Abstract
In order to transport an intact and complete paternal genome to the oocyte, spermatozoa are characterized by an extremely compacted nuclear DNA as compared to the nucleus of somatic cells. Such packaging of the chromatin is obtained through a dramatic nuclear reorganization occurring in developing spermatids leading to an almost complete replacement of histones with protamines. During the epididymal transit, spermatozoa complete the process of chromatin packaging by formation of disulphide bridges in nucleoproteins [1]. This process of sperm maturation is critical for male fertility. Indeed, it has been demonstrated that alterations in chromatin structure could impact male fertility potential compromising both in vivo and in vitro fertilization and the subsequent embryo development [2, 3].
17.1 Introduction
In order to transport an intact and complete paternal genome to the oocyte, spermatozoa are characterized by an extremely compacted nuclear DNA as compared to the nucleus of somatic cells. Such packaging of the chromatin is obtained through a dramatic nuclear reorganization occurring in developing spermatids leading to an almost complete replacement of histones with protamines. During the epididymal transit, spermatozoa complete the process of chromatin packaging by formation of disulphide bridges in nucleoproteins [1]. This process of sperm maturation is critical for male fertility. Indeed, it has been demonstrated that alterations in chromatin structure could impact male fertility potential compromising both in vivo and in vitro fertilization and the subsequent embryo development [2, 3].
For this reason, in recent years, assays to evaluate the sperm chromatin status have gained increased value in order to find a possible marker useful in male infertility work-up. Among the several methods that have been proposed, staining with chromomycin A3 (CMA3, 4) is commonly used. This method gives an indication of the degree of sperm protamination. In a recent paper, it has been demonstrated that in an adjusted model for confounding factors including female age and female factors, the sperm chromatin status evaluated by CMA3 is able to predict the achievement of good quality embryos with 78 percent sensitivity and 65 percent specificity [5]. Although the possible use of CMA3 in clinical practice is still debated, it could represent a simple and promising tool to be added to routine semen analysis for evaluation of male reproductive health before infertility treatment.
17.2 Principle
Chromomycin A3 (CMA3, Figure 17.1) is an antibiotic glycoside produced by the fermentation of a certain strain of Streptomyces griseous. In the presence of bivalent metal ions, CMA3 binds reversibly to DNA, preferentially to contiguous G/C base pairs. As mentioned, the probe is used as a fluorescent DNA stain for detection of protamine deficiency in sperm chromatin, since it competes with protamines for binding to DNA minor grooves.
When bound to DNA, CMA3 has a maximum excitation wavelength of 445 nm and a maximum emission wavelength of 575 nm. Two types of staining patterns are identified with fluorescence microscopy: bright green fluorescence (reflecting abnormal chromatin packaging) and weak green staining (normal chromatin packaging) of the sperm head (Figure 17.2A).
Figure 17.1 Chromomycin A3 chemical structure
Figure 17.2 (A) Staining with CMA3 of human spermatozoa. Two patterns are identified at fluorescence microscopy: bright green fluorescence of sperm head (A, abnormal chromatin packaging) and weak green staining (B, normal chromatin packaging). (B) CMA3 staining and is an index of protamine content and, consequently a sign of sperm chromatin maturity.
17.3 Protocol
17.3.1 Equipment Needed
‐ Fluorescence microscope with filters able to match the spectral excitation and emission characteristics of the fluorophore, CMA3, (excitation wavelength of 445 nm and emission wavelength of 575 nm);
‐ Slides.
17.3.2 Reagent Needed
‐ Chromomycin A3 (empirical formula: C57H82O26; molecular weight: 1183.25);
‐ Medium for washing spermatozoa (human tubal fluid, etc.);
‐ Paraformaldehyde solution 4 percent in phosphate buffer;
‐ Phosphate buffered saline (PBS);
‐ Mc Ilvaine Buffer (0.2 M Na2HPO4 and 0.1 M citric acid, pH7.00) added with 10 mM MgCl2;
‐ PBS/glycerol solution (1:1).
17.3.3 Sperm Preparation
The percentage of spermatozoa stained with CMA3 can be evaluated either in whole semen or selected (Swim up or PureSperm) spermatozoa after sperm separation.
Procedure:
‐ take 1 × 106 spermatozoa;
‐ in case of whole semen, wash spermatozoa twice in sperm culture medium (centrifuge at 500×g for five minutes, room temperature). In case of selected spermatozoa centrifuge once at 500×g for five minutes;
‐ remove supernatant and fix the pellet in 50 µL of 4 percent paraformaldehyde for 30 minutes at room temperature;
‐ take 20 µL of the sample and centrifuge at 300×g for seven minutes, room temperature;
‐ remove supernatant and wash once in PBS by centrifuging at 300×g for seven minutes, room temperature.
17.3.4 Incubation of Spermatozoa with CMA3 Solution
To prepare stock solution of CMA3 (0.5 mg/mL), 5 mg of CMA3 powder should be dissolved in 10 mL of Mc Ilvaine buffer added with 10 mM MgCl2. Aliquots of CMA3 stock solution can be stored at −20°C.
At the time of use, dilute aliquots 1:1 in Mc Ilvaine buffer added with 10 mM MgCl2 to obtain a final concentration of CMA3 of 0.25 mg/mL. Add 100 µL CMA3 solution (0.25 mg/mL) to sperm pellet (after washing and fixation in paraformaldehyde) and incubate for 20 minutes at room temperature. Then, add 200 µL Mc Ilvaine buffer added with 10 mM MgCl2 and centrifuge at 300×g for seven minutes at room temperature, discard supernatant and re-suspend the pellet in 10 µL Mc Ilvaine buffer added with 10 mM MgCl2.
17.3.5 Mounting of Slides
After staining and washing, make a spot on a slide, dry the slide on air, add a drop of PBS: glycerol and place a coverslip over.
17.3.6 Analysis by Fluorescence Microscopy
Spermatozoa exhibiting a bright green fluorescence in the head are scored as CMA3-positive. At least 200 spermatozoa from each sample should be scored and the percentage of CMA3-positive sperm calculated.
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