Dapi 2

Human normozoospermic A- and B-SPZ fractions were used for Chromomycin A3 (CMA3) and Aniline blue staining, to evaluate protamination and histone rates, respectively. In brief, for CMA3 staining sperm cells were fixed in methanol/acetic solution (n=4 different samples for each experimental group) and spread on Superfrost slides air-dried at RT overnight. Cells were subsequently incubated in 0.25 mg/ml of CMA3 solution in McIlvaine buffer (pH 7) for 20 min and washed twice for 2 min in McIlvaine buffer. Slides were then mounted with DAPI II (Abbott Laboratories) and a minimum of 200 cells were evaluated for each sample. For Aniline blue staining, sperm cells were fixed in 3% glutaraldehyde solution and dried on slides. Slides were hydrated 5 min in water, stained in 5% aniline blue solution diluted in 4% acetic acid solution, rinsed twice for 2 min in water, dehydrated through ethanol bath series (70, 90, and 100% ethanol solutions), and finally fixed in toluene for 2 min. Then, slides were mounted with Eukitt^®. At least 200 cells were evaluated with a Nikon Eclipse 80i epifluorescence microscope with a 100X oil objective. All assays were performed on four different replicates (n=4) for both A- and B-SPZ.

Slides were incubated for 10 min in HCl 0.1N/pepsin (0.5%) at 37 °C, and then washed in PBS. Slides were incubated in formaldehyde (1%) for 10 min, dehydrated in ethanol 70°, 90° and 100° (3 min each) and dried. Denaturation and hybridization were performed with a thermobrite (Leica Biosystems, Wetzlar, Germany). Denaturation was performed following manufacturer’s instructions: 10 min at 75 °C for ROS1 probe (zytolight SPEC ROS1 Dual-Color Break-Apart probe (PL101), zytovision, Bremerhaven, Germany), 4 min at 75 °C for ALK probe (Vysis LSI ALK Dual-Color Break-Appart, rearrangement probe, Abbott molecular, Des Plaines, IL, USA). Hybridization was performed overnight at 37 °C. Slides were washed in a buffered bath (saline–sodium citrate buffer 0.4Xat 72 °C for 2 min, then twice at room temperature for 30 s). Nuclei were stained with DAPI for 10 min at room temperature (DAPI II, Abbott, Chicago, IL, USA). Slides were mounted using FluorSave™ reagent (Merck Millipore, Burlington, USA) and observed using a fluorescence microscope (Leica, Wetzlar, Germany).

Semen samples were collected by masturbation in a sterile container and maintained at 37 °C for 30 min to allow liquefaction. We evaluated sperm concentration, motility, and morphology according to World Health Organization criteria for human semen analysis [40 ].
Each sperm sample was treated as previously described [41 (link)]. Briefly, samples were washed in PBS 1×, fixed in methanol/acetic acid (3:1, v/v) solution, spread on slides, decondensed in NaOH 1 M solution, and dehydrated in ethanol solutions. Each sample was co-denatured with its specific probes mix for 2 min at 75 °C and then hybridized for 18 h in a HYBrite^® system (Abbott Laboratories, Chicago, IL, USA). Slides were then washed according to manufacturer specifications and then mounted with DAPI II (Abbott Laboratories) to counterstain sperm nuclei. All probes and mixes used for each patient are presented in the Supplementary Materials Table S8.