Effects of mild ozonisation on gene expression and nuclear domains organization in vitro.

In the last two decades, the use of ozone (O3) as a complementary medical approach has progressively been increasing; however, its application is still limited due to the numerous doubts about its possible toxicity, despite the low concentrations used in therapy. For an appropriate and safe clinical application of a potentially toxic agent such as O3, it is crucial to elucidate the cellular response to its administration. Molecular analyses and transmission electron microscopy were here combined to investigate in vitro the effects of O3 administration on transcriptional activity and nuclear domains organization of cultured SH-SY5Y neuronal cells; low O3 concentrations were used as those currently administered in clinical practice. Mild ozonisation did not affect cell proliferation or death, while molecular analyses showed an O3-induced modulation of some genes involved in the cell response to stress (HMOX1, ERCC4, CDKN1A) and in the transcription machinery (CTDSP1). Ultrastructural cytochemistry after experiments of bromouridine incorporation consistently demonstrated an increased transcriptional rate at both the nucleoplasmic (mRNA) and the nucleolar (rRNA) level. No ultrastructural alteration of nuclear domains was observed. Our molecular, ultrastructural and cytochemical data demonstrate that a mild toxic stimulus such as mild ozonisation stimulate cell protective pathways and nuclear transcription, without altering cell viability. This could possibly account for the positive effects observed in ozone-treated patients.

Fine structural cytochemical analysis of homologous chromosome recognition, alignment, and pairing in Guinea pig spermatogonia and spermatocytes.

The nuclei of guinea pig spermatogonia and spermatocytes were studied by means of quantitative autoradiography and electron microscopic methods such as high-resolution cytochemistry, immunocytochemistry, and in situ hybridization. Our observations reveal, in the nucleus of spermatogonia type B, small lampbrush structures of extended chromatin not found in nonmeiotic cells. During meiotic interphase, pairs of parallel lampbrush structures become associated by numerous filaments. The formation of the synaptonemal complex is simultaneous with the extension of chromosomal axes in a continuous leptotene-zygotene stage. Some chromosomes do not recognize their homologs before the onset of the leptotene-zygotene stage and undergo classical leptotene and zygotene stages. The immunocytochemical localization of Dmc1 and Rad51 supports the idea that these proteins are not involved in homology search and final pairing. Immunolocalization of DNA, RNA polymerase II, heterogeneous nuclear ribonucleoproteins, small nuclear ribonucleoproteins, and the trimethyl-guanosin cap of small nuclear RNAs suggests that the chromatin of lampbrush structures transcribe hnRNA and that splicing is scarce. The results of quantitative autoradiography after [3H]uridine labeling show an intense transcription accompanied by a very slow export of RNA. In situ hybridization demonstrates the presence of RNA in the regions of homology recognition and pairing. These results lead us to propose that the RNA synthesized in the lampbrush structures is involved in the process of homology searching and recognition.