Semen samples from 10 control donors aged between 24 and 50, and 16 PWS fathers from 32 to 60 years of age were obtained. Donors were volunteers recruited from the general population. All subjects had normal karyotypes and were normozoospermic. To our knowledge, none of them had been exposed to genotoxic agents, and no history of chemotherapy, radiotherapy or chronic illness was recorded.
All subjects gave their informed consent in writing to participate in the study and the protocol was approved by the Institutional Ethics Committee of the Universitat Autònoma de Barcelona.
Samples were processed as described previously by our group [28
]. Briefly, the sperm fraction was resuspended in hypotonic solution (0.075 M KCl) for 30 minutes at 37°C and fixed in methanol:acetic acid (3:1). Spermatozoa were spread on a slide and kept at -20°C until processed.
Three BAC clones were selected using the resources of the Genome Browser database (UCSC Assembly; February 2009). All clones were obtained from the Children's Hospital Oakland Research Institute, BACPAC resources (Oakland, CA 94609 USA). BAC DNA extraction was performed using the QIAprep Miniprep kit (Qiagen GmbH; Hilden, Germany) following manufacturer's instructions.
BAC clones RP11-1122J3, RP11-322N14 and RP11-230M20 were fluorescently labeled with Spectrum Green-dUTPs (Abbott Molecular; Abbott Park, IL, USA), Spectrum Orange-dUTPs (Abbott Molecular) and DEAC-dUTPs (Perkin Elmer Inc; Boston, USA), respectively, by Nick Translation (Roche; Mannheim, Germany). Probes were mixed with 10 μg of Cot 1 DNA (Invitrogen; Carlsbad, USA), ethanol precipitated and resuspended in hybridization buffer (50% Formamide, 1xSSC and 10% dextran sulphate) (Abbott Molecular).
Probe positions were verified on lymphocyte metaphase chromosomes. Hybridization in spermatozoa was determined by the evaluation of 1,000 sperm nuclei per probe in three different FISH experiments.
Fluorescence in situ Hybridization on sperm (sperm-FISH)
Prior to hybridization, sperm nuclei were decondensed by slide incubation at 37°C in Tris buffer containing 25 mmol/ml dithiothreitol and 1% Triton X-100 for 45 minutes.
A triple-color FISH using the three BAC clones differentially labeled was performed following standard procedures. Briefly, probes were denatured at 80°C for 8 minutes and pre-annealed at 37°C for 15 minutes. Sperm nuclei were denatured at 73°C in 70% formamide in 2xSSC for 5 minutes. Hybridization was carried out by adding 5 μl of the corresponding probe mixture (200 ng of each probe) to the sperm preparation and incubating the slides in a moist chamber at 37°C for 48 hours. Post-hybridization washes were performed in 1xSSC with 0.3% NP-40 at 73°C followed by 2xSSC with 0.1% NP-40 at room temperature, for one minute in each solution. Slides were mounted with antifade solution (Abbott Molecular).
Analyses were performed using an Olympus BX-60 epifluorescence microscope equipped with a triple-band pass filter, and specific filters for Aqua, FITC and Cy3.
A minimum of 1,000 informative sperm nuclei were analyzed per sample.
Confocal Laser-Scanning Microscopy
To confirm the existence of normal and inverted haplotypes, 80 sperm-nuclei were captured and analyzed using a confocal-laser scanning TCS-SP5 AOBS microscope (Leica Microsystems, Heidelberg Gmbh; Mannheim, Germany).
Spectrum Orange fluorochromes were excited with the 561-nm line of a DPSS laser and observed in the red channel at an emission range of 569-nm to 671-nm. Spectrum Green fluorochromes were excited with the 405-nm line of a diode laser and viewed in the green channel at 502-nm to 551-nm. Finally, DEAC fluorochromes were excited with the 488-nm line of an Argon laser and observed in the blue channel at an emission range of 444-nm to 500-nm. Stacks of 16 to 20 sections every 0.3 μm were acquired.
Image combining and processing were performed with the IMARIS software package version 2.7 (Bitplane AG; Zürich, Switzerland).
Data obtained were statistically analyzed using SPSS version 15.0 (SPSS Inc; Chicago, IL, USA) under the advice of the statistical service of the Universitat Autònoma de Barcelona.
To assess the relationship between the two inverted haplotypes, the following analyses were performed:
• The mean frequencies of Type-1 and Type-2 inversions were compared in both populations by a Wilcoxon test.
• A Pearson's correlation test between the frequencies of the two inverted haplotypes was performed.
To assess the susceptibility of the 15q11-q13 region to generate inversions, the following statistical tests were performed:
• Population level: The mean population frequency of 15q11q13 inversions were compared between controls and PWS fathers by means of the Mann-Whitney test.
• Individual level: A Chi-square test comparing the inversion frequencies of every single PWS father with the mean frequency of inversions observed in the control population was performed.
• Age effect: A Pearson's correlation test between the sum of inversions and the age of all subjects was performed.
To assess a possible relationship between the frequency of inversions and the frequency of deletions previously found by our group [18
• Spearman's correlations between the frequency of the total inversions (Type-1 + Type-2) and the frequency of deletions were performed for both the control population and the PWS fathers.
Differences and correlations were considered statistically significant when P < 0.05.