This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.
Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.
I. Sample processing and cell fixation
II. Decondensation
III. Hybridization
IV. Washes post-hybridization
V. Visualization
The preparations can be evaluated under an epifluorescence microscope equipped with a triple-band filter for DAPI/Texas Red/FITC and single-band filters for Aqua, FITC and Texas Red. Standard assessment criteria must be followed for the correct evaluation of the sperm nuclei 1.
The preparation hybridized with probes for chromosomes X, Y and 18 should display signals for these three chromosomes. Every normal spermatozoa must show one blue signal (corresponding to the chromosome 18), and a green signal (X-bearing spermatozoa) or a red signal (Y-bearing spermatozoa).
In the preparation hybridized with probes for chromosomes 13 and 21, a green signal for chromosome 13 and a red signal for chromosome 21 should be distinguished in every normal spermatozoa.
This protocol describes how to process human semen samples for obtaining sperm FISH preparations. Using this protocol it is possible to analyze chromosomal abnormalities in male gametes. Spermatozoa aneuploidy screening has applications either in the context of basic research and in the reproductive advice given to infertile males. Although in clinical diagnosis the most widely studied chromosomes are X, Y, 13, 18 and 21, other commercial probes for a wide range of chromosomes and loci are available and can also be used in these experiments.
The authors have nothing to disclose.
This work was supported by the Generalitat de Catalunya (2005-SGR00437). The authors thank to J. Blanco and F. Vidal for their supervising and encouragement, and J. Lucas for his technical assistance.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
1,4-Dithiothreitol (DTT) | Material | Roche | 10197777001 | |
2-Amino-2-(hydroxymethyl)-1,3-propanediol | Material | Roche | 10708976001 | |
Acetic acid | Material | Merck | 100.063 | |
AneuVysion® Multicolor DNA Probe Kit
|
Material | Vysis Inc. | 32-161075
|
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Centrifuge 5804R | Tool | Eppendorf | ||
Centrifuge tube | Material | Nunc | 347856 | |
Coplin jar (glass) | Material | Barloworld Scientific | Hellendahl (ZCT278) | |
Coplin jar (plastic) | Material | Deltalab | 191087 | |
Cover slips (15×15) | Material | Knittel gläser | 4600115 | |
Cover slips (18×18) | Material | Knittel gläser | 4600118 | |
Diamond pencil | Material | Hammacher | 335117010 | |
Ethanol | Material | Merck | 100.983 | |
Formamide | Material | Roche | 11814320001 | |
Freezer | Tool | Zanussi | ||
Gloves | Material | Sanyc | 101/3300 | |
HYBrite™ | Tool | Vysis Inc. | ||
Immersion Oil | Material | Olympus | 35505 | |
Incubator | Tool | Heraeus | ||
Methanol | Material | Merck | 106.009 | |
Micropipette | Material | Labsystems | Finnpipette (4500000) | |
Micropipette replacement tip | Material | Daslab | 16-2001 | |
Nail varnish | Material | Quo-cosmetics | ||
Olympus BX60 epifluorescence microscope | Tool | Olympus | Equipped with a triple-band filter for DAPI/Texas Red/FITC and single-band filters for Aqua, FITC and Texas Red. | |
Pasteur Pipette | Material | Rubilabor | 211.0230 | |
Phase contrast microscope | Tool | Nikon Diaphot | ||
Plastic pipette (3ml) | Material | Deltalab | 200007 | |
Potassium chloride (KCl) | Material | Fluka | 60128 | |
Rubber cement | Material | Best-test | ||
Slides | Material | Knittel gläser | 4520022 | |
Slide Saver Boxes | Material | Deltalab | 19276.1 | |
Sterile container | Material | Deltalab | 409726 | |
Syringe | Material | PentaFerte | 002022300 | |
Thermometer | Material | Comark Instruments, Inc | KM12 | |
Tri-distilled water | Material | |||
Triton X-100 | Material | Sigma | 9002-93-1 | |
Tweezers | Material | B/Braun | Aesculap (BD224R) | |
Vertical laminar flow hood | Tool | Burdinola | OR-ST 1500 | |
Vortex mixer | Tool | Fisher Scientific | FB15012 | |
Water bath | Tool | Raypa® |