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From the * Departamento de Biología,
Universidad Autónoma de Madrid, Madrid, Spain; the
Sección de Genética y Unidad de
Investigación, Complejo Hospitalario Universitario Juan Canalejo,
Coruña, Spain; and the School of Animal
Studies, The University of Queensland, Gatton, Australia.
| Correspondence to: Dr Jaime Gosálvez, Departamento de Biología, Unidad de Genética, Edificio de Biología, Universidad Autónoma de Madrid, C/Darwin no 2, 28049 Madrid, Spain (e-mail: jaime.gosalvez{at}uam.es). |
| Received for publication November 27, 2006; accepted for publication February 5, 2007. |
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Abstract |
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Key words: Sperm chromatin structure, spermatogenesis, DNA damage, sperm nuclear proteins, reproduction
It is well established that sperm DNA is packaged to arrange the chromatin into a highly compact and stable form (Ward and Coffey, 1991). To arrive at such a structure, the proteinaceous components of the nucleus must undergo a remarkable series of structural and biochemical changes during spermiogenesis in which they are first replaced by intermediate proteins and then finally by protamines characteristic of the mature sperm of the individual species (Kimmins and Sassone-Corsi, 2005).
Studies on the relationship between alterations in sperm nuclear proteins and their effects on DNA are primarily concerned with alterations to protamines or transition proteins and have been associated with abnormal sperm chromatin organization, an increased number of DNA strand breaks, and decreased fertility (Balhorn et al, 1988; Yu et al, 2000; Cho et al, 2001; Zhao et al, 2001; Cho et al, 2003, Meistrich et al, 2003; Shirley et al, 2004; Aoki et al, 2005). However, it remains to be determined if the nuclear protein fraction is adversely affected when the DNA is fragmented and whether this will also cause a subsequent decrease in fertility.
Recently a new technique, the sperm chromatin dispersion (SCD) test, was developed for determining sperm DNA fragmentation in a range of mammalian species (Fernández et al, 2003, 2005; Enciso et al, 2006). This protocol is a simple, reliable procedure that does not require elaborate or expensive methodologies. For example, the procedure for visualizing DNA fragmentation in boar spermatozoa consists of a brief incubation of agarose-embedded cells in a lysing solution which results in protein removal in sperm cells. After DNA staining, those sperm cells with DNA fragmentation display large peripheral halos of diffusion of DNA fragments and are easily discriminated from spermatozoa without DNA fragmentation (Enciso et al, 2006; Pérez-Llano et al, 2006). Since this method relies on the effective partial depletion of protein, protein staining can be employed to examine the remnant nuclear proteinaceous matrix. The ability to simultaneously stain for both protein composition and DNA fragmentation was exploited in this study for the first time in boar spermatozoa and provided an opportunity to study the synergetic relationship between these 2 parameters.
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Materials and Methods |
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DNA Fragmentation and Protein Assay
The DNA fragmentation assay was performed using a commercial variant of the
SCD test (Sperm-Sus-Halomax; ChromaCell SL, Madrid, Spain). Gelled
aliquots (50 µL) of low–melting-point agarose in Eppendorf tubes
(provided in the kit) were placed in a water bath at 90°C to 100°C for
5 minutes to melt the agarose and then equilibrated for 5 minutes in a water
bath at 37°C prior to the addition of 60 µL of spermatozoa. The
semen-agarose (20 µL) mixture was then rapidly pipetted onto an
agarose-precoated slide (provided in the kit) and covered with a 22 x 22
mm coverslip. The slide was then placed on a cold metal plate (at 4°C) in
the refrigerator for 5 minutes to allow the agarose to set and produce a thin
microgel. The coverslips were gently removed, and half of the slide was
treated with lysing solution (provided in the kit) for 5 minutes to remove
sperm membranes and partially deproteinize nuclei. After washing in distilled
water for 5 minutes, the slides were dehydrated in an increasing series of
ethanol baths (70%, 90%, and 100%) for 2 minutes each and subsequently
air-dried. The slides were then stained for fluorescence microscopy by
incubating with 2,7-dibrom-4-hydroxy-mercuryfluorescein di-sodium salt
(Sigma-Aldrich, St Louis, Mo) for total protein staining, followed by
propidium iodide in Vectashield Mounting Medium H-1000 (Vector Laboratories,
Burlingame, Calif). The dual emission fluorochrome combination allowed
simultaneous visualization of DNA and total proteins (red for DNA and green
for proteins) using a dual-band pass fluorescence filter block; alternatively
single emission could be observed using a single-band pass fluorescence filter
block.
Image Analysis and Constraints
The main aim of this study was to explore differential protein removal
associated with sperm DNA fragmentation and to visualize these differences via
image analysis. Given that digital image analysis is only semiquantitative, it
was important to have a rigid internal control within the same slide to
minimize any technical environmental differences. This precaution reduced any
inherent variations in the SCD technique associated with microgel thickness.
Two independent sperm samples were prepared in the microgel per slide. One
sample was not processed for lysis and was referred to as the
control-untreated sample; the remaining sample underwent SCD lysis. The SCD
approach allowed the discrimination of both unfragmented and fragmented sperm
cells. These cell types will be referred as lysis-treated unfragmented sperm
cells and lysis-treated fragmented sperm cells, respectively. Additionally,
all sperm images were captured the same day, under the same conditions, using
a fixed time for image exposure and a constant 5-second fading prior to image
digitalization.
Digital images were produced as TIFF 12-bit images using a black and white cooled Leica DCF 300 camera mounted onto a Leica DM microscope using single-band pass filters (FITC-3540B-536/617; Cy5-4040A-492/516; Semrock, Rochester, NY). Digital image analysis was performed using Leica Q-Win software (Leica Microsystems, Barcelona, Spain). Three different predefined cells were used for the analysis of the assay according to halo morphology following visualization of the DNA: type 1: control-untreated sperm cells; type 2: treated sperm with unfragmented DNA with the absence of chromatin dispersion halos; and type 3: treated sperm with fragmented DNA with large halos of diffused DNA fragments. To quantify the amount of DNA and protein in each of the sperm head types, each sperm head (type 1: n = 35; type 2: n = 39; type 3: n = 38) was digitally analyzed after background extraction for red area (R-A) and whole red intensity (R-I) for DNA and green area (G-A) and total green intensity (G-I) to quantify protein. Images were transformed into color by converting TIFF 12-bit images into 8 bits using Photoshop 7.0 (Adobe Systems Inc, San Jose, Calif). Color assignment to each of the 8-bit gray images was also conducted using Photoshop with the red code for DNA and the green for proteins. This color code was selected to improve visual discrimination between both channels, but alternative color combinations are possible.
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Results |
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Fresh sperm samples evaluated with the SCD test showed 2 distinctive sperm nuclear morphologies when studied for DNA distribution. Cells without DNA fragmentation (type 2) showed a homogeneous core with an absence of halos or with a very small halo of chromatin dispersion, specifically located at the caudal extremity (close to the flagellum) of each sperm head (Figure 1b). The amount of nuclear protein staining in type 2 cells diminished compared with that in control-untreated cells but retained a similar staining pattern of distribution (Figure 1b').
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Sperm which had been incubated in H2O2 for 48 hours at 37°C and processed under standard SCD test conditions showed a quite different staining pattern. The majority of the sperm heads had halo-associated nuclei morphology, although halo size varied from cell to cell in association with a noticeable and progressive core disruption (Figure 1d through f). Under these experimental conditions, halo DNA tended to disperse because of massive DNA breakage after oxidative stressful conditions such that the apparent sizes of the halos were diminished (Figure 1d through f). In parallel to the level of DNA fragmentation, there was also a sequential lose of protein remnant; the smaller the halo, the higher the core disruption and the more protein remnant that was lost (Figure 1d' to f'). The final protein component that remained anchored to the sperm head was closely associated with the base of the postacrosomal region in close proximity to the implantation fossa (Figure 1 f and f').
Four different variables defining DNA (R-A and R-I) and protein (G-A and G-I) fluorescence were used to quantitatively discriminate among the 3 predefined cell types. The descriptive statistics of this analysis are described in Table 1. No significant differences were found which could be attributed to the presence of different cell subpopulations within each cell type. However, differences (P < .05) were observed among cell types for R-A, R-I, and G-I. Significant differences were also observed for G-A between cell type 3 and types 1 and 2 but not between types 1 and 2. Type 3 cells showed 65% of the G-A and 34% of the G-I observed in control-untreated cells, whereas type 2 cells showed 93% of the G-A and 43% of the G-I observed in controls.
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To determine which of these variables were required to reach the best possible classification of the 3 different cell types, a discriminant analysis was performed. All variables were integrated into a model composed of just 2 discriminant functions. The first discriminant function distinguished between untreated and treated cells (Table 2). This first function gave more weight to G-I (protein) and to a lesser extent, R-I (DNA) (Table 3). According to centroid positioning (Table 2), those cells with high G-I and low R-I were classified as untreated (type 1), while those with low G-I and high R-I were defined as treated (types 2 and 3). The second function distinguished between fragmented and unfragmented sperm cells within the treated cell group. This function gave more weight to R-A (DNA) and G-A (protein). According to centroid positioning (Table 2), those cells with high R-A and low G-A were classified as fragmented (Figure 3). The first function explained 77.2% of the variability and the second function 22.8%. For different boars or different charge-coupled devices, the actual raw numeric data may vary but not the general profile of the plot (data not shown). Differences among boars or experimental conditions are of lesser magnitude than those existing among cell types.
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Discussion |
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In sperm nuclei of eutherian mammals, the chromatin is stabilized by intermolecular and intramolecular covalent disulfide bonds between protamines. The SCD test involves treatment with an agent that reduces protamine disulfide bonds, relaxes the compact structure of the DNA, and produces nucleoids with a central core and restricted halos of DNA loops; the latter is a consequence of the successful extraction of protamines (Tsanev and Avramova, 1981). Those nuclei containing fragmented DNA have nucleoids with markedly larger halos of chromatin diffusion.
This study showed that the amount of nuclear protein diminished in lysis-treated unfragmented cells compared with control-untreated cells, and this loss is indicative of the successful extraction of protamines following lysis. However, despite the loss of protein, unfragmented lysis-treated sperm still retained a similar distributional pattern of protein remnant compared to that seen in untreated cells. For example, they still exhibited a high level of staining in the caudal extremity of the nucleus and the equatorial region is clearly visible (compare Figure 1a' to b'). Sperm DNA is organized into loop domains that are attached by specific sequences to the structural component of the nucleus known as the sperm nuclear matrix (Ward, 1994; Kramer and Krawetz, 1996). The fact that a similar pattern of protein remnant distribution is observed, in spite of a differential extraction of protamines (treated unfragmented compared with untreated cells), indicates that this characteristic distribution may be dependent on proteins from the nuclear matrix that are resistant to extraction by the lysis agent.
In contrast, the distributional pattern of protein, which is normally stable to reducing factors, disintegrates in those lysis-treated nuclei containing fragmented DNA and consequently those sperm showing large halos of chromatin diffusion (Figure 1c'). Perhaps the loss of regional protein distribution in fragmented sperm is related to a differential stability of the nuclear matrix in these cells compared with unfragmented sperm nuclei.
This study has shown a clear relationship between nuclear protein loss and sperm DNA fragmentation, such that simultaneous observation of protein and DNA is likely to give an improved measure and understanding of DNA fragmentation damage. However, it should be noted that simultaneous detection of altered protein conformation and DNA damage is not currently feasible with other methods used to assess DNA fragmentation such as the sperm chromatin structure assay (Evenson et al, 2002), terminal deoxynucleotidyl transferase nick end labeling (Schlegel and Paduch, 2005), or comet assay test (Agarwal and Allamaneni, 2005; Schlegel and Paduch, 2005). Damaged or altered proteins remain undetected in these systems because these tests were developed to only target abnormalities in continuous double-stranded DNA conformation and do not account for proteins. Variations of proteins and DNA damage can be examined together using the current methodology and can now be used to pose and answer important new questions relating to the effect of abnormal sperm protein assembly, its relationship to DNA fragmentation, and their combined effect on fertility.
The results of this study show that the protein fraction and amount of residual proteins in the core following SCD have the potential to be used as an indirect and complementary indicator of sperm DNA fragmentation. We recently used a similar protocol to examine the relationship between DNA fragmentation and protein lysis in human spermatozoa (Santiso et al, 2006) and found that sperm cells without DNA fragmentation showed almost complete removal of nuclear matrix proteins, whereas spermatozoa with DNA fragmentation tended to retain residual nucleoskeletal proteins in a collapsed and condensed state. It is likely, therefore, that the relationship of protein lysis and DNA fragmentation needs to be examined on a species-specific basis.
Interestingly, when sperm samples were incubated in the presence of H2O2 at 37°C for 48 hours prior to SCD processing to increase DNA fragmentation by induction of oxidative stress, the chromatin appeared to decondense more intensively and eventually completely dispersed. A higher proportion of protein remnant was lost in those spermatozoa showing a greater degree of DNA decondensation (Figure 1d' through f'). For spermatozoa in which the core had been disrupted, the expanded DNA appeared to remain attached to a crescent-shaped protein component anchored to the sperm flagellum. This might be related to a postacrosomal structure similar to the nuclear annulus described in the hamster spermatozoon by Ward and Coffey (1989). These authors found a very small representation of protamines in the nuclear annulus, comprising about 3% of the total protein content, and which were structurally separate from the bulk of the protamines. It was proposed that these protamines are protected from proteolytic degradation and may be involved in the organization of the decondensing sperm DNA. Under the strong protein depletion condition experienced after SCD treatment in this experiment, the protein fraction at the distal base of the boar sperm also appeared to be the regional domain most resistant to degradation.
Sperm DNA fragmentation has been correlated with increasing rates of reactive oxygen species (Twigg et al, 1998, Aitken and Krausz, 2001), the triggering of autonomous nucleases that cleave DNA within the dying cell (Chen et al, 2004), and even variations in telomere length caused by the absence of telomerase activity (Rodríguez et al, 2005). In the same way, deficiency in protamines may also result in abnormal sperm chromatin organization and decreased fertility; this requires evaluation in future experiments and new species. For example, mutations in transition protein TP1 or TP2 that result in an increased number of DNA strand breaks (Zhao et al, 2001) should now be investigated.
Working with boar semen, Huang et al (2000) concluded that heat shock protein 90 (HSP90) is responsible for a reduction of sperm motility, and Yue et al (1999) suggested that a small change in HSP90 function, such as those generated by point mutations, could lead to infertility in Drosophila. Epigenetic effects in certain protein residues may also lead to DNA conformational changes in the sperm nuclei. For example, phosphorylation/dephosphorylation of the DNA-TP2 complex is highly correlated with under-condensation of chromatin (Meetei et al, 2002). The results of the current study highlight the importance of the synergistic effect of nuclear protein alteration and DNA damage. It is likely that some sperm cells are more inherently susceptible than others to DNA breakage as a consequence of altered chromatin structure, which makes these spermatozoa more accessible to DNA cleavage. Endogenous DNA breakage releases proteins, thus giving rise to conformational changes in chromatin, which makes it even more susceptible to further DNA alterations. While it is difficult to establish which damage is occurring first, it is likely that these factors are working together through a positive cascade/feedback effect and thereby generating what might be regarded as a DNA fragmentation vortex.
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Footnotes |
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, unfragmented treated cells (type 2); 
, fragmented treated cells
(type 3); and 
, group centroids for each cell type.