Morphology of the femoral glands in the lizard Ameiva ameiva (teiidae)

JOURNAL OF MORPHOLOGY 00:000–000 (2007)
Morphology of the Femoral Glands in the Lizard
Ameiva ameiva (Teiidae) and Their Possible
Role in Semiochemical Dispersion
Beatriz A. Imparato,1 Marta M. Antoniazzi,1 Miguel T. Rodrigues,2 and Carlos Jared1,2*
1
2
Laboratório de Biologia Celular, Instituto Butantan, CEP 05503-900, São Paulo, Brazil
Departamento de Zoologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil
ABSTRACT Many lizards have epidermal glands in
the cloacal or femoral region with semiochemical function related to sexual behavior and/or territorial demarcation. Externally, these glands are recognized as a row
of pores, opening individually in the center of a modified
scale. In many species the pores are used as systematic
characters. They form a glandular cord or, in some species, a row of glandular beads below the dermis, and are
connected to the exterior through the ducts, which continuously liberate a solid secretion. Dead cells, desquamated from the secretory epithelium, constitute the
secretion, known as ‘‘a secretion plug.’’ The present work
focusses on the morphology of the femoral glands of the
teiid lizard Ameiva ameiva, correlating it to the way in
which the secretion is deposited in the environment. The
results here obtained are compared to those available
for other lizards and amphisbaenians. We observed that
the diameter of the glandular pores did not show significant differences between males and females. The glands
comprise germinative and secretory cells, which pass
through at least three stages of differentiation, during
which an accumulation of cytoplasmic granules, with a
glycoprotein content, occurs. The cells eventually die
and desquamate from the secretory epithelium, forming
a secretory plug mostly constituted by juxtaposed nonfragmented secretory cells. Because of the arrangement
of 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, these
scales may be involved in the breakage of their respective plugs, depositing tiny portions on the substrate. In
this manner, it seems that the method for signal dispersion in this species involves specifically adapted structures and does not simply involve the chance breakage
of the plug, as the gland secretes it. Signal dispersion
must also be intimately associated with the animal’s
movement within its territory. J. Morphol. 00:000–000,
2007. Ó 2007 Wiley-Liss, Inc.
KEY WORDS: squamata; lizards; teiidae; femoral pores;
epidermal glands; Ameiva ameiva; pheromone
Among the Squamata, semiochemicals have an
important role in sexual behavior, defense, predation, territorial marking, orientation, intraspecific
aggregation, and parental care (Halpern, 1992).
Besides their importance in squamate biology, the
morphology of the structures related to semiocomÓ 2007 WILEY-LISS, INC.
munication are poorly known. Most morphological
papers 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; Dujsebayeva, 1998). More recently, the morphology of
pre-cloacal glands in an amphisbaenian (Amphisbaena alba) was reported and their role in the natural history of this species was analyzed (Antoniazzi et al., 1993, 1994; Jared et al., 1999).
Femoral glands are the most common semiochemical sources in lizards (Jullien and RenousLécuru, 1973) although some glandular scales or
‘‘generation’’ glands (Maderson, 1967, 1968) have
been described in cordylids in other regions of the
body (van Wyk et al., 1992). Femoral glands are
localized in one or two rows on the ventral side of
each thigh. The first morphological studies on femoral glands date from the end of XIX century and
beginning of XX century (Ábrahám, 1930), and
concerned primarily European species. The subject
was ignored by morphologists until the 1960s, when
papers 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 very
important in lizard biology, studies focusing on
these aspects are rare (Mason, 1992; Cooper, 1994;
Cooper et al., 1996). However, many experiments
have demonstrated that several species are able to
recognize chemical signals secreted by femoral
glands (Cooper and Vitt, 1984; Alberts, 1989, 1993;
Lopez et al., 1998, Martin and Lopez, 2000).
The localization of glands in both lizards and
amphisbaenians suggests that signals are pas-
*Correspondence to: C. Jared, Instituto Butantan, Laboratório de
Biologia Celular, Av. Vital Brasil 1500, CEP 05503-900, São Paulo,
Brazil. E-mail: [email protected]
Published online in
Wiley InterScience (www.interscience.wiley.com)
DOI: 10.1002/jmor.10473
2
B.A. IMPARATO ET AL.
sively deposited in the environment during locomotion of the animals within their territory (Maderson,
1986; Jared et al., 1999). Among lizards possessing
femoral glands, signal dispersion involves the
breakage of small portions of the secretion plugs.
These remain on the substrate, releasing chemical
signals for a period of time that must be related to
both the natural history and environment of a particular species (Alberts, 1993).
Ameiva ameiva is a teiid lizard with a broad geographic distribution in Central and South Americas, from Panamá to South Brazil and North Argentina (Vanzolini et al., 1980; Colli, 1991). The
aim of the present paper was the study of the morphology and some aspects of the histochemistry of
the femoral glands of the teiid Ameiva ameiva,
and the comparison of the results with those
obtained for other lizards and amphibaenians. We
hope to contribute to the integrative knowledge of
the natural history of lizards, especially in relation
to chemical communication. Our data suggest that
in Ameiva ameiva the differentiated scales containing the femoral pores and the secretion plugs
are structures adapted to the dispersion of chemical signal on the substrate and seem to be associated with the locomotion of these animals throughout their territory.
MATERIALS AND METHODS
Animals
Adult individuals of Ameiva ameiva Linnaeus 1758 were collected at Palmas (State of Tocantins, Brazil) and maintained for
2 years in the vivarium of the Laboratory of Cellular Biology of
the Instituto Butantan. They were housed in terraria containing humid earth at the bottom with dry branches above and
pieces of plastic pipe for shelter. They were daily exposed to
sunlight during the morning period. Food was supplied weekly
and was composed of a variation of live items (beetles - Tenebrio
molitor, crickets - Gryllus sp, cockroaches - Pycnocellus surinamensis), fruits, eggs, and minced beef.
For the morphological studies, six animals were sacrificed
with a intraperitoneal overdose of sodic thiopental just after
being collected from nature. Samples of the skin of the femoral
region containing the femoral glands were removed and processed as described below.
Voucher specimens were deposited in the collection of the
Museum of Zoology, University of São Paulo (MZUSP).
Histology
The skin containing the femoral glands was cut in strips of
about 3 3 5 mm2 using a razor blade and fixed in 4% paraformaldehyde in PBS 0.1 M, pH 7.2 for 24 h. The material was
dehydrated and embedded either in historesin (glycol methacrylate, Leica) or paraffin (at 608C).
For general study of the tissues, 2 lm historesin sections
were stained with toluidine-fuchsin (Junqueira, 1995) and 5–7
lm 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 muscular
fibers, and the Weigert’s resorcin-fuchsin stain (Bancroft and
Stevens, 1996), for the identification of elastic fibers.
Journal of Morphology DOI 10.1002/jmor
Histochemistry
Historesin and paraffin sections were subjected to the following histochemical staining procedures (according to Bancroft
and Stevens, 1996): periodic acid-Schiff (PAS) and alcian blue
pH 2.5, for identification of neutral and acidic mucosubstances,
respectively, and bromophenol blue, for identification of proteins. Sudan black B was used for identification of lipidic substances.
Photomicrography
Photographs were obtained by using a Leica DMLB microscope equipped with a Leica MPS60 photographic system.
Scanning Electron Microscopy
A strip of skin from the femoral region containing 5–7 femoral pores was removed from the animals, fixed in Karnovsky solution (Karnovsky, 1965) for 24 h, post-fixed in 1% osmium tetroxide, incubated in 1% tannin, and postfixed once more in 1%
osmium tetroxide (Mizuhira and Futaesaku, 1974; Simionescu
and Simionescu, 1976). The material was dried by the critical
point method, coated with gold, and examined in a JEOL SM
6100 and in a JEOL JSM 5300, operating between 15 and 25 kV.
Transmission Electron Microscopy
Fragments of the femoral glands (separated from the skin) of
about 1 mm3 were fixed in Karnovsky solution, pH 7.2 (Karnovsky, 1965) for 24 h, post-fixed in 1% osmium tetroxide, contrasted in 2% uranyl acetate, dehydrated in ethanol and embedded in epoxy resin. Ultrathin sections (60 lm) were obtained in
a Sorvall MT 6000 ultramicrotome and were examined with a
LEO 906E, operating at 80 kV.
Morphometrical Study
Adult male (N ¼ 14) and female (N ¼ 14) specimens of
Ameiva ameiva from the zoological collection of Museu de Zoologia da Universidade de São Paulo, all from the same population, were studied. The number and the diameter of the pores
in the femoral region of males and females were quantified and
possible differences in both sexes were analyzed by Student
test. The diameters of the pores of three regions in the pore row
(the two extreme regions, named P1 and P3, and the middle
region, named P2) were measured. Because data were normally
distributed and had homogeneous variances, they were analyzed by ANOVA test.
RESULTS
General Anatomy and Histology of the
Femoral Glands
In Ameiva ameiva (Fig. 1), the ventral skin of
the thighs, in both males and females, shows a single row of pores (Fig. 2), whose number varies
from 17 to 23 in males and from 16 to 22 in
females. Each pore opens in the center of a modified scale, resembling a rosette (Fig. 3). Because of
their prominence, the row of pore-bearing scales
stand out from the neighboring scales and, for this
reason, are easily recognized (Figs. 2, 3). Studying
the skin from the dermal side reveals that each of
the pores corresponds to a gland. The row of
glands, superimposed on one another, forms a deli-
Figures 1–3 (legend on page 4)
Figures 4–6 (legend on page 4)
4
B.A. IMPARATO ET AL.
cate yellowish cord (Fig. 4), which is juxtaposed to
the dermis by delicate connective tissue. When
this tissue is removed, it is possible to observe that
each one of the glands is branched and flat (Fig.
4). In sections of this region, the glandular bodies
are cut in different planes and internal ductules
appear among the lobules (Fig. 5). The main ducts
connecting the glandular bodies to the exterior are
visible in sections cut transverse or longitudinal to
the axis of the femur. The ducts are always filled
with a solid plug comprising a yellowish and brittle secretion (Fig. 5).
A thin layer of connective tissue, which is supplied by many blood vessels, covers each of the
femoral glands. The connective tissue forms septa
dividing the gland into lobules (Fig. 6). Each lobule
comprises a holocrine epithelium that converges to
the ductules, which finally connect to the main
duct.
The main duct is lined by a stratified epithelium, which is continuous with the skin epidermis
(Fig. 5). The holocrine secretion that fills the duct
comprises small units with an affinity for toluidine
blue (in toluidine blue-fuchsin staining) (Fig. 6) or
for eosin (in hematoxylin–eosin staining).
Scanning Electron Microscopy of the
Glandular Pore Region
The scale containing the glandular pore comprise three or four plates arranged like the petals
of a flower (Fig. 7). One of the plates is always
larger and more prominent when compared to the
others (Fig. 7, insert). Each one of the scale plates,
specially the larger one, shows a fine arched internal border, which contacts the plug (Fig. 7).
Figs. 1–3 Fig. 1. A male specimen. Fig. 2. Ventral side of
the thighs showing the pair of femoral pore rows (arrows). Fig.
3. Macrophotography of the femoral pores. The glandular ducts
open in the center of a row of modified scales (arrows) resembling rosettes. The duct lumina are filled by the secretion plugs
(*).
Figs. 4–6 Fig. 4. Dermal side of the skin in the region of
the femoral pores. The femoral glands are packed close together
forming a delicate yellowish cord. The arrows point to some
branched glandular bodies, while the arrowheads indicate the
glandular ducts. Fig. 5. Histological section of the skin in the
region of the femoral pores. Part of the glandular cord is visible
beneath the dermis, with two glandular bodies sectioned in different planes, longitudinal (G1) and transversal (G2), and two
main ducts transversally sectioned (D1 and D2), full of secretion. The whole is enveloped in a delicate connective tissue
(arrowheads). The asterisks indicate internal ductules in the
distal portion of one of the glands. One main duct (D3) opens
through 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 into
lobules separated by loose connective tissue (ct). The lobules are
constituted by germinative cells (arrowheads), in the periphery,
and secretory cells in different stages of differentiation (*).
Arrows, blood vessels; d, ductule. Glycol methacrylate, toluidine-fuchsin stain.
Journal of Morphology DOI 10.1002/jmor
The secretion plug emerges from the pore in the
form of a rod, the tip of which is usually about the
same level of the skin surface. While a smooth
cover (Fig. 7) envelops its lateral aspect, small
units corresponding to the dead cells, which are
desquamated from the holocrine epithelium, form
its distal surface (Fig. 8). Most of these dead cells
are intact and show no signs of abrasion from contact with the substrate (Fig. 8).
Light Microscopy of the
Secretory Epithelium
Germinative cells and secretory cells (Fig. 9A–F)
comprise the glands. To facilitate the morphological description of the differentiation process, the
secretory cells were divided into three subsequential stages (S1, S2, and S3), according to their
structural characteristics.
The germinative cells (G) are localized at the periphery of the lobules. They are elongated and
have voluminous nuclei (Fig. 9A). The cells in the
first stage of differentiation (S1) have the same
localization as the germinative cells but are generally more spherical. Their nuclei are also spherical
and voluminous and usually have one or two conspicuous nucleoli. During the first stage, the cytoplasm is lightly stained and does not possess secretion granules (Fig. 9A).
The second stage of differentiation (S2) is characterized by cells with a larger volume and observed
in different phases of the secretory process. These
phases can be identified by the increase in the
number of secretory granules and were named P1,
P2, and P3. In P1, the cells are usually spherical
and the cytoplasm has a number of discrete granules (Fig. 9B). In P2, small granules that are sometimes metachromatic (Fig. 9B) characterize the
cells. In the last phase (P3), the granules occupy a
large portion of the cytoplasm, are larger and
spherical, but are heterogeneous in size and
in their tinctorial properties (Fig. 9C). Irregular
granules containing heterogeneous secretion (Fig.
9D) which usually forms a half-stained pattern
within the granule distinguish late P3. In the
most differentiated cells, the irregular nuclei tend
to be displaced to the periphery, and are smaller
in relation to the total cell volume. The cells
remain spherical.
In the third and last stage (S3), the secretory
cells, already close to the duct, assume a flat form
and the cytoplasm is full of irregular granules with
heterogeneous secretion. The peripheral nuclei are
pycnotic (Fig. 9E). At the end of this stage, the cells
desquamate from the secretory epithelium, enter
the duct, and become part of the solid plug.
During the differentiation process, cells of a different type may be seen among the secretory cells.
They contain dense inclusions suggesting clusters
of secretion granules lying inside a large vacuole,
FEMORAL GLANDS IN THE LIZARD AMEIVA AMEIVA
5
Figs. 7 and 8. Fig. 7. The femoral pore, showing the cylindrical secretory plug (P), with its smooth cover (Co), emerging through
a rosette of scales. The rosette is composed of a larger plate (**) and three small plates (*). This arrangement is best observed in
the insert. The arrows point to the arched internal border of the larger plate. SEM. Fig. 8. The plug’s surface (P), at higher magnification, 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.
and occupy a considerable part of the cell cytoplasm (Fig. 9C,F).
Histochemical Observations
of the Femoral Glands
The secretory cells showed a strong and homogeneous PAS positivity (before and after amylase
treatment) either in the secretion granules or in
the plug. Similarly, granules and plug were posi-
tive to bromophenol blue, although the granules
are 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 glandular
structures.
Light Microscopy of the Rosette
The skin of Ameiva ameiva comprises a cornified
epidermis and a dermis essentially constituted by
Journal of Morphology DOI 10.1002/jmor
Figure 9
Figures 10–13
FEMORAL GLANDS IN THE LIZARD AMEIVA AMEIVA
dense regular connective tissue in a basket arrangement with intermingled blood vessels.
Longitudinal sections of femoral skin show the general structure of the modified scales (rosettes) containing the pores, which may also be seen in transverse section (Figs. 10–13). Picro Sirius staining
showed that the dermal connective tissue is basically
formed by collagen fibers, which stain bright red.
N.M.C. trichrome staining revealed the characteristic
arrangement of dermal collagen fibers (which appear
in deep blue color) within the rosette. The dermis
comprises an exceptionally well-developed deep layer
and a poorly developed superficial layer, immediately
below the epidermis (Figs. 10 and 11). Immediately
around the duct the collagen fibers are thinner and
are circularly arranged (Fig. 11). Between the deep
dermis and the circular fibers, there is zone of loose
and less organized connective tissue well irrigated by
blood vessels (Fig. 11).
When Weigert’s Fucsine-Resorcine is applied to the
sections, delicate purple elastic fibers are revealed
among the collagen fibers, mainly in the well developed
deep layer in the form of a net (Fig. 12) and following a
circular arrangement around the duct (Fig. 13).
Transmission Electron Microscopy of the
Femoral Glands
At the ultrastructural level, the germinative
cells are polygonal or spindle-shaped (Fig. 14A). As
Fig. 9. Histological sections at high large magnification
showing the internal arrangement of the glandular lobules.
Gland cells are seen in different stages of differentiation. Germinative cells (G) lie at the periphery (A–C, E and F). Secretory cells in the first stage of differentiation (S1) have no granules (A, D, and F). Secretory cells in the second stage of differentiation, divided in three subsequent phases (P1, P2 e P3), in
which the granules are secreted, increase in number, and
undergoes maturation (B–D). Secretory cells in the third stage
of differentiation (S3), in which they acquire a flat form, present
evident signals of decline and finally desquamate from the secretory epithelium (E). Cells with dense inclusions (*), observed
among the other cells (C, D, and F). Arrowheads point to pycnotic nuclei. Glycol methacrylate, toluidine-fuchsin stain.
Figs. 10–13. Fig. 10. Histological section through a rosetteshaped scale containing a femoral pore transversally sectioned.
The arrangement of the dermis in the scale is better observed
in Figure 11, which corresponds to a high magnification of the
area indicated by the rectangle. The dermis comprises a thin
superficial layer immediately below the epidermis (Ep), a welldeveloped deep layer (dt) mainly formed by collagen fibers, and
a circular layer (ct), also mainly formed by collagen fibers,
around the duct. Between the deep layer and the circular layer
there are areas mainly composed of loose and less organized
connective tissue (lt) with many blood vessels (arrows). P, secretion plug; *, duct epithelium. Paraffin. N.M.C. trichrome stain.
Fig. 11. Magnified image of the area indicated by the rectangle
in Figure 10. Fig. 12. Details of a histological section through
one rosette-shaped scale containing a femoral pore transversally
sectioned. The section was stained to demonstrate the delicate
elastic fibers (arrows) which form a disorganized net among the
collagen fibers in the deep layer. Ep, epidermis; *, duct epithelium. Paraffin. Weigert’s resorcine-fuchsin. Fig. 13. The circular
arrangement of collagen fibers around the duct.
7
in other epithelial tissue, the cell membranes show
many interdigitations and desmosomes. The cytoplasm of germinative cells is quite electron lucent
and is rich in mitochondria, granular and agranular endoplasmic reticulum, free ribosomes, and
Golgi apparatus (Fig. 14B). Bundles of intermediate filaments (tonofilaments) occur through the
cytoplasm or linked to desmosomes (Fig. 14A,B).
The spherical nuclei with one to three nucleoli
occupy a large volume of the cell and present predominantly loose chromatin (Fig. 14A).
The secretory cells in the first stage of differentiation (S1) can be distinguished from the germinative cells by their more spherical shape, and especially by the small and scarce secretion granules
in the cytoplasm (Fig. 14C). These granules are
spherical and homogeneous. Similar to the germinative cells, the cytoplasm is still quite electron
lucent and tonofilaments and desmosomes are
abundant. An increase in the granular endoplasmic reticulum (GER) and Golgi apparatus occurs
(Fig. 14D). The nuclei is spherical, with predominantly loose chromatin, and is less voluminous
within the cell when compared to the situation in
germinative cells (Fig. 14C).
The second stage (S2) is characterized by nuclei
with irregular shapes, an increase in cytoplasmic
volume and in the number of the secretion granules. It is possible to distinguish at least three
phases in this stage: (P1) In the initial phase, cells
have spherical, homogeneous granules of different
sizes occupying part of the cytoplasm (Fig. 14E).
Lamellae of the granular endoplasmic reticum are
quite developed and dilated and Golgi cisternae
were often seen (Fig. 14F). (P2) The cytoplasm of
the cells contains many large spherical granules,
some of them with heterogeneous content (half
electron dense, half electron lucent). The GER is
well developed and is still dilated (Fig. 15A). Other
organelles are not so visible and, as the number of
granules increases, nuclei are more electron dense
and are pushed towards the cell periphery. (P3) In
the last phase of S2, the cytoplasm is replete with
secretion granules with heterogeneous content
(Fig. 15B). The nuclei often appear already
shrunk, are electron dense and have an irregular
shape. While most organelles are barely observed
among the granules, it seems that there is an
increase in the number of lipidic inclusions, with
medium electron density (Fig. 15B).
In the last stage of differentiation (S3), the cells
show evident signs of cytoplasmic disorganization
and acquire a flatter form (Fig. 15C). The granules
occupy practically the whole volume of the cells.
The nuclei are pycnotic or absent. There is an
increase in the number of lipidic inclusions (Fig.
15C). The cells in this stage are beginning to desquamate from the secretory epithelium, or are already desquamated and lie loose in the duct, forming the secretion plug. Ultrastructure of the plug
Journal of Morphology DOI 10.1002/jmor
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B.A. IMPARATO ET AL.
Figure 14
Journal of Morphology DOI 10.1002/jmor
FEMORAL GLANDS IN THE LIZARD AMEIVA AMEIVA
shows that it comprises electron-dense polyhedral
units, measuring about 1.3 lm in the larger axis.
A few secretory cells, mainly in the first stages
of differentiation, contain dense inclusions in their
cytoplasm (Fig. 15D).
Morphometrical Study
The number of pores in males and females did not
show differences (meanmales ¼ 20.27, meanfemales ¼
19.67, N ¼ 28, t ¼ 0.0159). The comparison of pore
diameters among regions P1, P2, and P3 did not show
differences in males (meanP1 ¼ 0.51 mm, meanP2 ¼
0.57 mm, meanP3 ¼ 0.49 mm, F ¼ 3.15, N ¼ 42) as
well as in females (meanP1 ¼ 0.34 mm, meanP2 ¼
0.36 mm, meanP3 ¼ 0.35 mm, F ¼ 0.52, N ¼ 42).
The comparison of pore diameters between males
and females for regions P1, P2, and P3 also did not
show differences (tP1 ¼ 3.63E08, tP2 ¼ 1.93E07,
tP3 ¼ 3.25E05).
DISCUSSION
Epidermal glands in pre-anal or femoral regions
are wellknown in several lizard families (Jullien
and Renous-Lécuru, 1973; Rastogi, 1975) although
they can be found in other body regions (Simon,
1983; Dujsebayeva, 1998). Many behavioral and
biochemical studies conducted in recent decades
have demonstrated the role of these glands in lizard
chemical communication (Pough et al., 2001). There
is a considerable external variation of epidermal
glands in relation to their distribution in the body
(Kluge, 1967a,b; Dujsebayeva, 1998), the shape of
the scales containing the pores (Blasco, 1975; van
Wyk et al., 1992), and the coloration of the secretion plug (Cole, 1966b; Blasco, 1975). Internally
these glands can also present anatomical and color
Fig. 14. The cells constituting the femoral gland lobules.
(TEM). A: Polygonal germinative cell with a medium electronlucent cytoplasm and a voluminous nucleus (N), with loose chromatin and a well developed nucleolus (n). The cell is connected
to the neighboring cells by many desmosomes (arrows). B:
Higher magnification of a germinative cell. The cytoplasm is
rich in tonofilaments (arrows), mitochondria (m), agranular
endoplasmic reticulum (*), small vesicles (arrowheads) and free
ribosomes spread, among the other organelles. C: First stage of
differentiation 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 the
cell. The first secretion granules (arrows) lie close to both the
granular endoplasmic reticulum (GER) and Golgi apparatus
(Go). n, nucleolus. D: Higher magnification of rectangle marked
in (C), showing details of the Golgi apparatus (Go), surrounded
by many small vesicles (arrowheads), and of the GER. G, secretion granule. E: First phase P1 of the second stage of differentiation. The total cell volume is enlarged and the number of secretory granules (G) has increased. Arrow, bundle of tonofilaments; *, Golgi apparatus. F: Enlarged area of a cell in P1. The
secretion granules are spherical with dense, homogeneous content. Go, Golgi apparatus; GRE, granular endoplasmic reticulum.
9
variations (Gasc et al., 1970; Rastogi, 1975). Some
of these differences may be related to the way epidermal glands are used for chemical communication within each group.
Rosette-like differentiated scales are not exclusive of teiids but can also be found in other lizard
families such as iguanids and lacertids (Cole,
1966a; Blasco, 1975). Our histological observations
on Ameiva ameiva indicate that this structure possibly derives from the fragmentation of one single
scale (Fig. 5).
Femoral pores have been used as systematic
characters for a long time (Linnaeus, 1758;
Duméril and Bibron, 1834). They are also used to
define sexual dimorphism (Cole, 1966a). In the
case of captive Ameiva ameiva we have not
observed any significant circannual modification in
the external morphology of femoral glands
although the animals remained sexually active,
with copulations being frequently observed. Our
morphometrical results indicated that in Ameiva
ameiva no sexual dimorphism can be observed in
relation to the number or to the diameter of pores.
This apparent similarity in the glands of both
sexes could indicate that they may be used by all
individuals for territorial demarcation. However,
differences in the biochemical composition of gland
secretion between males and females cannot be
discarded.
In relation to their internal anatomy, femoral
glands of Ameiva ameiva correspond to the model
proposed by Gabe and Saint Girons (1965) for lizards in general. In this model, the glands are flat
and branched, with an internal ramification of
ductules connected to a main duct, which leads to
the secretion to the exterior. The solid secretion
plug is generally the same color of the gland in
which it is produced (Cole, 1966b). In Ameiva
ameiva, femoral glands and secretion plugs all
have the same yellowish coloration.
Femoral glands in Ameiva ameiva, as in other
squamates, are holocrine. The extrusion of the
solid secretion plug through the pore is due to the
pressure exerted by the distal proliferation and
differentiation of the secretory cells within tubules
(Antoniazzi et al., 1993). This process, however,
does not explain the lateral aspect of the plug, visible by scanning electron microscopy (Figs. 7, 8).
This seems to be a structure formed by the accumulation of desquamated epithelial layers since,
according to Maderson (1972), the gland duct epithelia do not show cyclic changes of the type that
are associated with periodic shedding.
The ultrastructure of the germinative cells is
typical of epidermal cells, showing large numbers
of tonofilaments and desmosomes. These cells
reveal an intense metabolic activity, which is morphologically evidenced by the cytoplasm rich in different organelles. The observation of histological
and ultrastructural differences among the secreJournal of Morphology DOI 10.1002/jmor
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B.A. IMPARATO ET AL.
Fig. 15. The cells constituting the femoral gland lobules. (TEM). A: Second phase P2 of the second stage of differentiation of a
secretory cell. The secretion granules (G) increase in volume and in number and the contents of some of them (arrows) are not as
homogeneous as phase 1 (F and G). The GER remains well developed. N, nucleus. B: Third phase P3 of the second stage of differentiation. 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). Lipid
inclusions lie in the cytoplasm among the granules (arrowheads). N, nucleus. C: Third stage of differentiation S3 of the secretory
cells. 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) occupy
most of it. D: Dense inclusions (I) resembling clusters of secreted material are quite common in the cytoplasm of many secretory
cells, mainly in the first stages of differentiation.
tory cells made it possible to distinguish at least
three different stages of differentiation. In the
third stage, small inclusions with medium electron
density seem to be lipidic in nature. Furthermore,
we often observed in all differentiation stages the
Journal of Morphology DOI 10.1002/jmor
presence of large inclusions that are apparently
the accumulation of secretory material in the form
of vesicles whose destiny in the cells we have not
being able yet to determine. All these differentiation processes resemble what has been observed in
FEMORAL GLANDS IN THE LIZARD AMEIVA AMEIVA
the epidermal glands of other lizards (Cole, 1966a;
Gasc et al., 1970; Maderson, 1972) and amphisbaenians (Antoniazzi et al., 1993, 1994; Jared et al., 1999).
In relation to the chemical nature of Ameiva
ameiva femoral gland secretion, our histochemical
and ultrastructural data indicate that it consists of
neutral mucosubstances and proteins, probably
forming glycoproteins. The same results were
obtained 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 negative result obtained with the use of Sudan Black B
for detection of lipids, at the ultrastructural level,
in advanced stages of cell differentiation, we have
observed small inclusions in the secretory cell cytoplasm that seem to be lipidic in nature. Lipids are
common among substances involved in chemical
communication. (Alberts, 1990; Alberts et al., 1992;
Halpern, 1992). Alberts (1993), working with the
chemistry of pheromones in Dipsosaurus dorsalis,
discussed environmental influence on the composition of the femoral gland secretion of this iguanid.
In Iguana iguana and Dipsosaurus dorsalis, the
low concentration of lipids in their glandular secretion could only be detected by gas chromatography
(Alberts, 1990; Weldon et al., 1990). Thus,
although undetectable by histochemical techniques, small amounts of lipid might be present in
teiid femoral gland secretions.
The ventral localization of the epidermal glands
on the body of lizards and amphisbaenians suggests that their secretions are passively deposited
in the environment (Maderson, 1986). Recent
papers have demonstrated that in fact many species of lizards respond to secretions derived from
these glands (Cooper and Vitt, 1986; Cooper et al.,
1994; Cooper et al., 1996). However, the method
used by lizards for depositing chemical signals in
the environment has not been studied so far. In
amphisbaenians, (Antoniazzi et al., 1993, 1994),
studying the morphology of the pre-cloacal glands
of Amphisbaena alba, demonstrated that, as a
result of the position and arrangement of these
glands, the secretion plugs, perpendicular to the
body, make direct contact with the tunnel walls
where this species lives. These authors suggest
that, during locomotion, the plugs are naturally
abraded and secretion fragments are deposited on
the substrate, forming a trail. In fact, Jared et al.
(1999) experimentally demonstrated the formation
of a secretion trail during locomotion of A. alba.
Scanning electron microscopic examination, both
of the trail and of the abraded surface of the plug,
showed that the fragments of the trail correspond
to the secretion granules of the secretory cells. The
trail then may constitute an extensive area for volatilization of chemical signals, which may be an efficient way for intraspecific (or interspecific) communication within tunnels.
11
Differently from amphisbaenians, in lizards it
has already been observed that distal portions of
femoral gland secretion plugs are fractured and
deposited on the substrate, where they remain in
the form of small secretion blocks (Alberts, 1993).
According to our observations, that seems also to
be valid in Ameiva ameiva.
Scanning electron microscopy showed that the
secretion plugs in this species are composed of
dead cells that desquamate from the femoral gland
secretory epithelium. The fact that most of the
cells seen on the plug’s surface are intact suggests
that, rather unlike amphisbaenians, they are not
abraded by the substrate. Rather, the microscopical appearance of the surface suggests that the
frail plug is probably fractured, falling in small
pieces on the substrate. On the other hand, the
epithelial cover may function to maintain the cells’
cohesion as a plug, favoring the prolongation of
signal effect by preventing rapid secretion volatilization. Unlikely, in Amphisbaena alba, the surface
of the secretion plug shows a different arrangement, in the manner of a cigar, with interspersed
layers of keratinocytes and secretory cells desquamated from the glandular epithelium. In this case,
the arrangement of the plug possibly helps in the
gradual fragmentation of the secretion during the
trail formation, modulating friction through the
presence of the interspersed keratinocyte layers
(Jared et al., 1999). In Ameiva ameiva, on the
other hand, we have observed that femoral pores
only touch the substrate when the animal rests
the internal side of its thighs on the ground. In a
resting position, it probably presses the row of
femoral pores against the substrate. The frail nature of the secretion, resulting from the juxtaposition of the dead secretion cells, without being
interspersed with keratinocytes (as is the case in
amphisbaenians) may favor transverse fracturing
of the plug and the deposition of secretion tiny portions in the environment. This process may be
facilitated by the rosette-like shape and histological arrangement of the pore-bearing scales.
Microscopic examination of the row of femoral
pores in Ameiva ameiva shows that some plugs
are more prominent than others relative to the
skin surface. These differences in plug lengths are
an indication of the size of the tiny pieces of secretion deposited on the substrate. The plug deposition system may function each time the animal
rests relaxing its legs on the ground. Then, in this
species, the method for dispersing the pheromone
seems not to consist of the chance breakage of the
plug but is much more complex. It involves both
morphological characteristics of the rosettes and
the structural and mechanical characteristics of
the plug itself. These seem to be adaptations for
making dispersion more efficient through the constant deposition on the substrate of tiny portions
of secretion every time the animal rests. Although
Journal of Morphology DOI 10.1002/jmor
12
B.A. IMPARATO ET AL.
having its own peculiarities, this method of signal
dispersion in A. ameiva, as occurs in amphisbaenians, is intimately dependent on the locomotion
of the animal within its territory.
ACKNOWLEDGMENTS
The authors are thankful to Dr. Ida S. SanoMartins and Dr. José Roberto M.C. Silva for the
use of the photomicroscopes. Thanks to Tatiana L.
Fernandes, Laurinda A. Soares, and Maria Helena
Ferreira for their kind technical assistance, to
Dr. Hana Suzuki and Leonardo de Oliveira for
their critical revision and to Alberto Barbosa de
Carvalho for the help with the color figures. Thanks
are also due to Brazilian National Research Council
CNPq, FAPESP, and Fundação Butantan. Specimens
were collected under IBAMA permits Procs. n8
02001.000636/01-86 and 02001.002792/98-03.
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