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Ultrastructural patterns of secretory activity in serous cutaneous glands of the pacific tree‐frog hyla regilla (Anura, hylidae) a
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Rossana Brizzi , Claudia Corti , Giovanni Delfino & Robert Drewes
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Dipartimento di Biologia animale e Genetica , Via Romana 17, Firenze, I‐50125, Italy b
Dipartimento di Biologia animale e Genetica , Via Romana 17, Firenze, I‐50125, Italy E-mail: c
Department of Herpetology , California Academy of Sciences , Golden Gate Park, San Francisco, CA, 94118, USA E-mail: Published online: 22 Nov 2010.
To cite this article: Rossana Brizzi , Claudia Corti , Giovanni Delfino & Robert Drewes (2004) Ultrastructural patterns of secretory activity in serous cutaneous glands of the pacific tree‐frog hyla regilla (Anura, hylidae), Italian Journal of Zoology, 71:S2, 77-80, DOI: 10.1080/11250000409356611 To link to this article: http://dx.doi.org/10.1080/11250000409356611
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Ital. J. Zool., Suppl. 2: 77-80 (2004)
Ultrastructural patterns of secretory activity in serous cutaneous glands of the Pacific tree-frog Hyla regilla (Anura, Hylidae) ROSSANA BRIZZI CLAUDIA CORTI GIOVANNI DELFINO Dipartimento di Biologia animale e Genetica, Via Romana 17, I-50125 Firenze (Italy) E-mail:
[email protected]
ROBERT DREWES
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Department of Herpetology, California Academy of Sciences, Golden Gate Park, San Francisco, CA 94118 (USA) E-mail:
[email protected]
INTRODUCTION Serous (or poison) glands in hyline tree-frogs consist of discrete storage bodies (secretory granules) with a recurrent substructure, resulting from close aggregation of discrete subunits (modules), with random (Delfino et al, 2001b, in Scinax acuminata, S. fuscovaria and S. nasicd) to highly ordered, parallel arrangements (Delfino et al., 1994; in the Italian Tree Frog Hyla intermedia, formerly ascribed to H. arbored). Ultrastructural investigation on anuran species provided with different types of cutaneous serous glands showed that their distinctive granules derive from peculiar biosynthetic as well as maturational processes, which, in turn, are expressed by specific developmental programmes (Delfino, 1977; Delfino et al., 1998). These morphological traits may be indicative of taxonomic relationships between the above taxa (Delfino et al., 2001a). Following this viewpoint, we are collecting data on Hylidae, Leptodactylidae and Pseudidae, which are contiguous families in current dendro-cladograms of Neobatrachia (Lynch, 1973; Ford & Cannatella, 1993). The present study fits this phylogenetic perspective and aims to provide transmission electron microscope (TEM) data on the serous product of the Pacific treefrog H. regilla, to be discussed both in comparison with species of the same family and different families.
MATERIALS AND METHODS Skin specimens of dorsal trunk and hindlimb of H. regilla were pre-fixed in the Department of Herpetology, California Academy of Sciences, San Francisco, according to current procedures (Delfino et al., 2001a). The same standards were employed in succeeding steps in TEM analysis (post-fixation, embedding, ultramicrotomy, electron-dense staining and observation) which were followed in the Dipartimento di Biologia animale e Genetica, Universita di Firenze.
RESULTS AND DISCUSSION
ABSTRACT Biosynthesis and maturation processes are described in Hyla regilla serous cutaneous glands, which produce secretory granules provided with a recurrent substructure, consisting of thick aggregations of globular and rod-shaped subunits. Patterns observed fit those detected in the congeneric Italian tree frog H. intermedia and resemble secretory features in larval Scinax nasica and Physalaemus biligonigerus. These findings suggest that serous glands in species of the same genus may produce granules with identical features through identical secretory steps. In addition, genera of the same family or related families, may share early steps of serous biosynthesis. KEY WORDS: Hylidae - infrastructure - Cutaneous Glands. (Received 11 November 2003 - Accepted 2 March 2004)
Preliminary light microscope (LM) observations were carried out on the leg (zeugopodium) skin, where serous glands form large accumulations (Delfino et al., 1982). Therefore, because of the large numbers, different stages of secretory processes are expected. We selected glands with secretory granules characterised by variable degrees of staining, due to maturational processes affecting the serous products. Under the TEM, these granules display a wide density range, and are contained in a syncytial secretory unit (Fig. 1A); the gland lumen is an exiguous, empty cavity just beneath the intercalary tract (or neck) of the gland (Fig. 1A). This is the regenerative gland compartment and consists of discrete stem cells, with a high nucleo-plasmatic ratio (Fig. 1A), and is continuous with the basal epidermal layer. A single mantle of spindle-like myocytes (myoep-
R. BRIZZI, C. CORTI, G. DELFINO, R. DREWES
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;.250nm,-.> Fig. 1 - Ultrastructural features of serous glands in H. regilla. A, low power magnification of serous gland: the secretory unit exhibits an exiguous, empty lumen (arrow) and contains large amounts of granules in its syncytial cytoplasm (lacking any plasma membrane between nuclei). Thick myocytes (mec) of the contractile sheath and the stem cell compartment at the upper gland pole (arrowheads) are seen. B, neurite (ne) inserted between secretory (upper) and contractile (lower) compartments; rough endoplasmic profiles (rer) occur in the syncytial cytoplasm. C, the serous biosynthesis machinery includes rough profiles (rer) and Golgi stacks (G); arrow points to a dense particle, corresponding to the early secretory product. Also note a glomerular-like, mature granule (arrowhead). D, distally from the Golgi apparatus, the dense particles progressively fade out and give rise to fuzzy material. E, this rearrangement of the secretory material proceeds along with elongation of the post-Golgian particles. F, in an intermediate stage of serous maturation, elongated (arrowhead) and globular (arrow) particles coexist in the same secretory granule. G, peripheral area of the secretory syncytium: a glomerular-like granule (arrowhead) as well as granules with ordered patterns of globular subunits (arrow) are seen; small arrows point to merging processes between serous deposits. H, occurrence of elongated (arrow) and globular (arrowhead) profiles in the same granule possibly derives from random arrangement of bowed, rod-like secretory subunits.
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SECRETORY ACTIVITY IN CUTANEOUS GLANDS OF HYLA REGILLA
ithelial cells or mecs) ensheathes the secretory unit (Fig. 1A); mecs are responsible for the secretory release, under control from neurites (Fig. IB), which in anurans are orthosympathetic (adrenergic) in nature (Dockray & Hopkins, 1975; Delfino et al, 1982). Starting from the boundary zone with myoepithelium, the secretory unit discloses a centripetal, functional gradient, involving slender, somewhat parallel cisterns of the rough endoplasmic reticulum (rer), and stacks of the Golgi apparatus. The former hold a moderately opaque material (Fig. 1B-C), the latter minute dense particles in their trans saccules (Fig. 1C), to be released within secretory vesicles (Fig. ID). These patterns are usual in anurans which produce proteinaceous skin poisons (Delfino, 1991), and closely resemble secretory processes described in the congeneric H. intermedia (Delfino et al, 1994). Proceeding from the Golgi areas towards the centre of the syncytium, the secretory particles undergo a decrease in their electron density, whereas a fuzz-like product becomes obvious around them (Fig. ID). In the meantime, merging processes between secretory vesicles (Delfino et al., 1994) lead to larger aggregates of minute densities in a fuzzy background (Fig. IE). Formation of this structureless product occurs by fading as well as elongation of the minute particles (Fig. 1E-F). Both processes are observed in intermediate stages of poison maturation, until elongated secretory particles tend to become more numerous than round particles; eventually, there is an ultimate decline in their density. Thus relatively opaque rods and globules - which are formerly assembled in glomerular-like aggregates and later in a closer, ordered arrangement - become real modular subunits of mature granules involved in merging processes (Fig. 1G). When wide cytoplasmic areas are observed, it appears that this recurrent substructure, typical of the mature poisons in Hylinae, is consistent throughout the syncytium, from its periphery (Fig. 1G), to the upper pole. At this level, serous deposits aggregate against the small gland lumen (Fig. 1A). Serous subunits display dimorphic features, since globular and rod-shaped modules occur in different granules, and also alternate in the same granule (Fig. 1H), although the latter pattern could be a section effect, due to random aggregation of bowed, rod-shaped modules. The close similarity detected between serous products of H. regilla and H. intermedia suggests that serous glands in species of the same genus may produce granules with identical features through identical secretory steps, and confirms previous investigations on various genera (Alytes: Delfino, 1979; Delfino et al., 2001b; Bombina, formely Bombinator. Bertossi, 1937; Faraggiana, 1937; Delfino et al, 1990; Bufo: Delfino et al., 1995; Discoglossus: Delfino, 1979; Delfino et al, 2001b; Physalaemus: Delfino et al, 1999; Terreni et al, 2001; Scinax: Delfino et al, 2001b; Terreni et al, 2002). A comparison between the functional sequence of poison manufacturing in H. regilla and formation of
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glomerular-like granules in Physalaemus biligonigerus (Delfino et al, 1999) as well as in H. nana, S. acuminata, S. fuscovaria, S. nasica, and Pseudis paradoxa (Terreni et al, 2002) reveals several differences. In the latter species, the repeating aggregation of rather transparent rods and globules into glomerular granules usually results from multi-point condensation of dispersed secretory material contained in post-Golgian vesicles. This might suggest that the remarkable ultrastructural similarities detected in the species under comparison represent the results of convergence from different secretory repertories. However, the proper approach to analyse these similarities could be the investigation of larval glands in tadpoles. During the development of serous glands in the skin of larval specimens of P. biligonigerus, small electron-dense particles are produced (Delfino et al, 2001a), closely resembling those described in H. regilla and H. intermedia. Instead of assembling as discrete subunits into serous deposits, these particles merge together to form larger dense granules that undergo inner rearrangement, leading to labyrinthine and later to glomerular-like patterns. In S. nasica, larval glands produce dense particles as well as secretory vesicles with dispersed material, both changing into glomerular-like structures, through labyrinthine rearrangement and multi-point condensation, respectively (Terreni et al, 2003). Adopting the recapitulation paradigm, we assume that secretory processes in early developmental stages may foreshadow shared repertories, both biosynthetic and maturational, inherited from a common ancestor. Accordingly, similarities in the early and ultimate features of the serous products confirm the phylogenetic relationships between Hylinae (H. intermedia, H. regilla and S. nasica) and extend these relationships to Leptodactylinae (P. biligonigerus). These species belong to different, but related bufonoid families, Hylidae and Leptodactylidae, which usually appear on contiguous branches of current anuran phylogenetic trees (Lynch, 1973; Ford & Cannatella, 1993).
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