Angiogenesis and vascular network of teratocarcinoma from embryonic stem cell transplant into seminiferous tubules.

BACKGROUND: Carcinoma in situ (CIS) of the testis is considered to be a precancerous germinal cell lesion, but the precise cellular and molecular mechanisms underlying transformation of CIS into invasive pluripotent cancer cells remain to be elucidated. Moreover, a satisfactory animal model for the experimental study of germinal tumours has not been developed to date. METHODS: We have developed a tumour model that involves the microinjection of green fluorescent protein-labelled embryonic stem (ES) cells (which are functionally equivalent to CIS cells) into syngenic mouse seminiferous tubules, a unique cell microenvironment in which germinal cells mature and CIS arise. To characterise the vascularisation of teratocarcinomas, which arise after cell transplant, we used immunohistochemistry, together with a qualitative and quantitative analysis of scanning electron microscopy images of corrosion casting samples. RESULTS: Embryonic stem cells transplanted into seminiferous tubules did not differentiate into germinal cells, but rather they behaved as invasive embryonal carcinoma (EC) stem cells. The vascular pattern of the experimental teratocarcinomas showed a highly disorganised architecture, and some of the neoplastic capillaries were derived, at least in part, from the original transplanted ES cells. CONCLUSION: The transplantation of pluripotent ES cells into seminiferous tubules efficiently recapitulates the early stages of development of teratocarcinomas. Consequently, this method constitutes a novel in vivo model to study the mechanisms of invasion and progression of experimental germinal tumours.

Structural and functional preservation of specific sequences of DNA and mRNA in apoptotic bodies from ES cells.

Retinoic acid-induced apoptosis of embryonic stem (ES) cells is an experimental system which resembles the physiological programmed cell death that occurs during differentiation in embryonic development. Our aim was to analyze the involvement of epigenetic modifications such as DNA methylation and chromatin structure in the apoptotic process and to investigate the metabolic activity of apoptotic bodies. We found a relationship between DNA methylation and apoptosis, shown by a dose-dependent induction of apoptosis after treatment with the inhibitor of DNA methylation 5-aza-2'-deoxycytidine. Interestingly, we found a slight demethylation of specific sequences of the U2afl-rs1 imprinted gene in those RA treated cells which were specifically undergoing apoptosis. In addition, apoptotic bodies exhibited an unexpected open chromatin conformation accessible to the endonuclease DNase-I. Furthermore, we observed a structural and functional preservation of specific DNA sequences and mRNA. These results suggest that biological activities, such as transcription or protein synthesis, could be maintained even towards the end of the apoptotic process.

Nucleoplasmin binds to nuclear pore filaments and accumulates in specific regions of the nucleolar cortex.

Nucleoplasmin is a karyophilic protein that is involved in nucleosome formation and decondensation of chromatin, although other precise functions and modes of action of this molecule are still poorly understood. In the present paper we describe a novel nucleocytoplasmic transport assay that has enabled us to study the nuclear distribution of nucleoplasmin following its transport into the nucleus. Single Xenopus laevis oocyte nuclei were isolated and incubated with Xenopus egg extract containing colloidal gold-conjugated nucleoplasmin. After a period of incubation, each individual nucleus was processed for electron microscopy. The nuclear accumulation of nucleoplasmin was dependent upon the karyophilic properties of the protein, since BSA-conjugated gold particles did not enter the nuclear interior under the same experimental conditions. Once inside the nucleus, nucleoplasmin was detected in tracks emanating from the nuclear pores and reaching the nucleolus. Additionally, we found a striking accumulation of nucleoplasmin in specific areas of the nucleolar cortex. These perinucleolar regions were surrounded by areas of electron density similar to that of the fibrillar centers. Our results indicate that nucleoplasmin may play an important role in the transcription of ribosomal precursors. Moreover, this nucleocytoplasmic transport assay will enable the determination of the precise intranuclear localization of other karyophilic proteins.

The nuclear basket of the nuclear pore complex is part of a higher-order filamentous network that is related to chromatin.

We have achieved well-preserved nuclear basket structures in amphibian nuclear envelope spreads dried by the critical point method after tannic acid fixation, and we have compared these images with those obtained from conventional sections of amphibian oocyte nuclei. In cross sections, bundles of filaments from adjacent nuclear pore complexes were interconnected at regular distances, building up a higher-order network. Sometimes these bundles were observed to extend inward to amplified nucleoli located near the nuclear envelope. Furthermore, immunoelectron analysis indicated that DNA and histones were localized at these intranuclear filaments, suggesting a close relationship between chromatin and nuclear pore complexes. A model is proposed by which the intranuclear filaments associated with adjacent nuclear pore complexes create a regular higher-order network, which extends into the nucleus.

The Turbidity of Cell Nuclei in Suspension: A Complex Case of Light Scattering.

The turbidity of a suspension of cell nuclei isolated from animal tissue homogenates is a complex case of non-Rayleigh scattering. As a first approximation to this system, we have characterized a number of factors that may contribute to the observed turbidity: cation-dependent chromatin condensation, thermal denaturation of chromatin, nuclear shrinking, and changes in the optical properties of the membrane bilayer. Small differences in cation concentration, particularly in the case of divalent cations, lead to large changes in chromatin supramolecular organization, thus to large turbidity effects; thermally-induced changes in turbidity have a similar origin, although they are less pronounced. Under certain circumstances, either salts or heat may induce condensation of chromatin, the latter being connected to the inner side of the nuclear envelope, nuclear shrinking ensues, and this in turn modifies the suspension turbidity. Finally, changes in the physical properties of the lipid bilayers or of the phospholipids in the nuclear envelopes may also have significant effects, though smaller than chromatin changes, in the overall turbidity of the nuclear suspension.