This protocol describes the fluorescence in situ hybridization (FISH) of DNA probes on mitotic chromosome spreads optimized for two super-resolution microscopy approaches-structured illumination microscopy (SIM) and stimulated emission depletion (STED). It is based on traditional DNA FISH methods that can be combined with immunofluorescence labeling (Immuno-FISH). This technique previously allowed us to visualize ribosomal DNA linkages between human acrocentric chromosomes and provided information about the activity status of linked rDNA loci. Compared to the conventional wide-field and confocal microscopy, the quality of SIM and STED data depends a lot more on the optimal specimen preparation, choice of fluorophores, and quality of the fluorescent labeling. This protocol highlights details that make specimens suitable for super-resolution microscopy and tips for good imaging practices.

Although most XYY men have normal sperm counts and are fertile (supposedly due to the loss of the extra Y before meiosis), there is a minority who are infertile. In these cases, the XYY spermatocytes are able to enter meiosis and form different synaptic configurations. With regard to mosaics, there is scarce well-defined information on the presence of the second Y and its meiotic behaviour. In this study, the chromosome constitution and the synaptic behaviour of pachytene spermatocytes from an azoospermic man with testicular hypotrophy and non-mosaic 47,XYY karyotype were analysed. Furthermore, we determined the chromosome constitution of the somatic Sertoli cells. Five karyotypically normal men with obstructive azoospermia, but having complete spermatogenesis, were included as controls. Immuno-FISH using specific protein markers of synapsis and recombination (SYCP3, SYCP1, BRCA1, MLH1, CREST) and a specific Yq12 DNA probe were used. In addition, we used the newly developed Super-Resolution Structured Illumination Microscopy (SR-SIM) to clearly define the synaptic configurations. FISH analysis was also performed on Sertoli cells. The histopathological analysis showed variable degrees of spermatogenesis development in the testicular tissue of the propositus. Immuno-FISH analysis showed that most of the primary spermatocytes were euploid 46, XY. The use of SR-SIM confirmed the existence of this euploidy. Only a few pachytene spermatocytes showed an aneuploid X + YY constitution. Sertoli cells showed two different populations with one or two Y chromosomes, in similar proportions. Thus an abnormal niche of sex-trisomic Sertoli cells should be also considered when searching for the origin of spermatogenesis failure in XYY men.

Preservation of genome integrity by complete, error-free DNA duplication prior to cell division and by correct DNA damage repair is paramount for the development and maintenance of an organism. This holds true not only for protein-encoding genes, but also it applies to repetitive DNA elements, which make up more than half of the human genome. Here, we focused on the replication and repair kinetics of interspersed and tandem repetitive DNA elements.
We integrated genomic population level data with a single cell immunofluorescence in situ hybridization approach to simultaneously label replication/repair and repetitive DNA elements. We found that: (1) the euchromatic Alu element was replicated during early S-phase; (2) LINE-1, which is associated with AT-rich genomic regions, was replicated throughout S-phase, with the majority being replicated according to their particular histone marks; (3) satellite III, which constitutes pericentromeric heterochromatin, was replicated exclusively during the mid-to-late S-phase. As for the DNA double-strand break repair process, we observed that Alu elements followed the global genome repair kinetics, while LINE-1 elements repaired at a slower rate. Finally, satellite III repeats were repaired at later time points.
We conclude that the histone modifications in the specific repeat element predominantly determine its replication and repair timing. Thus, Alu elements, which are characterized by euchromatic chromatin features, are repaired and replicated the earliest, followed by LINE-1 elements, including more variegated eu/heterochromatic features and, lastly, satellite tandem repeats, which are homogeneously characterized by heterochromatic features and extend over megabase-long genomic regions. Altogether, this work reemphasizes the need for complementary approaches to achieve an integrated and comprehensive investigation of genomic processes.

Monosomy-3 in primary uveal melanoma (UM) is associated with a high risk of metastasis and mortality. Although circulating melanoma cells (CMC) can be found in most UM patients, only approximately 50% of the patients develop metastases. We utilized a novel immuno-FISH assay to detect chromosome-3 in intact CMC isolated by dual immunomagnetic enrichment. Circulating melanoma cells were detected in 91% of the patients (n = 44) with primary non-metastatic UM, of which 58% were positive for monosomy-3. The monosomy-3 status of CMC corresponded to the monosomy-3 status of the primary tumor in 10 of the 11 patients where this could be tested. Monosomy-3 in the CMC was associated with an advanced tumor stage (P = 0.046) and was detected in all four patients who developed metastasis within the follow-up period of 4 yr. This non-invasive technique may enable the identification of UM patients at risk for metastasis particularly when a primary tumor specimen is unavailable.