Morphological Changes of the Epididymis and Description of the Excurrent Ducts of Phrynops geoffroanus (Testudines: Chelidae) During the Reproductive Cycle
Article first published online: 2 DEC 2010
Copyright © 2010 Wiley-Liss, Inc.
The Anatomical Record
Volume 294, Issue 1, pages 145–155, January 2011
How to Cite
Pagliarini Cabral, S. R., Zieri, R., Franco-Belussi, L., De Souza Santos, L. R., Saranz Zago, C. E., Taboga, S. R. and Oliveira, C. D. (2011), Morphological Changes of the Epididymis and Description of the Excurrent Ducts of Phrynops geoffroanus (Testudines: Chelidae) During the Reproductive Cycle. Anat Rec, 294: 145–155. doi: 10.1002/ar.21302
- Issue published online: 13 DEC 2010
- Article first published online: 2 DEC 2010
- Manuscript Received: 10 DEC 2010
- Manuscript Accepted: 29 SEP 2010
- Manuscript Revised: 27 SEP 2010
- Sao Paulo Research Fundation (FAPESP). Grant Numbers: 05/02919-5, 08/52389-0
- Coordenação de Aperfeiçoamento de Pessoal de Nível Superior [(CAPES), Master's grant]
- Phrynops geoffroanus;
- deferent duct;
- efferent ductules;
The seminal ducts (efferent ductule, epididymis, and deferent duct) in adults of Phrynops geoffroanus were examined using light microscopy. A series of tubules (efferent ductules) connect the testes to the epididymides. The efferent ductules are formed by a rete of small tubules of varying diameters, with simple columnar epithelium formed by the ciliated cells, nonciliated cells, and few basal cells. The epididymis is a simple, long and highly convoluted tubule that receives the efferent ductules throughout its extension. It is covered by a pseudostratified columnar epithelium with three cellular types: the principal cells, which are the most abundant, basal cells, and a small narrow cell. The histological differences in the epididymis region (cranial, medial, and caudal), as well as the differences in the epithelium throughout the reproductive cycle, are discussed. The deferent ducts consist of a low pseudostratified epithelium with two cellular types: the principal and basal cells. During the months analyzed, spermatozoa were stored in the epididymis, and deferent ducts were found. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.
The extratesticular seminal ducts are composed of efferent ductules, epididymides, and deferent ducts. It is generally accepted that these structures originate from the archinephric duct, and in anamniotes, they have function of transporting urine and spermatozoa (Romer and Parsons,1985; Orr,1985; Hickman et al.,2004). In the amniotes, the ducts are used exclusively for transportation of spermatozoa (Jollie, 1962; Romer and Parsons,1985).
The epididymides and other spermatic ducts are considered as adaptive organs, involved in the transition of vertebrates from aquatic to terrestrial environment, and they are related to internal fertilization in tetrapods (Orr,1985; Pough et al.,2003). The spermatic ducts are often involved in maturation, storage, transportation, and gametes defense (Romer and Parsons,1985; Jones,1999; Chan and Zhang,2005).
Testudines represent an early vertebrate group that uses epididymides to store spermatozoa for long periods of time (Holmes and Gist,2004). Throughout the chelonians from temperate regions that reproduce seasonally, the spermatozoa produced during the summer are stored in the epididymides and are used in matings that occur throughout the year (Jones,1999; Gist et al.,2000).
Many previous studies deal with the structure, function, and regulation of the epididymal ducts of mammals and birds (Hamilton and Cooper,1978; Calvo et al.,1987; Stefanini et al.,1999; Aire and Stoley,2000; Chimming and Vicentini,2001; Aire,2002a; Simões et al.,2004; Gist et al.,2007). However, few studies emphasize the morphology of the excurrent ducts of reptiles. For testudines, studies are even scarcer, and they highlight only species from temperate regions (Christiansen and Dunham,1972; Guix et al.,1989; Etchenber and Stovall,1990; Meyland et al.,2002; Gribbins et al.,2003; Holmes and Gist,2004; Akbarsha et al.,2006; Takahashi et al.,2008).
Phrynops geoffroanus, commonly known as Geoffroy's side-necked turtle, is a small animal, with the male averaging approximately 21 cm in length and the female averaging 35 cm. They are frequently found in rivers, lakes, and ponds with slow currents, and have a wide distribution, including South American countries as Colombia, Venezuela, Guyana, the far southern region of Paraguay, and the southeastern, central-western, and northeastern regions of Brazil (Pritchard,1979; Ernest and Barbour,1989). In P. geoffroanus, studies are related to the description of reproductive behavior and nesting; there are no studies related to the description of reproductive systems. Studies using captive P. geoffroanus are helping researchers to understand the reproductive behavior, aggressiveness interaction, and also aspects of the species (Guix et al.,1989; Molina,1992,1998). The aim of this study was to characterize morphologically the extratesticular seminal ducts of P. geoffroanus (Schweigger, 1812; Testudines, Chelidae), and to verify if there are histological differences occurring in the epithelium thickness and in the thickness of the epididymal muscular tunic during the reproductive cycle.
MATERIALS AND METHODS
Sixteen adult males of P. geoffroanus (Testudines, Chelidae) were collected monthly in the Felicidade stream (20°46′20.6″S, 49°21′18.0″W) in São José do Rio Preto, São Paulo state, Brazil, (061/2005-RAN/IBAMA), between April 2005 and May 2006.
In the laboratory, the specimens were anesthetized and euthanized using injectable ketamine hydrochloride (Auricchio and Salomão,2002), and they were then submitted to morphological studies. Animal handling and experiments were done according to the ethical guidelines of São Paulo State University (UNESP), following the Guide for Care and Use of Laboratory Animals.
Dissection was realized using a vibratory circular saw to remove plastron and to access the epididymides and deferent ducts. For histological processing, fragments (3 mm3) were removed from the middle third of the left deferent duct and the caudal and cranial endings, and they were removed from the middle portion of the left epididymis.
The fragments were fixed in Bouin for 24 hr, dehydrated in an alcohol gradient (Hopwood,1990), and embedded in Methacrilato glicol Historesin® (Leica, Germany) or Paraplast® (Oxford Labware). Thick sections of 3 μm were stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) and examined under an Olympus BX 60 microscope (Olympus America Inc., Melville, NY) using the program Image-Pro Plus 4.5.
Some testicular fragments were obtained, and the final portion of the intratesticular seminiferous pathways was studied to develop a morphological description. Cross-sections from the cranial, middle, and caudal portions of the epididymis were randomly selected, and eight measurements were taken in each region, totalizing 24 measurements per animal. To check for the existence of morphometric differences in epididymis regions throughout the year, measurements of the epithelium height and the thickness of muscular tunic were taken in five animals captured during the reproductive season (October to April) and other five captured during the nonreproductive season (September to December), using the 20× objective lens.
Morphological differences were found in the thickness of the epithelium and in the diameter of the muscular tunic. Shapiro–Wilk tests were performed on the data to calculate the outliers and the normal distribution. All nonparametric data were normalized using square roots. The data were also analyzed using one-way ANOVA and the Kruskal–Wallis test, complemented by the Tukey test. To verify the occurrence of variations of parameters considered over time, the Mann–Whitney test was applied. To attribute statistical signification, the P value was set at P ≤ 0.05 (Zar,1999).
P. geoffroanus presents body weight 1.113 ± 360 g and measuring 19.8 ± 1.6 mm, obtained from plastron length from 16 animals. The anatomical organization of the extratesticular seminal pathway as well as the histology of the ducts in all of the segments making up the pathway were described morphologically. The morphological changes during the reproductive cycle were analyzed only for the epididymis.
The efferent ductule of P. geoffroanus emerge from the dorsal face of each testis as a tangle of small, lightly winding tubes and are supported by the connective tissue, which surrounds them and flow into the epididymides ducts throughout the extent of the epididymis (Fig. 1A and B).
The efferent ductules are surrounded by a thin layer of smooth muscle tissue (Fig. 1C), and they have a pseudostratified columnar epithelium, which alternates between ciliated, nonciliated, and few basal cells. The ciliated cells have a spherical nucleus, located in either the base or the central region of the cell. The nucleolus is prominent, and the cytoplasm does not have granulations visible through light microscopy. They have long cilia, which are inside the ductules and in contact with the luminal contents. The cilia begin as basal corpuscle, which take up the stain intensely. The nonciliated cells are morphologically similar to the ciliated cells: they possess a spherical nucleus and are basal or centrally located. However, they take up stains less intensely (Fig. 1C).
The lumen of efferent ductules showed varied diameter and irregular outline, and they were usually empty (Fig. 1C).
Histologically, the epididymis of P. geoffroanus is lined by a pseudostratified epithelium, which is covered by layers of smooth muscle tissue with a longitudinal arrangement of their fibers, surrounded by a layer of connective tissue where the blood vessels are located (Fig. 2A).
Principal cells and vesicular cells were identified in the epithelium, followed by basal and narrow cells (Fig. 2B, C, and E). The principal cells, in larger numbers, are column shaped and have a large cytoplasm and an oval shaped nucleus with a prominent nucleolus located in the basal pole (Fig. 2C). The cytoplasm and the apical part of the principal cells reacted positively to the PAS (Fig. 2A and D). The basal cells are located under the vesicular cells and are supported by the basal membrane (Fig. 2B and D). They are short when compared with the principal cells and are round or triangular in shape, generally with an elongated nucleus. Another cellular type that occurs in small amounts between the principal cells is the narrow cell. These cells are column shaped and elongated and possess a bright-colored nucleus, which contrasts with the nuclei of the principal cells (Fig. 2E).
The epididymis of P. geoffroanus possesses a thicker epithelium in the cranial portion, but decrease in thickness in the middle and caudal portions (Fig. 3). Significant differences related to the epithelium height were observed in the cranial portion when compared with the middle and caudal portions during the reproductive season (one-way ANOVA: F = 16.24 and P = 0.000); however, between the middle and caudal portions, significant differences were not verified (P > 0.05) (Fig. 3). During the nonreproductive season, differences between the height of cranial and middle portions of the epithelium were not observed; however, the caudal portion had a thinner epithelium when compared with the other two regions (Kruskal–Wallis: H = 28.69 and P = 0.0001; Fig. 4).
During the reproductive phase, the following averages were found: 58.19 ± 15.71 μm for the cranial portion; 39.07 ± 11.02 μm for the middle portion, and 37.93 ± 15 μm for the caudal portion (Fig. 3). However, in the nonreproductive season, the epithelium height was lower in all portions: 39.07 ± 20.60 μm for the cranial portion, 28.78 ± 5.60 μm for the middle portion, and 20.48 ± 5.26 μm for the caudal portion (Fig. 4). These values indicate significant differences among the three structures analyzed during the two phases (Mann–Whitney: U = 125, P = 0.0003; U = 121, P = 0.0002; U = 119, P = 0.0002; Fig. 5).
The smooth muscle layer, which surrounds the epididymal ducts, has longitudinal arrangement, and its thickness increases gradually from the cranial portion to the caudal portion (Fig. 6). There is no significant difference in the thickness of the muscular layer in the cranial and middle portions; however, there is a significant difference between these two regions compared with the caudal portion when the seasons are separately analyzed (Kruskal–Wallis: H = 31.68 and P = 0.0001; ANOVA: F = 7.69 and P = 0.0013; Fig. 6). Between the reproductive and nonreproductive seasons, the analyses revealed that there are no differences in the thickness of muscular layers for the cranial, middle, and caudal portions (Mann–Whitney: U = 239.5, P = 0.15; U = 260, P = 0.31; U = 231, P = 0.11; Fig. 6). During all months analyzed, the presence of a large amount of spermatozoa stored in the epididymides was detected.
Starting from the caudal region of each epididymis, as the epididymal ducts become less convoluted, the deferent ducts arise and flow into the penis. Histologically, the wall of the deferent ducts includes a mucus membrane, an intermediate muscular tunic and an external adventitia (Fig. 7).
The mucosa is formed by a pseudostratified epithelium, consisting of principal and basal cells, similar to the epithelium of caudal portions of the epididymides. The principal cells are column shaped and extend from the base to the tubular lumen. The nucleus is basal, elongated or spherical, with one or two nucleoli (Fig. 7B). The basal cells are located next to the basal membrane and never reach the tubular lumen. Their nucleus is oval shaped, with parallel orientation to the basal membrane (Fig. 7C). The lumen of deferent ducts showed uniform outlines. There were no mucosal imaginations, and there were spermatozoa inside the ducts of all of the analyzed samples.
The excurrent ducts of P. geoffroanus constituted of efferent ductules, epididymides, and deferent ducts. This basic standard of organization has been described for other reptiles (Jollie, 1962; Ilio and Hess,1994; Holmes and Gist,2004; Akbarsha et al.,2006), birds (Artoni et al.,1999; Aire and Stoley,2000; Simões et al.,2004; Orsi et al.,2007) and mammals (Ilio and Hess,1994); however, the morphology of the seminal ducts can differ significantly between given groups of animals (Holmes and Gist,2004).
The histological analyses of testes of P. geoffroanus did not reveal regions corresponding to the histological description of rete testis, as observed in Chrysemys picta (Holmes and Gist,2004), Caimam crocodilus (Guerrero et al.,2004), and Sitana ponticeriana (Akbarsha et al.,2006) and which was characterized by Holmes and Gist (2004) as tubules formed by a simple layer of cubic epithelial tissue and covered by two or three layers of muscle tissue.
The histological analysis of epididymal ducts of P. geoffroanus revealed a considerable morphological analogy to other species studied. The structure, which consists of a pseudostratified columnar epithelium surrounded by a thin lamina propria and externally covered by a smooth muscle tissue, has been described for reptiles, birds, and mammals (Holmes and Gist,2004; Guerrero et al.,2004; Simões et al.,2004; Akbarsha et al.,2006; Orsi et al.,2007).
In this study, we consider the height of the epididymal epithelium as well as the thickness of the muscular layer between cranial, middle, and caudal segments to verify the occurrence of histological differences. The morphological analyses of the epididymal ducts of P. geoffroanus showed an increase in the thickness of the smooth tissue layer from the cranial to the caudal region, and revealed that, when the data were separated based on the reproductive and nonreproductive seasons, there was no significant variation of the averages. These results indicate that the size of the musculature remains unchanged during the reproductive cycle. Our observations confirm that the organization of the muscularis mucosa seems to be similar to what is generally proposed for mammals (Bloom and Fawcett,1977; Cormack,1991).
As for the height of the epididymal epithelium, the analyses showed a variation between the cranial and middle regions (which had a higher epithelium), and the caudal region (which had a thinner epithelium). The epithelium is significantly higher during periods of reproductive activity. Cyclical alterations of epididymal ducts, as well as other ducts of the extratesticular seminiferous system, are influenced by the levels of sexual hormones, as observed in other species of reptiles (Mesure et al.,1991; Ferreira et al.,2002; Guerrero et al.,2004).
The cellular types of the epididymal epithelium of P. geoffroanus correspond to the principal, basal, and narrow cells. The diversity and the amount of cells that make up the epididymal epithelium vary among the vertebrates. In Lacerta vivipara (Mesure et al.,1991) and Tropidurus itamberere (Ferreira et al.,2002), principal and basal cells were identified. In the lizard S. ponticeriana (Akbarsha et al.,2006), six different cellular types were identified: principal, basal, narrow, clear, apical cells, and intraepithelial leucocytes. In the freshwater testudine C. picta (Holmes and Gist,2004), four types of cells were identified: vesicular, basal, narrow, and cells rich in mitochondria. However, according to Holmes and Gist (2004), cells that can be conclusively identified by light microscopy are the principal, basal, and narrow cells, and the other types are effectively differentiated through ultrastructural analysis only.
In P. geoffroanus, the principal cells are the most abundant, and they are found in all regions of the epididymis. These cellular types are often described among vertebrates. The histochemical and ultrastructural analyses indicate secretory and absorptive activity (Guerrero et al.,2004; Holmes and Gist,2004). In C. picta (Holmes and Gist,2004), the vesicular cells are structurally comparable with the principal cells found in P. geoffroanus. The same authors report that the cells do not have cilia or stereocilia, and they have tight junctions and desmosomes. They also concluded that its main function is absorption, and they suggest that secretory activity also occurs. The columnar cells described in C. crocodilus (Guerrero et al.,2004) seem to be similar to vesicular and principal cells. Due to structural similarities between the vesicular and columnar cells and the principal cells of P. geoffroanus, we can infer that they are the same cellular types with only different denominations. Spermatozoa or their fragments were occasionally found connected to the apical surface of the cell, and wide vacuoles usually occupied a supranuclear portion, results which were also found by Holmes and Gist (2004) in C. picta.
The basal cells were frequently found in the caudal region of epididymis and were always connected to the basal membrane. According to Holmes and Gist (2004), the basal cells present interdigitations with the vesicular cells, mainly in the caudal region, and their number varies in accordance with the stage of the reproductive cycle (their values increase during spermiogenesis). Studies indicate three important roles for basal cells: support, electrophile neutralization, and immune surveillance of spermatozoa (Veri et al.,1993; Yeung et al.,1994; Seiler et al.,1998).
The rare occurrence of narrow cells in the epididymis of reptiles was initially reported in C. picta (Holmes and Gist,2004) and later in S. ponticeriana (Akbarsha et al.,2006). As was found in these previously studied species, the narrow cells of P. geoffroanus have elongated, narrow and more strongly stained nuclei, and they are also extremely rare.
Findings on the reproductive biology of the P. geoffroanus, Acanthochelis radiolata, and Acanthochelis spixii in captivity indicate that the reproductive activity of males occurs between October and April (Guix et al.,1989; Molina,1992), coinciding with the high production of gametes analyzed in this study. Nevertheless, spermatozoa were found in three regions of the epididymis throughout the year. As with the majority of species, spermatogenesis occurs at a certain interval, which is generally in the summer. There is an advantage in storing spermatozoa after this period; when the testes are spermatogenically inactive, which is where reproduction can still occur (Christiansen and Dunhan,1972; Etchemberg and Stoval,1990; Mahmoud and Cyrus,1992; Gribbins et al.,2003). This situation is particularly common among reptiles and birds (Gist and Jones,1987).
Studies related to the physiological characteristics of spermatozoa, which were obtained from testudines epididymides, revealed that the motility and the swimming velocity are lower than that in other species. Additionally, these studies concluded that these sexual cells remain stable for long periods of time, and that the physiological characteristics of spermatozoa (motility, swimming velocity, and response to chemical stimulator) stored in the epididymides for five months are similar to those produced during the high point of spermatogenesis. They also concluded that the low metabolism presented by the spermatozoa can be an advantageous characteristic for sperm that is stored for prolonged periods (Gist et al.,2000,2001).
In spite of evidence indicating that the chelonian epididymides hold a primary role in storage, histological analyses suggest a large structural similarity among the epididymides of vertebrate, and they can be physiologically and ultrastructurally analogous. Therefore, the epididymides can be considered more than a mere storage organ, because they are also involved in the maturation or activation of spermatozoa.
The efferent ductules conduct sperm from the testes to the epididymides and consist of a system of tubules with unique characteristics, because it is the only segment of the male reproductive tract with ciliated epithelium (Bloom and Fawcett,1977; Ilio and Hess,1994). Histologically, the efferent ductules of P. geoffroanus are similar to those that have been described for other species (Ilio and Hess,1994). The connection of the efferent ductules to the epididymides occurs at the point where the medial–caudal region of the testes meets the caudal region of the epididymides, but ductules were found in the histological sections of cranial region of the epididymides as well as in the conjunctive tissue that covers the deferent ductules. According to Ilio and Hess (1994), there are few histological differences in the features of efferent ductules. Among the species that were already studied, the epithelium was classified as pseudostratified columnar or cubical, depending on the species.
The cellular types identified in this study (ciliated, nonciliated, and basal cells), are present in the amniotes vertebrates studied to date, and the main cellular difference among the species is the presence of vacuoles and granulations (Ilio and Hess,1994; Holmes and Gist,2004; Akbarsha et al.,2006). The cellular granulations were one of the criteria used to classify the ductules in different regions (Holmes and Gist,2004; Simões et al.,2004). However, such granulations were not identified in P. geoffroanus.
Histological differences throughout the extent of the ductules have been described for several mammal species (Ilio and Hess,1994), birds (Simões et al.,2004; Orsi et al.,2007), and reptiles (Holmes and Gist,2004; Akbarsha et al.,2006). The P. geoffroanus had a ductule diameter that varied significantly in size.
The ciliated cells of P. geoffroanus are unique because of the length of the cilium that projects into the lumen, maintaining contact with the luminal content. The presence of long cilia was also described for C. picta and C. crocodilus (Guerrero et al,2004; Holmes and Gist,2004).
The histological observations show that there is a morphological heterogeneity in the shape and size of the cells and also the shape, size, position, and degree of affinity of the nucleus to the stains. Because of these differences, we can infer the possibility of ciliated and nonciliated cells subtypes, as reported by Ilio and Hess (1994) in their review of efferent ductules. Among birds, other cellular types were identified. In Columba livia (Stefanini et al.,1999) ciliated cells, clear, dark, and angular nonciliated cells, and halo cells were found. In the apical region of nonciliated cells of Struthio camelus (Aire and Stoley,2000) and Anasplathyrynchos (Simões et al.,2004), microvilli were found. Data regarding morphological changes in the deferent ductules of birds suggest that histological alterations occur in the ductules throughout the reproductive cycle (Aire,2002a,b). These findings were also reported by Guerrero et al. (2004) in their research on C. crocodilus.
Several functions have been attributed to the efferent ductules, including the transportation of spermatozoan and fluid from the testes to the epididymides, the reabsorption of large amounts of liquid, the synthesis and metabolism of steroid and other compounds, and the phagocytosis of spermatozoa, particularly in perturbed epithelium (Bloom and Fawcett,1977; Cormack,1991; Ilio and Hess,1994; Holmes and Gist,2004). During our analyses, we did not find spermatozoa in the lumen of efferent ductules throughout most of the year. However, these cells were often seen during the months of more frequent reproductive activity, as reported by Guix et al. (1989) and Molina (1998). When we consider that the epididymides store spermatozoa for long periods of time, we can conclude that the efferent ductule rete of P. geoffroanus is involved in the transportation of sperm, and that they are not involved in storage.
The deferent ducts correspond to the distal and less convoluted portion of epididymal duct. In P. geoffroanus, the deferent duct is histologically characterized by three layers of tissue, an internal mucosa, an intermediate muscular tunic, and an external adventitia of connective tissue. This same pattern of organization is described for other vertebrates (Bloom and Fawcett,1977; Hamilton and Cooper,1978; Chimming,2001).
The mucosa of the deferent ducts is made up of three layers of smooth muscle tissue, with space arrangement of fibers similar to what was described for other species (Bloom and Fawcett,1977). Musculature peristaltic contractions are responsible for sperm elimination (Aires,1989).
The parallel ductules, identified in the adventitia of deferent ducts, are similar to the efferent ductules. In C. crocodilus (Guerrero et al.,2004), parallel ducts were also identified; however, the connection to the testes was not evident.
The crenulated or fenestrated features generally found in the mucosa of deferent ducts of mammals were not found in this study. In all duct segments analyzed, stored sperm was found.
The deferent duct epithelium was pseudostratified, columnar, and nonciliated, and two cellular types were found: the principal and basal cells. These same cellular types were found in other vertebrate groups (Bloom and Fawcett,1977; Hamilton and Cooper,1978; Chimming,2001). In Cebus apella monkeys and other mammals, subtypes of principal cells, called apical and dark cells, were identified. These cells were not identified in P. geoffroanus and were not reported in C. crocodilus either (Chimming,2001; Guerrero et al.,2004).
The basal cells have a morphology that is similar to those reported for other species. In his research, Chimming (2001) argues that the basal cells are undifferentiated cells, capable of differentiation, which gives rise to the other cellular types of the duct epithelium.
The studies about deferent ducts mainly involve mammal species. Some information in the literature is brief, incomplete, and even sometimes contradictory, which makes it difficult to obtain accurate knowledge about the organ. Nevertheless, the use of sophisticated techniques such as histochemistry, physiology, and ultrastructure in some studies confirm that the organ is more than a mere conductor of spermatozoa.
The authors are thankful to Danielle Deremo for help with the translation of the manuscript.
- 2000. The surface of the epithelial lining of the ducts of the epididymis of the ostrich (Struthio camelus). Anat Histol Embryol 29: 119–126. , .
- 2002a. Cyclical reproductive changes in the non-ciliated epithelia of the epididymis of birds. Anat Histol Embryol 31: 113–118. .
- 2002b. Morphological changes in the efferent ducts during the main phases of the reproductive cycle of birds. J Morphol 253: 64–75. .
- 1989. Fisiologia. 2nd ed. Rio de Janeiro: Editora Guanabara Koogan. p 939. .
- 2006. Histological variation along and ultrastructural organization of the epithelium of the ducts epididymidis of the fan-throated lizard Sitana ponticeriana Cuvier. Acta Zool (Stockholm) 87: 181–196. , , .
- 1999. Morphometric evaluation of the epididymal area and efferent ducts and epididymal ducts in the domestic quail, throughout the year. Braz J Vet Res Anim Sci 36: 283–289. , , , , .
- 2002. Técnicas de coleta e preparação de vertebrados para fins científicos e didáticos. São Paulo: Instituto Pau Brasil de História Natural. p 89–93. , .
- 1977. Tratado de Histologia. 1st ed. Rio de Janeiro: Interamericana. p 509–527. , .
- 1987. Morphological and histochemical changes in the epididymis of hamsters (Mesocricetus auratus) subjected to short photoperiod. J Anat 191: 77–88. , , .
- 2005. Epididymial defensis and sperm maturation. Andrologia 37: 200–201. , .
- 2001. Ultrastructural features in the epididymis of the dog (Canis familiaris L.). Anat Histol Embryol 30: 327–332. , .
- 2001. Morphological study of the vas deferens in the tuffed capuchin monkeys, Cebus apella. Rev Chil Anat 19: 311–316. .
- 1972. Reproduction of the yellow mud turtle (Kinosternon flavescens flavescens) in New Mexico. Herpetologica 28: 130–137. , .
- 1991. Ham Histologia. 9th ed. Rio de Janeiro: Guanabara Koogan. p 738–783. .
- 1989. Turtles of the World. Washington, DC: Smithsonian Institution Press. , .
- 1990. Seasonal variation in the testicular cycle of the loggerhead musk turtle, Sternotherus minor minor, from central Florida. Can J Zool 68: 1071–1074. , .
- 2002. Reproductive cycle of male green iguanas, Iguana iguana (Reptilia:Sáuria:Iguanidae), in the pantanal of Brazil. Braz J Morphol Sci 19: 23–28. , , .
- 2007. Estrogen response system in the reproductive tract of the male turtle: an immunocytochemical study. Gen Comp Endocrinol 151: 27–33. , , , , .
- 2001. Sperm storage in the turtle: a male perspective. J Exp Zool 292: 180–186. , , , ,
- 1987. Storage of sperm in reptilian oviduct. Scan Microsc 1: 1839–1849 , .
- 2000. Chemical and thermal effects on the viability and motility of spermatozoa from the turtle epididymis. J Reprod Fertil 119: 271–277. , , .
- 2003. Cytological evaluation of spermatogenesis and organization of the germinal epithelium in the male slider turtle, Trachemys scripta. J Morphol 255: 337–347. , , .
- 2004. Morphology of the male reproductive duct system of Caiman crocodiles (Crocodylia, Alligatoridae). Ann Anat 186: 235–245. , , , .
- 1989. Aspectos da reprodução de Phrynops geoffroanus (Schweigger, 1812) em cativeiro (Testudines, Chelidae). Grupo de Estudos Ecológicos, Série Documentos. 1: 1–19. , , , .
- 1978. Gross and histological variation along the length of the rat vas deferens. Anat Rec 190: 795–810. , .
- 2004. Princípios integrados de zoologia. 11th ed. Rio de Janeiro: Guanabara Koogan. p 531–538. , , .
- 1990. Fixation e fixatives. Cap 2:21–42. In: BancroftJD, StevensA. Foreword by DavidRT. 1990. Theory and practice of histological techniques. 3rd ed. Edinburgh, London, Melbourne and New York: Churchill Livingstone Inc. 726 p.
- 2004. Excurrent duct system of the male turtle Crysemys picta. J Morphol 261: 312–322. , .
- 1994. Structure and function of the ductuli efferentes: a review. Microsc Res Tech 29: 432–467. , .
- 1999. To store or mature spermatozoa? The primary role of the epididymis. Int J Androl 22: 57–67.
- 1992. The testicular cycle of the common snapping turtle, Chelydra serpentina, in Wisconsin. Herpetologica 48: 193–201. , .
- 1962. Chordate morphology. Reinhold Books in the Biological Sciences. Pittsburg, Pennsylvania: Reinhold Publishing Corporation. 41 p. .
- 1991. Structure and ultrastructure of the epididymis of the viviparous lizard during the annual hormonal cycle: changes of the epithelium related to secretory activity. J Morphol 210: 133–145. , , , , .
- 2002. Spermatogenic cycle of the florida softshell turtle, Apalone ferox. Copeia 3: 779–786. , , .
- 1992. Observações sobre o comportamento agonístico de cágados Phrynops geoffroanus (Schweigger, 1812) (Reptilia, Testudines, Chelidae) em cativeiro. Biotemas 5: 79–84. .
- 1998. Comportamento e biologia reprodutiva dos cágados Phrynops geoffroanus, Acanthochelis radiolata e Acanthochelis spixii (Testudines, Chelidae) em cativeiro. Rev Bras Etol 25–40; Special number. .
- 1985. Biologia dos Vertebrados. 5th edição. São Paulo: Livraria Rocca Ltda. 508 p. .
- 2007. Variabilidade sazonal no ducto epididimário de codorna doméstica: observações morfológicas. Pesquisa Veterinária Brasileira 27: 495–500. , , , , .
- 2003. A Vida dos Vertebrados. 3rd ed. São Paulo: Atheneu. p 270–290. , , .
- 1979. Encyclopedia of Turtle. New York: TFH Publication Ltd. .
- 1985. Anatomia comparada dos vertebrados. 5th ed. São Paulo: Atheneu. 599 p. .
- 1998. The appearance of basal cells in the developing murine epididymis and their temporal expression of macrophage antigens. Int J Androl 21: 217–226. , , , , , .
- 2004. Structural features of the epididymal region of the domestic duck (Anas plathyrynchos). Bras J Vet Res Anim Sci 41: 92–97. , , , , , .
- 1999. Morphologic study of the efferent ductules of the pigeon (Columba livia). J Morphol 242: 247–255. , , , , .
- 2008. Systematic review of late Pleistocene Turtles (Reptilia: Chelonii) from the Ryukyu Archipelago, Japan, with special reference to paleogeographical implications. Pacific Sci 62: 395–402. , , .
- 1993. Immunocytochemical localization of the Yf subunit of glutathione S-transferase P shows regional variation in the staining of epithelial cells of the testis, efferent ducts and epididymis of the male rat. J Androl 14: 23–44. , , .
- 1994. Basal cells of the human epididymis—antigenic and ultrastructural similarities to tissue-macrophages. Biol Reprod 50: 917–926. , , , , , , .
- 1999. Biostatistical analyses. New Jersey: Printice Hall, 663 p. .