Anatomia, Histologia, Embryologia
ORIGINAL ARTICLE
Anatomy and Histology of the Male Reproductive Tract
and Spermatogenesis Fine Structure in the Lesser Anteater
(Tamandua tetradactyla, Myrmecophagidae, Xenarthra):
Morphological Evidences of Reproductive Functions
L. F. Rossi1, J. P. Luaces1, H. J. Aldana Marcos2, P. D. Cetica3, G. Perez Jimeno4 and M. S. Merani1*
Addresses of authors: 1 Laboratorio de Biologı́a Cromosómica, Facultad de Medicina, Universidad de Buenos Aires, C1121ABG, C.A.B.A.,
Argentina;
2
Laboratorio de Histologı́a, Facultad de Ciencias Exactas y Naturales, Universidad de Belgrano, C1426DQG, C.A.B.A., Argentina;
3
Cátedra de Quı́mica Biológica, Facultad de Ciencias Veterinarias, Instituto de Investigación y Tecnologı́a en Reproducción Animal, Universidad
de Buenos Aires, C1427CWO, C.A.B.A., Argentina;
4
Proyecto de Conservación Oso Hormiguero Gigante, Zoo Florencio Varela, Florencio Varela, C1188DWG, Buenos Aires, Argentina
*Correspondence:
Tel.: +54 11 5950 9500 int. 2153;
Fax: +54 11 5950 9612;
e-mail: mmerani@fmed.uba.ar
With 5 figures and 1 table
Received March 2012; accepted for
publication August 2012
doi: 10.1111/ahe.12008
Summary
The anatomy and histology of the male genital tract of the lesser anteater were
studied. Fine details of spermatozoa regarding their genesis and morphology
were also studied in six adult specimens. The testes lie in the pelvic cavity. The
deferent duct emerges from the epididymis and opens into the ejaculatory
duct, which drains into the membranous urethra. Accessory glands (prostate,
seminal vesicle and bulbourethral gland) are histologically similar to those
described in other mammals. The short penis presents an urethral orifice, while
the corpus spongiosum becomes thinner at the end indicating the absence of a
histologically defined glans. The seminiferous epithelium shows: (1) Sertoli cells
with deep nuclear indentations, (2) spermatogonia with crusty-like chromatin,
(3) spermatocytes at different stages of maturation and (4) three morphologically distinct stages of spermatid differentiation according to nuclear shape,
acrosome development and chromatin condensation. Sperm heads appear oval.
The length of the spermatozoa averages 67.33 ± 1.60 lm. Two specimens with
inactive spermatogenesis were azoospermic. Their testes and epididymis presented sizes smaller than those with active spermatogenesis. These studies
together with others in anteaters may contribute to successful breeding in
conservation programmes.
Introduction
Xenarthra’s reproductive tract exhibits several peculiar
features. Among them, the occurrence of internal testes
(Kaudern, 1914; Grassé, 1955) and the extraordinary
shape of the spermatozoa’s head in males (Cetica et al.,
1998) stand out. The presence of polyovular follicles and
urogenital sinus in females (Cetica et al., 2005) is also
another unusual characteristic. There are several methodologies for assisted reproduction (e.g. means of sperm
preservation or the detection of the stage of the oestrous
© 2012 Blackwell Verlag GmbH
Anat. Histol. Embryol.
cycle by vaginal cytology) that would be interesting to
apply to these species.
The lesser anteater (Tamandua tetradactyla), one species of Xenarthra, belongs to the family of the anteaters
(Mymercophagidae). This family constitutes an important
group for conservation, because some species, as the giant
anteater (Myrmecophaga tridactyla), present a ‘vulnerable’
status according to the International Union for Conservation of Nature (IUCN; Superina et al., 2010).
Although the first anatomical studies of the male genital
system in T. tetradactyla were carried out about 100 years
1
Reproductive Tract of Male Lesser Anteater
ago (Kaudern, 1914; Grassé, 1955), histological data are
still scanty. Some important issues like the histological
details of the glands and ducts remain unknown. Transverse sections of the penis were illustrated by Kaudern
(1914); however, the arrangement of the erectile tissues
remains unclear. No work has been focused on the penile
functional morphology, and nothing is known about the
roles and behaviour of erectile tissues.
Even though species with internal testes are unusual
among mammals, no studies about testes have been carried out in the lesser anteater yet. Furthermore, nothing
is known about the fine structure of the cells of the seminiferous epithelium.
In spite of the continuous spermatogenesis suggested
for the giant anteater (Bartmann et al., 1991), rest and rut
periods were reported by Grassé (1955) for the lesser anteater. This indicates that further studies are required. For
other xenarthrans, like the common long-nosed armadillo
(Dasypus novemcinctus), testicular cycles with changes in
size, either with continuous spermatogenesis along the
year (Mc Cusker, 1985) or with seasonal spermatogenesis
(Torres et al., 1983), have been demonstrated. Finally,
there is a fragmentary knowledge about spermatology (i.e.
cell shape and size) in this family. Only the morphology
and morphometry of the male gametes were commented
for the lesser anteater (Hay et al., 1994), and no illustrations were provided.
Characterizing the morphology of the reproductive
tract and the male gamete is critical to develop and optimize techniques for assisted reproduction. With this
scope, we studied the anatomical and histological characteristics of the male reproductive tract of the lesser anteater (fine structure of the seminiferous epithelium
together with some morphological sperm features). This
information would contribute to protocols for applying
reproductive biotechnology to diverse programmes related
to its conservation.
Material and Methods
Taking into account the vulnerable status of the species, a
main difficulty of this survey was to obtain well-preserved
samples to accomplish fine morphological studies. Such
conditions were met by four dead T. tetradactyla supplied
by Argentinian zoos (see Table 1): Roque Saenz Peña
Zoo, Province of Chaco (male 1); Mendoza Zoo, Province of Mendoza (male 2); Rosario Zoo, Province of
Santa Fe (male 3); and Florencio Varela Zoo, Province of
Buenos Aires (male 4).
Reproductive tracts were dissected and submitted to
macroscopic observation, identification and the pertinent
measurements. Tracts were preserved by immersion in 4%
formaldehyde. Testicular volumes were calculated using the
2
L. F. Rossi et al.
Hansen
formula
(Hansen
and
With,
1952)
[V (volume) = L (length) 9 W2 (width) 9 0.52). After
dissection, the tracts were marked serially for identification
of corresponding histological sections (Fig. 1c). Samples
from the different regions were dehydrated and embedded
in paraffin. Microtome sections of 5-lm thickness were
stained with haematoxylin-eosin (H-E), Crossman trichrome or periodic-acid-Schiff (PAS) reaction for
identification of glycoconjugates containing hydroxyl
groups. Microscopic details were observed and digitalized
with a Leica optic microscope connected to a Leica camera
DFC300 (Leica Microsystem, Wetzlar, Germany). Images
were taken using Leica Application Suite, version 3.6.0,
Leica Microsystem.
For fine structure studies, transmission electron
microscopy (TEM) was carried out. A small piece of
testicular parenchyma was excised and fixed in 2.5%
glutaraldehyde in 0.1 M phosphate buffer (pH 6.9) at
room temperature for 2 h and post-fixed in 0.1% OsO4.
The samples were embedded in Maraglas, and serial sections were picked up in single-hole grids and stained with
uranyl acetate and lead citrate. Electron micrographs were
obtained with a Zeiss EM–109T electron microscope
(Carl Zeiss Inc., Oberkochen, Germany).
For sperm morphology studies, samples were obtained
from the cauda epididymis of males 3 and 4. The epididymis was removed and then minced in phosphate-buffered saline medium; the suspension of spermatozoa was
centrifuged for 10 min at 600 rpm, the supernatant was
discarded, and a small amount of the suspension was
placed on a slide. The linear dimensions of the length
and width of the head, tail length, midpiece length and
total sperm length of 150 spermatozoa were measured
from photographs obtained by the silver nitrate staining
method (Cetica et al., 1993). An estimate of the chromatin volume was obtained multiplying the average nuclear
thickness calculated in 30 different points (acrosomal,
equatorial segment and post-acrosomal region), by the
average area of the sperm head measured in micrographs
of five spermatozoa arising from sperm fixed, embedded
and observed by TEM as previously described (Cetica
et al., 1993, 1997). Area measurements were made from
light microscopy photographs using the Adobe Photoshop
CS3 software (Adobe Systems Inc., San Jose, CA, USA).
In addition, external genitalia of two live animals from
Bioparque Temaikén (males 5 and 6, see Table 1), Province of Buenos Aires, were photographed for morphological descriptions. Furthermore, these animals were scanned
using a Medison SA 600 ultrasound scanner (7.5 MHz
convex; Samsung Medison Co., Seoul, South Korea) for
internal morphological traits.
According to the configuration of the seminiferous
epithelium, all animals were classified as sexually mature
© 2012 Blackwell Verlag GmbH
Anat. Histol. Embryol.
L. F. Rossi et al.
Reproductive Tract of Male Lesser Anteater
Table 1. Morphometry of the genital tract of the studied specimens of Tamandua tetradactyla
Testes
Epididymis
Penis
Male no.
Weight (kg)
Season of the study
Lenght
(cm)
Width
(cm)
Depth
(cm)
Volume
(cm3)
Diameter
(cm3)
length
(cm3)
Spermatozoa
1
2
3
4
5
6
4.1
4.0
5.2
4.0
4.5
4.5
Summer (12 February 1999)a
Winter (1 August 1999)a
Winter (21/07/1999)a
Autumn (4 April 2009)a
Summer (18 February 2010)
Summer (18 February 2010)
2.0
2.0
3.5
2.7
3.1
3.6
1.0
1.3
2.0
1.8
1.9
2.4
0.8
0.8
1.5
1.3
NA
NA
1.0
1.8
7.3
4.5
5.8
10.8
0.4
0.4
0.6
0.6
NA
NA
2.0
2.6
NA
3.6
3.3
3.5
No
No
Yes
Yes
NA
NA
NA, not available.
Testes and epididymes dimensions are expressed as media of the left and right testis and epididymus.
a
Date of dead.
(i.e. presence of Sertoli cells, spermatogonia, spermatocytes
and in some cases spermatids). Additionally, the specimens’ weight corresponded to adults (Wetzel, 1985).
Results
Anatomy and histology of the genital tract in Tamandua
tetradactyla
The general anatomy of the male genital tract of the lesser
anteater is shown in Fig. 1a–c. The testes are found in the
pelvic cavity, joined medially to each other by a thin layer
of visceral peritoneum, between the bladder and the rectum
(Fig. 1a). The kidneys are located next to the testes and cranially to them. Renal veins were observed coming off the
caudal vena cava at an acute angle, denoting the caudal
position of the kidneys in this species (Fig. 1b, see inset).
(a)
(b)
Testes are ovoid structures, elongated in a cephalocaudal direction. Both are approximately of the same size
(Fig. 1a-c). They are lined by a well-developed layer of
connective tissue, corresponding to the tunica albuginea.
The rete testis constitutes a system of interconnected
channels extending from the mediastinum to the tunica
albuginea. From the seminiferous tubules to the rete
testis, there is a transitional epithelium extended from the
typical testicular stratified germinal type (Fig. 2a) to a
single layer of cuboidal cell type. Efferent ducts, which
lead from the rete testis to the epididymis, emerge from
the testicular cranial pole becoming highly convoluted
before opening into the epididymis.
The epididymides are demarcated into a convex caput that
covers the upper pole of the testes, connected by a corpus to a
caudal prominence which projects from the caudal border of
the testes (Fig. 1c). They are positioned on the internal side of
(c)
Fig. 1. Anatomical gross features of the male genital tract of Tamandua tetradactyla (ventral view). (a) Significant gross features of the genital
tract, arrowhead marks the visceral peritoneum that connects both testes. (b) Position of the testes in the pelvic cavity. Inset: detail illustrating the
left renal vein (arrow); arrowhead marks the left testicular vein. (c) Schematic drawing of the genital tract showing the sites where histological
sections were made. Scale bars: 2 cm. Abbreviations: r, rectum; bl, bladder; k, kidney; t, testis; e, epididymis; dd, duct deference; p, prostate;
sv, seminal vesicle; bu, bulbourethral glands; pn, penis; uo, urethral orifice; mu, membranous urethra.
© 2012 Blackwell Verlag GmbH
Anat. Histol. Embryol.
3
Reproductive Tract of Male Lesser Anteater
(a)
L. F. Rossi et al.
(d)
(e)
(b)
(f)
(c)
(g)
Fig. 2. Anatomy and histology of the testis (a, d), epididymis (b–d) and accessory sex glands (d–f) of Tamandua tetradactyla. (a) Cross section of
the testis showing seminiferous tubules, with spermatids lying around the lumen ready to be released and interstitium (arrowhead marks Leydig
cells). H-E staining. (b) Cross section of the epididymis showing PAS-positive granules in the lumen. PAS staining. (c) Epididymis with active secretion. Crossman trichrome staining. (d) Dissected genital tract denoting glands (dorsal view). (e) Seminal vesicle. H-E staining. (f) Prostate. H-E staining. (g) Bulbourethral gland showing PAS-positive cell mucus secretion. PAS staining. Scale bars: 20 lm (a), 100 lm (b, c), 3 cm (d), 50 lm (e–g).
Abbreviations: t, testis; e, epididymis; dd, duct deference; u, ureter; bl, bladder; p, prostate gland; sv, seminal vesicles; bu, bulbourethral glands;
mu, membranous urethra.
the testes. As a consequence, epididymides remain in contact
with each other (Fig. 1a,c). The epididymal duct of the caput
is lined by a pseudostratified epithelium with tall columnar
and basal epithelial cells surrounded by a layer of smooth
muscle cells (Fig. 2b,c). The lumen increases in diameter
through the corpus, while the epithelium decreases in height.
The ducts of the caudal region are greatly distended, lined by
a low epithelium with flattened nuclei.
The deferent ducts are long (4.25–4.49 cm in length),
convoluted, star shaped in cross section, lined by a
columnar pseudostratified epithelium and emerge from
the cauda epididymis (Fig. 1c). The deferent ducts have a
typical lamina propria and are surrounded by two thin
layers of smooth muscle and a fibroelastic adventitia. The
ductus deferens open into the ejaculatory ducts, which
drain into the membranous urethra (Fig. 2d), at the colliculus seminalis.
The urethra is lined by a polymorphic epithelium
(Fig. 3g–i). It is divided into two segments: the membranous urethra (2.8 cm in length), which leads from the
bladder to the base of the penis, and the star-shaped
penile urethra (4.6 cm in length; Fig. 3g–i), which runs
along the length of the penis on its ventral surface.
The seminal vesicles (1.8–2.3 cm in length) are
elongated, thin and coiled glands located dorsally to the
prostate gland (Fig. 2d). These glands are composed of
alveoli lined by cuboidal or columnar epithelial cells
(Fig. 2e). The lumen is filled with secretory droplets that
are mainly acidophilic. The alveoli drain into the main
4
collecting ducts, which open into the ejaculatory duct
(Fig 2d) caudally to the openings of the ductus deferens.
The prostate gland (2.4–2.8 9 1.0–1.4 9 0.4–0.7 cm)
consists of a bilobed gland situated dorsally to the
membranous urethra (Fig. 2d). It is composed of alveoli that are lined with cuboidal to columnar epithelial
cells (Fig. 2f). The alveoli drain into a main collecting
duct that runs through the urethral tissue for a short
distance and then joins the duct on the opposite side
to form a common duct that ends into the membranous urethra.
The bulbourethral or Cowper glands are a pair of flattened ovoid bodies (0.9–1.2 9 0.7–0.8, 0.5–0.6 cm) situated on the ventral surface of the penile urethra
(Fig. 2d). The internal surface is divided by broad connective tissue partitions into numerous small alveoli.
The epithelial lining is composed of simple cuboidal
cells with nuclei characterized by a PAS-positive
secretory product near the apical surface (Fig. 2g). The
glandular ducts arise from the medial surface of the
glands into the penile urethra ventrally on each side of
the midline. Each gland is smoothly encapsulated by
connective tissue and striated fibres of the bulbocavernosus muscle.
The penis is directed backwards, located ventrally and
very close to the anus without a noticeable perineum
(Fig. 3a–d). In the specimens studied, its length and
diameter were approximately 2.0–3.6 and 1.6–2.0 cm,
respectively (Table 1, Fig. 3a–c). The organ is conically
© 2012 Blackwell Verlag GmbH
Anat. Histol. Embryol.
L. F. Rossi et al.
Reproductive Tract of Male Lesser Anteater
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
Fig. 3. External genitalia and penis histology of Tamandua tetradactyla. (a) Penis in lateral view. (b) Penis in front view showing tip (*). (c) Penis in
front view showing urethral orifice (arrowhead). (d, e) Transverse sections of the penis at the base (d) and middle portion (e). (f) Histological transverse section at the penis tip. H-E staining. (g) Penile urethra showing star-shaped lumen. H-E staining. (h) Proximal section of the penis showing
cavernous sinus (arrowhead). H-E staining. I. Distal section of the penis showing small cavernous sinus (arrowhead). H-E staining. Scale bars 1 cm
(a–f), 100 lm (g–i). Abbreviations: pn, penis; a, anus; cc, corpus cavernosum; cs, corpus spongiosum; u, urethra.
shaped with a central fold (0.8 cm in length) that ends in
the external urethral orifice next to the summit (Fig. 3c).
In histological cross sections, the bulk of the penis was
formed by dense connective tissue. The erectile tissues
(corpus cavernosum and spongiosum) were centrally
located at the base of the penis and more dorsally located
in the middle portion. The corpus cavernosum is horseshoe shaped and surrounds the small corpus spongiosum
except at penile ventral part (a general view of the penile
structures is shown in Fig. 3d–f). The tunica albuginea
that surrounds the corpus cavernosum is continuous,
with no clear limit, with the dense connective tissue of
the bulk of the penis. The corpus spongiosum surrounds
the urethra and is present along the penile length, becoming thinner and weaker at the upper body (Fig. 3d–i).
As a consequence, its distal end is not expanded to form
a true glans penis. No skin receptacle or prepuce was
observed. The erectile tissues are constituted of caverns
lined by endothelium and surrounded by dense irregular
connective tissue with very few smooth muscle cells
(Fig. 3f–i).
© 2012 Blackwell Verlag GmbH
Anat. Histol. Embryol.
Testis histology and spermatogenesis fine structure
The testicular parenchyma is formed by the seminiferous
tubules and the interstitium (with Leydig and stromal cells;
Fig. 2a). The seminiferous epithelium is composed by Sertoli cells and four or five concentric layers of germ cells
(Fig. 4a). Unexpectedly, periodic indentations of the basal
membrane inside the seminiferous tubules were observed.
Sertoli cells show an ovoid nucleus, mostly close to the basal
membrane, with indentations that extend into the nucleoplasm deep down the long axis (Fig. 4b). The chromatin of
this cells is dispersed within the nucleus and presents small
and flat heterochromatin ‘clumps’ attached to the nuclear
envelope (Fig. 4b,c); the nucleolar complex is not apparent,
a fact that was also reported in other mammalian species. In
these cells, lipid droplets can be observed in the cytoplasm
(Fig. 4b,c). Next to the Sertoli cells, B-like spermatogonia
containing crusty-like chromatin are lying over the basal
membrane. Their round nucleus is characterized by dark
chromatin clumps mostly attached to the nuclear envelope
(Fig. 4c). Spermatocytes at different stages of maturation
5
Reproductive Tract of Male Lesser Anteater
(a)
L. F. Rossi et al.
(b)
(c)
(d)
(e)
(f)
Fig. 4. Histology (a) and fine structure (b–e) of the seminiferous epithelium and spermatozoa’s morphology (f) of Tamandua tetradactyla. (a) Seminiferous tubule in transverse section, arrowhead marks spermatogonial mitosis. PAS staining. (b) Sertoli cells with nuclear indentations (*) and
lipid droplets (arrowhead), spermatocyte (inset shows the synaptonemal complexes) and myoepithelial cell at the basal membrane (arrow). (c) Sertoli cell with lipid droplets (arrowhead) and crusty spermatogonia cells. Arrow marks basal membrane indentations. (d) Spermatids with ovalshaped nucleus and proacrosomic dense material (arrowhead) extending laterally, acrosomic sac (arrow). (e) Elongated spermatids with nucleus
capped by a well-developed acrosome (arrow), homogeneous chromatin (*) and midpiece with noticeable mitochondria (arrowhead). (f) Left,
spermatozoa in phase contrast; right, spermatozoa stained with silver nitrate. Arrowhead marks end of middle piece. Scale bars: 10 lm (a); 3 lm
(b–e); 10 lm (f). Abbreviations: Se, sertoli cell; S, spermatocyte; sC, synaptonemal complexes; M, myoepithelial cell; cG, crusty spermatogonia;
Sp, spermatids; AB, acrosome boundary; PAR, post-acrosomal region; PP, principal piece.
can be recognized, and the pachytene stage could be clearly
identified because well-defined synaptonemal complexes
were observed (Fig. 4b, see inset). In the adluminal compartment, round spermatids are found with a single large
granule nearby the nucleus signalling the beginning of the
6
Golgi phase of the acrosome development. Round spermatids show little clumps of heterochromatin all over the
nucleus (data not shown). In other seminiferous tubules,
spermatids with oval-shaped nucleus were observed, with
proacrosomic dense material extending laterally and cover© 2012 Blackwell Verlag GmbH
Anat. Histol. Embryol.
L. F. Rossi et al.
Reproductive Tract of Male Lesser Anteater
(a)
(b)
Fig. 5. Testis (a) and epididymis (b) of an inactive individual (male 1). (a) Cross section of an inactive testis showing partial degeneration of
seminiferous tubules. Inset shows free cells in lumen with apoptotic figures. H-E staining. (b) Cross section of the epididymis of an individual with
inactive gonads showing no spermatozoa in the lumen. H-E staining. Scale bars 100 lm.
ing a small nuclear region. This is the beginning of the cap
phase of spermatids (Fig. 4d). In some tubules, a second
layer of spermatids is observed, showing an elongated
nucleus capped with a well-developed acrosome (Fig. 4e).
The chromatin is homogeneous and grey stained, indicating
an advanced stage of maturation (Fig. 4e).
Sperm morphology
The sperm heads are oval shaped, with a central tail
insertion (Fig. 4f). The mean and standard deviations for
the linear dimensions obtained for the length and width
of the sperm heads were 9.50 ± 0.66 and 4.65 ± 0.34 lm,
respectively (Fig. 4f). The acrosome covers a large portion
of the head, accounting for 70.3% of the total head length
(Fig. 4f). The tail length was 59.18 ± 1.78 lm, and midpiece length was 14.67 ± 0.76 lm (Fig. 4f). The average
of the total length was 67.33 ± 1.60 lm (Fig. 4f). The
TEM study showed that the nuclear thickness of the
sperm head averaged 0.23 ± 0.07 lm and increased from
the tip to the base. The sperm nuclear volume was estimated to be 9.98 ± 0.63 lm3.
Morphological variations of the genital tract between
individuals
Males 1 and 2 presented no spermatozoa in the epididymis and showed a disorganized seminiferous epithelium, with abundant apoptotic figures and presence of
free cells in the lumen (Fig. 5a), which lacked late spermatogenic cell types (i.e. round and elongated spermatids). Testes of these specimens were classified as inactive,
and testicular volumes were lesser respect to males 3, 4, 5
and 6 (Table 1). Finally, epididymal diameters in males 1
and 2 were lesser compared to males 3 and 4 (Table 1).
The epididymis’ histology showed lower lumen diameter
in the tubules and rich intertubular connective tissue in
males 1 and 2 (Fig. 5b) with respect to males 3 and 4.
© 2012 Blackwell Verlag GmbH
Anat. Histol. Embryol.
Discussion
In this article, we describe for the first time, the histology
of the male reproductive tract and the fine structure of
the spermatogenesis in the lesser anteater (T. tetradactyla). Our histomorphological studies allowed a correct
recognition of the accessory glands and a detailed description of the penile structure in this species. The spermatozoon was illustrated. Additionally, while we were
assessing the reproductive condition, reproductively inactive individuals were found.
Testes in primitive mammals (except for marsupials) are
placed in the abdominal cavity next to the kidneys (condition of testicondia; Werdelin and Nilsonne, 1999). As illustrated in the present study (Fig. 1a,b), testes in the lesser
anteater, even when they are internal, are found more caudally into the pelvic cavity. This condition, different from
testicondia, was reported for other groups of xenarthrans
like armadillos and sloths (Grassé, 1955). Joined testes,
which were found in the lesser anteater, are also present in
the giant anteater (Bartmann et al., 1991) and the sloth
Bradypus torquatus (Dos Santos Martins, 2003). However,
in the latter, testes are asymmetrically disposed (one positioned more cephalical than the other; Dos Santos Martins,
2003). On the other hand, testes in armadillos are not
joined one to another (Grassé, 1955), and further studies
are needed to see whether joined testes is a shared feature
of all species of anteaters and sloths (order Pilosa).
Our anatomical observations of the male accessory
sexual glands are in agreement with previous reports for
this species (Kaudern, 1914; Grassé, 1955). As described
in the giant anteater (Bartmann et al., 1991) and sloths
(Dos Santos Martins, 2003), the presence of a colliculus
seminalis was observed in this species. The histological
characterization of the male accessory sexual glands
(prostate, seminal vesicle and bulbourethral glands) in the
lesser anteater showed no important differences from
those of other mammals (Banks, 1986).
7
Reproductive Tract of Male Lesser Anteater
The short and conical-shaped copulatory organ of the
lesser anteater is comparable to that found in the giant
anteater (Bartmann et al., 1991) and sloths (Dos Santos
Martins, 2003). Because of the shortness of the penis in
this species, copulation could be explained by a shallow
penetration. This is in agreement with the proposal of
Bartmann et al. (1991) for the giant anteater. Additionally, as previously proposed, the hymen in females of the
lesser anteater might not be perforated during copulation
(Rossi et al., 2011).
By our histological analyses, some clues of the erection
and intromission mechanisms could be ascribed: (1) the
arrangement of erectile structures of the penis (i.e. the corpus cavernosum and the corpus spongiosum) vary depending on the species: in the lesser anteater, the corpus
cavernosum forms a unit, which is largest proximally and
extends laterally covering partially the ventrally placed urethra, while the corpus spongiosum is poorly developed.
The stretched disposition of the corpus cavernosum and
the urethra suggests a protection of the latter and the maintenance of the lumen aperture during intromission in this
species. (2) Smooth muscle elements are not well developed
in the lesser anteater. At a large number of species, the septum that circumscribes the areolas of the corpus cavernosum contains smooth muscle elements (Stanley and
Hillemann, 1960; Cook, 1965; Nickel et al., 1981; Banks,
1986; Wrobel and Bergmann, 2006). The penile erectile tissue, specifically the cavernous smooth muscle cells, plays a
key role in the erectile process (Dean and Lue, 2005).
Because corporeal smooth muscle cells control the vascular
event leading to erection, the presence of few smooth
muscle cells in the erectile tissues of the lesser anteater can
be expected to affect a good erectile response. (3) Additionally, according to Wrobel and Bergmann (2006), based on
the predominance of erectile tissue over the connective tissue and vice versa, penises can be classified into three types,
namely the vascular type, where the caverns predominate;
the fibroelastic type, where the connective tissue prevails;
and the intermediate type, which is in-between the previous two types. The penis of the lesser anteater was suitably
considered in ‘fibroelastic type penis’. In the cross sections,
it was observed that dense connective tissue occupies most
of the penis. This great development of dense connective
tissue also limits the erectile process. However, as the exact
length of the penis during the erectile process can only be
demonstrated in live animals, more studies are yet to be
performed to clarify the strategy for transferring gametes
from one individual to the other in this and the in rest of
anteaters species.
In contrast with the anatomical observations made by
Kaudern (1914), who described a tiny glans for the lesser
anteater, no defined glans was observed in our histological
study. Also, for the giant anteater, Bartmann et al. (1991)
8
L. F. Rossi et al.
described the pigmented tip of the penis as a glans by anatomical studies. Among xenarthrans, a true histological
glans has been described only for the large hairy armadillo
Chaetophractus villosus (Affanni et al., 2001). A preputial
opening in the site of the urethra ending has also been
described in the giant anteater (Bartmann et al., 1991). As
our present observations indicate the absence of a glans,
and consequently the absence of a true prepuce, we consider the term urethral opening more adequate (instead of
preputial opening) for the lesser anteater.
Testis histology and spermatogenesis fine structure
The main ultrastructural aspects of the cells of the
seminiferous epithelium were described in this work, this
information constitutes the basis to understand the
process of spermatogenesis of the lesser anteater. In
Xenarthra, the fine structure of the cells of the seminiferous epithelium has been reported only for the common
long-nosed armadillo Dasypus novemcintus (Nagy and
Edmonds, 1973; Weaker, 1977). The spermatogonia presently found were similar to that described as B spermatogonia by Weaker (1977). Sertoli cells present typical
aspect with deep nuclear indentations. The general aspects
of the process of spermiogenesis can also be derived from
this work. The typical phases of development could be
established for the spermatid cell according to nuclear
shape and chromatin condensation. The description of
the normal configuration of the seminiferous epithelium
was necessary to confront with the one found in some
individuals with incomplete spermatogenesis. Finally,
protrusions of the basal lamina pointing towards the
nuclei of the Sertoli cells were described only in pathological conditions in humans (Haider et al., 1986),
Sperm morphology
Sperm shape was consistent with the shape previously
reported by Hay et al. (1994) for this species, but tail
length was significantly shorter in our studies. Total
sperm length (67 ± 1.60 lm) was similar to the average
eutherian spermatozoa (69.23 ± 4.13 lm), except rodents
(101 ± 3.17 lm, Roldan et al., 1992). We base our observations on several measurements of mature and well-preserved spermatozoa from two individuals. Additionally,
we provide illustrations to support these data. As a consequence, we consider that the size of the male gamete of
this report is the correct one. Sperm morphology in the
lesser anteater is similar to that found in Bradypus
tridactylus, but the spermatozoon is longer than that
found in the sloth (33.2 ± 0.75 lm; Peres et al., 2008). In
other xenarthrans, like the armadillos, the shape and size
of the spermatozoa were similar to those of the phyloge© 2012 Blackwell Verlag GmbH
Anat. Histol. Embryol.
L. F. Rossi et al.
netically basal species (the genus Dasypus; Cetica et al.,
1998; Moller-Krull et al., 2007). This was different from
those of the more derived ones (Priodontes maximus,
Cabassous unicinctus, Chaetrophractus vellerosus, Chaetrophractus villosus, Zaedyus pichiy, Euphractus sexcinctus;
Cetica et al., 1998; Moller-Krull et al., 2007), which present extremely thin and large spoon-like sperm heads
(Cetica et al.,1998).
Morphological variations of the genital tract between
individuals
Notorious differences in the size of the genital tract
organs were observed. Specimens with active gonads had
bigger testicular volumes and larger epididymal diameters
than those with inactive gonads, probably suggesting different mating or heat periods. This observation is in
agreement with the sketches showed by Grassé (1955).
The histological study of the testes of the lesser anteater
exhibited complete spermatogenesis in the specimens with
active gonads. Abnormal spermatogenesis was present in
specimens with inactive gonads. This was correlated with
variation in testicular size and presence of spermatozoa in
these individuals (see Table 1). In agreement with our
observations in the lesser anteater, significative variations
of testicular size and weight have also been reported in
other xenarthrans, like the common long-nosed armadillo
(Mc Cusker, 1985). To establish whether the lesser anteater has a reproductive seasonality, an increased number
of individuals in their natural state must be studied.
As showed in the present work, the lesser anteater has
intrapelvic testes, a feature that is shared with other xenarthrans; some of the studied specimens of the lesser anteater exhibit a gonadal regression that is reflected on
other structures of the reproductive tract. We expect that
our results will contribute to guide further studies on the
reproductive systems of this species and this family,
aimed to improve successful breeding in conservation
programmes.
Acknowledgements
We thank Dr. Jorge Mario Affanni, Dr. Juan Donati,
Dr. Julio Correa and Dr. Maria Ines Pigozzi for valuable
comments and advice. We thank DMV Pablo Fernandez
for the Temaiken Zoo for doing the ecographs. This work
was supported by PICT 1198 (MSM) and PIP 0204
(MSM).
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