Germ cell cysts, a fetal feature in mammals, are constitutively present in the adult armadillo.

Formation and subsequent break down of ovarian germ cell (GC) cysts is a key and an evolutionary-conserved developmental event, described in phylogenetically diverse species of invertebrates and vertebrates. In mammals, cyst break down (CBD) ends at the time of, or soon after, birth with the formation of primordial follicles enclosing single oocytes, which constitute the sole reservoir of gametes available through the whole female's reproductive life. In this study, we challenge this paradigm demonstrating the constitutive presence of a large number of cysts, enclosing two-thirty GCs, in the ovary of the adult armadillo Chaetophractus villosus, belonging to the superorder Xenarthra, one of the earliest offshoots among placentals. We also describe that (a) GCs enclosed within cysts are connected by intercellular bridges-intercellular bridges-markers of their clonal origin; (b) CBD occurs through four main phases, ending with primordial follicles containing single oocytes; (c) GCs encompass meiotic prophase I stages, from leptotene to diplotene; (d) seasonal variations in the number of GCs enclosed within cysts, suggesting the presence of a GC multiplying activity. The armadillo C. villosus''s ovary emerges as an extraordinary resource to investigate folliculogenesis and to explore the evolutionary past of the mammalian ovary.

Sertoli-immature spermatids disengagement during testis regression in the armadillo.

In nature, mammalian seasonal breeders undergo spermatogenetic arrest during the non-breeding season. In the large hairy armadillo Chaetophractus villosus, testis regression initiates with immature post-meiotic germ cells sloughing into the tubule lumen and continues with the death of the remaining spermatocytes. At the end of the regression period, only spermatogonia and Sertoli cells persist in the seminiferous epithelium. It has been suggested that cell sloughing is determined by changes in the adhesion complexes between Sertoli cells and spermatids, which are mediated by low intra-testicular testosterone levels. By immunofluorescence and Western blotting we studied key proteins of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks that contribute to the complex Sertoli/spermatid adhesion system throughout the eight stages of the seminiferous epithelium cycle in the comparison between active and regressing testes. In active testis, B1-integrin, laminin G3, N-cadherin, B-catenin, P-B-catenin-Tyr654, FAK, P-FAK-Tyr397, SRC, P-SRC-Tyr416 proteins present a spermatogenetic cycle-dependent localisation pattern, unmaintained in regressing testes. In the latter, quantitative variations and changes in the phosphorylation state of protein FAK, SRC and B-catenin contribute to the disassembly of the N-cadherin/N-cadherin and A6B1-integrin/laminin interlocks, thus promoting the massive release of immature spermatids.

Cytogenetic damage in peripheral blood cultures of Chaetophractus villosus exposed in vivo to a glyphosate formulation (Roundup).

Different concentrations of a glyphosate formulation, Roundup® Full II (66.2% glyphosate) were tested in culture peripheral blood of armadillo Chaetophractus villosus with cytogenetic biomarkers like mitotic index (MI), chromosomal aberrations (CA), sister chromatid exchange (SCE) and cell proliferation kinetics (CPK) by means of replication index. Adults animals of both sexes were exposed to RU at four concentrations ranging from 0.026 mL RU solution to 0.379 mL RU daily in oral treatment with the same volume (0.2 mL) during 7 days. We analyzed the induced damage at different times considering T0 as control value, one (T1), seven (T7) and 30 days (T30). One day after, only the higher concentration shows MI significant differences (p < 0.05), at T7 the frequency increases and at T30 it decreases reaching T0 values. The analysis of CA frequencies shows that only 0.106 mL RU/day exhibit significant differences vs T0 values. A great variability is expressed in the values of standard deviation (SD) and in the wide confidence intervals of the media. One day after treatments (T1) all four concentrations shows significant differences in SCE vs T0 values. Replication Index (RI) does not show significant differences. The dose-response behavior was not observed in either CA or SCE. The consistency of the findings obtained with the same biomarkers in vitro support the idea of expanding studies in order to characterize the risk doses for these mammals.

Genotoxic effects of Roundup Full II® on lymphocytes of Chaetophractus villosus (Xenarthra, Mammalia): In vitro studies.

In Argentina, Chaetophractus villosus has a wide distribution that overlaps with agricultural areas where soybean is the predominant crop. In such areas the pesticide Roundup Full II® (RU) is widely applied. The genotoxic effect of its active ingredient glyphosate (RU is 66.2% glyphosate) on the peripheral blood lymphocytes of C. villosus was tested over a range of concentrations (280, 420, 560, 1120 µmol/L). Culture medium without glyphosate served as negative control, while medium containing mitomycin C served as positive control. Genetic damage was characterized in terms of the percentage of cells with chromosome aberrations (CA), the mean number of sister chromatid exchanges (SCE) per cell, and the modification of cell proliferation kinetics via the calculation of the replication index. Significant increases (p < 0.0001) were seen in the CA frequency and the mean number of SCEs per cell compared to negative controls at all the RU concentrations tested. Chromatid breaks, the only form of CA observed, under the 560 µmol/L RU conditions and in presence of mitomycin C were four to five times more common than at lower concentrations, while no viable cells were seen in the 1120 µmol/L treatment. The mean number of SCEs per cell was significantly higher under the 280 µmol/L RU conditions than the 420 or 560 µmol/L RU conditions; cells cultivated in the presence of MMC also showed significantly more SCEs. All the RU concentrations tested (except in the 1120 µmol/L RU treatment [no viable cells]) induced a significant reduction in the replication index (p < 0.0001). The present results confirm the genotoxic effects of RU on C. villosus lymphocytes in vitro, strongly suggesting that exposure to RU could induce DNA damage in C. villosus wildlife.

Chaetophractus villosus as a sentinel organism: Baseline values of mitotic index, chromosome aberrations and sister chromatid exchanges.

Sentinel species are useful tools for studying the deleterious effects of xenobiotics on wildlife. The large hairy armadillo (Chaetophractus villosus) is the most abundant and widely distributed mammal in Argentina. It is a long-lived, omnivorous, burrowing species, with fairly restricted home ranges. To evaluate the level of spontaneous genetic damage in this mammal, we determined the baseline values of several genotoxicity biomarkers. The study included 20 C. villosus adults of both sexes from eight pristine localities within its geographic distribution range. Genotoxicity analysis was performed on 72-h lymphocyte cultures, using mitomycin C as positive control. We obtained the baseline values of mitotic index (MI=10.52±0.30 metaphases/total cells, n=20), chromosome aberrations (CA=0.13±0.22, n=20), sister chromatid exchanges (SCE)=6.55±0.26, n=6) and replication index (RI=1.66, n=6). MI and CA did not show significant differences (P>0.05) among localities or between sexes. No significant differences in MI, CA, SCE, and RI (P>0.05) were found between values from the pristine localities and historical data. There were significant differences in CA, SCE, and RI (P<0.05) between lymphocyte cultures from pristine localities and those exposed to mitomycin C. We propose the large hairy armadillo as a sentinel organism for environmental biomonitoring of genotoxic chemicals due to its abundance, easy manipulation, well-known biology, the fact that it is usually exposed to different mixtures and concentrations of environmental contaminants, and the baseline values of genetic damage characterized by MI, CA, SCE and RI as biomarkers.

Loss of Sertoli-germ cell adhesion determines the rapid germ cell elimination during the seasonal regression of the seminiferous epithelium of the large hairy armadillo Chaetophractus villosus.

The armadillo Chaetophractus villosus is a seasonal breeder whose seminiferous epithelium undergoes rapid regression with massive germ cell loss, leaving the tubules with only Sertoli cells and spermatogonia. Here, we addressed the question of whether this regression entails 1) the disassembly of cell junctions (immunolocalization of nectin-3, Cadm1, N-cadherin, and beta-catenin, and transmission electron microscopy [TEM]); 2) apoptosis (immunolocalization of cytochrome c and caspase 3; TUNEL assay); and 3) the involvement of Sertoli cells in germ cell phagocytosis (TEM). We showed a dramatic reduction in the extension of vimentin filaments associated with desmosomelike junctions at the interface between Sertoli and germ cells, and an increased diffusion of the immunosignals of nectin-3, Cadm1, N-cadherin, and beta-catenin. Together, these results suggest loss of Sertoli-germ cell adhesion, which in turn might determine postmeiotic cell sloughing at the beginning of epithelium regression. Then, loss of Sertoli-germ cell adhesion triggers cell death. Cytochrome c is released from mitochondria, but although postmeiotic cells were negative for late apoptotic markers, at advanced regression spermatocytes were positive for all apoptotic markers. Transmission electron microscopy analysis showed cytoplasmic engulfment of cell debris and lipid droplets within Sertoli cells, a sign of their phagocytic activity, which contributes to the elimination of the residual meiocytes still present in the latest regression phases. These findings are novel and add new players to the mechanisms of seminiferous epithelium regression occurring in seasonal breeders, and they introduce the armadillo as an interesting model for studying seasonal spermatogenesis.