serous cutaneous glands of the pacific tree-frog hyla regilla (anura

Tissue and Cell 38 (2006) 65–77

Serous cutaneous glands of the Pacific tree-frogHyla regilla (Anura,Hylidae): Patterns of secretory release induced by nor-epinephrine

G. Delfinoa,∗, R.C. Drewesb, S. Magherinia, C. Malentacchic, D. Nosid, A. Terrenie

a Dipartimento di Biologia Animale e Genetica dell’Universita, via Romana 17, 50125 Firenze, Italyb Department of Herpetology, California Academy of Sciences, 875 Howard St, San Francisco, CA 94103, USA

c Dipartimento di Fisiopatologia clinica dell’Universita, Unita di Genetica umana, viale Pieraccini 6, 50139 Firenze, Italyd Dipartimento di Anatomia, Istologia e Medicina legale dell’Universita, viale Morgagni 85, 50134 Firenze, Italy

e Laboratorio Centrale di Analisi Biochimico-Cliniche, Azienda Ospedaliera di Careggi, viale Morgagni 85, 50134 Firenze, Italy

Received 17 May 2005; received in revised form 21 November 2005; accepted 22 November 2005

Abstract

The serous (poison) cutaneous glands of the Pacific tree-frogHyla regilla were induced to release their product by 10−3 M nor-epinephrines urthermore,t ope analysis.A tion of thep xpulsion oft rmed by them revealedt ts, acquired©

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timulation. After discharge structural and ultrastructural features of the cutaneous glands involved in release were observed. Fhe discharged product, consisting of discrete, secretory granules, was collected and processed for transmission electron microscs indicated by patterns found in the myoepithelium encircling the syncytial secretory unit, gland discharge is caused by contraceripheral myocytes. Muscle cell compression dramatically affects the syncytium and results in degenerative changes, including e

he secretory unit nuclei. Therefore, the structural collapse in depleted glands has been ascribed to the mechanical activity perfoyoepithelium during discharge, rather than cytoplasm involution described in conventional, holocrine glands. TEM investigation

hat the secretory granules collected after discharge maintain their peculiar traits: they consist of recurrent patterns of thin subuniduring serous maturation and provided with remarkable structural stability.2005 Elsevier Ltd. All rights reserved.

eywords: Serous glands; Skin;Hyla regilla; Adrenergic stimulation; Ultrastructure

. Introduction

Biosynthesis and maturation patterns described in serouspoison) cutaneous glands ofHyla regilla (Brizzi et al., 2004)roceed along the same pathways as those reported in the con-eneric ItalianHyla intermedia (formelyH. arborea, Delfinot al., 1994). In both species the mature serous granulesxhibit a noticeably repeating substructure resulting from thelose aggregation of subunits (serous modules), subsphericalo cylindrical in shape, which derive from dense particleseleased by the Golgi apparatus. During the post-Golgian,aturational phases, these serous subunits become less densend arrange themselves according to a typical recurrentattern. In the Italian tree-frog, single serous subunits are

∗ Corresponding author. Tel.: +39 055 2288 295; fax: +39 055 2288 299.

involved in merocrine processes (Delfino et al., 1994) thatfollow typical exocytotis. Exocytosis of discrete amouof serous product is unusual in anuran serous glands,ventionally regarded as holocrine organs (Faraggiana, 1931939). Holocriny is a constitutive, apoptotic process affecsecretory cells: it involves endogenous changes in theplasm which is converted into secretory product. In anurdegenerative changes in serous secretory units are duecompression exerted, under neural control, by the myoeplial cells (mecs)which form a contractile sheath or myoepitlium encircling them. These changes are, therefore, induand exogenous in nature. Indeed, poison release fromran glands induced by this contractile response hasdescribed as bulk discharge, since it involves the greateof the product as well as cytoplasm (Delfino, 1980, 1991Delfino et al., 1990, 1996) and does not include degenera

040-8166/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.tice.2005.11.002

66 G. Delfino et al. / Tissue and Cell 38 (2006) 65–77

change due to secretory activity. Nor-epinephrine was usedto reproduce ortho-sympathetic control on the gland myoep-ithelium in vitro and to evoke serous discharge (Dockray andHopkins, 1975; Delfino, 1980; Bols et al., 1986; Flucher et al.,1986; Barberio et al., 1987; Mastromei et al., 1991; Balboniet al., 1992; Sanna et al., 1993; Delfino et al., 2002; Nosiet al., 2002). In pharmacological studies, serous dischargeproved to be a dose-dependent, regulated process, since it is amulti-factorial response influenced by the number of myoep-ithelial cells affected by stimulation, antagonists, and/or ions(Holmes et al., 1977; Holmes and Balls, 1978; Delfino et al.,1982).

After gland discharge induced by catecholamines, theserous products can be collected from the experimentalmedium to be analysed under the transmission electronmicroscope (TEM). Under the TEM, the poisons consist ofgranules that closely resembled those contained in the glands(Dockray and Hopkins, 1975, in Xenopus laevis; Delfino etal., 2002, in Osteopilus septentrionalis; Nosi et al., 2002, inPhyllomedusa hypochondrialis). From these experiments itappears that neither the mechanical activity of the myoep-ithelium nor conditions in the experimental medium alterthe granule substructure. However, the secretory granules inthe species considered above contain dense products whichaccount for structural stability. It therefore seemed perti-nent to extend our investigation to the serous deposits ofH eirt rtherg ruc-t s byn or ac

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animals (a total of eight strips) were immersed in the Ringersaline and processed in the same manner as the experimentalones.

2.2. Preparation for transmission electron microscopy

After secretory discharge, the skin strips (both exper-imental and control) were prepared for TEM examina-tion as described below. In addition, the discharged secre-tory product was collected with a Pasteur pipette from thesaline and suspended in Eppendorf test tubes containing1 ml of amphibian Ringer solution. The secretory productwas pelleted at 10,000 g for 5 min by centrifugation andthen processed like the skin strips (Dockray and Hopkins,1975). Experimental and control skin specimens, as well asthe pellets, were treated for prefixation (3 h, 4◦C) with aglutaraldehyde-paraformaldehyde mixture in 0.1 M, pH 7.0cacodylate (Karnovsky, 1965) and washed in the same buffer.The skin strips were then reduced into smaller strips (2 mm2

in surface area) and postfixed (1 h, 30 min) in OsO4 (1% incacodylate). After rinsing in this buffer, the samples weredehydrated in graded ethanol, soaked in propylene oxide andinfiltrated in Epon 812. The Eppendorf test tubes contain-ing the secretory product were centrifuged during washingand dehydration steps and used as sample holders duringsoaking, infiltration and polymerisation. After polymerisa-t a-m ins (1%b M)o esh,u droal-c ml,l per-f ope.T ringo (e.g.p dlingt

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. regilla, to ascertain the morphological stability of thhin, elaborate substructures during discharge. As a fuoal, this study aims to provide structural and ultrast

ural reports on changes induced in the serous glandor-epinephrine stimulation, including mucous glands fomparison.

. Materials and methods

.1. Specimens and pharmacological treatment

Adult specimens ofH. regilla were collected near Sanlara, California and kept for several days in the Depent of Herpetology, California Academy of Science,rancisco. Six frogs were carried by hand to the Dipento di Biologia Animale e Genetica (Universita di Firenze

taly), where they were acclimatised to laboratory conditiour specimens were chosen for our experiments andt 4◦C until their responses to external stimulations wbolished, before they were sacrificed by decapitation.rocedure eliminated any interference from anaesthetic

ng pharmacological tests while minimising stress andn the frogs. For experimental purposes, five skin stripmall surface areas (25–36 mm2) were removed from thacks of each frog, immersed in amphibian Ringer con

ng 10−3 M nor-epinephrine (Delfino et al., 1982), and theiesponses checked under a digital video-camera until sory release ceased (which usually occurs within 15–20t room temperature). Control skin samples from the s

ion, the Epon blocks were cut with a NOVA LKB ultricrotome into 0.5–1.5�m semithin sections and ultrath

ections. Semithin sections were stained with bufferedorax) toluidine blue, and used for light microscope (Lbservations. Ultrathin sections were collected on 300 mncoated copper grids, and stained with a saturated, hyoholic solution of uranyl acetate, followed by 2 mg/ead citrate alkaline solution. TEM observations wereormed (at 80 kV) with a Siemens 101 electron microsco exclude non-specific patterns of gland stimulation dubservations, we avoided analysing any sample areaseripheral ones) that could have been affected by han

he skin strips.

. Results

.1. Patterns of secretory discharge at low poweragnification

Under the video-camera the control specimens showesual features of anuran skin, including clusters of intraal chromatophores (Fig. 1A). Once the skin strips in th

xperimental bath began to release their product fromeriphery, the whole surface was engaged in discharge0 min (Fig. 1B). Gentle stirring of the solution causedecretory material resting on the skin surface to separatehe tissue strips and accumulate on the floor of the incubell. The material discharged by single glands appear

hin, coiled threads with a whitish colour (Fig. 1C).

G. Delfino et al. / Tissue and Cell 38 (2006) 65–77 67

Fig. 1. Skin strips under video-camera. (A) Control specimen: arrows indi-cate chromatophore clusters. (B) Treated specimen: arrows indicate serousproduct accumulations on the skin surface after discharge. (C) Treated spec-imens: notice serous product (arrows) on the floor of the experimentalwell.

3.2. Light microscope features

3.2.1. Control specimensThe cutaneous glands in control specimens conformed

to the main patterns observed in all anurans under nor-mal conditions, demonstrating that handling proceduresemployed in this study do not evoke any response mim-icking pharmacological stimulation. Observation of closelycontiguous serous and mucous glands (Fig. 2A–C) showedtheir differential traits (see below) and allowed comparativeanalysis of their particular behaviours under experimentalconditions.

As a common trait in anurans, serous secretory unitsare syncytial in nature, and their multi-nucleated cytoplasmlacks any real lumen. The serous product is therefore con-

tained in the syncytium, and encircled by a single row ofnuclei located at the periphery (Fig. 2A–C). In semithinsections, it appeared as a rather amorphous mass contain-ing sparse, relatively large granules (Fig. 2A and B), thatare only detectable by their different densities (Fig. 2C).As demonstrated in serial sections, both serous and mucousglands share most structural characters in common: secre-tory unit, contractile sheath (myoepithelium), sub-epidermalintercalary tract, and intraepidermal duct (Fig. 2A and B).Differential traits could be detected between the secretoryunits: the mucous glands were characterised by a large vari-ety of secretory patterns, in sharp contrast to the homoge-neous features of serous glands. As a rule, the lumen wasevident in the mucous secretory units, its width varyingwith the height of the surrounding, discrete secretory cells(mucocytes) (Fig. 2A and C). Furthermore, secretory activ-ity in mucocytes was not synchronous, so that their apicesheld different amounts of the product with variable densities(Fig. 2A–C).

3.2.2. Treated specimensNor-epinephrine treatment evoked a wide range of con-

tractile responses in serous gland myoepithelia. Along withscanty poison glands, whose features were comparable tothose of the control glands, or consisted of slight structuralchanges, most secretory units appeared to be affected bym nsesf toryp iph-es tionc theiri reh ithe-l d oft eiw um,w

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yoepithelium contraction. As a rule, moderate resporom muscle cells were consistent with reduced secreroduct content and dislocation of nuclei from the perry toward the centre of the syncytium (Fig. 2D). Glandshowing intermediate degrees of myoepithelium contracontained both nuclei and residual secretory granules innner portions (Fig. 2E). On the contrary, glands that weighly affected by stimulation showed contracted myoep

ia as well as irregular profiles, and were wholly depleteheir secretory granules (Fig. 2F). The secretory unit nuclere crowded towards the inner portion of the syncytihich was distinctly narrowed (Fig. 2F).Compared with serous glands, glands of the mucous

n stimulated specimens retained usual features. Theirble patterns were due to the usual asynchronous secctivity which characterised mucocytes in different glaFig. 2F) as well as in the same gland (Fig. 2D–F).

.3. Electron-microscope patterns

.3.1. Control specimensTEM analysis provided detailed patterns of the gran

ontained in the syncytial cytoplasm; these are roundedes characterised by a fine substructure, detectable evow power magnification (Fig. 3A). The secretory units weurrounded by a continuous sheath of myoepithelialf relatively flat shape and moderately opaque cytoplaFig. 3A). Higher magnifications of the peripheral cylasm revealed the usual, morpho-functional polarisatioerous units: mitochondria and rough endoplasmic retic


(rer) cisterns were distributed near the contractile-secretoryboundary, whereas Golgi stacks were found in a slightly morecentral position (Fig. 3B). Stacked Golgi saccules producedminute, dense particles contained in small secretory vesicles,

which merged together to form larger aggregates (Fig. 3B).As a result of post-Golgian maturational changes, the denseparticles turned into light subunits arranged in a repeatingpattern (Fig. 3B).


Fig. 3. Ultrastructural features of serous glands in control specimens. G, golgi stacks; m, mitochondria; mec, myoepithelial cell; rer, rough endoplasmicreticulum; (s-s5), serous product in sequential maturational stages. (A) Secretory unit and myoepithelium: notice peripheral myocytes and inner syncytialcytoplasm with serous product in various maturational stages; only a single nucleus (arrow) is detectable in this semi-tangential section. (B) Thisperipheralarea of the secretory unit reveals rough endoplasmic reticulum, Golgi stacks,mitochondria and serous products that acquire a repeating pattern in advancedmaturational stages (arrows).

3.3.2. Treated specimensIn serous glands, which gave no or weak response to stim-

ulation, the secretory units retained the usual ultrastructuralfeatures described above (Fig. 4A). However, because of theaccumulation of large amounts of serous product, the secre-tory granules shifted towards the myoepithelium (Fig. 4B).They exhibited a peculiar substructure, with serous subunitsaggregated in a repeating thick pattern (Fig. 4A and B).Evident changes could, however, be seen in the myoepithe-lial cells of these glands, consistent with nor-epinephrinestimulation. The peripheral myocytes showed contractile fila-ments aggregated in thick masses of slightly variable density(Fig. 4C). Myoepithelial cells were arranged in a wave-likepattern (Fig. 4D), and their profiles differed depending onwhether the external (stromal) or inner (secretory) sides wereobserved. The stromal profile appeared with a somewhat,

comb-like arrangement of alternating in- and evaginations(Fig. 4B). On the opposite side, the mec profile was charac-terised by small bulgings which penetrated into the secretorysyncytium (Fig. 4C) and displayed a lighter cytoplasm dueto the lack of contractile filaments (Fig. 4B).

Intermediate degrees of mec stimulation enhanced the pat-terns described above. There was a sharper re-distributionof the contractile apparatus, which formed a remarkablyelectron-dense mass in the centre of the mec and leavedlarger electron-lucent zones at the boundary with the secre-tory syncytium (Fig. 5A). Contiguous mec portions, devoidof contractile filaments, appeared in section as discrete areaswith irregular, interwoven profiles (Fig. 5B). As a result ofintense contraction responses, mec nuclei were pushed intothe transparent sarcoplasm portions inserted into the densesecretory syncytium cytoplasm (Fig. 5C).

Fig. 2. Light microscope micrographs of cutaneous glands from control (A–C) and experimental specimens (D–F). mec, myoepithelial cell/s; lu, lumen. (A)Contiguous serous (left) and mucous (right) glands. The left secretory unit is encircled by thick myoepithelial cells, is syncytial in nature and lacks a lumen. Asin (B and C), the arrow indicates dilation of the interstitium between secretory and contractile compartments. Mucous glands possess a thin myoepithelium andmucocytes in various secretory phases, encircling a wide lumen. (B) The same as in (A) in serial section: arrowhead shows that the cell cluster on the top of thesecretory unit (gland neck) possesses a proper lumen. (C) Compared with (A and B), the serous gland exhibits a wider variability of secretory granule density.(D) Patterns of moderate stimulation are obvious in this serous gland: wave-like arrangement of myoepithelial cells and nuclei (arrows) pushed towards thecentre of the syncytium, still containing large amounts of granules (arrowheads); notice the duct lumen (large arrow). (E) Intermediate degree of stimulationinvolves crowding of pyknotic nuclei in the centre of the syncytium (arrows). Arrowheads point to residual granules. (F) As a result of intense stimulation,t y conta ot ap
hese serous glands exhibit contracted myoepithelia and syncytia onlharmacological treatment.
ining degenerating nuclei (arrows). Notice that mucous glands are nffected by


Fig. 4. Ultrastructural patterns of glands weakly affected by stimulation. mec, myoepithelial cell; rer, rough endoplasmic reticulum. (A) Common features ofserous glands are evident in this peripheral area of a secretory unit, namely organelles as well secretory granules. (B) Detail of the boundary regionbetweencontractile and secretory compartments, showing rough endoplasmic reticulum and mature granules. (C) Longitudinal section of serous gland: myoepithelialcells exhibit some thickenings of myofilaments (arrowheads): they are absent in small, translucent areas at the secretory unit side (arrows). (D) Wave-like profileof contracted myoepithelial cell. Arrows point to deep invaginations on the stromal side.


Fig. 5. Ultrastructural changes in myoepithelial cells in glands affected by intermediate degree of stimulation. mec, myoepithelial cell. (A) The myofilamentsform a thick band (white arrow) in the centre of this myoepithelial cell, while the cytoplasm bulging towards the syncytium has an electron lucent backgrounddue to the lack of any contractile apparatus (asterisk). Arrows point to indentations of the stromal profile. (B) Light myoepithelial cell cytoplasms, closelycontiguous to secretory granules. Arrows point to opposite cell boundaries. (C) In some instances contraction pushes myoepithelial cell nuclei towards thesecretory syncytium inside the light cytoplasm regions (arrowhead).

Glands that released all their secretory products along withlarge portions of their syncytia were characterised by thickmecs which in semi-tangential sections alternated with resid-ual areas of cytoplasm (Fig. 6A). In the absence of secretorygranules, the cytoplasm contained several vesicles. Some ofthese were relatively large (diameter about 4�m), and con-tained degenerating mitochondria along with microtubules(Fig. 6B). Smaller vesicles possessed a double limiting mem-brane, and structureless inner material, resembling degener-ated mitochondria free in the cytoplasm (Fig. 6C). Finally,several vesicles were bounded by single membrane unitswith associated ribosomes and exhibited a pale compartment:these were complements of the rough endoplasmic reticulum(Fig. 6C). Near the secretory-contractile interface, discreteprofiles contained minute vesicles, bundled microtubules andsmall mitochondria with dense matrix (Fig. 6D): these wereneurite endings trapped inside the syncytial cytoplasm.

Nuclei in the residual syncytium displayed obvious pat-terns of hyaline degeneration, i.e. transparent nuclear sapand clumped chromatin (Fig. 7A). These degenerative traitswere in slight contrast to the normal features of nuclei per-taining to discrete cells, inserted in the residual syncytiumcytoplasm (Fig. 7A), namely adenoblasts from the stem poolin the inner zone of the intercalary tract. Their undifferenti-ated nature was confirmed by the high nucleo-plasmatic ratioand relevant amounts of free ribosomes responsible for the

marked electrondensity of their cytoplasm (Fig. 7B). Whileinner intercalary tract cells migrated into the residual syn-cytium cytoplasm, the peripheral ones retained their position,providing stable insertion to the mecs (Fig. 7C).

TEM investigation showed that pharmacological treat-ment only slightly affected the mucous glands, or indeednot at all. The myofilament apparatuses in contiguous mecsshowed different degrees of thickening (Fig. 8A). This sug-gests that mucous gland myoepithelia give a weak contractileresponse, but the mucocyte apices showed no significantsecretory release (Fig. 8A and B).

3.3.3. Secretory product collected after dischargeThe pelleted material consisted of discrete aggregates of

secretory product, closely resembling granules described inserous glands of this species (Fig. 9A). These aggregationsmaintained their peculiar substructure, resulting from theclose association of minute subunits in a recurrent pattern,despite the fact that most of the granules collected afterrelease did not retain their limiting membranes (Fig. 9B).Furthermore, when discharge caused granule de-aggregation,single subunits were found (Fig. 9B) that displayed their usualshape (sub-spherical) as well as size (about 50 nm). Limitingmembranes were preserved in patches where closely con-tiguous granules adhered together (Fig. 9C), and in the entireextension around immature granules (Fig. 9D). Several nuclei


Fig. 6. Ultrastructural patterns in depleted glands. mec, myoepithelial cell; ne, nerve ending; rer, rough endoplasic reticulum. (A) Low power magnification ofa tangential section, showing a small portion of syncytium enclosed in its contractile sheath. Notice several vesicles in the syncytial cytoplasm ofthe secretorycompartment. (B) Small and large vesicular profiles in the secretory unit cytoplasm: the large ones contain mitochondria (arrowheads) and microtubules(arrows). (C) Residual organelles in the secretory syncytium consist of roundish, rough endoplasmic reticulum cisterns and degenerating mitochondria: arrowsindicate double mitochondrial membranes, arrowheads indicate remnants of the cristae. (D) Mec contraction caused nerve endings to be dislocated into thesecretory syncytium. Arrows point to small mitochondria with dense matrix inside the nerve endings.


Fig. 7. Stem cells in depleted glands. ab, adenoblast; inc, inner cell of the gland neck; mec, myoepithelial cell; onc, outer cell of the gland neck; sy,secretorysyncytium. (A) Closely contiguous nuclei of undifferentiated secretory cell (adenoblast) and syncytium, notice different chromatin patterns. (B) Detail of (A):arrow indicates local dilation of the perinuclear cistern in the secretory syncytium nucleus (a possible artifact resulting from compression), opposite-facingarrowheads the boundary between adenoblast and syncytium. (C) Outer and inner cells of the gland neck, with myoepithelial cells inserted between.


Fig. 8. Ultrastructural features of mucous glands in treated specimens. fc, fibrocyte. (A) As a possible weak response to stimulation, these myoepithelial cellsshow different degrees of thickening in their contractile apparatuses (closed and open arrowheads). (B) No secretory release is detectable at the apices ofmucocytes.

of the secretory syncytium could be detected dispersed in thedischarged material (Fig. 9D), but in some instances theyadhered together (Fig. 9E) as a possible effect of mec com-pression and/or centrifugation during specimen preparation.These nuclei exhibited obvious patterns of karyolysis includ-ing wide dilatations of the compartment inside the nuclearcistern (Fig. 9E).

4. Discussion

Cutaneous poison discharge in anurans is part of the phys-iological and behavioural repertory of vertebrates (flight orfight response) based on theortho-sympathetic (adrener-gic) control of several autonomic activities: increase in heartrate, opisthotonic reflex (Unkenreflex, Haberl and Wilkinson,1997), escape or aggressive behaviours. Poison productionand release would appear to be an expensive strategy, sinceanuran skin lacks any stinging apparatus and their chemi-cal defence is based on massive (bulk) emission, involvinga large number of cutaneous glands. On the other hand,many pharmacologically active molecules in cutaneous poi-sons (peptides and biogenic amines) may also play ancestral

physiological roles in the skin microenvironment, includingion and water homeostasis as well as regulation of cutaneousblood flow. These molecules could have been produced sec-ondarily in excessive amounts for chemical defence (Daly etal., 1987) according to the cost-benefit ratio optimisation.

Our findings in this study illustrate changes in the con-tractile sheath of serous glands, that are typical in mecsundergoing contraction (Delfino, 1980; Delfino et al., 1990;Nosi et al., 2002). On the other hand, structural patternsdetected in the secretory syncytium closely resemble thosedescribed under LM in serous glands after external compres-sion (Faraggiana, 1938, 1939). Under such conditions, thesecretory syncytium appears devoid of pre-existing secretorymaterial and exhibits degenerative changes (Toledo et al.,1992). Several vesicles of various size can be seen (Delfinoet al., 1990): the smaller ones, which exhibit a light innercompartment containing structureless material, are swollenmitochondria (Toledo et al., 1992) and remnants of the endo-plasmic reticulum; the larger ones, enclosing mitochondria,hyaloplasmic matrix and microtubules, derive from mem-brane patches – possibly from degenerated organelles – whichhave fused together (Neuwirth et al., 1979). The lack of secre-tory material in the residual syncytium of stimulated glands


Fig. 9. Serous gland content collected after release. (A) Serous aggregates resembling secretory granules and sparse subunits (arrows). (B) Detailof secretorygranule lacking limiting membrane; notice secretory subunits, both aggregated and sparse (arrowheads and arrows, respectively). (C) Patch of limiting membranepreserved between closely contiguous granules (arrowheads). (D) Different degrees of karyolysis and secretory granule with limiting membrane (arrow); theelectron-dense particles in the granule suggest it corresponds to immature serous product. (E) These nuclei closely adhere to each other, possibly due tocompression during discharge or preparative centrifugation.

underlines the mechanical effectiveness of mec compression,as also supported by the occurrence of nuclei in the productwe have collected after discharge. This pattern was actuallypredictable, since nuclei passing through the neck lumen dur-ing secretory release have been already described (Delfino,1980). Furthermore, cytoplasm fractions (i.e. residual com-plements of the rer) have been detected in the saline afterdischarge (Delfino et al., 2002in Osteopilus septentrionalis).The expulsion of syncytium portions allows release of activemolecules stored in the cytoplasm without packaging themin vesicles or granules (Bols et al., 1986). However, bulkdischarge requires regenerative processes that are mostly per-formed by stem cells in the intercalary tract. These cellsfirst evolve as adenoblasts, then differentiate into adeno-cytes and later merge into the syncytium (Faraggiana, 1938,

1939; Delfino et al., 1988; Flucher et al., 1986). Our datasuggest that the regenerative activity involves the inner inter-calated cells contiguous to the residual syncytium, whereasthe peripheral, keratinocyte-like ones perform a mechanicalrole.

Pharmacological stimulation is a suitable method forobtaining cutaneous poisons under conditions that mimicphysiological response. When nor-epinephrine is adminis-tered into the lymph-sacs of living specimens, the procedureallows collecting large amounts of product for biochemi-cal/biological analysis (Barberio et al., 1987; Mastromei etal., 1991; Balboni et al., 1992; Sanna et al., 1993) as theprocedure can be repeated on the same animals after glandrehabilitation. Pharmacological stimulation of serous glandsmay sometimes be unsuccessful (Lacombe et al., 2000, in


Phyllomedusa bicolor), and when un-reactive glands repre-sent a distinct type in species with two or more gland lines(Delfino, 1980; Delfino et al., 1982, in Bombina variegata),the opportunity exists to collect serous products differentially(Barberio et al., 1987, in B. variegata; Nosi et al., 2002, in P.hypochondrialis).

Remarkable differences between mec responses can usu-ally be detected when comparing serous and mucous glands.The apparent sluggishness of mucous glands is consistentwith the lack of any direct, neural control on their myoepithe-lium (Whitear, 1974), although the mild contraction patternsobserved in these muscle cells suggest some response toneurotransmitters, possibly diffusing from contiguous nerveendings across a 0.5–1�m, stromal gap (Sjoberg and Flock,1976).

The TEM features of the product released into the exper-imental medium reveals that the substructure ofH. regillasecretory granules is stable and the small particles are firmlyjoined together. Indeed, we detected granules that retainedtheir inner arrangement even when the limiting membranewas destroyed. This demonstrates that the post-Golgian mat-urational phase effectively stabilises the architecture of theseserous aggregates. Furthermore, whenever granule degra-dation was observed, the secretory product still consistedin discrete subunits. This is possibly a transitory phase ofgranule disintegration which may also occur under normalc enlyd

pre-s culesi nce.T be inte tremece lti-p in seve andP 5T tura-t winga

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L las,ds of

onditions, allowing the serous product to perform evistributed activities on the body surface.

The modular substructure of the poison granules reents a satisfactory arrangement for storing active molenvolved in skin homeostasis as well as chemical defehe serous product, released through exocytosis, may

he form of discrete particles (Delfino et al., 1994, 1999) orntire granules when maturational change leads to exondensation and loss of any modular substructure (Delfinot al., 1999). This repeating substructure derives from muoint condensation processes and has been developedral families: Dendrobatidae, Hylidae, Leptodactylidaeseudidae (Terreni et al., 2002, 2003; Alvarez et al., 200).herefore, it seems to be the result of a successful ma

ional strategy in anuran cutaneous gland evolution, alloflexible use of serous products.

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serous cutaneous glands of the pacific tree-frog hyla regilla (anura

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