Damaged sperm chromatin may impair the capability of the spermatozoa to fertilize, decrease in vitro fertilization or insemination success, cause abortion or foetal abnormalities and even reduce offspring fitness (Virro et al. 2004). The SCSA®, SCSA diagnostics, Brookings, South Dakota, USA (sperm chromatin stability assay) is the most widespread test for assessing sperm chromatin by flow cytometry. This test is relatively simple to perform: sperm samples are submitted to a DNA denaturation step, mixed with an acridine orange (AO) solution and analysed by flow cytometry (Evenson and Jost 2000). AO is a metachromatic fluorochrome that readily intercalates in the DNA. When associated to double-stranded DNA (dsDNA) it fluoresces green, whereas it fluoresces red when associated to single-stranded DNA (ssDNA). The denaturation step induces the formation of ssDNA from breakages, therefore each sperm head yield a mixture of green and red fluorescence when interrogated with a 488-nm laser, depending on the DNA fragmentation (number of nicks) and the susceptibility of chromatin to denaturation (Fig. 2). Data is processed to obtain, for each spermatozoon, the red/[total fluorescence] ratio, called the DNA fragmentation index (DFI). A series of cut-off values can be set for DFI, to obtain the percentage of spermatozoa with moderate DFI (cut-off at 0.25 DFI) and with high DFI (cut-off at 0.75 DFI). It is important to consider that the DFI acronym is used both to refer to the red/total fluorescence ratio of individual spermatozoa (formerly termed αt) and to refer to the percentage of spermatozoa with moderate and high DFI (formerly termed COMPαt). Evenson et al. (2002) also defined high DNA stainability [HDS, formerly termed ‘HIGRN’ (Evenson and Jost 2000)] as a measure of the condensation degree of the sperm chromatin, and possibly related to chromatin alterations and infertility (Virro et al. 2004). A high DFI has been related to reduced fertility, longer times to pregnancy and higher spontaneous miscarriage rates in humans (Virro et al. 2004; Evenson and Wixon 2006), and it has shown a relation with fertility and prolificacy in domestic animals [bull: Waterhouse et al. (2006), García-Macías et al. (2007); boar: Boe-Hansen et al. (2008)]. We have used the SCSA® in ram, Iberian red deer, bull, domestic dog and bear, finding it sensitive for detecting seasonal changes in chromatin condensation (García-Macías et al. 2006a), comparing epididymal and ejaculated spermatozoa (García-Macías et al. 2006b) and assessing the effect of refrigeration or cryopreservation on spermatozoa (Martínez-Pastor et al. 2004, 2009b; Fernández-Santos et al. 2009a,b,c). A caveat to researchers implementing this test is to carefully follow the developers’ guidelines (Evenson and Jost 2000; Evenson et al. 2002). Although the SCSA® is relatively easy to follow, many factors can influence its results (Boe-Hansen et al. 2005). Thus, the preparation of a standard sample, denaturation and staining conditions and times must be strictly controlled, and the AO must be of the highest quality (chromatographically purified). Adjustment of the sperm concentration and dilution with the AO solution must be carefully performed, because AO must be at equilibrium with the sperm sample (regarding the molar relationship among AO and base pairs).
Figure 2. Assessment of sperm chromatin. Cytograms (a) and (b) were obtained after carrying out the SCSA® protocol. Acridine orange yields different amounts of red and green fluorescence depending on the dsDNA and ssDNA present in the nucleus. Cytogram (a) correspond to a sample with few spermatozoa showing medium or high DFI (red fluorescence/total fluorescence). Cells plotted to the right of the diagonal line have increased DFI, also termed ‘COMP’ (cells out of the main population). The inset shows the histogram of DFI, with most events within the low-DFI peak. Conversely, the cytogram (b) shows a higher number of spermatozoa in the COMP region, because of increasing DFI levels (as shown in the inset). Debris (events with low green fluorescence and in the low-green/low-red region) was previously removed from the analysis. Cytogram (c) shows spermatozoa treated with the TUNEL protocol and counterstained with PI. Note that PI allows debris (events with low red fluorescence) and aggregated spermatozoa (events with increasing red fluorescence above the main sperm population) to be removed. Green fluorescence, in this case because of an anti-BrdUTP antibody conjugated to FITC, increases with the occurrence of DNA single strand breaks and can be easily analysed using a histogram of the distribution of green fluorescence (inset), after gating out debris and aggregates, and setting the TUNEL+ marker by using positive and negative controls
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Another DNA assay that has been adapted to flow cytometry is the TUNEL assay (terminal transferase dUTP nick end labelling). Originally devised to detect DNA degradation in apoptotic somatic cells, it uses a terminal deoxynucleotidyl transferase to add deoxyuridine triphosphate nucleotides to the 3′-hydroxyl ends resulting from DNA breaks. The 3′-hydroxyl ends are labelled either by using fluorescent-tagged dUTP or by using 5-bromo-dUTP and incubating the marked sample with an anti-BrdUTP antibody conjugated with a fluorochrome (Fig. 2c). This assay is more expensive and more complex to perform than the SCSA®, but it provides precise information about the degree of sperm fragmentation (because fluorescence increases with the number of 3′-hydroxyl ends). Thus, the TUNEL is a promising technique that has demonstrated a good relationship with SCSA® and with fertility (Waterhouse et al. 2006; Benchaib et al. 2007). We have shown that the TUNEL assay combined with flow cytometry could discriminate among different sperm treatments that could cause an increase in DNA breaks in red deer spermatozoa (Domínguez-Rebolledo et al. 2009a, 2010). We have used PI to remove debris and sperm aggregates, better defining the sperm population (Fig. 2c). As other authors reported previously (Muratori et al. 2008), we have noticed changes in the PI fluorescence intensity of spermatozoa when high DNA damage was present. We must highlight the need of employing negative and positive controls, in a similar manner than for SCSA®. The positive control can be easily made by incubating the spermatozoa with DNase I. Moreover, washing steps can greatly decrease the sperm concentration if not carried out carefully, causing artefacts when analysing the samples.
Recently, other methods have been adapted for flow cytometry. Chromatin thiol status and compaction may be assessed by using monobromobimane (mBBr) and chromomycin A3 (CMA3), respectively (Zubkova et al. 2005). A high degree of DNA compaction in the sperm head limits the accessibility of DNA-damaging agents and depends on the number of disulphide bonds within and between protamine molecules. Faulty spermatogenesis or maturation can impair chromatin compaction. Thus, Zubkova et al. (2005) found a decrease in chromatin compaction (CMA3) with age in rat sperm and an increase in thiol oxidation after oxidative stress. Therefore, these probes could be useful in the quality assessment of semen doses from domestic animals.