The fetomaternal interface in the placenta of three species of armadillos (Eutheria, Xenarthra, Dasypodidae)
© Rezende et al.; licensee BioMed Central Ltd. 2012
Received: 28 February 2012
Accepted: 24 April 2012
Published: 4 May 2012
Placental characters vary among Xenarthra, one of four supraordinal clades of Eutheria. Armadillos are known for villous, haemochorial placentas similar to humans. Only the nine-banded armadillo has been well studied so far.
Placentas of three species of armadillos were investigated by means of histology, immunohistochemistry including proliferation marker, and transmission and scanning electron microscopy.
The gross anatomy differed: Euphractus sexcinctus and Chaetophractus villosus had extended, zonary placentas, whereas Chaetophractus vellerosus had a disk. All taxa had complex villous areas within the maternal blood sinuses of the endometrium. Immunohistochemistry indicated the validity of former interpretations that the endothelium of the sinuses was largely intact. Tips of the villi and the columns entering the maternal tissue possessed trophoblast cell clusters with proliferation activity. Elsewhere, the feto-maternal barrier was syncytial haemochorial with fetal vessels near the surface.
Differences among armadillos occurred in regard to the extension of the placenta, whereas the fine structure was similar. Parallels to the human suggest that armadillos are likely to be useful animal models for human placentation.
Molecular cladistics divides eutherian mammals into four supraordinal clades. Xenarthra likely has a basal position and may influence interpretations of character transformations [1–4]. Phylogenetics have stimulated evolutionary studies as on the placental system [5–9]. Distinct placental types have been recognized with regard to invasiveness, shape and internal organization [6, 8]. However, only about 1% of eutherian species have been investigated, varying on ordinal and family levels . Attention to rather exotic species such as armadillos were drawn from the middle of 20th century on [11, 12], indicating that placental characters vary within Xenarthra. Armadillos are known for haemochorial, villous placentas [11–14] similar to the human , indicating that they could be used as animal models . Only the nine-banded armadillo Dasypus novemcinctus from North America is well studied in regard to placental development [11–14, 17–20]. Data show a peculiar condition in that the developing villi entered pre-existing maternal blood sinuses and enlarged them while leaving the maternal vessels endothelium largely intact [11, 13, 14]. The sinuses were supplied by derivatives of the spiral arteries that were passing in endometrial septae and opened into the intervillous space [11, 13, 14]. However, data for other armadillo species are sparse [21–23]. We investigated armadillos from Brazil and Argentina, the large hairy armadillo Chaetophractus villosus desmarest, 1904, the small hairy armadillo Chaetophractus vellerosus gray, 1865, and the six-banded armadillo Euphractus sexcinctus, linnaeus, 1758. The species studied here exhibit litter sizes of 1 to 3 young and gestation periods of 60 to 70 days and inhabit pampas, cerrado and chaco vegetations [24–28].
Term placentas of Chaetophractus villosus and Chaetophractus vellerosus were obtained from the Museum of the University of La Plata, Argentina. Two pregnant females of Euphractus sexcinctus, both in the second half of gestation, were obtained from wild specimens found dead on a Brazilian highway. Tissues were processed similar to former studies (see [29–31] for details). Material for histology, fixed in 10% formalin in 0.1 M phosphate buffer, was sectioned at 5 μm and stained with hematoxylin and eosin (HE), Masson´s Trichrome and periodic acid-Schiff reaction (PAS). Samples for SEM were fixed in 2.5% glutaraldehyde, post-fixed in 2% phosphate-buffered osmium tetroxide, critical point dried and gold sputtered. TEM samples were embedded in Spurr’s Resin; ultrathin sections were contrasted with 2% uranyl acetate and 0.5% lead citrate. We used TEM only for the feto-maternal barrier, because the quality of the material derived from museums and dead animals was not the best. Immunohistochemistry (see [29–31]) was performed in Chaetophractus villosus for vimentin (mouse monoclonal anti-human antibody: RTU-VimV9; ready to use concentration; novocastra, Wetzlar, Germany) and cytokeratin (rabbit polyclonal antibody: wide spectrum screening N1512; ready to use concentration; dako, Cytomation, Carpinteria, USA) and as a proliferation marker a mouse monoclonal antibody to human anti-PCNA (proliferation cell nuclear antigen, clone PC10; 1:300; sigma, St Louis, USA); then a biotinylated secondary antibody and streptavidin-HRP (Dako) were applied and detection was done by Envision and a DAB and substrate chromogen system (dako). Negative controls were performed using bovine serum albumin to substitute the primary antibody. The research was approved by the Ethical Committee at the Faculty of Veterinary Medicine and Animal Science of the University of Sao Paulo.
Differences in armadillos have been revealed in regard to the form of the definitive placenta. Extended, zonary placentas occurred in Chaetophractus villosus, Euphractus sexcinctus as well as in Cabassous chacoensis Tolypeutes matacus and Dasypus hybridus. Dasypus novemcinctus possessed a restricted zonary placenta [11, 13]. A disk occurred in Chaetophractus vellerosus. For Chaetophractus villosus Chaetophractus vellerosus and the other species this represented the term state, whereas for Euphractus sexcinctus we did not have material from late gestation. All armadillo species studied so far had a complex villous area [11–14, 17–23]. The application of immunohistochemistry in Chaetophractus villosus indicated the validity of former interpretations derived from Dasypus novemcinctus, that the developing villi entered maternal blood sinuses without fully destroying and replacing the vessels endothelium [11, 13, 14]. In addition, our results proved the expression of a proliferation marker in the trophoblast cell clusters especially occurring at the tips of the villi [11–13, 19–23]. Thus, proliferation can be assumed as general activity of these cells, as was suggested by former work on morphology only [11–13]. In the human, the syncytiotrophoblast is formed from proliferative cytotrophoblast cells [14, 32, 33]. We suggest that these cytotrophoblast cells in armadillos may have a similar function. Elsewhere in the villi, the interhaemal barrier was haemomonochorial, syncytial and thin in places; these areas were associated with hypertrophied mesenchymal cells and capillaries that were near the surface [11–14, 19–23]. These features exhibit parallels to humans . The way in which the villous areas were established was different in primates and armadillos. In humans invasive trophoblast cells migrate deeply into the maternal tissues and remove the endothelium of the arteries [32–35]. However, armadillos are among the very few mammals with villous and invasive placentas. Thus, they may play a role as additional animal models for human placentation .
Principal differences between three armadillo species have been revealed in regard to the extension of the placenta, whereas the fine structure was mostly similar. Major parallels occurred compared to the human, suggesting that armadillos may play a role as additional animal models for particular questions.
We thank Dr. Antonio Carlini, Fernando Galiari and Cecilia Krmpotic, Department of Vertebrate Palaeontology at the University of La Plata, for providing the specimens of Chaetophractus. Prof. Jussara R. Ferreira and Prof. Selma A. Souza Kückelhaus, University of Brasilia, provided the material of Euphractus sexcinctus. We thank Prof. Anthony Carter, University of Odense in Denmark, for comments on a former version of the manuscript. Anthony Carter and Dr. Karl Klisch, University of Nottingham in Great Britain, helped with the English. This project was supported by CNPq and FAPESP.
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