Telomere distribution was assessed by fluorescence in situ hybridization (FISH) using a biotin-labeled pan-telomere probe (Cambio) following the manufacturer’s protocol. Then, 2D fluorescence microscopy was carried out at magnification of 100× using a BX61 Olympus microscope equipped with a CCD camera (C10600-10B—ORCA-R2, Hamamatsu) and appropriate filters. Images of at least 100 sperm nuclei per patient were captured using SmartCapture3 (Digital Scientific). Next, 3D fluorescence microscopy was carried out using an Olympus IX71 microscope. After identification of the best plane of focus for the center of the nucleus, 61 images were captured in 0.1 µm increments throughout the nucleus (30 images below and 30 above the central plane) using Metamorph software.
Telomeres are the only parts of the sperm nucleus that remain histone bound and not protamine bound [72 (link)], so the cells were not swollen prior to FISH. This had the benefit of allowing for morphological analysis without extreme distortion of the nuclear shape, while retaining accessibility of the FISH probes to the majority of the telomeres.
Telomeres are the only parts of the sperm nucleus that remain histone bound and not protamine bound [72 (link)], so the cells were not swollen prior to FISH. This had the benefit of allowing for morphological analysis without extreme distortion of the nuclear shape, while retaining accessibility of the FISH probes to the majority of the telomeres.
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