Glycosylation of the materno-foetal interface in the pregnant viviparous placentotrophic lizard Chalcides chalcides: a lectin histochemical study.

Glycosylation of the foeto-maternal interface of the skink Chalcides chalcides has been examined at various stages of gestation using lectin histochemistry. Specimens of incubatory chamber or placenta from early, mid-, late- and near-term pregnancy were fixed and embedded in epoxy resin. Areas of foeto-maternal apposition were probed with a panel of biotinylated lectins followed by an avidin-peroxidase revealing system to identify various classes of glycan at the interface. Both the external epithelium of unspecialized bilaminar omphalopleure, which forms by early pregnancy, and chorioallantoic membrane which develops by mid-pregnancy, were composed of two phenotypes, one of which secreted a wide range of glycans including high mannose and complex N-glycan, N-acetyl glucosamine, lactosamine and galactosamine, which became less prominent from mid-pregnancy onwards. The uterine epithelium also contained a well-developed secretory apparatus producing a similar range of glycans and there were indications that glycosylated secretions were taken up by the overlying chorioallantois. Foetal vasculature was well developed while maternal vessels appeared more contracted, and both were richly sialylated like their therian equivalents. Our findings indicate that this reptile has evolved a true epitheliochorial placenta with many aspects in common with its therian counterparts but also with unique features of its own.

Evidence of H beta 58, a gene involved in mammalian placental development, in the three-toed skink, Chalcides chalcides (Squamata: Scincidae), a viviparous placentotrophic reptile.

The H beta 58 gene, whose disruption in mice causes reabsorption of the embryo at 9.5 days post-conception, is believed to be essential for development of the placenta. Although the H beta 58 gene is well conserved in some Amniota, nothing is known about its presence in reptiles, some species of which have developed a chorioallantoic placenta. In this work, we investigated the expression of H beta 58 mRNA and protein in the three-toed skink, Chalcides chalcides. H beta 58 protein expression was found in the uterine epithelium beginning from the peri-ovulatory stage. However, it increased strongly at the moment of placental formation, when a high level of expression of mRNA and protein was also observed in the extra-embryonic membranes. The expression of H beta 58 mRNA and protein was maintained, although to a lesser degree, in the placenta during late pregnancy. It was also present in the early embryo. Finally, cloning and sequencing of a gene fragment revealed strong homology of the reptile gene with that of mammals. The high degree of conservation of the gene in amniote vertebrates and its presence in a viviparous squamate reptile (as in mammals) indicates an important role of this gene in the chorioallantoic placenta formation and development.

Localization of FMRFamide-like immunoreactivity in the brain of the viviparous skink (Chalcides chalcides).

Neuroanatomical distribution of FMRFamide-like immunoreactivity was investigated in the brain and olfactory system of the viviparous skink, Chalcides chalcides. In the adult brain FMRFamide immunoreactive (ir) perikarya were observed in the diagonal band of Broca, medial septal nucleus, accumbens nucleus, bed nucleus of the anterior commissure, periventricular hypothalamic nucleus, lateral forebrain bundle, and lateral preoptic, subcommissural, suprachiasmatic and lateral hypothalamic areas. This pattern was seen in both male and female brains. Though all major brain areas showed FMRFamide-ir innervation, the densest ir fiber network was observed in the hypothalamus. During development, ir elements were observed for the first time in embryos at mid-pregnancy. FMRFamide perikarya were located along the ventral surface of the vomeronasal nerve, in the olfactory peduncle mediobasally, as well as in the anterior olfactory nucleus and olfactory tubercle. Furthermore, some ir neurons were observed in the rhombencephalic reticular substance; however, the ir fiber network was poorly developed. Later in development FMRFamide-ir neurons appeared also in the bed nucleus of the anterior commissure as well as the rhombencephalic nucleus of solitary tract and the dorsal motor nucleus of vagus nerve. In juveniles, the distribution profile of FMRFamide immunoreactivity was substantially similar to that of the adults, with a less widespread neuronal distribution and a more developed fiber network. Ontogenetic presence of FMRFamide immunoreactivity in the nasal area has been linked to the presence of a nervus terminalis in this reptile.

Molecular analysis of arterial stenosis in rat carotids.

A new model of surgical injury for the induction and development of stenosis in common rat carotids is described. This model differs from balloon angioplasty or vein graft systems currently applied on animals to develop stenosis, since it involves the entire vessel wall layers and mimics the injury occurring during arterial grafts, endarterectomy or organ transplantation. At different times following arterial damage, the pattern of expression of genes already known to be involved in the proliferation, differentiation, and apoptosis of smooth muscle cells (c-myc, Angiotensin II receptor 1, Bcl-2 and Bax alpha), as well as of Rb and Rb2 genes, whose pattern of expression after arterial injury has not yet been reported, was analyzed by semi-quantitative reverse transcription-polymerase chain reaction technique. Histological and histochemical analysis on carotid sections shows the morphological changes which occurred 30 days after surgical injury in the vessel wall. Molecular and histological data demonstrate that this model of surgical injury induces neointimal proliferation in about 30% of rats. In about 70% of the remaining rats, it induces the processes responsible for negative remodelling, namely the significant accumulation of extracellular matrix and fibers and disorganization of arterial tunics. This model is therefore available for further studies on the expression of genes involved in the arterial stenotic process, as well as for testing drugs aimed at limiting this recurrent pathophysiological phenomenon.

Detection of DNA in ancient bones using histochemical methods.

We describe histochemical techniques for detecting DNA within the osteocytic lacunae of ancient bones. The bones examined were fragments of femurs from two human individuals found in the Pompeian C. I. Polybius house and fragments of metacarpals from two horses (Equus sp.) found in the Pompeian "Casti Amanti" house. Both buildings were buried by the 79 A. D. Vesuvius eruption. Fragments of femurs from a modern horse, a modern swine and a modern amphibian also were studied as controls. Some bone sections were stained with two different DNA-specific fluorochromes, 4'-'6-diamidino-2-phenylindole (DAPI) and chromomycin A3 (CMA), while others were stained by the Feulgen reaction. All of the techniques gave a positive reaction within the osteocytic lacunae. Histological analysis of the undecalcified, ground and unstained sections agreed well with results of bone sections stained with either the fluorochromes or the Feulgen reaction. Bones showing good histology also were positive by our DNA-specific stain. Histochemical and histological analyses correlated well with the success of DNA extraction and amplification. Using conventional DNA-specific histochemical techniques in conjunction with histological analysis can be useful in the study of DNA extracted from ancient bone remains while reducing both the amount of time and cost.

Histological analysis and ancient DNA amplification of human bone remains found in caius iulius polybius house in pompeii.

Thirteen skeletons found in the Caius Iulius Polybius house, which has been the object of intensive study since its discovery in Pompeii 250 years ago, have provided an opportunity to study either bone diagenesis by histological investigation or ancient DNA by polymerase chain reaction analysis. DNA analysis was done by amplifying both X- and Y-chromosomes amelogenin loci and Y-specific alphoid repeat locus. The von Willebrand factor (vWF) microsatellite locus on chromosome 12 was also analyzed for personal identification in two individuals showing alleles with 10/11 and 12/12 TCTA repeats, respectively. Technical problems were the scarcity of DNA content from osteocytes, DNA molecule fragmentation, microbial contamination which change bone structure, contaminating human DNA which results from mishandling, and frequent presence of Taq DNA polymerase inhibiting molecules like polyphenols and heavy metals. The results suggest that the remains contain endogenous human DNA that can be amplified and analyzed. The amplifiability of DNA corresponds to the bone preservation and dynamics of the burial conditions subsequent to the 79 A.D. eruption.

Endocrine activity of the corpus luteum and placenta during pregnancy in Chalcides chalcides (Reptilia, Squamata).

The structure of the corpus luteum and the steroidogenic activity of the corpus luteum and placenta in the viviparous reptile Chalcides chalcides have been investigated. The corpus luteum has a compact structure, almost without internal vascularized connective septa. It begins to degenerate after the middle of pregnancy, when plasma progesterone (P) remains high. The sections of the corpora lutea taken during early pregnancy showed an intense 3beta-HSDH reaction, whereas the sections taken in late pregnancy gave weak reactions localized exclusively in the peripheral luteal cells. In contrast, sections of placentae taken at the beginning and in the middle of pregnancy always gave negative 3beta-HSDH reactions, whereas those of late pregnancy were always strongly positive, localized in the maternal component of the placenta. In vitro, the corpora lutea from early pregnancy secreted significant amounts of P, whereas appreciable amounts of P were not detected in incubates of early pregnancy placentae. Near the time of delivery, P levels decreased in the culture medium of the corpora lutea, but increased in that of the placentae. The addition of pregnenolone (a precursor of P biosynthesis) to the culture medium caused an increase in the luteal and placental P levels, whereas the addition of trilostane (an inhibitor of 3beta-HSDH) reduced them. The placenta of C. chalcides is suggested to have an endocrine function and to replace the corpus luteum in the production of P when the gland degenerates in late pregnancy.

Cytokines in the viviparous reproduction of squamate reptiles: interleukin-1 alpha (IL-1 alpha) and IL-1 beta in placental structures of a skink.

Placental viviparity is known in many species of squamate reptiles. Among these, some scincids have developed an epithelio-chorial chorio-allantoic placenta which in the structure of its central ridged zone is similar to those of certain therian mammalian species. A broad range of immunoregulatory peptides, cytokines, has been identified at the maternofetal interface of several species of mammals, either with invasive or non-invasive types of placenta. Thus we began to study whether interleukin-1, which is considered to play a crucial role in mammalian pregnancy, might also be involved in the viviparity of reptilian species. Placentae of Chalcides chalcides L. were processed by immunohistochemistry and incubated in a culture medium for different times. A very strong immunoreactivity for interleukin-1 alpha (IL-1 alpha) and for interleukin-1 beta (IL-1 beta) was present in the chorial epiblast and in uterine epithelial cells, with varying degree and localization in different periods of pregnancy. IL-1 beta was also released into the medium at different amounts during incubation. In light of the mammalian data, our results suggest that the role of cytokines in pregnancy may represent a significant event in the evolution of placental viviparity.