morphology of the femoral glands in the lizard ameiva ... et al._2007_morphology of the... ·...

Morphology of the Femoral Glands in the LizardAmeiva ameiva (Teiidae) and Their PossibleRole in Semiochemical Dispersion

Beatriz A. Imparato,1 Marta M. Antoniazzi,1 Miguel T. Rodrigues,2 and Carlos Jared1,2*

1Laboratorio de Biologia Celular, Instituto Butantan, CEP 05503-900, Sao Paulo, Brazil2Departamento de Zoologia, Instituto de Biociencias, Universidade de Sao Paulo, Sao Paulo, Brazil

ABSTRACT Many lizards have epidermal glands inthe cloacal or femoral region with semiochemical func-tion related to sexual behavior and/or territorial demar-cation. Externally, these glands are recognized as a rowof pores, opening individually in the center of a modifiedscale. In many species the pores are used as systematiccharacters. They form a glandular cord or, in some spe-cies, a row of glandular beads below the dermis, and areconnected to the exterior through the ducts, which con-tinuously liberate a solid secretion. Dead cells, desqua-mated from the secretory epithelium, constitute thesecretion, known as ‘‘a secretion plug.’’ The present workfocusses on the morphology of the femoral glands of theteiid lizard Ameiva ameiva, correlating it to the way inwhich the secretion is deposited in the environment. Theresults here obtained are compared to those availablefor other lizards and amphisbaenians. We observed thatthe diameter of the glandular pores did not show signifi-cant differences between males and females. The glandscomprise germinative and secretory cells, which passthrough at least three stages of differentiation, duringwhich an accumulation of cytoplasmic granules, with aglycoprotein content, occurs. The cells eventually dieand desquamate from the secretory epithelium, forminga secretory plug mostly constituted by juxtaposed non-fragmented secretory cells. Because of the arrangementof the rosette-like scales surrounding the femoral pores,we suggest that when the animal is in a resting position,with its femoral regions touching the ground, thesescales may be involved in the breakage of their respec-tive plugs, depositing tiny portions on the substrate. Inthis manner, it seems that the method for signal disper-sion in this species involves specifically adapted struc-tures and does not simply involve the chance breakageof the plug, as the gland secretes it. Signal dispersionmust also be intimately associated with the animal’smovement within its territory. J. Morphol. 00:000–000,2007. � 2007 Wiley-Liss, Inc.

KEY WORDS: squamata; lizards; teiidae; femoral pores;epidermal glands; Ameiva ameiva; pheromone

Among the Squamata, semiochemicals have animportant role in sexual behavior, defense, preda-tion, territorial marking, orientation, intraspecificaggregation, and parental care (Halpern, 1992).Besides their importance in squamate biology, themorphology of the structures related to semiocom-

munication are poorly known. Most morphologicalpapers on this subject deal with European, Asian,Australian, and African species (Cole, 1966a,b;Maderson, 1968, 1970, 1985; Maderson and Chiu,1970, 1981; Chiu and Maderson, 1975; Schaefer,1901, 1902; Cohn, 1904; van Wyk et al., 1992; Duj-sebayeva, 1998). More recently, the morphology ofpre-cloacal glands in an amphisbaenian (Amphis-baena alba) was reported and their role in the nat-ural history of this species was analyzed (Anto-niazzi et al., 1993, 1994; Jared et al., 1999).

Femoral glands are the most common semio-chemical sources in lizards (Jullien and Renous-Lecuru, 1973) although some glandular scales or‘‘generation’’ glands (Maderson, 1967, 1968) havebeen described in cordylids in other regions of thebody (van Wyk et al., 1992). Femoral glands arelocalized in one or two rows on the ventral side ofeach thigh. The first morphological studies on fem-oral glands date from the end of XIX century andbeginning of XX century (Abraham, 1930), andconcerned primarily European species. The subjectwas ignored by morphologists until the 1960s, whenpapers by Gabe and Saint-Girons (1965) and Cole(1966a,b) were published. Uva (1967), Gasc et al.(1970), Chiu and Maderson (1975) and Chauhan(1986) then reported additional data on other species.

Although semiochemical communication is veryimportant in lizard biology, studies focusing onthese aspects are rare (Mason, 1992; Cooper, 1994;Cooper et al., 1996). However, many experimentshave demonstrated that several species are able torecognize chemical signals secreted by femoralglands (Cooper and Vitt, 1984; Alberts, 1989, 1993;Lopez et al., 1998, Martin and Lopez, 2000).

The localization of glands in both lizards andamphisbaenians suggests that signals are pas-

*Correspondence to: C. Jared, Instituto Butantan, Laboratorio deBiologia Celular, Av. Vital Brasil 1500, CEP 05503-900, Sao Paulo,Brazil. E-mail: [email protected]

Published online inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/jmor.10473

JOURNAL OF MORPHOLOGY 00:000–000 (2007)

� 2007 WILEY-LISS, INC.

sively deposited in the environment during locomo-tion of the animals within their territory (Maderson,1986; Jared et al., 1999). Among lizards possessingfemoral glands, signal dispersion involves thebreakage of small portions of the secretion plugs.These remain on the substrate, releasing chemicalsignals for a period of time that must be related toboth the natural history and environment of a par-ticular species (Alberts, 1993).

Ameiva ameiva is a teiid lizard with a broad geo-graphic distribution in Central and South Ameri-cas, from Panama to South Brazil and North Ar-gentina (Vanzolini et al., 1980; Colli, 1991). Theaim of the present paper was the study of the mor-phology and some aspects of the histochemistry ofthe femoral glands of the teiid Ameiva ameiva,and the comparison of the results with thoseobtained for other lizards and amphibaenians. Wehope to contribute to the integrative knowledge ofthe natural history of lizards, especially in relationto chemical communication. Our data suggest thatin Ameiva ameiva the differentiated scales con-taining the femoral pores and the secretion plugsare structures adapted to the dispersion of chemi-cal signal on the substrate and seem to be associ-ated with the locomotion of these animals through-out their territory.

MATERIALS AND METHODS

Animals

Adult individuals of Ameiva ameiva Linnaeus 1758 were col-lected at Palmas (State of Tocantins, Brazil) and maintained for2 years in the vivarium of the Laboratory of Cellular Biology ofthe Instituto Butantan. They were housed in terraria contain-ing humid earth at the bottom with dry branches above andpieces of plastic pipe for shelter. They were daily exposed tosunlight during the morning period. Food was supplied weeklyand was composed of a variation of live items (beetles - Tenebriomolitor, crickets - Gryllus sp, cockroaches - Pycnocellus surina-mensis), fruits, eggs, and minced beef.

For the morphological studies, six animals were sacrificedwith a intraperitoneal overdose of sodic thiopental just afterbeing collected from nature. Samples of the skin of the femoralregion containing the femoral glands were removed and pro-cessed as described below.

Voucher specimens were deposited in the collection of theMuseum of Zoology, University of Sao Paulo (MZUSP).

Histology

The skin containing the femoral glands was cut in strips ofabout 3 3 5 mm2 using a razor blade and fixed in 4% parafor-maldehyde in PBS 0.1 M, pH 7.2 for 24 h. The material wasdehydrated and embedded either in historesin (glycol methacry-late, Leica) or paraffin (at 608C).

For general study of the tissues, 2 lm historesin sectionswere stained with toluidine-fuchsin (Junqueira, 1995) and 5–7lm paraffin sections were stained with hematoxylin–eosin.Picro-Sirius and N.M.C. trichrome stains (Junqueira et al.,1979) were used for the identification of collagen and muscularfibers, and the Weigert’s resorcin-fuchsin stain (Bancroft andStevens, 1996), for the identification of elastic fibers.

Histochemistry

Historesin and paraffin sections were subjected to the follow-ing histochemical staining procedures (according to Bancroftand Stevens, 1996): periodic acid-Schiff (PAS) and alcian bluepH 2.5, for identification of neutral and acidic mucosubstances,respectively, and bromophenol blue, for identification of pro-teins. Sudan black B was used for identification of lipidic sub-stances.

Photomicrography

Photographs were obtained by using a Leica DMLB micro-scope equipped with a Leica MPS60 photographic system.

Scanning Electron Microscopy

A strip of skin from the femoral region containing 5–7 femo-ral pores was removed from the animals, fixed in Karnovsky so-lution (Karnovsky, 1965) for 24 h, post-fixed in 1% osmium te-troxide, incubated in 1% tannin, and postfixed once more in 1%osmium tetroxide (Mizuhira and Futaesaku, 1974; Simionescuand Simionescu, 1976). The material was dried by the criticalpoint method, coated with gold, and examined in a JEOL SM6100 and in a JEOL JSM 5300, operating between 15 and 25 kV.

Transmission Electron Microscopy

Fragments of the femoral glands (separated from the skin) ofabout 1 mm3 were fixed in Karnovsky solution, pH 7.2 (Kar-novsky, 1965) for 24 h, post-fixed in 1% osmium tetroxide, con-trasted in 2% uranyl acetate, dehydrated in ethanol and embed-ded in epoxy resin. Ultrathin sections (60 lm) were obtained ina Sorvall MT 6000 ultramicrotome and were examined with aLEO 906E, operating at 80 kV.

Morphometrical Study

Adult male (N ¼ 14) and female (N ¼ 14) specimens ofAmeiva ameiva from the zoological collection of Museu de Zoo-logia da Universidade de Sao Paulo, all from the same popula-tion, were studied. The number and the diameter of the poresin the femoral region of males and females were quantified andpossible differences in both sexes were analyzed by Studenttest. The diameters of the pores of three regions in the pore row(the two extreme regions, named P1 and P3, and the middleregion, named P2) were measured. Because data were normallydistributed and had homogeneous variances, they were ana-lyzed by ANOVA test.

RESULTSGeneral Anatomy and Histology of theFemoral Glands

In Ameiva ameiva (Fig. 1), the ventral skin ofthe thighs, in both males and females, shows a sin-gle row of pores (Fig. 2), whose number variesfrom 17 to 23 in males and from 16 to 22 infemales. Each pore opens in the center of a modi-fied scale, resembling a rosette (Fig. 3). Because oftheir prominence, the row of pore-bearing scalesstand out from the neighboring scales and, for thisreason, are easily recognized (Figs. 2, 3). Studyingthe skin from the dermal side reveals that each ofthe pores corresponds to a gland. The row ofglands, superimposed on one another, forms a deli-

2 B.A. IMPARATO ET AL.

Journal of Morphology DOI 10.1002/jmor

Figures 1–3 (legend on page 4)

Figures 4–6 (legend on page 4)

cate yellowish cord (Fig. 4), which is juxtaposed tothe dermis by delicate connective tissue. Whenthis tissue is removed, it is possible to observe thateach one of the glands is branched and flat (Fig.4). In sections of this region, the glandular bodiesare cut in different planes and internal ductulesappear among the lobules (Fig. 5). The main ductsconnecting the glandular bodies to the exterior arevisible in sections cut transverse or longitudinal tothe axis of the femur. The ducts are always filledwith a solid plug comprising a yellowish and brit-tle secretion (Fig. 5).

A thin layer of connective tissue, which is sup-plied by many blood vessels, covers each of thefemoral glands. The connective tissue forms septadividing the gland into lobules (Fig. 6). Each lobulecomprises a holocrine epithelium that converges tothe ductules, which finally connect to the mainduct.

The main duct is lined by a stratified epithe-lium, which is continuous with the skin epidermis(Fig. 5). The holocrine secretion that fills the ductcomprises small units with an affinity for toluidineblue (in toluidine blue-fuchsin staining) (Fig. 6) orfor eosin (in hematoxylin–eosin staining).

Scanning Electron Microscopy of theGlandular Pore Region

The scale containing the glandular pore com-prise three or four plates arranged like the petalsof a flower (Fig. 7). One of the plates is alwayslarger and more prominent when compared to theothers (Fig. 7, insert). Each one of the scale plates,specially the larger one, shows a fine arched inter-nal border, which contacts the plug (Fig. 7).

The secretion plug emerges from the pore in theform of a rod, the tip of which is usually about thesame level of the skin surface. While a smoothcover (Fig. 7) envelops its lateral aspect, smallunits corresponding to the dead cells, which aredesquamated from the holocrine epithelium, formits distal surface (Fig. 8). Most of these dead cellsare intact and show no signs of abrasion from con-tact with the substrate (Fig. 8).

Light Microscopy of theSecretory Epithelium

Germinative cells and secretory cells (Fig. 9A–F)comprise the glands. To facilitate the morphologi-cal description of the differentiation process, thesecretory cells were divided into three subsequen-tial stages (S1, S2, and S3), according to theirstructural characteristics.

The germinative cells (G) are localized at the pe-riphery of the lobules. They are elongated andhave voluminous nuclei (Fig. 9A). The cells in thefirst stage of differentiation (S1) have the samelocalization as the germinative cells but are gener-ally more spherical. Their nuclei are also sphericaland voluminous and usually have one or two con-spicuous nucleoli. During the first stage, the cyto-plasm is lightly stained and does not possess secre-tion granules (Fig. 9A).

The second stage of differentiation (S2) is charac-terized by cells with a larger volume and observedin different phases of the secretory process. Thesephases can be identified by the increase in thenumber of secretory granules and were named P1,P2, and P3. In P1, the cells are usually sphericaland the cytoplasm has a number of discrete gran-ules (Fig. 9B). In P2, small granules that are some-times metachromatic (Fig. 9B) characterize thecells. In the last phase (P3), the granules occupy alarge portion of the cytoplasm, are larger andspherical, but are heterogeneous in size andin their tinctorial properties (Fig. 9C). Irregulargranules containing heterogeneous secretion (Fig.9D) which usually forms a half-stained patternwithin the granule distinguish late P3. In themost differentiated cells, the irregular nuclei tendto be displaced to the periphery, and are smallerin relation to the total cell volume. The cellsremain spherical.

In the third and last stage (S3), the secretorycells, already close to the duct, assume a flat formand the cytoplasm is full of irregular granules withheterogeneous secretion. The peripheral nuclei arepycnotic (Fig. 9E). At the end of this stage, the cellsdesquamate from the secretory epithelium, enterthe duct, and become part of the solid plug.

During the differentiation process, cells of a dif-ferent type may be seen among the secretory cells.They contain dense inclusions suggesting clustersof secretion granules lying inside a large vacuole,

Figs. 4–6 Fig. 4. Dermal side of the skin in the region ofthe femoral pores. The femoral glands are packed close togetherforming a delicate yellowish cord. The arrows point to somebranched glandular bodies, while the arrowheads indicate theglandular ducts. Fig. 5. Histological section of the skin in theregion of the femoral pores. Part of the glandular cord is visiblebeneath the dermis, with two glandular bodies sectioned in dif-ferent planes, longitudinal (G1) and transversal (G2), and twomain ducts transversally sectioned (D1 and D2), full of secre-tion. The whole is enveloped in a delicate connective tissue(arrowheads). The asterisks indicate internal ductules in thedistal portion of one of the glands. One main duct (D3) opensthrough the epidermis (Ep). De, dermis. Glycol methacrylate,toluidine-fuchsin stain. Fig. 6. Magnification of part of Figure 5,focusing on a transverse section of gland body. It is divided intolobules separated by loose connective tissue (ct). The lobules areconstituted by germinative cells (arrowheads), in the periphery,and secretory cells in different stages of differentiation (*).Arrows, blood vessels; d, ductule. Glycol methacrylate, tolui-dine-fuchsin stain.

Figs. 1–3 Fig. 1. A male specimen. Fig. 2. Ventral side ofthe thighs showing the pair of femoral pore rows (arrows). Fig.3. Macrophotography of the femoral pores. The glandular ductsopen in the center of a row of modified scales (arrows) resem-bling rosettes. The duct lumina are filled by the secretion plugs(*).



and occupy a considerable part of the cell cyto-plasm (Fig. 9C,F).

Histochemical Observationsof the Femoral Glands

The secretory cells showed a strong and homoge-neous PAS positivity (before and after amylasetreatment) either in the secretion granules or inthe plug. Similarly, granules and plug were posi-

tive to bromophenol blue, although the granulesare not all positive to the same intensity.

Alcian Blue at different pHs (1.0, 2.5, and 3.1)and Sudan Black were negative for all glandularstructures.

Light Microscopy of the Rosette

The skin of Ameiva ameiva comprises a cornifiedepidermis and a dermis essentially constituted by

Figs. 7 and 8. Fig. 7. The femoral pore, showing the cylindrical secretory plug (P), with its smooth cover (Co), emerging througha rosette of scales. The rosette is composed of a larger plate (**) and three small plates (*). This arrangement is best observed inthe insert. The arrows point to the arched internal border of the larger plate. SEM. Fig. 8. The plug’s surface (P), at higher magni-fication, where many dead secretory cells (*) may be observed. The arrangement of the cells, which are juxtaposed to one another,gives an idea of the friability of this structure. SEM.

FEMORAL GLANDS IN THE LIZARD AMEIVA AMEIVA 5


Figure 9

Figures 10–13

dense regular connective tissue in a basket arrange-ment with intermingled blood vessels.

Longitudinal sections of femoral skin show the gen-eral structure of the modified scales (rosettes) con-taining the pores, which may also be seen in trans-verse section (Figs. 10–13). Picro Sirius stainingshowed that the dermal connective tissue is basicallyformed by collagen fibers, which stain bright red.N.M.C. trichrome staining revealed the characteristicarrangement of dermal collagen fibers (which appearin deep blue color) within the rosette. The dermiscomprises an exceptionally well-developed deep layerand a poorly developed superficial layer, immediatelybelow the epidermis (Figs. 10 and 11). Immediatelyaround the duct the collagen fibers are thinner andare circularly arranged (Fig. 11). Between the deepdermis and the circular fibers, there is zone of looseand less organized connective tissue well irrigated byblood vessels (Fig. 11).

When Weigert’s Fucsine-Resorcine is applied to thesections, delicate purple elastic fibers are revealedamong the collagen fibers, mainly in the well developeddeep layer in the form of a net (Fig. 12) and following acircular arrangement around the duct (Fig. 13).

Transmission Electron Microscopy of theFemoral Glands

At the ultrastructural level, the germinativecells are polygonal or spindle-shaped (Fig. 14A). As

in other epithelial tissue, the cell membranes showmany interdigitations and desmosomes. The cyto-plasm of germinative cells is quite electron lucentand is rich in mitochondria, granular and agranu-lar endoplasmic reticulum, free ribosomes, andGolgi apparatus (Fig. 14B). Bundles of intermedi-ate filaments (tonofilaments) occur through thecytoplasm or linked to desmosomes (Fig. 14A,B).The spherical nuclei with one to three nucleolioccupy a large volume of the cell and present pre-dominantly loose chromatin (Fig. 14A).

The secretory cells in the first stage of differen-tiation (S1) can be distinguished from the germina-tive cells by their more spherical shape, and espe-cially by the small and scarce secretion granulesin the cytoplasm (Fig. 14C). These granules arespherical and homogeneous. Similar to the germi-native cells, the cytoplasm is still quite electronlucent and tonofilaments and desmosomes areabundant. An increase in the granular endoplas-mic reticulum (GER) and Golgi apparatus occurs(Fig. 14D). The nuclei is spherical, with predomi-nantly loose chromatin, and is less voluminouswithin the cell when compared to the situation ingerminative cells (Fig. 14C).

The second stage (S2) is characterized by nucleiwith irregular shapes, an increase in cytoplasmicvolume and in the number of the secretion gran-ules. It is possible to distinguish at least threephases in this stage: (P1) In the initial phase, cellshave spherical, homogeneous granules of differentsizes occupying part of the cytoplasm (Fig. 14E).Lamellae of the granular endoplasmic reticum arequite developed and dilated and Golgi cisternaewere often seen (Fig. 14F). (P2) The cytoplasm ofthe cells contains many large spherical granules,some of them with heterogeneous content (halfelectron dense, half electron lucent). The GER iswell developed and is still dilated (Fig. 15A). Otherorganelles are not so visible and, as the number ofgranules increases, nuclei are more electron denseand are pushed towards the cell periphery. (P3) Inthe last phase of S2, the cytoplasm is replete withsecretion granules with heterogeneous content(Fig. 15B). The nuclei often appear alreadyshrunk, are electron dense and have an irregularshape. While most organelles are barely observedamong the granules, it seems that there is anincrease in the number of lipidic inclusions, withmedium electron density (Fig. 15B).

In the last stage of differentiation (S3), the cellsshow evident signs of cytoplasmic disorganizationand acquire a flatter form (Fig. 15C). The granulesoccupy practically the whole volume of the cells.The nuclei are pycnotic or absent. There is anincrease in the number of lipidic inclusions (Fig.15C). The cells in this stage are beginning to des-quamate from the secretory epithelium, or are al-ready desquamated and lie loose in the duct, form-ing the secretion plug. Ultrastructure of the plug

Fig. 9. Histological sections at high large magnificationshowing the internal arrangement of the glandular lobules.Gland cells are seen in different stages of differentiation. Ger-minative cells (G) lie at the periphery (A–C, E and F). Secre-tory cells in the first stage of differentiation (S1) have no gran-ules (A, D, and F). Secretory cells in the second stage of differ-entiation, divided in three subsequent phases (P1, P2 e P3), inwhich the granules are secreted, increase in number, andundergoes maturation (B–D). Secretory cells in the third stageof differentiation (S3), in which they acquire a flat form, presentevident signals of decline and finally desquamate from the se-cretory epithelium (E). Cells with dense inclusions (*), observedamong the other cells (C, D, and F). Arrowheads point to pyc-notic nuclei. Glycol methacrylate, toluidine-fuchsin stain.

Figs. 10–13. Fig. 10. Histological section through a rosette-shaped scale containing a femoral pore transversally sectioned.The arrangement of the dermis in the scale is better observedin Figure 11, which corresponds to a high magnification of thearea indicated by the rectangle. The dermis comprises a thinsuperficial layer immediately below the epidermis (Ep), a well-developed deep layer (dt) mainly formed by collagen fibers, anda circular layer (ct), also mainly formed by collagen fibers,around the duct. Between the deep layer and the circular layerthere are areas mainly composed of loose and less organizedconnective tissue (lt) with many blood vessels (arrows). P, secre-tion plug; *, duct epithelium. Paraffin. N.M.C. trichrome stain.Fig. 11. Magnified image of the area indicated by the rectanglein Figure 10. Fig. 12. Details of a histological section throughone rosette-shaped scale containing a femoral pore transversallysectioned. The section was stained to demonstrate the delicateelastic fibers (arrows) which form a disorganized net among thecollagen fibers in the deep layer. Ep, epidermis; *, duct epithe-lium. Paraffin. Weigert’s resorcine-fuchsin. Fig. 13. The circulararrangement of collagen fibers around the duct.



Figure 14



shows that it comprises electron-dense polyhedralunits, measuring about 1.3 lm in the larger axis.

A few secretory cells, mainly in the first stagesof differentiation, contain dense inclusions in theircytoplasm (Fig. 15D).

Morphometrical Study

The number of pores in males and females did notshow differences (meanmales ¼ 20.27, meanfemales ¼19.67, N ¼ 28, t ¼ 0.0159). The comparison of porediameters among regions P1, P2, and P3 did not showdifferences in males (meanP1 ¼ 0.51 mm, meanP2 ¼0.57 mm, meanP3 ¼ 0.49 mm, F ¼ 3.15, N ¼ 42) aswell as in females (meanP1 ¼ 0.34 mm, meanP2 ¼0.36 mm, meanP3 ¼ 0.35 mm, F ¼ 0.52, N ¼ 42).The comparison of pore diameters between malesand females for regions P1, P2, and P3 also did notshow differences (tP1 ¼ 3.63E�08, tP2 ¼ 1.93E�07,tP3 ¼ 3.25E�05).

DISCUSSION

Epidermal glands in pre-anal or femoral regionsare wellknown in several lizard families (Jullienand Renous-Lecuru, 1973; Rastogi, 1975) althoughthey can be found in other body regions (Simon,1983; Dujsebayeva, 1998). Many behavioral andbiochemical studies conducted in recent decadeshave demonstrated the role of these glands in lizardchemical communication (Pough et al., 2001). Thereis a considerable external variation of epidermalglands in relation to their distribution in the body(Kluge, 1967a,b; Dujsebayeva, 1998), the shape ofthe scales containing the pores (Blasco, 1975; vanWyk et al., 1992), and the coloration of the secre-tion plug (Cole, 1966b; Blasco, 1975). Internallythese glands can also present anatomical and color

variations (Gasc et al., 1970; Rastogi, 1975). Someof these differences may be related to the way epi-dermal glands are used for chemical communica-tion within each group.

Rosette-like differentiated scales are not exclu-sive of teiids but can also be found in other lizardfamilies such as iguanids and lacertids (Cole,1966a; Blasco, 1975). Our histological observationson Ameiva ameiva indicate that this structure pos-sibly derives from the fragmentation of one singlescale (Fig. 5).

Femoral pores have been used as systematiccharacters for a long time (Linnaeus, 1758;Dumeril and Bibron, 1834). They are also used todefine sexual dimorphism (Cole, 1966a). In thecase of captive Ameiva ameiva we have notobserved any significant circannual modification inthe external morphology of femoral glandsalthough the animals remained sexually active,with copulations being frequently observed. Ourmorphometrical results indicated that in Ameivaameiva no sexual dimorphism can be observed inrelation to the number or to the diameter of pores.This apparent similarity in the glands of bothsexes could indicate that they may be used by allindividuals for territorial demarcation. However,differences in the biochemical composition of glandsecretion between males and females cannot bediscarded.

In relation to their internal anatomy, femoralglands of Ameiva ameiva correspond to the modelproposed by Gabe and Saint Girons (1965) for liz-ards in general. In this model, the glands are flatand branched, with an internal ramification ofductules connected to a main duct, which leads tothe secretion to the exterior. The solid secretionplug is generally the same color of the gland inwhich it is produced (Cole, 1966b). In Ameivaameiva, femoral glands and secretion plugs allhave the same yellowish coloration.

Femoral glands in Ameiva ameiva, as in othersquamates, are holocrine. The extrusion of thesolid secretion plug through the pore is due to thepressure exerted by the distal proliferation anddifferentiation of the secretory cells within tubules(Antoniazzi et al., 1993). This process, however,does not explain the lateral aspect of the plug, visi-ble by scanning electron microscopy (Figs. 7, 8).This seems to be a structure formed by the accu-mulation of desquamated epithelial layers since,according to Maderson (1972), the gland duct epi-thelia do not show cyclic changes of the type thatare associated with periodic shedding.

The ultrastructure of the germinative cells istypical of epidermal cells, showing large numbersof tonofilaments and desmosomes. These cellsreveal an intense metabolic activity, which is mor-phologically evidenced by the cytoplasm rich in dif-ferent organelles. The observation of histologicaland ultrastructural differences among the secre-

Fig. 14. The cells constituting the femoral gland lobules.(TEM). A: Polygonal germinative cell with a medium electronlu-cent cytoplasm and a voluminous nucleus (N), with loose chro-matin and a well developed nucleolus (n). The cell is connectedto the neighboring cells by many desmosomes (arrows). B:Higher magnification of a germinative cell. The cytoplasm isrich in tonofilaments (arrows), mitochondria (m), agranularendoplasmic reticulum (*), small vesicles (arrowheads) and freeribosomes spread, among the other organelles. C: First stage ofdifferentiation S1 of a secretory cell. As in the germinative cell,the cytoplasm is medium electronlucent and the nucleus (N),with a prominent nucleolus (n), occupies a large volume of thecell. The first secretion granules (arrows) lie close to both thegranular endoplasmic reticulum (GER) and Golgi apparatus(Go). n, nucleolus. D: Higher magnification of rectangle markedin (C), showing details of the Golgi apparatus (Go), surroundedby many small vesicles (arrowheads), and of the GER. G, secre-tion granule. E: First phase P1 of the second stage of differen-tiation. The total cell volume is enlarged and the number of se-cretory granules (G) has increased. Arrow, bundle of tonofila-ments; *, Golgi apparatus. F: Enlarged area of a cell in P1. Thesecretion granules are spherical with dense, homogeneous con-tent. Go, Golgi apparatus; GRE, granular endoplasmic reticu-lum.



tory cells made it possible to distinguish at leastthree different stages of differentiation. In thethird stage, small inclusions with medium electrondensity seem to be lipidic in nature. Furthermore,we often observed in all differentiation stages the

presence of large inclusions that are apparentlythe accumulation of secretory material in the formof vesicles whose destiny in the cells we have notbeing able yet to determine. All these differentia-tion processes resemble what has been observed in

Fig. 15. The cells constituting the femoral gland lobules. (TEM). A: Second phase P2 of the second stage of differentiation of asecretory cell. The secretion granules (G) increase in volume and in number and the contents of some of them (arrows) are not ashomogeneous as phase 1 (F and G). The GER remains well developed. N, nucleus. B: Third phase P3 of the second stage of differ-entiation. The cytoplasm shows the first signs of disorganization although the main organelles, such as the GER are still visible.The contents of the secretion granules (G) are heterogeneous, sometimes half electrondense and half electronlucent (arrows). Lipidinclusions lie in the cytoplasm among the granules (arrowheads). N, nucleus. C: Third stage of differentiation S3 of the secretorycells. The cell is slightly flattened and the nucleus (N) lies in the periphery, shrinks as it becomes electron-dense and irregular.The cytoplasm is quite disorganized and the heterogeneous secretion granules, together with lipid inclusions (arrowheads) occupymost of it. D: Dense inclusions (I) resembling clusters of secreted material are quite common in the cytoplasm of many secretorycells, mainly in the first stages of differentiation.



the epidermal glands of other lizards (Cole, 1966a;Gasc et al., 1970; Maderson, 1972) and amphisbae-nians (Antoniazzi et al., 1993, 1994; Jared et al., 1999).

In relation to the chemical nature of Ameivaameiva femoral gland secretion, our histochemicaland ultrastructural data indicate that it consists ofneutral mucosubstances and proteins, probablyforming glycoproteins. The same results wereobtained for other lizards (Gabe and Saint Girons,1965; Cole, 1966b; Uva, 1967; Gasc et al., 1970;Rastogi, 1975; Alberts, 1993) and amphisbaenians(Antoniazzi et al., 1993, 1994). Despite the nega-tive result obtained with the use of Sudan Black Bfor detection of lipids, at the ultrastructural level,in advanced stages of cell differentiation, we haveobserved small inclusions in the secretory cell cyto-plasm that seem to be lipidic in nature. Lipids arecommon among substances involved in chemicalcommunication. (Alberts, 1990; Alberts et al., 1992;Halpern, 1992). Alberts (1993), working with thechemistry of pheromones in Dipsosaurus dorsalis,discussed environmental influence on the composi-tion of the femoral gland secretion of this iguanid.In Iguana iguana and Dipsosaurus dorsalis, thelow concentration of lipids in their glandular secre-tion could only be detected by gas chromatography(Alberts, 1990; Weldon et al., 1990). Thus,although undetectable by histochemical techni-ques, small amounts of lipid might be present inteiid femoral gland secretions.

The ventral localization of the epidermal glandson the body of lizards and amphisbaenians sug-gests that their secretions are passively depositedin the environment (Maderson, 1986). Recentpapers have demonstrated that in fact many spe-cies of lizards respond to secretions derived fromthese glands (Cooper and Vitt, 1986; Cooper et al.,1994; Cooper et al., 1996). However, the methodused by lizards for depositing chemical signals inthe environment has not been studied so far. Inamphisbaenians, (Antoniazzi et al., 1993, 1994),studying the morphology of the pre-cloacal glandsof Amphisbaena alba, demonstrated that, as aresult of the position and arrangement of theseglands, the secretion plugs, perpendicular to thebody, make direct contact with the tunnel wallswhere this species lives. These authors suggestthat, during locomotion, the plugs are naturallyabraded and secretion fragments are deposited onthe substrate, forming a trail. In fact, Jared et al.(1999) experimentally demonstrated the formationof a secretion trail during locomotion of A. alba.Scanning electron microscopic examination, bothof the trail and of the abraded surface of the plug,showed that the fragments of the trail correspondto the secretion granules of the secretory cells. Thetrail then may constitute an extensive area for vol-atilization of chemical signals, which may be an ef-ficient way for intraspecific (or interspecific) com-munication within tunnels.

Differently from amphisbaenians, in lizards ithas already been observed that distal portions offemoral gland secretion plugs are fractured anddeposited on the substrate, where they remain inthe form of small secretion blocks (Alberts, 1993).According to our observations, that seems also tobe valid in Ameiva ameiva.

Scanning electron microscopy showed that thesecretion plugs in this species are composed ofdead cells that desquamate from the femoral glandsecretory epithelium. The fact that most of thecells seen on the plug’s surface are intact suggeststhat, rather unlike amphisbaenians, they are notabraded by the substrate. Rather, the microscopi-cal appearance of the surface suggests that thefrail plug is probably fractured, falling in smallpieces on the substrate. On the other hand, theepithelial cover may function to maintain the cells’cohesion as a plug, favoring the prolongation ofsignal effect by preventing rapid secretion volatili-zation. Unlikely, in Amphisbaena alba, the surfaceof the secretion plug shows a different arrange-ment, in the manner of a cigar, with interspersedlayers of keratinocytes and secretory cells desqua-mated from the glandular epithelium. In this case,the arrangement of the plug possibly helps in thegradual fragmentation of the secretion during thetrail formation, modulating friction through thepresence of the interspersed keratinocyte layers(Jared et al., 1999). In Ameiva ameiva, on theother hand, we have observed that femoral poresonly touch the substrate when the animal reststhe internal side of its thighs on the ground. In aresting position, it probably presses the row offemoral pores against the substrate. The frail na-ture of the secretion, resulting from the juxtaposi-tion of the dead secretion cells, without beinginterspersed with keratinocytes (as is the case inamphisbaenians) may favor transverse fracturingof the plug and the deposition of secretion tiny por-tions in the environment. This process may befacilitated by the rosette-like shape and histologi-cal arrangement of the pore-bearing scales.

Microscopic examination of the row of femoralpores in Ameiva ameiva shows that some plugsare more prominent than others relative to theskin surface. These differences in plug lengths arean indication of the size of the tiny pieces of secre-tion deposited on the substrate. The plug deposi-tion system may function each time the animalrests relaxing its legs on the ground. Then, in thisspecies, the method for dispersing the pheromoneseems not to consist of the chance breakage of theplug but is much more complex. It involves bothmorphological characteristics of the rosettes andthe structural and mechanical characteristics ofthe plug itself. These seem to be adaptations formaking dispersion more efficient through the con-stant deposition on the substrate of tiny portionsof secretion every time the animal rests. Although



having its own peculiarities, this method of signaldispersion in A. ameiva, as occurs in amphisbae-nians, is intimately dependent on the locomotionof the animal within its territory.

ACKNOWLEDGMENTS

The authors are thankful to Dr. Ida S. Sano-Martins and Dr. Jose Roberto M.C. Silva for theuse of the photomicroscopes. Thanks to Tatiana L.Fernandes, Laurinda A. Soares, and Maria HelenaFerreira for their kind technical assistance, toDr. Hana Suzuki and Leonardo de Oliveira fortheir critical revision and to Alberto Barbosa deCarvalho for the help with the color figures. Thanksare also due to Brazilian National Research CouncilCNPq, FAPESP, and Fundacao Butantan. Specimenswere collected under IBAMA permits Procs. n802001.000636/01-86 and 02001.002792/98-03.

LITERATURE CITED

Abraham A. 1930. Uber die schenkeldrusen der archaeo-undneolacerten. Stud Zool Budapest 1:204–225.

Alberts AC. 1989. Ultraviolet visual sensitivity in desert igua-nas: Implications for pheromone detection. Anim Behav38:129–137.

Alberts AC. 1990. Chemical properties of femoral gland secre-tions in the desert iguana Dipsosaurus dorsalis. J Chem Ecol16:13–25.

Alberts AC. 1993. Chemical and behavioral studies of femoralgland secretions in iguanid lizards. Brain Behav Evol 41:255–260.

Alberts AC, Pratt NC, Phillips JA. 1992. Seasonal productivityof lizard femoral glands: Relationship to social dominanceand androgen levels. Physiol Behav 51:729–733.

Antoniazzi MM, Jared C, Pellegrini CMR, Macha N. 1993. Epi-dermal glands in Squamata: Morphology and histochemistryof the pre-cloacal glands in Amphisbaena alba (Reptilia,Squamata, Amphisbaenia). Zoomorphology 113:199–203.

Antoniazzi MM, Jared C, Junqueira LCU. 1994. Epidermalglands in Squamata: Fine structure of pre-cloacal glands inAmphisbaena alba (Amphisbaenia, Amphisbaenidae). J Mor-phol 221:101–109.

Bancroft JD, Stevens A. 1996. Theory and Practice of Histologi-cal Techniques, 4th ed. New York: Churchill & Livingstone.776 p.

Blasco M. 1975. El dimorfismo sexual en cinco especies de lafamilia Lacertidae (Reptilia). Bol R Soc Esp Hist Nat SeccBiol 73:237–242.

Chauhan NB. 1986. Histological and structural observations onpre-anal glands of the gekkonid lizard Hemidactylus flaviri-dis. J Anat 144:93–98.

Chiu KW, Maderson PFA. 1975. The microscopic anatomy of epi-dermal glands in two species of gekkonidae lizards, with someobservations on testicular activity. J Morphol 147:23–40.

Cohn L. 1904. Die schenkeldrusen des Cnemidophorus lemnis-catus—Daud. Zool Anz 27:185–192.

Cole CJ. 1966a. Femoral glands of the lizard Crotaphytus colla-ris. J Morphol 118:119–136.

Cole CJ. 1996b. Femoral glands in lizards: A review. Herpeto-logica 22:199–206.

Colli GR. 1991. Reproductive ecology of Ameiva ameiva (Sauria:Teiidae) in the cerrado of central Brazil. Copeia 1991:1002–1012.

Cooper WE Jr. 1994. Multiple functions of extraoral lingualbehaviour in iguanian lizards: Prey capture, grooming andswallowing, but not prey detection. Anim Behav 47:765–775.

Cooper WE Jr, Vitt LJ. 1984. Conspecific odor detection by themale broad-headed skink, Eumeces laticeps: Effects the sexand site of odor source and of male reproductive conditions. JExp Zool 230:199–209.

Cooper WE Jr, Vitt LJ. 1986. Lizards pheromones: Behavioralresponses and adaptative significance in skinks of the genusEumeces. In: Durvall D, Muller-Schwarze D, Silverstein RM,editors. Chemical Signals in Vertebrates: Ecology, Evolutionand Comparative Biology, Vol 4. New York: Plenum. pp 323–340.

Cooper WE Jr, Lopez P, Salvador A. 1994. Pheromone detectionby an amphisbaenian. Anim Behav 47:1401–1411.

Cooper WE Jr, van Wyk JH, Mouton PFN. 1996. Pheromonaldetection and sex discrimination of conspecific substrate depos-its by the rock-dwelling cordilid lizard. Copeia 1996:839–845.

Dujsebayeva TN. 1998. The histology of callous scales of themales of Asian rock agamas, Laudakia caucasia and Lauda-kia himalayana (Reptilia; Agamidae). Russ J Herpetol 5:160–164.

Dumeril AMC, Bibron G. 1834. Erpetologie generale ou histoirenaturelle complete des reptiles. Vol 1, Paris: Roret.

Gabe M, Saint-Girons H. 1965. Contribution a la morphologiecomparee du cloaque et des glandes epidermoides de la regioncloacale chez les lepidosoriens. Mem Mus Natn Hist Nat Paris33:149–292.

Gasc JP, Lageron A, Schlumberger J. 1970. Morphologie, histo-logie et histochimie des glandes femorales chez un individumale de Ctenosaura acanthura (Shaw) (Reptilia, Sauria, Igua-nidae), suivi des reflexions sur le role des glandes femoraleschez les lezards. Gegenbaurs Morphol Jb 114:572–590.

Halpern M. 1992. Nasal chemical senses in reptiles: Structureand function. In: Gans C, editor. Biology of the Reptilia: Hor-mones, Brain and Behavior, Vol 18. Chicago: The Universityof Chicago Press. pp 423–525.

Jared C, Antoniazzi MM, Silva JRMC, Freymuller E. 1999. Epi-dermal glands in Squamata: Microscopical examination ofprecloacal glands in Amphisbaena alba (Amphisbaenia,Amphisbaenidae). J Morphol 241:197–206.

Jullien R, Renous-Lecuru S. 1973. Etude de la repartition despores femoraux et ventraux chez les Lacertiliens (Reptilia).Bul Mus Nat d’Hist Nat 104:1–32.

Junqueira LCU. 1995. Histology revisited—Technical improve-ment promoted by the use of hydrophilic resin embedding.Cienc Cult 47:92–95.

Junqueira LCU, Bignolas G, Brentani R. 1979. Picrosiriusstaining plus polarization microscopy, a specific method forcollagen detection in tissue sections. Histochem J 11:447–455.

Karnovsky MJ. 1965. A formaldehyde–glutaraldehyde fixativeof high osmolality for use in electron microscopy. J Cell Biol27A:137,138.

Kluge,AG. 1967a. Higher taxonomic categories of gekkonid liz-ards and their evolution. Bull Am Nat Hist 135:1–60.

Kluge AG. 1967b. Systematics, phylogeny and zoogeography ofthe lizard genus Diplodactylus Gray (Gekkonidae). Aust JZool 15:1007–1108.

Linnaeus C. 1758. Systema Naturae, Vol 1, 10th ed. Stockholm:Salvii. 824 p.

Lopez P, Aragon P, Martın J. 1998. Iberian rock lizards (Lacertamonticola) assess conspecific information using composite sig-nals from faecal pellets. Ethology 104:809–820.

Maderson PFA. 1967. The histology of the escutcheon scales ofGonatods (Gekkonidae) with a comment on the squamatesloughing cycle. Copeia 4:743–752.

Maderson PFA. 1968. The epidermal glands of Lygodactylus(Gekkonidae, Lacertilia). Brev Mus Comp Zool 288:1–35.

Maderson PFA. 1970. Lizard glands and lizard hands: Modelsfor evolutionary study. Forma et Functio 3:179–204.

Maderson PFA. 1972. The structure and evolution of holocrineepidermal glands in sphaerodactyline and eublepharine gek-konid lizards. Copeia 3:559–571.

Maderson PFA. 1985. Some developmental problems of the rep-tilian integument. In: Gans C, Billett F, Maderson PFA, edi-tors. Biology of the Reptilia: Development. New York: Wiley.



Maderson PFA. 1986. The tetrapod epidermis: A system pro-toadapted as a semiochemical source. Chem Sig Vert 4:13–25.

Maderson PFA, Chiu KW. 1970. Epidermal glands in gekko-nid lizards: Evolution and phylogeny. Herpetologica 26:233–238.

Maderson PFA, Chiu KW. 1981. The effects of androgens on theb-glands of the tokay (Gekko gekko): Modification of an hy-pothesis. J Morphol 167:109–118.

Martin J, Lopez P. 2000. Chemoreception, symmetry and matechoice in lizards. R Soc Lond B 267:1265–1269.

Mason RT. 1992. Reptilian pheromones. In: Gans C, editor. Biol-ogy of the Reptilia: Hormones, Brain and Behavior, Vol 18.Chicago: The University of Chicago Press. pp 114–228.

Mizuhira V, Futaesaku Y. 1974. New fixation for biologicalmembranes using tannic acids. Acta Histochem Cytochem5:233–236.

Pough H, Andrews RM, Cadle JE, Crump ML, Savitzky AH,Wells KD. 2001. Herpetology, 2nd ed. New Jersey: PrenticeHall. 577 p.

Rastogi AK. 1975. Femoral glands and their inter-relationshipwith endocrine organs. Ind J Zool 15:23–35.

Schaefer F. 1901. Uber die schenkelporen der Haeertilier. ZoolAnz 24:308–309.

Schaefer F. 1902. Uber die schenkelporen der Eidechsen. ArchNat 63:27–64.

Simionescu N, Simionescu M. 1976. Galloyl-glucoses of low mo-lecular weight as mordant in electron microscopy, Part 1: Pro-cedure and evidence for mordating effect. J Cell Biol 70:608–621.

Simon CA. 1983. A review of lizard chemoreception. In: HueyRB, Pianka ER, Schoener TW, editors. Lizard Ecology: Stud-ies of a Model Organism. Cambridge: Harvard UniversityPress. pp 119–133.

Uva B. 1967. I pori femorali in Lacerta podargis sicula. BollMus Ist Biol Univ Genova 35:169–183.

Vanzolini PE, Ramos-Costa AMM, Vitt LJ. 1980. Repteis dasCaatingas. Brasil: Acad Bras Cien.

van Wyk JH, Mouton P, Le FN. 1992. Glandular epidermalstructures in cordylid lizards. Amphib Reptil 13:1–12.

Weldon PJ, Dunn BS Jr, McDaniel CA, Werner DI. 1990. Lipidsin the femoral gland secretions of the green iguana (Iguanaiguana). Comp Biochem Physiol 95:541–543.



morphology of the femoral glands in the lizard ameiva ... et al._2007_morphology of the... ·...

Documents