Morphology of female genital tracts in Dasypodidae (Xenarthra, Mammalia): a comparative survey
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- Cetica, P.D., Aldana Marcos, H.J. & Merani, M.S. Zoomorphology (2005) 124: 57. doi:10.1007/s00435-005-0111-5
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Previous works about comparative spermatology in Dasypodidae determined that sperm morphology is a striking variable among genera. It was suggested that this sperm feature may be related to specific morphologies of the female reproductive tract. The present comparative study of the morphology of the female genital tract from seven species corresponding to six genera of Dasypodidae is aimed to determine the main similarities and differences between the species and to establish a possible correlation with the sperm shapes and sizes. Genital tracts were studied macroscopically and histologically. Dasypus hybridus has disk-shaped ovaries and the cortex occupies almost all the organ with a single oocyte in each follicle. Tolypeutes matacus, Chaetophractus villosus, Chaetophractus vellerosus, Zaedyus pichiy, Cabassous chacoensis and Clamyphorus truncatus possess ovoid and elongated ovaries, with both longitudinally polarized cortex and medulla, and the peculiar presence of several oocytes in the same follicle. D. hybridus and T. matacus have a simple pear-shaped uterus, but in the other species the uterus is pyramid shaped and bicornuate. The uterine cervix is very long in all studied species. Only T. matacus presents a true vagina as in most eutherian mammals; on the other hand, in the other species a urogenital sinus is observed. The structure of female reproductive tracts in Dasypodidae contains a mixture of assumedly primary and other derived features. According to the different morphologies of the regions analyzed, a classification of the female genital tracts in three groups can be performed (group 1: Dasypus; group 2: Tolypeutes; group 3: Chaetophractus, Zaedyus, Cabassous, Clamyphorus) and a correlation between each group and a specific sperm morphology can be established.
The reproductive biology of Dasypodidae is poorly known and the scarce available data show that these species stand out among eutherians as regards this subject (Galbreath 1985; Storrs et al. 1989; Laughry et al. 1998). Our previous work about comparative spermatology in Dasypodidae determined that sperm shape and size are not constant across taxa, contrary to what has been observed in most eutherian taxa (Cetica et al. 1993, 1997). An important evolutive differentiation was established on sperm morphology and morphometry between the different taxa in Dasypodidae, determining four different sperm types (Cetica et al. 1998).
Spermatozoa are likely to be under intense selective pressure given their crucial role in reproduction. The evolution of the features such as sperm shape and size is probably the result of two major selective forces, namely sperm competition between males and female reproductive functional features (Roldan et al. 1992a, b).
In animals with internal fertilization, females from different species show a wide range of internal environments and of genitalia, and this has lead to the evolution of a surprising variety of sperm morphologies. Spermatozoa appear to have undergone functional, metabolic and morphological changes as a consequence of the need to survive in and progress through the female tract. This relationship suggests some sperm adaptations to the environment and structures that they encounter within the female reproductive system (Sivinski 1984). Although it has been proposed that sperm shape and dimension could be related to specific morphologies of the female reproductive tract (Smith and Yanagimachi 1990; Roldan et al. 1992a, b; Gomendio and Roldan 1994), only a few studies in Coleoptera and Passeriformes have examined the relationship between sperm size and female genital tract dimensions (Dybas and Dybas 1981, Briskie and Montgomerie 1992).
In spite of the basal position of the group, there is a scarcity of papers dealing with the structure of the whole female reproductive tracts in Dasypodidae and only in the genus Dasypus (Altmann 1924; Talmage and Buchanan 1954; Enders and Buchanan 1959). Macroscopic differences in uterine shapes were observed among some species of Dasypodidae (Galbreath 1985). Different morphologies of the female reproductive tracts could be correlated with the different sperm types described in Dasypodidae.
The aim of this work was to perform a comparative study of the main macroscopic and histological characteristics of the female genital tract in seven species of Dasypodidae to determine the main similarities and differences between the studied species, and to establish a possible correlation with the sperm shapes and sizes.
Materials and methods
Eighteen adult female genital tracts were used in this study: 3 Dasypus hybridus Desmarest, 1804 (southern lesser long-nosed armadillo), 4 Tolypeutes matacus Desmarest, 1804 (Azara’s domed armadillo), 4 Chaetophractus villosus Desmarest, 1804 (large hairy armadillo), 1 Chaetophractus vellerosus Gray, 1865 (lesser hairy or crying armadillo), 1 Zaedyus pichiy Desmarest, 1804 (pichi), 1 Cabassous chacoensis Wetzel, 1980 (chacoan naked-tailed armadillo) and 4 Clamyphorus truncatus Harlan, 1825 (pink fairy armadillo). The animals were obtained from different sources: (1) D. hybridus (†15/06/94; †15/08/94; †21/12/94), Ch. villosus (†10/03/96; †23/11/96; †02/07/97; †14/08/97), Ch. vellerosus (†20/10/96) and Z. pichiy (†09/11/96) from the Instituto de Neurociencias, CONICET, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (Argentina), (2) T. matacus (†19/04/95; †19/08/95; †25/07/95; †18/10/95) and C. chacoensis (†11/08/96) were found dead in the fields of Chaco and Santiago del Estero Provinces, respectively (Argentina) and (3) C. truncatus (#199; 208, 584) from the museum collection of the Museo Municipal de Historia Natural de San Rafael (Mendoza) and (#118) from the mammalian collection of the Museo Argentino de Ciencias Naturales Bernardino Rivadavia (Buenos Aires).
Ovaries and some remnants of the reproductive tract from C. chacoensis were not recovered by the field collector and samples from C. truncatus were not in excellent fixation conditions, but they were studied due to the scarce possibilities to get these threatened species, which are classified as vulnerable in the “Libro Rojo de Mamíferos Amenazados de la Argentina” (Red Book of Threatened Mammals from Argentina) (Díaz and Ojeda 2000). In this book, D. hybridus and T. matacus are considered potentially vulnerable.
The reproductive tracts had been kept in 10% formalin. Macroscopic identification of the different regions of the genital tracts was made under stereomicroscopic observation.
For histological studies, the genital tracts and surrounding tissues were dissected and further re-fixed in 10% formalin overnight. Samples from the different regions were dehydrated and embedded in paraffin. Sections of 5-μm thickness were stained with hematoxylin-eosin (H-E), Masson’s trichrome, orcein for elastic fiber detection and Periodic-Acid-Schiff (PAS) reaction for carbohydrate localization.
The oviducts are coiled and filiform, and their lengths from the infundibulum to their insertion into the uterine wall are 2.0 to 2.5 cm in Ch. villosus, 1.5 cm in Ch. vellerosus, 2.0 cm in Z. pichiy, 1.5 cm in C. chacoensis, 0.6 to 0.7 cm in C. truncatus, 2.0 cm in T. matacus and 2.5 to 3.0 cm in D. hybridus. Oviducts possess the three characteristic tubal parts present in most eutherians (infundibulum, ampulla and isthmus) and insert into each side of the fundic portion of the uterus (Fig. 2b). Histologically the wall consists of three layers: a tunica mucosa, a tunica muscularis and a tunica serosa. The mucosa is characterized by an epithelium that shows a typical pseudoestratified epithelium with ciliated, non-ciliated columnar (secretory) and basal cells, surrounded by a lamina propria. The ampulla has high-branched mucosal folds (Fig. 1b), and the isthmus has few folds and is more muscular (Fig. 2b). In D. hybridus, the mucosa of the isthmus region close to the uterus is very similar to that observed in the endometrium.
The uterine cervix is one of the longer regions of the genital system in the studied Dasypodidae, occupying about 60–70% of the overall length of the uterus. It is a slightly arched anteroposterior muscular tube with longitudinal folds lined by a cubic to columnar simple epithelium (Fig. 4c), which bears a prominent distal fringe of PAS positive mucus. In the anterior region of the cervix of Ch. villosus and Z. pichiy the epithelium and lamina propria are so extensively folded that they appear to be “glandular” (Fig. 4c). Only in D. hybridus, scarce epithelial glands occur throughout the length of the tube (Fig. 4d). In all species, the lamina propria contains well-developed sinusoids and loose connective tissue. The muscularis externa contains longitudinal, circular and obliquely oriented smooth muscle bundles, which are a continuation of the outer layers of the myometrium.
Lower portion of the genital tract
On the other hand, in Ch. villosus, Ch. vellerosus, Z. pichiy, C. chacoensis, C. truncatus and D. hybridus, the cervix leads to a tubular structure where the columnar epithelium changes abruptly to transitional epithelium (Figs. 4g, 5a–d, f), forming a urogenital sinus rather than a true vagina (Fig. 4h). The length of the urogenital sinus is about 1.8 to 2.5 cm in Ch. villosus, 1.2 cm in Ch. vellerosus, 1.8 cm in Z. pichiy, 1.8 cm in C. chacoensis, 0.4 to 0.5 cm in C. truncatus and 1.5 to 2.0 cm in D. hybridus. The urogenital sinus in Ch. villosus is a relatively long tube with two zones, a caudal or ascendent one and a cranial or descendent one, with the opening of the cervix as a borderline separating them. The urethra opens on the dorsal wall of the middle portion of the descendent zone (Fig. 5a). In Ch. vellerosus and Z. pichiy, the urogenital sinus is a long straight tube with a small final dilatation. It is joined to two tubes, a ventral tube, the urethra, and a dorsal tube, the cervix (Fig. 5c, d). In C. truncatus, the urogenital sinus is like an inverted “s” and the cervix and urethra open to its end (Fig. 5f). The urogenital sinus of D. hybridus is a large and slightly arched tube; in the middle region, it joins to the dorsal cervix and close to the end the opening of the urethra can be seen (Fig. 5b). For C. chacoensis, it was not possible to sketch the complete genital tract. Despite this, the analysis of the transversal histological sections clearly showed the presence of a urogenital sinus.
Both the vagina or the urogenital sinus are flaccid and distensible cavities, and the lumen shows broad, longitudinal folded outlines covered by the epithelium and dense collagen connective tissue with scarce elastic fibers. The lamina propria contains a highly vascular erectile tissue of the vestibulo-vaginal bulb that also surrounds the urethra (Fig. 4e). This series of sinusoids anastomose freely and connect with cervical sinusoids. Close to the external orifice, the epithelium of the vagina or the urogenital sinus changes gradually to stratified keratinized epithelium.
The vulva in all studied species is large. The length ranges in D. hybridus 0.8 to 1.0 cm, T. matacus 1.1 to 1.5 cm, Ch. villosus from 2.0 cm to 3.0 cm, Ch. vellerosus 0.8 cm, Z. pichiy 1.2 cm, C. chacoensis 1.0 cm and C. truncatus 0.4 to 0.5 cm. In studied specimens, the clitoris may be partially located in a cutaneous pocket or appear slightly out of it. A histological cross section denotes the presence of two ventral fascicles of striated retractor clitoridis muscle and the erectile tissue structure corpus cavernosum clitoridis (Fig. 4i). The surface of the clitoris is covered with stratified squamous keratinized epithelium.
From the comparative analysis of the macro and microscopical features of female genital tracts in Dasypodidae, we observed two main different morphologic types in the following structures: ovaries, uterus and the lower portion of the reproductive tract.
Ovary type 1 (D. hybridus) is disk-shaped and the cortex occupies almost all the organ, with only scarce medulla-like tissue in the convex surface. Only follicles with a single oocyte are present in the cortex. These features are very similar to those previously described in D. novemcinctus Linné, 1758 (Enders and Buchanan 1959). Ovary type 2 (T. matacus, Ch. villosus, Ch. vellerosus, Z. pichiy and C. truncatus) is ovoid, elongated and it has a longitudinal polarization in the cortex and medulla. Usually, ovarian follicles contain a single oocyte in mammals (Weichert 1967; Kluge 1977; McDonald 1989), but in these species several follicles contain groups of oocytes surrounded by a common follicular cell-layer, especially those at early stages of development. Codón and Casanave (2000) have observed the same polyovular follicles in Ch. villosus, Ch. vellerosus and Z. pichiy. The follicular dynamics and reproductive significance of these follicles is difficult to explain at the present time, and further studies about ovarian physiology have to be carried out in Dasypodidae in order to determine its function.
Both types of ovaries do not show the typical organization in central medulla and external cortex that is usual in most mammals (Weichert 1967; Kluge 1977; McDonald 1989). This feature was also reported in some species of Dasypodidae by other authors (Enders and Buchanan 1959; Codón and Casanave 1996, 2000). This particular medullary cortical distribution in ovaries from adult Dasypodidae resembles that observed in mammalian ovaries during early embryogenesis (van Wagenen and Simpson 1965; Hamilton and Mossman 1973; Carlson 2000). Probably, the ovaries of Dasypodidae could be related to more primary forms of mammalian female gonads, as it is also suggested by the presence of embryonic remnants not completely degenerated in most of them. In agreement, mitotic figures were observed in medullary cords in ovaries of D. novemcinctus (Enders and Buchanan 1959).
Uterus type 1 (D. hybridus and T. matacus) is pear-shaped and classified as simple, as it has also been observed in D. novemcinctus and Tolypeutes tricinctus (Enders and Buchanan 1959; Galbreath 1985). Uterus type 2 (Ch. villosus, Ch. vellerosus, Z. pichiy, C. chacoensis and C. truncatus) is pyramid-shaped, with a well-developed body and two small lateral horns. Due to the unsettled controversy between different authors with respect to the classification of eutherian uterine shapes (Grassé 1969; Nalbandov 1964; Weichert 1967; Kluge 1977; McDonald 1989), we considered the uterus type 2 as bicornuate because of the only presence of uterine horns. Galbreath (1985) also described the presence of a bicornuate uterus in Ch. villosus, Ch. vellerosus, Euphractus sexcintus Linné, 1758 and Cabassous centralis Miller, 1899.
It is generally assumed that in the evolution of eutherian mammals the uterus undergoes a change from double, through different bicornuate-like types, to the simple form, the former type being associated with a high number of offprings and the latter one with one or a low number of offprings (Newfang 1947; Kluge 1977). This relationship fits well in domestic animals (McDonald 1989), and in other Xenarthra that possess simple uteri (Myrmecophagidae) or unicornuate uteri (Bradypodidae and Melanonychidae) and have a single young (Bernirschke 2004). Nevertheless, the number of offprings reported for different species of Dasypodidae varies from 1 to 12 (Birabén 1951; Galbreath 1985; Laughry et al. 1998; Adamoli et al. 2001), independently of the uterine shape. Probably this feature may represent a reproductive strategy in these species. It has been suggested that independently of the uterine shape, all Dasypodidae possess “functionally simplex” uteri according to the implantation of the conceptus (Galbreath 1985) and that this shared function could partly be due to the fact that they share the same hemochorial placental type (Enders 1960; Adamoli et al. 2001).
The uterine cervix is very long in all studied species of Dasypodidae. Enders and Buchanan (1959) described the equivalent organ in D. novemcinctus as a vagina because on the basis of gross structure it is where the vagina would be expected to be to receive the seminal discharge. We consider this tubular structure as a cervix because the PAS positive mucus secretion, the type of epithelium, the lamina propia and the surrounding muscularis externa of smooth muscle continuation of myometrium are very similar to those observed in most mammalian cervixes (Corbeil et al. 1985; McDonald 1989), as it was also proposed in genus Dasypus by other authors (Altmann 1924; Newfang 1947). Furthermore, in many mammals the ejaculation is not necessarily vaginal, like in swines, which present intracervical seminal discharge (McDonald 1989).
Lower portion of the genital tract
In T. matacus, the tubular structure lined by a stratified squamous epithelium forms a true vagina, as described in most eutherian mammals (Weichert 1967; Corbeil et al. 1985; McDonald 1989). On the other hand, in D. hybridus, Ch. villosus, Ch. vellerosus, Z. pichiy, C. chacoensis and C. truncatus the tubular structure lined by a transitional epithelium determines a urogenital sinus. In D. novemcinctus was also described the presence of a urogenital sinus, which is considered a plesiomorphic feature in mammals (Newfang 1947; Talmage and Buchanan 1954; Enders and Buchanan 1959). Supporting the idea of the primary nature of this structure in Dasypodidae, urogenital sinuses were also described in monotremes and marsupials. Moreover, during embryonic development in eutherian mammals the cloaca is modified by the formation of a transverse septum that separates a dorsal digestive chamber and a ventral urogenital sinus, before the differentiation of the definitive vagina (Burns 1939; Weichert 1967; Kluge 1977).
A striking feature observed in all studied armadillos is the presence of two ventral fascicles of striated retractor clitoridis muscle. In other eutherian, like domestic mammals, this muscle is smooth (Sisson and Getty 1982).
Relation between sperm shape and female genital tract morphology
According to the different morphologic types of the reproductive tract regions analyzed above in Dasypodidae, a classification of these female genital tracts into three groups can be performed: group 1 (ovary type 1, uterus type 1 and urogenital sinus) for D. hybridus and D. novemcinctus, group 2 (ovary type 2, uterus type 1 and vagina) for T. matacus and group 3 (ovary type 2, uterus type 2 and urogenital sinus) for Ch. villosus, Ch. vellerosus, Z. pichiy, C. chacoensis and C. truncatus.
A correlation between the three groups of the female genital tracts and sperm morphology in Dasypodidae (Cetica et al. 1998) can be established. Species of Dasypodidae with reproductive tracts from group 1 present the sperm type 1 (short spermatozoa with paddle-shaped head: Dasypus); those from group 2 show the sperm type 2 (mid spermatozoa with paddle-shaped head and laterocaudal nuclear extension: Tolypeutes) and those from group 3 show the closely related sperm types 3 and 4 (long spermatozoa with spoon-shaped heads: Chaetophractus, Zaedyus, Cabassous).
From these observations, it may be assumed that different structural features of the genital tract in females from each genus have some relationship with sperm morphology and morphometry in Dasypodidae. There is not yet a comprehensive explanation of adaptive modifications in mammalian sperm shape and sizes, but it has been suggested that they would be related to specific morphologies of the female reproductive tract and to the particular features of the uterine and oviductal fluids (Smith and Yanagimachi 1990; Roldan et al. 1992a, b; Gomendio and Roldan 1994).
In Dasypodidae, female genital tracts could represent different functional and structural barriers for spermatozoa that would induce changes in sperm shape and dimensions, suggesting a process of co-evolution between female reproductive biology and male gamete.
We thank Dr. J.M. Affani from the Instituto de Neurociencia (CONICET), Dr. H. Lagiglia from Museo Municipal de Historia Natural de San Rafael, Dr. O. Vaccaro from Museo Argentino de Ciencias Naturales Bernardino Rivadavia and M.L. Bolcovich for help in obtaining the samples. We acknowledge Dr. A.J. Solari for the critical reading of the manuscript. This research was supported by grants of PICTR 00074.