Simultaneous Observation of DNA Fragmentation and ProteinLoss in the Boar Spermatozoon Following Application of theSperm Chromatin Dispersion (SCD) Test

JOAQUINA DE LA TORRE,* CARMEN LOPEZ-FERNANDEZ,* MIGUEL PITA,*

JOSE LUIS FERNANDEZ,{ STEVE D. JOHNSTON,{ AND JAIME GOSALVEZ*

From the *Departamento de Biologıa, Universidad Autonoma de Madrid, Madrid, Spain; the �Seccion de Genetica y

Unidad de Investigacion, Complejo Hospitalario Universitario Juan Canalejo, Coruna, Spain; and the `School of Animal

Studies, The University of Queensland, Gatton, Australia.

ABSTRACT: DNA fragmentation and the nuclear protein matrix in

boar spermatozoa were simultaneously assessed using a specific

variant of the sperm chromatin dispersion (SCD) test that allows

direct visualization of DNA and nuclear proteins under standard

conditions of chemical lysis. Nuclear proteins remaining after lysis

were stained with the fluorochrome 2,7-dibrom-4-hydroxy-mercury-

fluorescein for specific protein staining. DNA and nuclear protein

were stained in control-untreated (no lysis) and treated sperm cells

(lysis), resulting in the identification of 3 cell types: type 1: nonlysed

(control-untreated) cells; type 2: lysed cells showing nonfragmented

DNA; and type 3: lysed cells showing fragmented DNA. DNA

damage was also purposely induced by incubating the sperm in

0.015% H2O2 for 48 hours at 37uC; the cells were correspondingly

stained for DNA fragmentation and protein. Nonlysed control sperm

(type 1) nuclei showed no halos and stained strongly for protein in

the postacrosomal region. Lysed spermatozoa with nonfragmented

DNA (type 2) showed evidence of restricted DNA loop dispersions at

the caudal extremity of the sperm head and a more homogenous but

similar distribution of protein matrix in comparison with untreated

spermatozoa. Lysed spermatozoa with fragmented DNA (type 3)

exhibited large halos of DNA loops and a loss of the nuclear protein

matrix component. Sperm cells exposed to 48 hours’ incubation at

37uC and then treated with the lysing agent showed a concurrent and

progressive loss of nuclear protein in association with correspond-

ingly increased levels of DNA fragmentation. Discriminant analysis of

quantitative fluorescence using digital image analysis and conducted

after SCD processing revealed that DNA fragmentation and protein

could be evaluated in an automated system. Ninety-seven percent of

the total analyzed cells were accurately classified according to

previously defined cell types (1, 2, and 3). The results of the current

study demonstrated a synergistic relationship between that of

nuclear protein alteration and DNA damage in the boar sperm cell.

The importance of abnormal nuclear protein alteration to DNA

fragmentation and any related effect on fertility remains to be

investigated.

Key words: Sperm chromatin structure, spermatogenesis, DNA

damage, sperm nuclear proteins, reproduction.

J Androl 2007;28:533–540

The proportion of spermatozoa with fragmented

DNA is higher in infertile males compared with

fertile individuals (Ollero et al, 2001; Agarwal and Said,

2003). Moreover, individuals with abnormal seminal

parameters are more likely to show a higher incidence of

sperm nuclear DNA damage than those with normal

semen (Lopes et al, 1998; Irvine et al, 2000; Ollero et al,

2001; Sakkas et al, 2003). Hence, any procedure that

allows reliable determination of sperm DNA fragmen-

tation would be of great value in the predictive

assessment of male fertility (Evenson et al, 2002;

Agarwal and Said, 2003; Sakkas et al, 2003; Agarwal

and Allamaneni, 2005). To date, the assessment of

sperm nuclear damage has focused exclusively on DNA,

whereas the composition and structural organization of

nuclear protein has received little if any attention.

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

The work has been possible through grants from the Ministerio de

Ciencia y Tecnologıa (MCYT, Spain) (BOS2002-00232 and BOS2003-

04263).

Correspondence to: Dr Jaime Gosalvez, Departamento de Biologıa,

Unidad de Genetica, Edificio de Biologıa, Universidad Autonoma de

Madrid, C/ Darwin nu 2, 28049 Madrid, Spain (e-mail: jaime.gosalvez@

uam.es).

Received for publication November 27, 2006; accepted for

publication February 5, 2007.

DOI: 10.2164/jandrol.106.002246

Journal of Andrology, Vol. 28, No. 4, July/August 2007Copyright E American Society of Andrology

533

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 chromatindispersion (SCD) test, was developed for determining

sperm DNA fragmentation in a range of mammalian

species (Fernandez 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 fragmen-

tation in boar spermatozoa consists of a brief incubation

of agarose-embedded cells in a lysing solution whichresults 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 spermato-

zoa without DNA fragmentation (Enciso et al, 2006;

Perez-Llano et al, 2006). Since this method relies on the

effective partial depletion of protein, protein staining

can be employed to examine the remnant nuclearproteinaceous matrix. The ability to simultaneously

stain for both protein composition and DNA fragmen-

tation 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.

Materials and Methods

Semen Collection and DNA Degradation

Semen samples were collected in February 2006 from one 2-

year-old boar located in a commercial artificial insemination

center in Madrid, Spain. The boar was clinically healthy at the

time of semen collection and produced ejaculates of satisfac-

tory semen quality as assessed by sperm motility, concentra-

tion, vitality, and acrosomal status. The level of sperm DNA

fragmentation in the ejaculate did not exceed 5%. Control

semen samples were diluted in Acromax extender (GVP,

Madrid, Spain) and prepared for analysis within 1 hour of

collection. To induce severe DNA degradation via oxidative

stress, the semen sample was diluted to 107 cells/mL in

extender containing 0.015% (v/v) H2O2 and incubated for

48 hours in a 37uC water bath.

DNA Fragmentation and Protein Assay

The DNA fragmentation assay was performed using a com-

mercial variant of the SCD test (Sperm-Sus-Halomax;

ChromaCell SL, Madrid, Spain). Gelled aliquots (50 mL) of

low–melting-point agarose in Eppendorf tubes (provided in the

kit) were placed in a water bath at 90uC to 100uC for 5 minutes

to melt the agarose and then equilibrated for 5 minutes in

a water bath at 37uC prior to the addition of 60 mL of

spermatozoa. The semen-agarose (20 mL) mixture was then

rapidly pipetted onto an agarose-precoated slide (provided in

the kit) and covered with a 22 6 22 mm coverslip. The slide

was then placed on a cold metal plate (at 4uC) 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 fluoro-

chrome 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. Additional-

ly, 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, Roch-

ester, 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 visualiza-

tion 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

534 Journal of Andrology N July �August 2007

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 5 35; type 2: n 5

39; type 3: n 5 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.

Statistical Analysis

Statistical analysis was performed to discriminate among

sperm head types (1, 2, and 3) using SPSS version 13 for

Windows (SPSS Inc, Chicago, Ill). In this analysis, cell type

was the dependent variable and R-A, R-I, G-A, and G-I were

defined as the independent or discriminant variables.

Results

In control-untreated sperm (type 1), the DNA fluoro-

chrome revealed a brighter fluorescence at the caudal

extremity of the sperm nucleus, close to the flagellum

(Figure 1a). The protein fluorochrome provided non-

homogeneous staining of the sperm head with the

caudal portion displaying a brighter intensity of

fluorescence compared with the rest of the sperm head.

Most notable was the distinctive appearance of the

equatorial region and the postacrosomal sheath and

junction of the sperm head (Figure 1a9). The image

following protein staining in this experiment was similar

to that obtained with fixation in 2% glutaraldehyde and

phase-contrast microscopy.

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

Figure 1. Morphology of control (a) and sperm chromatin dispersion (SCD)–treated boar spermatozoa (b through f). Type 1 (control-untreated)sperm cell after DNA (a) and protein (a9) staining. Type 2 (SCD-treated unfragmented) sperm cell after DNA (b) and protein (b9) staining. Type 3(SCD-treated fragmented) sperm cell after DNA (c) and protein (c9) staining. (d through f), sperm morphology of induced DNA fragmentationafter incubation in H2O2 at 37uC for 48 hours. Note the varying levels of DNA staining (d through f) and their respective protein remnants afterSCD (d9 through f9).

de la Torre et al N DNA Damage and Protein Loss in Boar Sperm 535

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 1b9).

Sperm heads containing fragmented DNA (type 3)

showed a reduced but still highly fluorescent core

surrounded by halos of diffusion of variable size, mainly

formed by DNA residues that were protein depleted

after the lysing procedure was complete (Figures 1c and

2). The amount of fluorescence associated with protein

staining from degraded DNA (Figure 1d9 through f9)

varied from one cell to another but was always lower

than that in sperm with no lysis or those sperm nuclei

that were treated but contained unfragmented DNA

(Figures 1c9 through f9 and 2). The regional heteroge-

neous protein distribution obtained in control and

unfragmented sperm cells was not evident in cells with

fragmented DNA, but instead a homogeneous distribu-

tion of fluorescence was observed (Figure 1c9).

Sperm which had been incubated in H2O2 for

48 hours at 37uC and processed under standard SCD

Figure 2. Simultaneous visualization of DNA (red) and total protein (green) in control (a"") and sperm chromatin dispersion–treated (b""–d"")boar spermatozoa. Control (a) and different levels of DNA damage after DNA staining (b–d). Control (a9) and different levels of protein damageafter protein staining (b9–d9). Note the increasing size of the halo as the proteinaceous core diminishes (a"–d").

536 Journal of Andrology N July �August 2007

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 1d9 to f9). 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 implan-

tation fossa (Figure 1 f and f9).

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.

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.

Discussion

This experiment has clearly demonstrated that DNA

fragmentation in boar spermatozoa is directly associated

Table 1. Descriptive statistics for the analyzed variables of the 3 different boar sperm cell types*

Cell Type n3 R-A3 R-I3 G-A3 G-I3

1 35 1425 6 23 141 153 6 3242 1391 6 36 110 298 6 3834

2 39 3416 6 57 268 018 6 4015 1295 6 30 47 114 6 1463

3 38 7896 6 323 319 836 6 12 619 907 6 37 37 438 6 1967

* Cell types: 1: control-untreated sperm cells; 2: treated cells with unfragmented DNA and no halos of chromatin dispersion; 3: treated cells

with fragmented DNA and large halos of chromatin dispersion.

3 n indicates the number of cells for each cell type used for digital image analysis; R-A, red area; R-I, total red intensity; G-A, green area; and

G-I, total green intensity. Results are reported as mean 6 standard error.

Table 2. Sperm cell type centroid positioning in eachdiscriminant function

Cell Type

Function*

1 2

1 25.187 0.589

2 1.487 22.530

3 3.251 2.054

* Function 1 distinguishes between untreated (type 1) and treated

(types 2 and 3) cell types. Function 2 distinguishes between

unfragmented (type 2) and fragmented (type 3) cell types within

the treated cell group.

Table 3. Standardized coefficients of the discriminantfunctions

Independent

Variable

Function*

1 2

R-A3 0.355 1.087

R-I3 0.477 20.650

G-A3 0.466 20.922

G-I3 21.237 0.904

* Function 1 gives more weight to G-I and a lesser extent to R-I.

Function 2 gives more weight to R-A and G-A.

3 R-A indicates red area; R-I, total red intensity; G-A, green area;

and G-I, total green intensity.

de la Torre et al N DNA Damage and Protein Loss in Boar Sperm 537

with the simultaneous alteration of the nuclear matrix

protein fraction. The following points can be made: 1)

halos of chromatin dispersion in fragmented DNA

nuclei are partially protein depleted and there is a 30%

decrease in the protein fraction in terms of G-A and

a 20% decrease in G-I of fragmented sperm nuclei when

compared with unfragmented nuclei; 2) protein rem-

nants complexed with tightly packed DNA are in-

corporated into a core that is observed in the nuclei of

both fragmented and unfragmented DNA; 3) the size of

the protein core diminishes in an ordered sequence as

the level of DNA fragmentation increases; 4) while

DNA release after protein depletion commences at the

caudal postacrosomal region of the sperm head close to

the implantation fossa, the protein component of this

domain also appears to be the most resistant to chemical

lysis; and 5) the 4 variables analyzed for simultaneous

observation of DNA fragmentation and protein loss in

the present approach can be used via a discriminant

analysis for the automated evaluation of DNA frag-

mentation index. Interestingly, 97% of the total

analyzed cells were accurately classified according to

previously defined cell types (1, 2, and 3). In addition,

classification errors were biased toward treated cells—

8% of type 3 cells (3 out of 38) were misclassified as type

2, while no classification errors were observed for sperm

cell types 1 and 2.

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 struc-

ture 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, unfragmentedlysis-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 1a9 to b9). Sperm DNA is organized into loop

domains that are attached by specific sequences to the

structural component of the nucleus known as the spermnuclear 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 com-

pared with untreated cells), indicates that this charac-

teristic 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, disinte-

grates in those lysis-treated nuclei containing fragmen-

ted DNA and consequently those sperm showing large

halos of chromatin diffusion (Figure 1c9). 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 betweennuclear 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 etal, 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 ac-

count for proteins. Variations of proteins and DNAdamage can be examined together using the current

methodology and can now be used to pose and answer

Figure 3. Dispersion diagram of boar spermatozoa used fordiscriminant analysis on the plane defined by the 2 discriminantfunctions. Spermatozoa are identified by the corresponding cell type:o indicates control-untreated cells (type 1); D, unfragmented treatedcells (type 2); e, fragmented treated cells (type 3); and &, groupcentroids for each cell type.

538 Journal of Andrology N July �August 2007

important new questions relating to the effect of

abnormal sperm protein assembly, its relationship to

DNA fragmentation, and their combined effect onfertility.

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) andfound that sperm cells without DNA fragmentation

showed almost complete removal of nuclear matrix

proteins, whereas spermatozoa with DNA fragmenta-

tion 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 37uC for 48 hours prior to SCD

processing to increase DNA fragmentation by induction

of oxidative stress, the chromatin appeared to decon-

dense 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 1d9 through f9). Forspermatozoa 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 postacroso-

mal 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 bethe 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 (Rodrıguez et al, 2005). In the same way,

deficiency in protamines may also result in abnormalsperm 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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