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Hum. Reprod. Advance Access originally published online on April 15, 2008
Human Reproduction 2008 23(6):1263-1270; doi:10.1093/humrep/den112
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© The Author 2008. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Nuclear organization in human sperm: preliminary evidence for altered sex chromosome centromere position in infertile males

K.A. Finch1,2, K.G.L. Fonseka1, A. Abogrein1, D. Ioannou1, A.H. Handyside2, A.R. Thornhill2, N. Hickson1 and D.K. Griffin1,3

1 Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK 2 The London Bridge Fertility, Gynaecology and Genetics Centre, One St Thomas Street, London SE1 9RY, UK

3 Correspondence address. Tel: +44-1227-823022; Fax: +44-1227-763912; E-mail: d.k.griffin{at}kent.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Authors' contributions
 Funding
 References
 
BACKGROUND: Many genetic defects with a chromosomal basis affect male reproduction via a range of different mechanisms. Chromosome position is a well-known marker of nuclear organization, and alterations in standard patterns can lead to disease phenotypes such as cancer, laminopathies and epilepsy. It has been demonstrated that normal mammalian sperm adopt a pattern with the centromeres aligning towards the nuclear centre. The purpose of this study was to test the hypothesis that altered chromosome position in the sperm head is associated with male infertility.

METHODS: The average nuclear positions of fluorescence in-situ hybridization signals for three centromeric probes (for chromosomes X, Y and 18) were compared in normoozoospermic men and in men with compromised semen parameters.

RESULTS: In controls, the centromeres of chromosomes X, Y and 18 all occupied a central nuclear location. In infertile men the sex chromosomes appeared more likely to be distributed in a pattern not distinguishable from a random model.

CONCLUSIONS: Our findings cast doubt on the reliability of centromeric probes for aneuploidy screening. The analysis of chromosome position in sperm heads should be further investigated for the screening of infertile men.

Key words: male infertility/sperm/centromere/FISH


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Authors' contributions
 Funding
 References
 
A range of chromosomally related genetic defects has been associated with male infertility phenotypes (Chandley et al., 1979Go; de Braekeleer and Dao, 1991Go; Shah et al., 2003Go; Griffin and Finch, 2005Go). Constitutional structural abnormalities, e.g. inversions and balanced translocations (Chandley et al., 1979Go), Y chromosome deletions (Affara and Mitchell, 2000Go) and constitutional trisomy (mosaic or full blown) (e.g. Skakkebaek et al., 1973Go; Sheridan et al., 1989Go) are the most cited. While these different types of chromosome abnormalities lead to infertility via various different etiologies (e.g. some involve asynapsis and altered recombination patterns while others do not) collectively they highlight the continuing importance of cytogenetically based genotype–phenotype correlations in studies of male reproduction. More recently, the association between compromised semen parameters and increased sperm disomy (presumably as a result of aberrant meiotic behaviour) has become well documented with many authors reporting a 10–30-fold increase in the most extreme cases (Tempest and Griffin, 2004Go). However, to date a correlation between altered chromosome position in sperm head nuclei and male infertility has not yet been established.

Chromosome territory position in the interphase nucleus is commonly regarded as an indicator of nuclear organization in a range of cell types and developmental stages (Foster and Bridger, 2005Go). Spermatogenesis is a developmental process in which nuclear organization and architecture have been studied extensively (Zalensky et al., 1993Go, 1995Go; Tilgen et al., 2001Go; Sbracia et al., 2002Go; Mudrak et al., 2005Go). Sperm nuclear architecture may be important for spatial chromatin differentiation and may help to direct normal development of the fertilized egg. Such architecture is thought to have evolved alongside mammal-specific regulatory systems such as X inactivation and genomic imprinting (Greaves et al., 2003Go). During pachytene, the sex chromosomes are transcriptionally silenced and condensed to form the sex body, which is located towards the periphery of the nucleus (reviewed in Turner, 2007Go). Moreover, Foster et al. (2005)Go established in porcine testes that the sex chromosomes then reposition from the nuclear periphery to the nuclear centre, occuring between the secondary spermatocyte and round spermatid stages. During spermiogenesis, remodelling of the chromatin takes place as the histones are replaced, first by transition proteins, and then by protamines, leading to highly compacted chromatin (Kierszenbaum and Tres, 1975Go; Loir and Courtens, 1979Go; Meisrich et al., 1979Go, 2003Go; Kistler et al., 1996Go).

In the sperm of monotreme mammals, chromosomes are arranged in a specified order. Work by Greaves et al. (2003)Go suggested that the X chromosome lies in the region that makes first contact with the egg, and this position may be related to its predisposition for inactivation. Greaves et al. (2001)Go focused on the positions of chromosomes in marsupial sperm, demonstrating that chromosomes occupy fixed positions in both immature and mature sperm; this may be important in establishing patterns of gene activity in the developing embryo. In rats (Rattus rattus) telomeres in sperm heads are located peripherally, whereas peri-centromeres are more interior (Meyer-Ficca et al., 1998Go). Human sperm chromosome territories are organized into loop domains that are attached at specific sites to the sperm nuclear matrix (Kalandadze et al., 1990Go; Nadel et al., 1995Go; Kramer and Krawetz, 1996Go). Nuclear architecture in the mammalian sperm is thought to be characterized by the clustering of the 23 centromeres into a ‘chromo-centre’ which is positioned well inside the nucleus (Zalensky et al., 1993Go, 1995Go; Tilgen et al., 2001Go; Sbracia et al., 2002Go; Mudrak et al., 2005Go; Zalensky and Zalenskaya, 2007Go), although the nature and compactness of the chromo-centre may vary (Zalensky and Zalenskaya, 2007Go). Moreover the human X chromosome territory shows preference for a position in the anterior half of the nucleus (Luetjens et al., 1999Go; Hazzouri et al., 2000Go; Zalensky and Zalenskaya, 2007Go). Patterns of organization of the chromosome territory and its associated relative centromere and telomere positioning suggest that DNA loop domains may be mediated by the nuclear matrix (Ward and Zalensky, 1996Go); it has been further hypothesized that the spatial organization of the male-haploid genome is important in sperm function and early development (Sotolongo and Ward, 2000Go). Moreover, functionally abnormal sperm, in which the nuclear matrix has been chemically disrupted (and thus presumably the nuclear organization perturbed), are thought to be unable to produce viable offspring (Ward et al., 1999Go). However it has yet to be determined whether individual infertile men have compromised nuclear organization as measured by assays of chromosome territory position. The purpose of this study was to test the hypotheses (i) that three centromeric loci occupy a central position in a population of normozoospermic males and, (ii) if so, whether this pattern is altered in men with compromised semen parameters.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Authors' contributions
 Funding
 References
 
Source of samples
Control sperm nuclei preparations were made from nine chromosomally normal men (controls 1–9, Table I) with normal semen parameters (according to WHO criteria 1999). The infertile cohort comprised sperm samples from 15 men undergoing male factor IVF treatment (Patients 10–24). These patients were classified as oligozoospermic (O) if sperm concentration was <20 million/ml, severe oligozoospermic (sO) if <5 million/ml, asthenozoospermic (A) if <25% forward motility and teratozoospermic (T) if >85% abnormal forms. Thus, Patients 10–13 were classified ‘O’, Patient 14 as ‘A’, Patients 15–17 as ‘AT’, 18–19 were ‘OA’, 20 and 21 were ‘sO’, 22 was ‘sOA’ and 23–24 ‘sOAT’ (Table I). All samples were from men participating in sperm donation or under treatment for infertility at The London Bridge Fertility, Gynaecology and Genetics Centre in London, or at the National Infertility Centre, Abumeliana Fertility Clinic, Farah Fertility Centre or Misurata Infertility Centre in Libya. All men were chromosomally normal according to standard G-banded karyotyping, but none was tested for submicroscopic Y chromosome deletions. All participants gave informed consent for the use of their sperm for research purposes and this work was approved under the auspices of the treatment licence awarded by the HFEA to the London Bridge Fertility Centre, the Libyan Ministry of Health and the Local Research and Ethics committee of the University of Kent.


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  		Table I. Summary of location of three loci (18c, Xc and Yc) in controls (1–9) and infertile men (Patients 10–24).

 
Preparation of samples
Sperm samples were prepared from fresh ejaculate, washed in 10 mM NaCl + 10 mM Tris pH 7.0 sperm buffer solution, re-suspended and centrifuged at 300g for 5 min. The supernatant was removed to leave ~1 ml remaining, the pellet re-suspended and the process repeated up to five times. Samples were fixed drop-wise to a final volume of 5 ml using 3:1 methanol:acetic acid. The solution was re-suspended and centrifuged at 300g for 5 min. The supernatant was removed to leave ~1 ml remaining, the pellet re-suspended and the process repeated three times.

Fluorescence in-situ hybridization
Briefly, 5 µl of sample was spread over an area 20 x 20 mm on a Poly-L-lysine slide (VWR International, Leicestershire, UK) allowed to dry at room temperature, the optimal density checked and the sample area marked on the underside of the slide using a diamond pen. The slide was incubated on a hot plate at 65°C for 1.5 h. Sperm cells were swollen by exposure to 0.1 M DTT for 20 min, treated with 1% pepsin/0.01 M HCl for 20 min (39°C), fixed in 4% paraformaldehyde, dehydrated and air-dried. To increase the level of sperm swelling DTT incubations of 40 and 60 min were used. Either the pan-centromeric probe set or the pre-prepared triple colour Aneuvysion© probe set for the centromeres of chromosomes 18 (aqua), X (green) and Y (red) (Vysis, Abbott laboratories, UK) were applied to the nuclear DNA under a coverslip. This was sealed using rubber cement and both nuclear and probe DNA were denatured simultaneously using a ‘HYBrite’ (Vysis) hotplate for 5 min at 75°C. Hybridization at 37°C continued for 16–24 h and post-hybridization washes consisted of 0.4x standard saline citrate (SSC)/0.3% Tween-20 (Sigma) at 73°C for 3 min and 1 min in 2x SSC/0.1% Tween-20 at room temperature before dehydration in an alcohol series. Slides were then air-dried, mounted in Vectashield anti-fade medium (Vector Laboratories, CA, USA). FISH signals were evaluated using a BX61 (Olympus, Hertfordshire, UK) microscope equipped for epifluorescence; images were captured using either Cytovysion Software (Applied Imaging/Genetix, Hampshire, UK) or SmartCapture software (Digital Scientific UK, Cambridge, UK) and exported as JPEG and/or TIFF files to Paint Shop Pro 9.1 for aneuploidy and locus position analysis.

Scoring and interpretation
Signals were classified as representing separate hybridization events if two or more similarly sized fluorescent foci could be identified that were greater than one signal's diameter apart. If the loci were less than one signal's diameter apart, the signal was classed as a ‘split signal’ and thus scored as a single hybridization event. Only haploid, non-disomic nuclei were scored.

Analysis of locus position in interphase nuclei
Adaptations of previously published approaches for three-dimensional extrapolations from two-dimensional data were used. An approach very similar to that of Croft et al. (1999)Go and Boyle et al. (2001)Go was employed to assess chromosome location using two-dimensional images. A transparent five-circle template (Fig. 1) was used consisting of five concentric circles derived from concentric spheres of equal volume. Five spheres of linearly increasing volume were considered (from V = 1 to V = 5). The radius of each sphere was calculated from the equation: V = 4/3 {pi} r3.


Figure 1
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  		Figure 1: Sperm nuclei with FISH signals and 5 shell template overlaid.

The nuclear counterstain is DAPI (blue), the chromosome 18 centromeric probes is aqua, the X chromosome centromeric probe is green and the Y chromosome probe red.

 
Hence,


Formula

The template was overlaid on the nucleus image using the rotation, vertical and horizontal size editing functions to best fit the nucleus (Fig. 1). Chromosome loci signals were scored according to which of the five rings they appeared in: area (shell) one was defined as the outer-most ring and area (shell) five as the innermost. If a probe spanned more than one shell it was scored depending upon where the majority of that signal lay. Signal scores were compiled for each nucleus using Microsoft® Excel. Sperm heads were not of sufficient quality (particularly in infertile patients) to determine the polarity of the cell and hence the relative anterior/posterior chromosome territory position.

Statistical analysis
In testing the null hypothesis that distributions were not significantly different from what would be observed from a random pattern, our ‘expected’ values for each shell were derived from the number of signals scored divided by the number of rings (five). In essence, if a random pattern were present, we would expect all bars in the histogram to be roughly of the same height. For each locus, in each cell type our raw data (i.e. the number of signals that were actually scored) constituted our ‘observed’ values. From there, a chi-squared test was used to determine the presence any of non-random distributions. Given that there were five individual values (one for each shell) that made up the subsequent chi-squared total, P-values were determined at four degrees of freedom. Distributions were considered significantly different when compared with the nuclear counterstain at P ≤ 0.05 and ‘highly’ significantly different when P ≤ 0.01. This approach represents only a minor modification from previously described methodology (e.g. Croft et al., 1999Go; Bridger et al., 2000Go; Boyle et al., 2001Go; Meaburn et al., 2005aGo, bGo). Results were included only when at least 50 sperm could be scored.

Histogram presentation of results
Relative positions were presented by ‘% relative observed frequency’ in order to allow direct comparisons between graphs (Fig. 2). In addition to the chi-squared data therefore, the shape of the graphical distributions was taken into account including analysis of standard error of the mean (as depicted by the standard error bars in the histograms—Fig. 2). In order to determine whether the highest bar in the histogram was significantly greater than its neighbour(s), individual chi-square tests (1 df) were performed on the raw data. This (and visual inspection of the shape of the graph) allowed us to determine the ‘directivity’ of our distributions, i.e. whether the signal was distributed peripherally, medially or centrally. For the purposes of this study, a ‘central’ location was defined as a non-random distribution where the bar representing shell 5 (or 2 bars representing shells 4 and 5) was (were) significantly higher that its (their) nearest neighbour by chi-squared test. A central/medial location was defined as a non-random distribution where the bar representing shell 4 (or 3 bars representing shells 3–5) was (were) significantly higher that its (their) nearest neighbour by chi-squared test and so on. Therefore, in order to determine the mean nuclear position of each centromere we considered (i) whether the distribution was significantly different from that predicted by a random pattern and (ii) if so, in which shell (or shells) was (were) the signal seen most often.


Figure 2
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  		Figure 2: Distribution of loci (18c, Xc, Yc) in controls.

One example for each locus is given.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Authors' contributions
 Funding
 References
 
Position of centromeric loci in the sperm head using a pan-centromeric probe
A probe that recognizes all human chromosomes was applied to control human sperm heads and revealed that, unlike in mice, a clear and fixed chromocentre was not as easily discernable. Numerous spots appeared in the sperm head and visual inspection indicated a general tendency to the nuclear centre. However meaningful ‘shell’ analysis was not practicable due to our inability to discern overlapping signals with any accuracy. For this reason, shell/histogram analysis was continued only on individual centromeres.

Identification of the three individual centromeric loci in sperm nuclei of men with normal semen parameters
Our results provide evidence for a chromo-centre arrangement with all three loci occupying a central or central/medial location in the majority of normozoospermic control males (Table I). Figure 2 shows representative examples of the central positions for each of the centromeric loci in control patients. In addition our findings show that this central position of the centromeric array is maintained in a range of sperm head sizes under a range of swelling conditions (results not shown). Only 3 out of 27 analyses (11.1%) displayed a pattern for centromeric loci that differed from a non-random, central (or central/medial) pattern. Specifically: in control 1 the distribution of locus Xc was not significantly different from that predicted by a random distribution (Table I), control 3 displayed a significant ‘medial’ location (shell 3) for 18c and, for control 8, the Y chromosome centromere distribution observed was not significantly different from that predicted by a random pattern (Table I). All nine controls therefore showed a non-significant, central (or central-medial) pattern for at least two out of three centromeric loci (Table I).

Investigation of the position of three centromeric loci (X, Y and 18) in sperm nuclei of men with impaired semen parameters compared to the controls
Given that these men had fewer sperm and that hybridization efficiencies were less successful than in controls, a larger proportion of experiments were not successful despite repeated attempts. We were able to score all three signals in Patients 11, 14 and 22 and two signals in all other patients with the exception of Patient 17 where only the 18c signal was analysable (Table I). On average, hybridization efficiencies (i.e. the proportion of nuclei that displayed a discernable signal) were 96% for control samples (range 75–100%) and 66% for patients (range 0–98%).

The distribution of locus 18c (where sufficient numbers of cells could be analysed) was significantly non-random (P < 0.05) in all infertile men (Patients 10–24), the same as it was in all controls (1–9). In the control group, 18c deviated from the ‘usual’ central (or central/medial) position in only one male (11.1% - control 3); similarly among infertile men (Patients 10–24), the same locus deviated from the ‘usual’ pattern in 2 out of 14 patients (14.3%). For the centromere of chromosome 18, therefore, patterns observed in the patient group were not significantly different from those in the control group. However, among the infertile males (Patients 10–24) the pattern of signal distribution for the X chromosome centromere was not significantly different from that predicted by a random model in 4 out of 10 (40%) measurements and similarly for the Y chromosome centromere in three out of eight (37.5%) measurements. Therefore an apparently random pattern of signal distribution for the sex chromosome centromeres was seen in 7 out of 18 measurements (38.9%) (Table I). This ‘apparently random’ distribution among the sex chromosomes (both individually and collectively) was observed significantly more often (in fact over three times more frequently) (P < 0.01 by t-test) compared with the control group.

Significant non-random, central distributions of loci 18c, Xc and Yc were observed in all oligozoospermic men (Patients 10–13) with the exception of Yc for Patient 12 and 18c for Patient 10. The distribution of 18c was non-random and central/medial but not significantly different from that predicted by a random distribution for Xc and Yc in a single asthenozoospermic male (Patient 14). In asthenoteratozoospermic patients (15–17), all but one displayed a non-random distribution for the loci studied (see Fig. 3), while in oligoasthenozoospermic patients (18 and 19) significant non-random, central distributions for 18c were largely observed. Severe oligozoospermic patients (20 and 21) showed significant non-random distributions for loci 18c and Yc, appearing most frequently in medial and central/medial positions, respectively, though locus Xc displayed a pattern not significantly different from that predicted by a random distribution in Patient 20. In a single severe oligoasthenozoospermic patient (22), all three loci (18c, Xc and Yc) displayed significant non-random, central (or central/medial) distributions, and in severe oligoasthenoteratozoospermic (OAT) patients (23 and 24), loci 18c and Xc showed a significant non-random distribution at central/medial positions (see Fig. 4) with one (apparently random) exception. All of the above results are expressed in more detail in Table I.


Figure 3
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  		Figure 3: Distribution of loci (18c, Xc, Yc) in patient 17 (asthenoterotozoospermic).

 

Figure 4
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  		Figure 4: Distribution of loci (18c, Xc, Yc) in patient 23 (severe oligoasthenoteratozoospermic).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Authors' contributions
 Funding
 References
 
Our results provide evidence that the three centromeric loci analysed preferentially occupy a central position as predicted by Zalensky et al. (1993Go, 1995Go) and Zalenskaya and Zalensky (2004)Go with very little but nonetheless some inter-individual variation. However, the use of the pan-centromeric probe revealed that this pattern was not nearly as clear-cut as the defined and punctate chromo-centre seen in murine sperm. One obvious initial practical implication of our data is that probes which more commonly occupy the nuclear periphery are more likely to detect a higher proportion of disomies than probes which occupy the nuclear centre (such as the sex chromosome probes and the centromeric probes) because of the increased likelihood of signals at the nuclear centre overlapping. For the most part, the probes of choice for studies of male meiotic non-disjunction (for review see Tempest and Griffin, 2004Go) are those that recognize centromeric arrays, largely because of the bright signals that they produce. However, the observation that these probes are found more often at the nuclear centre calls into question their utility when drawing conclusions about the absolute levels of aneuploidy in the male gamete. Similarly although studies comparing data from the same chromosome between patients may well be valid, inter-chromosomal comparisons may not, e.g. if a peripherally located locus is compared with centrally located one. On another technical point it was also noteworthy that hybridization efficiencies were lower for infertile patients compared with normal controls. While there is no easy explanation for this observation it did mirror the overall quality of the semen samples used in this study.

Our results also provide some preliminary evidence in support of the hypothesis that, at least in certain men, altered nuclear organization for the sex chromosome is associated with male factor infertility. While a failure to establish significant evidence for a central or central/medial location of the X and Y chromosome probes (both individually and collectively) was not common in the control group, it was a regular occurrence in the cohort of infertile men, happening in nearly half of the cases (where the signals could be analysed) and three and a half to four times more frequently than in the control group. Altered chromosome position has been associated with a range of cell disorders including epilepsy, certain types of laminopathies and cancer (Borden and Manuelidis, 1988Go; Misteli, 2004Go; Meaburn et al., 2005aGo, bGo). To the best of our knowledge this is the first time that alterations in nuclear organization have been related to a human reproductive phenotype.

Our findings may suggest a relationship between impaired spermatogenic regulation and altered nuclear organization in certain men. Foster et al. (2005)Go first reported the migration of the sex chromosomes from the nuclear periphery to the nuclear centre in porcine spermatogenesis by the round spermatid stage. In parallel, the organization of chromosome territories in a ‘chromo-centre’ (also by the round spermatid stage) is now an accepted model in a number of species including mouse and human (Zalenskaya and Zalensky, 2004Go, 2007Go; Namekawa et al., 2006Go). Recently there has been clear evidence for the juxtaposition of the post-meiotic sex chromatin (PMSC) with the chromo-center in murine spermatogenesis (Namekawa et al., 2006Go). Migration to the nuclear centre at the round spermatid stage is accompanied by a reactivation of 33 X chromosome genes that had previously been inactivated by meiotic sex chromosome inactivation (MSCI) including Pctk1, Ube2a and Pdk3 (Namekawa et al., 2006Go). Turner et al. (2006)Go noted that MSCI is an inevitable consequence of asynapsis (e.g. of the sex chromosome bivalent) and is an example of the general mechanism of meiotic silencing of unsynapsed chromatin (MSUC). Turner (2007)Go goes on to argue that MSCI/MSUC is involved in protecting the gamete from generating excessive levels of aneuploidy (which also can be generated by synaptic errors). Compromised semen parameters are associated with increased levels of sperm aneuploidy (reviewed in Tempest and Griffin, 2004Go) and, according to our results, increased failure of migration of the PMSC to the nuclear centre by the round spermatid stage is also possible. It therefore prompts us to speculate that mechanisms involved in the genesis of male infertility and its association with increased levels of aneuploidy may well be related to MSCI/MSUC. A closer examination of chromosome position and its relationship between sperm aneuploidy and compromised semen parameters in a larger group of patients may enable us to determine this.

In infertility clinics, males are routinely screened for standard semen parameters such as concentration, motility and morphology. Increased sperm aneuploidy is associated with compromised semen parameters and was first reported in the late 1990s (Pang et al., 1999Go). Some infertility clinics are now adopting FISH aneuploidy screening in sperm as a standard protocol. Indeed we have argued that sperm aneuploidy levels should constitute another semen parameter (Griffin et al., 2003Go). If FISH experiments are already being performed to assess chromosome copy number, the additional time also to assess nuclear organization would be minimal. It should be stressed however that our results, as they stand, although showing a significant association between sex chromosome position in sperm heads and infertility, do not provide evidence that sex chromosome centromere position analysis constitutes an accurate diagnostic test for infertility. It remains to be seen whether assays for chromosome position will ultimately constitute yet another semen parameter. However, in the meantime, studies such as those involving three-dimensional analyses, would help to indicate whether altered nuclear organization is a general phenomenon, or whether it applies to a specific sub-set of patients.


   Authors' contributions
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Authors' contributions
Funding
 References
 
K.A.F., K.G.L.F., N.H. and D.K.G. performed the experiments, data collection and analysis. A.A., A.H.H. and A.R.T. provided patient material and critically appraised the manuscript. D.I. performed the experiments to address the reviewer comments. D.K.G. (with assistance from A.H.H. and A.R.T.) conceived and managed the project.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Authors' contributions
 Funding
References
 
K.A.F was funded by a BBSRC quota studentship awarded to the University of Kent. D.I. is funded by a BBSRC industrial CASE studentship awarded to Digital Scientific UK (Cambridge, UK). K.G.L.F., A.A. and N.H. are/were self-funded postgraduate students at the University of Kent.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Authors' contributions
 Funding
 References
 
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Submitted on January 3, 2008; resubmitted on February 28, 2008; accepted on March 13, 2008.


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