory patterns during ontogenesis G. Delfino', R. Brizzi 1, B. B. Alvarez 2, R. Kracke-Berndorff

Abstract. Three syncytial gland types (la, Ib and II) have been described in the skin of larval, juvenile and adult Phyllomedusa hypochondrialis, which share the ultrastructural traits common to the serous secretory units of anuran skin, although each manufactures a peculiar product. Type la secretion consists of dense granules provided with a peculiar substructure, type Ib of vesicles holding a lucent material, type II of lipid deposits. None of the developmental stages investigated showed intermediate features between any of the three cutaneous products, which accumulate in the syncytial cytoplasms of the secretory units following different biosynthetic pathways, consistent with each gland type. These findings confirm previous results on adult specimens of P. hypochondrialis and P. sauvagei and stress the polymorphism of the serous glands in the genus Phyllomedusa. This morphological variability reflects the wide adaptive flexibility of serous glands in anurans. Keywords: Serousskin glands, lipid glands, development, Phyllomedusa

Introduction The cutaneous gland sets in the Argentine tree frogs

Phyllomedusa hypochondrialis and P. sauvagei exhibit remarkable polymorphism, unique in the anurans so far studied (Delfino et al., submitted for publication). Among the common mucous glands, with radially arranged mucocytes, three secretory unit types have been described which display the syncytial arrangement characteristic of the serous (or poison) secretory lines in anuran skin (Faraggiana, 1938b; Delfino et al., 1988). Two serous gland types (type Ia and Ib) produce granules and vesicles respectively, and pertain to the main proteinaceous secretory line, IDipartimento di Biologia Animale e Genetica dell 'Universit&, via Romana 17, 50125 Firenze, Italy. ~Departamento de Biologia, Facultad de Ciencias Exactas Y Naturales Y Agrimensura, Universidad Nacional del Nordeste, 9 de julio 1449, 3400 Corrientes, Argentina. Received 30 June 1997 Accepted 23 September 1997 Correspondence to: Giovanni Delfino, Tel: +39 55 2288295; Fax: +39 55 222565; E-mail: delfino@dbag, unifi, it

phylogenetically continuous in the skin of lower vertebrates (Quay, 1972). A third syncytial gland type (type II), peculiar to Phyllomedusa, is involved in lipid production and release, regulating water loss through the epidermis (Blaylock et al., 1976). When secretory polymorphism occurs in serous cutaneous glands, their biosynthesis processes should be followed to evaluate whether the various products are manufactured by the same gland type, but in different secretory phases, or by distinct gland strains. In adult anurans, serous product manufacture proceeds at a very slow rate, since the poison glands are mostly engaged in maturation rather than in biosynthesis processes (Delfino, 1991). In several instances, this makes it more difficult to follow the specific secretory pathways of various serous gland types and to detect possible transition features between the different products. Previous studies on tadpoles of several families revealed that in anuran serous gland Anlagen, secretory cytodifferentiation coincides with product accumulation (GilloisChevalier, 1960; Vanable, 1964; Delfino et al., 1988; Seki et al., 1989), thus signalling the appropriate time to follow serous biosynthesis in tadpoles. This paper is a concise 30

SEROUS CUTANEOUS GLANDS IN AN ANURAN

Table

31

Specimens of P. hypochondriaIis collected for study

Stages Tadpoles (41-42) Tadpoles (43) Tadpoles (44) Juveniles (46) Subadults Adults

Snout-vent length (SVL)

Animals observed

LM, skin specimens observed

TEM, skin specimens observed

18 m m 20 m m 17 and 19 m m 18 and 2I m m 19 and 22 m m 33-38 m m

3 2 2 2 2 3

9 6 8 8 6 9

4 4 4 4 5

report of findings collected from the ultrastructural study of serous glands in larval and juvenile P. hypochondrialis. Both observation and discussion follow the morpho-functional criteria which emphasize the specific role of each component part of the serous glands in larval and adult anurans.

Materials and methods Specimens of Phyllomedusa hypochondrialis were collected from the outskirts of Resistencia (Chaco, Argentina). The Table records the total number of tadpoles, froglets and frogs studied and their distinctive traits. The number of skin specimens observed under light microscope (LM) and transmission electron microscope (TEM) are also recorded. In listing tadpoles we referred to the staging of Gosner (1960) which fits the development of all anurans with free swimming larvae. The tadpoles were reared in the Departamento de Biologia of the Universidad Nacional del Nordeste, Argentina, until they reached the required stages. Skin strips from the back (4-6 mm 2in surface area) were removed from animals (including adults, both sexually mature and immature) sacrificed with 0.2% chlorobutanol, and were treated (4 h, 4°C) with the aldehyde mixture after Karnovsky (1965). The tissue fragments were then washed with the fixative buffer (0. l M, pH 7 cacodylate) and sent in 2-4 ml of this solution (with the addition of a drop of glutaric aldehyde) to the Dipartimento di Biologia Animale e Genetica, University of Florence, Italy. The skin specimens were washed once more, reduced in size and postfixed (90 rain) in 1% OsO 4, again using the cacodylate buffer. After rinsing in the buffer, the samples were dehydrated in graded ethanol, soaked in propyleneoxide and infiltrated in Epon 812 to obtain flat blocks. These were reduced with a NOVA LKB ultramicrotome into semithin (1-2 gm) and ultrathin (yellow-white interference colour) sections. Once stained with buffered toluidine blue, the semithin sections served for light microscope observations. Ultrathin sections were collected on 300 mesh, uncoated copper grids and electron dense stained with a hydroalcoholic saturated solution of uranyl acetate, followed by an alkaline solution of lead citrate (2 mg/ml). These samples were then observed (80 kV) under a Siemens 101 electron microscope.

Results Preliminary LM investigations showed that intermediate tadpoles (41--42) already possess numerous cutaneous glands, with advanced patterns of maturation. On the other hand, several gland generations follow each other with development, so late tadpoles may exhibit adenomeres in early stages of secretory activity. Therefore we could not perfectly link our description to larval timing but could only refer to the major developmental steps (metamorphosis and postmetamorphic growth). Accordingly, we illustrated our results and arranged the figures following the sequence of biosynthesis steps, and the stages observed were indicated in detail in the legends. Adult glands were described only under the TEM, as their LM traits largely resemble those of larval specimens.

Light microscope observations In the ontogenetic phases of P. hypochondrialis investigated, type I and II glands can be recognized by the differential features of their secretory deposits (Fig. la), which resemble, or at least foreshadow, those described in adults. Type Ia secretory product consists of dense granules (ranging from 2 to 3 gm in diameter, Fig. lb), whereas type Ib glands produce large (up to 10 ~m) vesicles which contain a translucent material (Fig. la). The lipid deposits show a typical substructure resulting from the aggregation of elongated, transparent complements (Fig. lc). The most consistent differences between larval, juvenile and adult glands are the size of the secretory units (adenomeres) and the length of the duct, which develops according to the maturation of the epidermis. Serous adenomeres in tadpoles and newly metamorphosed froglets are smaller than in adults and contain relatively large amounts of secretory product. There are no obvious partitions between discrete adenocytes, since the secretory compartment has already acquired the syncytial arrangement typical of mature serous glands in anurans. The peripheral portion of the syncytium, free from secretory deposits and holding the nuclei, appears rather thick in larval and juvenile glands (Fig. lb, c), when compared to the mature secretory units. In type Ia glands, early secretory deposits can be recognized in this peripheral cytoplasm, in the shape of vesicles holding dense material within a relatively transparent matrix.

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Fig. 1 Serous glands in larvae and juveniles under the light microscope. Ia = type Ia serous glands, Ib = type Ib serous glands, II = type II serous glands, ch = cutaneous chromatic unit, d d = dense dermis (Stratum compactum), mg = mucous gland, a) Stage 43; in this complete set of cutaneous glands serous units largely prevail. Arrow points to a serous gland with contracted myoepithelium, x 350. b) Stage 41-42; type Ia serous product includes mature central granules and peripheral immature aggregates with varying structure, x 700. e) Stage 46 (juvenile); type II serous product consists of elaborated storage structures containing translucent material, x 800

Ultrastructural observations In larval specimens, the early type I serous product arises inside small vesicles dispatched from the Golgi apparatus, in the form of a finely dispersed material containing single dense particles (Fig. 2a, d). The serous product undergoes

elaborate condensation leading to variable morphology. Recurrent features are obvious, however, corresponding to dense spheroidal particles, embedded in a spongy background (Fig. 2b). In some instances, the particles merge together and form larger structures resembling horseshoes in section (Fig. 2b). Further consistent features are vesicles containing single spherical bodies (Fig. 2c, e), which on a larger scale look like the product dispatched by the Golgi apparatus, and possibly correspond to the vesicular profiles detected under the LM. Opaque, sponge-like aggregates (Fig. 2b), enclosing a paler (Fig. 2c) or denser (Fig. 2e) zone, are also detectable. Throughout the premetamorphic interval (41-44) the rough endoplasmic reticulum is prominent, including slender cisterns (Fig. 2d) and small vesicular complements (Fig. 2e), The former may contain a remarkably dense product (Fig. 2d), and often cluster with mitochondria at the peripherery of the secretory syncytium (Fig. 2f). As gland maturation proceeds, the evident polymorphism of the secretory product tends to decrease; in mature glands, most secretory deposits look like aggregates of opaque particles, contained in a paler matrix of variable density (Fig. 2g). The biosynthesis machinery is now restricted to the very periphery of the secretory syncytium and consists of small Golgi stacks (dictyosomes) and slender rer complements. In some instances the rough cisterns form closed structures, ring-shaped in section, and are most obvious when their compartment holds a dense material (Fig. 2g). Secretory differentiation in the syncytium fits evolution of myoepithelial cells, where myofilaments tend to occupy the whole cytoplasm (Fig. 2f). The syncytial and myoepithelial compartments are separated by an exiguous interstice, which enlarges remarkably to accommodate cells migrating from the stroma, provided with a lobated nucleus and heterogeneous intracytoplasmic inclusions (Fig. 2h). Throughout the ontogenetic span considered, the secretory vesicles contained in the central syncytial cytoplasm in type Ib glands display homogeneous features. They are subspherical or ellipsoidal in shape and enclose a finely dispersed material (Fig. 3a). The vesicles are engaged in multiple merging processes, so that the inner portion of the adenomere seems to consist of a multi-chambered secretory compartment. Whereas the secretory vesicles did not vary significantly in morphology, in the larval stages studied there was a remarkable development in the biosynthesis apparatus. In early glands, the peripheral cytoplasm

Fig. 2 Early and intermediate patterns of secretory activity in type Ia serous glands of larval specimens. G = Golgi stack; i = interstice between myoepithelium and secretory syncytium; rer = rough endoplasmic reticulum, a) Stage 44; minute electrondense particles (arrows) are obvious inside the Golgi compartment, x 19 000. b) Stage 41-42; recurrent patterns in type I serous maturation: dense sponge-like granules are associated with secretory aggregates holding a paler spongy background, where dense particles merge together to form horseshoe profiles in section, x 14 000. c) Stage 44; further consistent deposits are subspherical vesicles holding a finely dispersed material and a dense central core (small arrows point to merging patterns). Notice the opaque, sponge-like granules containing a paler zone (large arrow). × 16 000. d) Stage 44; rough endoplasmic cisterns containing electrondense material (small arrows); large arrow points to a secretory particle, x 22 000. e) Stage 41-42; widely diffuse rer with vesicular and tubular profiles; two serous aggregates are also obvious: a vesicle with an opaque core, and a thick sponge-like granule containing a remarkably dense zone (large arrow), x 10 000. f) Stage 41-42; notice rer-mitochondria associations (arrows) in the secretory syncytium and bundled myofilaments in the myoblast (arrowheads). x 16 000. g) Stage 41-42; resting secretory syncytia exhibit inactive Golgi stacks and large serous deposits. Arrow points to a cistern forming a close profile, x 14 000. h) Stage 41-42; the peripheral interstice contains macrophages with lobated nuclei and dense intracytoplasmic bodies, x 10 000.

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Fig. 3 Early and intermediate patterns of secretory activity in type Ib glands. Ia = type Ia serous gland, Ib = type Ib serous gland, ch = melanophore of a cutaneous chromatic unit, mec = myoepithelial cell/s, ne = nerve ending, s and s~= secretory product in various condensation stages, sv = synaptic vesicles; sy = secretory syncytium, a) Stage 43; close contiguous type Ia and Ib glands: in the latter the secretory vesicles are obvious, engaged in merging processes (arrows); arrowhead points to a vesicular endoplasmic reticulum cluster, x 2000. b) Stage 41-42; the syncytial structure of the secretory unit can be seen together with the characteristic vesicular profiles of the endoplasmic reticulum (arrowhead). x 7500. e) Stage 43; transition between vesicular and slender (flat or tubular) elements of the endoplasmic reticulum (arrows). The slender profiles are rough in nature, whereas the vesicular ones may be granular or agranular, x 30 000. d) Stage 43; the slender rer elements often form close structures, x 40 000. e) Stage 43; in advanced larval stages a structureless cytoplasm (asterisk) separates the secretory product from the peripheral biosynthesis apparatus (arrowhead). x 10 000. f) Stage 44; this labyrinthine membrane pattern corresponds to an active Golgi stack (compare with insert in 1) which produces secretory vesicles, x 11 000. g) The same as above; these secretory vesicles merge together (arrowhead) and with smaller vesicles (arrows) also dispatched by the Golgi apparatus, x 10 000. h) Stage 46 (juvenile); serous glands in just metamorphosed froglets possess a fully developed neuromuscular apparatus; notice dense-cored synaptic vesicles. x 20 000. i) Juvenile; after metamorphosis, the serous glands contain large amounts of secretory product deposits with a variable degree of density, which are engaged in merging processes (arrows). The nuclei pertain to undifferentiated cells of the gland neck. x 5000. I) Juvenile; the rough endoplasmic reticulum undergoes obvious reduction; rer remnants form whorls contiguous to mitochondria provided with a dense matrix, x 16 000. Golgi stacks can also be detected, although in a resting state (insert); compare with f). x 21 000

encircling the secretory deposits is provided with diffuse complements of both the rough and smooth reticula (rer cisterns and ser vesicles). The rer cisterns are subspherical, similar in shape and size (about 150-200 nm in diameter) to the smooth vesicles (Fig. 3b-e). Cisterns and vesicles form conspicuous aggregates around the nuclei, which show a very regular, ellipsoidal shape and are arranged in a single row (Fig. 3b). During gland maturation, the rough complements tend to prevail and assume a tubular shape as well as a parallel arrangement. We found evidence that the subspherical and tubular cisterns are continuous (Fig. 3c), which suggests that such rer evolution consists of transformation rather than substitution processes. As in type Ia glands, the elongated cisterns form closed profiles (Fig. 3d). During this secretory evolution, the biosynthesis machinery gradually concentrates at the very edge of the syncytium periphery, and a structureless intermediate cytoplasm separates the organelles from the secretory vesicles (Fig. 3e). At the border between these cytoplasmic zones, several Golgi complexes can be detected, characterized by elaborated features, which produce small to intermediate-sized (100 nm-1 ~tm) vesicles, containing a rather translucent material (Fig. 3f). These secretory formations repeatedly merge together, and the confluence processes cause slight changes in the density of the product they contain (Fig. 3g). As expected, gland maturation also involves the neuro-contractile apparatus at the periphery of the syncytium. The myoblasts ensheathing the secretory syncytium gradually acquire the structural traits typical of anuran myoepithelial cells and exhibit a widespread sarcoplasmic reticulum. After metamorphosis (stage 46) the neurite supply to the gland is fulfilled, with the formation of typical nerve endings, provided with dense-cored synaptic vesicles (Fig. 3h). In this stage, type Ib glands possess remarkable amounts of secretory product, which consists of large vesicles containing the usual thin material, although of varying electrondensity, and engaged in merging processes (Fig. 3i). Typical features of the biosynthesis machinery can be detected at the periphery, where slender rough cisterns are obvious, contiguous to mitochondria provided with a very dense matrix. These rer complements are arranged concentrically

and form characteristic ergastoplasmic whorls (Fig. 31), which possibly derive from the ring-shaped arrangement observed in the larval glands. Metamorphosis involves an obvious decrease in the number of rough cisterns, while the Golgi stacks reduce their activity considerably (insert in Fig. 31). Type II larval glands contain large accumulations of rer at the periphery of the syncytium, encircling large secretory aggregates (Fig. 4a). The rough endoplasmic reticulum consists of flattened cisterns, arranged in a closely parallel array (Fig. 4b). Small stacks of the Golgi apparatus can also be detected, consisting of flattened sacculi which dispatch minute vesicles (insert in Fig. 4b). The biosynthesis apparatus undergoes an obvious reduction during larval development, whilst the cytoplasm of the syncytium accommodates secretory aggregates. As a rule, structure and distribution of such secretory deposits follow an ordered pattern: the smaller are peripheral and consist of both minute particles and dense rods (shafts), whereas those in the center are larger and contain dissolving material, which forms moderately opaque to transparent shafts (Fig. 4c). When observed at low power magnification, typical patterns of anuran serous glands were obvious in these secretory units, based on the partition between a denser cytoplasm region at the periphery and a clearer one in the center of the syncytium, where large secretory aggregates crowd (Fig. 4d). Further morphofunctional features, typical of anuran serous glands, consist in sequential merging processes (Fig. 4d and f). Following these processes, the secretory aggregates increase considerably in size and acquire an elaborated shape. Scanty, minute rer cisterns and ribosomal aggregates could still be detected in the exiguous cytoplasm partitions separating secretory deposits (Fig. 4e). In type II glands, characteristic laminar cells with their nuclei located beneath the neck separate the secretory syncytium from the intercalary lumen, where single cells can often be observed (Fig. 4f), possibly migrating from the stroma. At higher power magnification, intricate cell interweavings can be detected in the thin cytoplasmic screen which borders the lumen (Fig. 4g). The plasma membranes of the cells involved in these labyrinthine relationships show only scanty desmosomes, and therefore may be involved in sliding movements during the growth of

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Fig. 4 Patterns of secretory activity in type II serous glands, lu = lumen, m = macrophage, mec = myoepithelial cell, rer = rough endoplasmic reticulum. a) Stage 41-42; the peripheral cytoplasm holds widespread rer and single secretory shafts, x 12 000. b) Detail of the selected area in a, the rough endoplasmic reticulum consists of slender, parallel cisterns, x 47 000. The insert shows a Golgi stack, x 30 000. e) Stage 41-42; in early secretory deposits, minute particles prevail (small arrows), whereas in more advanced stages of maturation, the secretory aggregates hold moderately opaque shafts with dissolving material (large arrows), x 15 000. d) Stage 44; a centripetal gradient, typical of anuran syncytial giands, is obvious in this secretory unit: proceeding from the periphery (left), the cytoplasm background of the syncytium becomes thinner and is less supplied with organelles, whereas the secretory aggregates increase in size through merging processes (arrows). x 7000. e) In these glands, slender cytoplasmic partitions remain between the large secretory deposits and contain remnants of the biosynthesis machinery, x 19 000. f) Stage 44; the large secretory deposits, involved in merging processes (arrows), crowd against the exiguous lumen bound by undifferentiated laminar cells. The intercalary lumen holds a migrating cell. x 4000. g) The same as above; the laminar adenoblasts adhere by means of intertwining plasma membranes (arrows) which allow cell sliding and widening of the lumen, x 27 000.

the larval glands and/or in variations of intercalary lumen width, often occurring in adults. The remarkable reduction of the biosynthesis machinery continues throughout adult life, whilst large amounts of product accumulate in the syncytia. In type Ia glands, the very peripheral cytoplasm holds residual organelles, such as associations between rer and mitochondria, resembling those described in larval glands (Fig. 5a). The secretory product has undergone a marked condensation and homogeneous, opaque granules are intermingled with secretory bodies similar to the larval and juvenile ones. However, the dense granules still display an obvious substructure at their periphery (Fig. 5a), reflecting that of the contiguous, less mature deposits, which are still engaged in merging processes (Fig. 5b). Similar patterns of confluence are the most remarkable functional trait in Type Ib glands (Fig. 5c), and involve serous deposits of various sizes, larger (mature) vesicles and smaller (immature) ones, which continue to be produced, although at a slower rate, by the Golgi stacks (Fig. 5d). The lipid product in type II glands retains the structural traits already described in tadpoles and froglets, with small, immature deposits at the periphery and large, mature storage bodies in the central cytoplasm (Fig. 5e). The lipid producing glands in adult frogs retain the biosynthesis machinery, mostly consisting of flat rough cisterns, contained in the dense peripheral cytoplasm (Fig. 5f).

Discussion Serous cutaneous glands in adult anurans possess a solid secretory unit provided with an exiguous lumen which always appears empty. Secretory products are stored in the syncytial cytoplasm (Dockray & Hopkins, 1975; Neuwirth et al., 1979) and undergo a post-Golgian phase of maturation, which affects the material dispatched by dictyosomes (Delfino et al., 1995). However, the secretory machinery is poorly developed in the mature serous glands, where residual secretory organelles are found in the dense, peripheral cytoplasm of the syncytium (Hostetler & Cannon, 1974; Cannon & Hostetler, 1976; Dockray & Hopkins, 1975; Delfino et al., 1990, 1994; Toledo et al., 1992). These residual organelles participate in gland recovery after secretory release, together with differentiating adenoblasts from the gland neck (Delfino, 1980).

In type II glands of P. hypochondrialis and P. sauvagei (Delfino et al., submitted for publication), complements of the biosynthesis apparatus and scanty aggregates of the product in intermediate phases of maturation can be detected at the very periphery of the syncytium. In these species, patterns of biosynthesis activity and minute secretory deposits have also been described in the adenoblasts of the neck region (intercalated tract), possibly after secretory discharge. Apart from the biosynthesis activity of neck adenoblasts, it is arduous to follow the secretory pathways in mature glands and, when different serous gland types occur, to evaluate possible transitional steps of biosynthesis between them. Our ontogenetic approach provides results relevant to elucidation of the remarkable serous gland polymorphism in P. hypochondrialis since the syncytial glands in tadpoles and froglets of this species are engaged in active biosynthesis. TEM results indicate that type I and II serous products are manufactured by different syncytial gland types, which can clearly be distinguished from the early secretory phases. Furthermore, consistent differences do exist between type Ia and b glands, which therefore should be regarded as variations of the fundamental poison gland line of anuran skin, producing granules (Delfino, 1976, 1979; Delfino et al., 1996), vesicles (Delfino et al., 1992) or more elaborate structures (Neuwirth et al., 1979; Delfino et al., 1994, 1995; Melis, 1995). Differences involve both the biosynthesis pathways and the early morphology of the secretory products. Type Ia granules are manufactured according to the route which characterizes the biosynthesis of dense, proteinaceous products reserved for exocytosis, and engage both rer and Golgi apparatus. Although the intermediate features of this secretory product are peculiar, its maturation consists of condensation and merging processes, according to a functional pattern observed in most anuran genera (Delfino, 1991). Type Ib product pertains to the fluid serous secretions of anuran skin, which undergo maturation without condensation, such as the 'large granules' of Bombina species (Bertossi, 1937; Faraggiana 1937; Delfino, 1976; Delfino et al., 1990) and vesicles of Pelobates cultripes (Delfino et al., 1992). The widespread rer occurring in immature type Ib glands underlines that polypeptides and/or proteins are fundamental component molecules of the material contained within the vesicles. On the other hand, the patterns of Golgian activity affecting type Ib product fit its

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Fig. 5 Syncytial glands in adult specimens, ab ---neck adenoblasts, i -- interstice between secretory syncytium and myoepithelium; rer -- rough endoplasmic reticulum, S = Ib serous product, a) Type Ia gland; maturation processes lead to the formation of dense granules, which however disclose a substructure (arrows) derived from that of immature aggregates. Note clusters of rer cisterns and mitochondria in the peripheral cytoplasm, x 14 000. b) Type Ia gland; immature serous deposits are engaged in merging processes (arrows). Notice accumulations of the rough endoplasmic reticulum in the paranuclear cytoplasm, x 13 000. e) Type Ib gland; subintercalar portion of the secretory syncytium, showing neck adenoblasts and confluence between large vesicles (arrows). × 7000. d) Type Ib gland; the Golgi stacks are still active and dispatch secretory vesicles containing material characterized by weak density, x 17 000. e) Type II gland; functional polarization is obvious in the secretory unit: the peripheral cytoplasm shows a dense background and contains secretory organelles as well as small lipid accumulations; the enclosed area is shown in detail in f. x 6500. f) The same as above; cluster of rough endoplasmic reticulum between secretory deposits, both immature (right) and mature (left). x i9 000

fluid appearance, since dictyosomes were never seen to perform condensation. According to the usual pathway of serous secretion, Golgi stacks modify the products manufactured in the rough reticulum compartment, and provide them with membrane complements. The occurrence of active Golgi stacks in adult specimens suggests a late participation of this organelle to the type Ib serous biosynthesis, by providing constituent molecules or enzymatic sets active in maturation processes (Neuwirth et al., 1979; Myers & Daly, 1983; Flucher et al., 1986). Current knowledge does not allow detection of specific participation of type Ia and Ib secretory products to the cutaneous poison of Phyllomedusa 'as a whole'. Nonetheless, it must be recalled that the skin extracts of species pertaining to this genus include polypeptides provided with remarkable pharmacological activities (Erspamer et al., 1989; Mignogna et al., 1992). On the other hand, consistent evidence (both pharmacological and immunocytochemical) stresses that the active compounds contained in anuran skin extracts correspond to the serous secretions (Dockray & Hopkins, 1975; Yoshie et al., 1985). Type II syncytial glands share a widespread rough endoplasmic reticulum with type Ia and b glands. This organelle may be engaged in the biosynthesis of protein or peptides, structural or functional in nature, associated with the lipid fractions which prevail in the secretory product of type II glands (Blaylock et al., 1976). Despite peculiar characteristics of their product and related biosynthesis pathways, type II secretory units display the basic arrangement of anuran serous glands, namely centripetal polarization of biosynthesis organelles, intracytoplasmic secretory maturation, direct nerve supply, and patterns of holocrine (bulk) discharge (Delfino et al., submitted for publication). This ontogenetic study suggests that type II glands should be considered as derived from the serous gland line, rather than belonging to a specific kind of cutaneous secretory units. In the secretory syncytium involved in lipid production, we have also detected the characteristic development patterns of the poison glands, which lead to a consistent reduction of biosynthesis machinery during product accumulation. Whatever the case, this reduction is not so dramatic in lipid glands as in other syncytial types. Mature type II glands retain their secretory apparatus since they produce the permanent surface layer involved in regulating water loss across the skin in adults. The occurrence of large amounts of type I (a and b) serous cutaneous products in 41--42 tadpoles suggests that these secretory materials should also play a role, both

regulative and defensive, in the aquatic environment. Remarkable accumulations of lipid deposits have been detected also in type II larval glands, which should represent stored material to be promptly used, after metamorphosis, in subaerial life. When compared with the Old World anurans, P. hypochondrialis tadpoles revealed unusual precocity in serous gland development. This finding seems to be consistent with the duration of their larval period, which is remarkably short, ranging from 1 to 2 weeks, depending on the environmental conditions. The precocity of serous units in this species includes the early arrangement of the neuromuscular apparatus, according to the usual developmental lines in anurans (Delfino et al., 1987). This system driving secretory release is adrenergic in nature (Holmes et al., 1977; Holmes & Balls, 1978; Delfino et al., 1982). The occurrence of migrating cells in the interstices and cavities of larval glands emphasizes their advanced level of development. These macrophages are common cell components in the spongy layer of the dermis in adult specimens, and have been observed migrating into the serous glands (Delfino et al., 1990, 1992). Macrophage activity consists of removing cytoplasm and secretory wastes from the gland and is a functional marker of fully working glands. Migration and phagocytosis are enhanced by gland depletion, and take part in secretory syncytium turnover, which includes proliferation and differentiation of neck adenoblasts (Faraggiana, 1938a; 1939; Delfino, 1980). The present findings provide ultimate evidence of the remarkable polymorphism of the serous glands in Phyllomedusa. Furthermore, this study confirms the adaptive flexibility of the syncytial glands of anuran skin, capable of performing a wide range of roles in the survival strategies of the order, in both the premetamorphic and postemetamorphic phases of the life cycle. REFERENCES Bertossi, F. 1937. Sulle ghiandole granulose cutanee di Bombinator igneus Laur. Monitore zool. it., 48, 341-344. Blaylock, L.A., Ruibal, R. and Platt-Aloia, K. 1976. Skin structure and wiping behaviour of phyllomedusine frogs. Copeia., 1976, 283-295. Cannon, M.S. and Hostetler, J.R. 1976. The anatomy of the parotoid gland in Bufonidae with some histochemical findings. I1. Bufo alvarius. J. Morph., 148, 137-160. Delfino, G. 1976. Structural and ultrastrnctural aspects of the cutaneous granular glands in Bombina variegata (L.) (Amphibia, Anura, Discoglossidae). Monitore zool. ital., 10, 421448. Delfino, G. 1979. Le ghiandole granulose cutanee diAlytes cisternasii Bosc~t e Discoglossus pictus Otth (Anfibi, Anuri, Discoglossidi): struttura, ultrastrnttura e alcuni dati citochimici. Archo ital. Anat. Embriol., 84, 81-106.

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