Origin of DNA damage in ejaculated human spermatozoa

Abstract

The molecular basis of many forms of male infertility is poorly defined. One area of research that has been studied intensely is the integrity of the DNA in the nucleus of mature ejaculated spermatozoa. It has been shown that, in men with abnormal sperm parameters, the DNA is more likely to possess strand breaks. However, how and why this DNA damage originates in certain males and how it may influence the genetic project of a mature spermatozoon is unknown. Two theories have been proposed to describe the origin of this DNA damage in mature spermatozoa. The first arises from studies performed in animal models and is linked to the unique manner in which mammalian sperm chromatin is packaged, while the second attributes the nuclear DNA damage in mature spermatozoa to apoptosis. One of the factors implicated in sperm apoptosis is the cell surface protein, Fas. In this review, we discuss the possible origins of DNA damage in ejaculated human spermatozoa, how these spermatozoa arrive in the ejaculate of some men, and what consequences they may have if they succeed in their genetic project.

Full text

© 1999 Journals of Reproduction and Fertility
1359-6004/99 $15.00
Reviews of Reproduction (1999) 4, 31–37
The formation of mature spermatozoa is a unique process
involving a series of meioses and mitoses, changes in cyto-
plasmic architecture, replacement of somatic cell-like histones
with transition proteins and the final addition of protamines,
leading to a highly packaged chromatin (Kumaroo et al., 1975;
Goldberg et al., 1977; Poccia, 1986). Mature mammalian sper-
matozoa contain high percentages of protamines, for example,
human and mouse sperm nuclei contain more than 85% and
95% protamines in their nucleoprotein component, respectively
(Gatewood et al., 1987; Bellvé et al., 1988; Debarle et al., 1995).
In mice, protamines allow the mature sperm nuclei to adopt a
volume 40 times less than that of normal somatic nuclei (Ward
and Coffey, 1991).
In many mammals, spermatogenesis leads to the production
of spermatozoa that appear highly homogeneous in form and
function. However, in humans, it is apparent that there are
large differences between the form and function of spermato-
zoa among males and within the ejaculate of an individual.
Classically, analyses of the differences in spermatozoa among
men have been measured by examining sperm concentration,
motility and morphology. Although this analysis gives a broad
clinical insight, it does not explain why and where differences
originate.
For a number of years, many laboratories have concentrated
on analysing differences in sperm populations by examining
chromatin structure. These studies have shown that the major
factor affecting chromatin packaging in ejaculated human sper-
matozoa appears to be linked to faulty or incomplete prota-
mine deposition during spermiogenesis. In numerous studies,
spermatozoa from infertile men were found to exhibit sperm
chromatin anomalies related to the deposition of protamines
(Balhorn, 1982; Foresta et al., 1992; Belokopytova et al., 1993;
de Yebra et al., 1993). These anomalies range from altered ratios
of protamine 1 and 2 (Balhorn et al., 1988; Belokopytova et al.,
1993) to the complete absence of protamine (de Yebra et al.,
1993).
During the 1990s, several groups have analysed the sperm
nucleus further by examining the integrity of the DNA in
mature human spermatozoa. This review summarizes the ac-
cumulated knowledge concerning DNA damage in mature
human spermatozoa and how this may be related to male infer-
tility. Furthermore, we will speculate on how and why DNA
damage may originate in certain males and how it influences
the genetic project of a mature spermatozoon.
DNA packaging in mammalian spermatozoa
The chromatin contained in the nuclei of mature mammalian
spermatozoa is an extremely compact and stable structure.
Sperm DNA must be organized in a specific manner (Fig. 1),
which differs substantially from that of somatic cells, to achieve
this unique condensed state (Poccia, 1986; Ward and Coffey,
1991). This DNA organization not only permits transfer of
the very tightly packaged genetic information to the egg, but
also ensures that the DNA is delivered in such a physical
and chemical form that the developing embryo can access
the genetic information.
Ward (1997) has proposed a model for sperm DNA pack-
aging on the basis of his work and that of other laboratories.
This model depicts a mock assembly of chromosomes starting
as long strands of DNA that are gradually packaged at four
levels of organization within the mature spermatozoon:
(i) chromosomal anchoring by the nuclear annulus, (ii) sperm
DNA loop domain organization, (iii) protamine decondensation,
Origin of DNA damage in ejaculated human spermatozoa
Denny Sakkas
1,2
, Ewa Mariethoz
2
, Giancarlo Manicardi
3
,
Davide Bizzaro
3
, Patrizia G. Bianchi
2
and Umberto Bianchi
3
1
Assisted Conception Unit, Birmingham Women’s Hospital, Birmingham B15 2TG, UK;
2
Clinic of Sterility,
Department of Obstetrics and Gynaecology, University Hospital of Geneva, Switzerland; and
3
Department of Animal Biology, University of Modena, Italy
The molecular basis of many forms of male infertility is poorly defined. One area of research
that has been studied intensely is the integrity of the DNA in the nucleus of mature ejaculated
spermatozoa. It has been shown that, in men with abnormal sperm parameters, the DNA
is more likely to possess strand breaks. However, how and why this DNA damage originates
in certain males and how it may influence the genetic project of a mature spermatozoon is
unknown. Two theories have been proposed to describe the origin of this DNA damage in
mature spermatozoa. The first arises from studies performed in animal models and is linked
to the unique manner in which mammalian sperm chromatin is packaged, while the second
attributes the nuclear DNA damage in mature spermatozoa to apoptosis. One of the factors
implicated in sperm apoptosis is the cell surface protein, Fas. In this review, we discuss the
possible origins of DNA damage in ejaculated human spermatozoa, how these spermatozoa
arrive in the ejaculate of some men, and what consequences they may have if they succeed in
their genetic project.

DNA
double helix
Sperm DNA packaging
Doughnut
Doughnut
loop
Nuclear
matrix
Protamines
added
Positioning of a single chromosome
Key
Nuclear annulus
Nuclear annulus–DNA
Telomere
Centromere
Matrix attachment
regions on nuclear
matrix
Single compacted
chromosome
Without
protamines
DNA
loops
Protamine
and (iv) chromosome organization. This model represents the
intricacies of the organization of the sperm nucleus and sup-
ports the assumption that anomalies in the DNA would most
likely extrapolate to anomalies in overall nuclear organization.
DNA damage in mature spermatozoa
Sperm DNA has been analysed by a number of laboratories. In
our laboratory, the integrity of the DNA in mature ejaculated
human spermatozoa has been analysed using in situ nick trans-
lation. These experiments were performed by omitting endo-
nuclease treatments, since, in the presence of pre-existing DNA
endogenous nicks, the DNA polymerase I, by virtue of its 5’–3’
exonucleotic activity can catalyse movement of the nicks along
the double helix (Sambrook et al., 1989). Our results have de-
scribed the percentage of spermatozoa possessing endogenous
DNA nicks in the ejaculates of men and have shown a cor-
relation with reduced fertility (Bianchi et al., 1993, 1996;
Manicardi et al., 1995, 1996; Sakkas et al., 1996). In most cases,
the spermatozoa of men with oligoasthenoteratozoospermia
display a higher percentage of nicks.
The terminal deoxynucleotidyl transferase (TUNEL) assay
has also been used to show the incidence of DNA frag-
mentation in human spermatozoa and a correlation between
abnormal sperm chromatin packaging (poorly protaminated
spermatozoa) and the presence of DNA strand breaks has been
shown (Gorczyca et al., 1993a; Sailer et al., 1995; Manicardi et al.,
1998). Sun et al. (1997), in a cohort of 298 patients, used TUNEL
labelling and fluorescence-activated cell sorting to show a
negative correlation between the percentage of DNA fragmen-
tation and the motility, morphology and concentration of ejacu-
lated spermatozoa. In 143 in vitro fertilization (IVF) samples, a
significant negative association was reported between the per-
centage of spermatozoa with DNA fragmentation and the fertil-
ization and embryo cleavage rates. Lopes et al. (1998) have also
shown that the percentage of spermatozoa with DNA fragmen-
tation was negatively correlated with fertilization rates ob-
tained with intracytoplasmic sperm injection (ICSI).
The differences in the mode of action between nick trans-
lation and the TUNEL technique can be explained when
considering that DNA polymerase, being primer- and template-
dependent, cannot label blunt-ended or 5’-recessed DNA frag-
ments, and that TUNEL, being template-independent, can label
all types of fragments at the hydroxylated 3’ ends (Gold et al.,
1994). However, data reveal that the two techniques cannot
distinguish differences in the presence of endogenous DNA
damage in human spermatozoa (Manicardi et al., 1998).
32 D. Sakkas et al.
Fig. 1. Levels of DNA packaging in sperm chromatin based on the
doughnut loop model. Protamine binds to the DNA lengthwise
along the double helix. The protamine-bound DNA is coiled into
concentric circles. These circles of one loop then collapse into a
doughnut into which the neutral DNA protamine complexes are
tightly packed. Each doughnut represents one DNA loop domain
attached to the sperm nuclear matrix. Single chromosomes are
organized with centromeres located in the centre of the nucleus.
Each chromosome is attached to the nuclear matrix at intervals of
about 40 kb leading to DNA loop domain formation. (Adapted
from Ward, 1993, 1997.)

The sperm chromatin structure assay (SCSA), which measures
the susceptibility of DNA to heat- or acid-induced denaturation
in situ, is also effective in identifying fertility potential (Evenson
et al., 1980). However, accurate measurement of DNA damage
in a single spermatozoon has proved difficult. Although tech-
niques such as in situ nick translation and the TUNEL assay can
be used to observe a single spermatozoon, there are limitations
to the sensitivity of these techniques, and it is possible that
many spermatozoa with damaged DNA escape detection.
Other methods for assessing the DNA of a single spermato-
zoon, such as the Comet assay (single cell gel electrophoresis),
are also being established (Hughes et al., 1996; Aravindan et al.,
1997). This assay has been recognized in many cell lines to be
one of the most sensitive techniques available for measuring
DNA strand breaks (Collins et al., 1997). Aravindan et al. (1997)
established a significant relationship between the Comet assay
for human spermatozoa and the SCSA and TUNEL assays. The
Comet assay has been used to examine the effect of various
chemicals, such as food mutagens and oestrogenic substances,
on spermatozoa in vitro (Anderson et al., 1997a,b) and may
prove to be one of the more sensitive tests for examining DNA
strand breaks in sperm chromatin.
Whether the DNA damage in spermatozoa is representative
of single- or double-stranded DNA breaks will also be of
interest. Single-stranded lesions should be repaired in the
oocyte upon fertilization and so should not be lethal. However,
if a fertilizing spermatozoon possesses single stranded DNA
breaks of significant size, these may prove difficult for the
oocyte to repair and may lead to failure in either the fertil-
ization process or later in development.
Origin of DNA damage in mature spermatozoa
In humans, it is clear that the population of spermatozoa in
an ejaculate can be highly heterogeneous. Unfortunately, this
appears to be more evident in patients whose sperm param-
eters fall below normal WHO values (WHO, 1992). The positive
relationship between poor sperm parameters and DNA dam-
age in spermatozoa points to inherent problems in spermato-
genesis in specific patients. Two theories have been proposed
to explain the phenomenon of why there are anomalies in the
DNA of ejaculated human spermatozoa.
The first theory arises from studies performed in animal
models and is linked to the unique manner in which mam-
malian sperm chromatin is packaged. Endogenous nicks in
DNA have been shown to be present normally at specific stages
of spermiogenesis in rats and mice, and are thought to have a
functional significance (McPherson and Longo, 1992, 1993a,b;
Sakkas et al., 1995). In the rodent species, endogenous nicks are
evident during late spermiogenesis (step 12–13 rat spermatids)
but are not observed once chromatin packaging is completed
(McPherson and Longo, 1993a; Sakkas et al., 1995). Therefore,
the presence of nicks is greatest during the transition from
round to elongated spermatids in the testis and occurs before
the completion of protamination in maturing rat and mouse
spermatozoa (McPherson and Longo, 1992, 1993a,b; Sakkas
et al., 1995).
In considering the remodelling of chromatin, McPherson
and Longo (1992, 1993a,b) postulated that chromatin packaging
may necessitate endogenous nuclease activity to create and
ligate nicks that facilitate protamination. They proposed that
the endogenous nuclease, topoisomerase II (topo II), may play
a role in both creating and ligating nicks during spermiogen-
esis. These nicks are thought to provide relief of torsional stress
and to aid chromatin rearrangement during the displacement
of histones by protamines (McPherson and Longo, 1992). Chen
and Longo (1996) have also shown that changes in DNA topo II
expression and localization patterns are consistent with the
involvement of topo II in mediating DNA modifications and
chromatin changes during rat spermatogenesis. Therefore, the
presence of endogenous nicks in ejaculated spermatozoa
indicates incomplete maturation during spermiogenesis. This
hypothesis is supported by observations that the presence of
DNA damage in mature spermatozoa is correlated with poor
chromatin packaging due to underprotamination (Gorczyca
et al., 1993a; Manicardi et al., 1995; Sailer et al., 1995).
The second theory proposes that the presence of endogen-
ous nicks in ejaculated human spermatozoa is characteristic of
programmed cell death, as seen in apoptosis of somatic cells,
and functional elimination of possibly defective germ cells from
the genetic pool (Gorczyca et al., 1993a,b).
Apoptosis during spermatogenesis
Spermatogenesis is the dynamic process of germ cell prolifer-
ation and differentiation from stem spermatogonia to mature
spermatozoa. In mammalian testes, germ cells expand clonally
through many rounds of mitosis before undergoing the dif-
ferentiation steps that result in mature spermatozoa. This
clonal expansion is excessive, requiring that there is a mech-
anism to match the number of germ cells with the supportive
capacity of the Sertoli cells. In a number of animal models,
overproliferation of early germ cells is tempered by selective
apoptosis of their progeny (Allan et al., 1992; Tapanainen
et al., 1993; Bartke, 1995; Billig et al., 1995; Sinha Hikim
et al., 1997; Furuchi et al., 1996; Rodriguez et al., 1997). Testicular
germ cell apoptosis occurs normally and continuously
throughout life. One factor postulated to be implicated in
sperm apoptosis is the cell surface protein, Fas (Lee et al.,
1997). Fas is a type I membrane protein that belongs to the
tumour necrosis factor–nerve growth factor receptor family,
and mediates apoptosis (Suda et al., 1993; Krammer et al., 1994;
Schulze-Osthoff, 1994). Binding of Fas ligand (FasL) or agon-
istic anti-Fas antibody to Fas kills cells by apoptosis (Suda et al.,
1993). In mice and rats, it has been shown that, in the normal
state, Sertoli cells express FasL and signal the killing of Fas-
positive germ cells, limiting the size of the germ cell population
to numbers they can support (Lee et al., 1997; Rodriguez et al.,
1997). In addition, after injury, FasL expression of Sertoli cells
increase to reach a new equilibrium state that matches the re-
duced capacity of the dysfunctional Sertoli cells with fewer
germ cells. Thus, upregulation of Fas in germ cells is seen as a
self-elimination process for cells that are destined to die be-
cause of inadequate support. The role of Fas during spermato-
genesis in humans is unclear. However, there are a number of
indications that apoptosis occurs during spermatogenesis in
humans. The observation that mature ejaculated human sper-
matozoa are positive for the TUNEL assay has lead to the
theory that apoptosis is occurring (Gorczyca et al., 1993a;
Baccetti et al., 1996; Sun et al., 1997; Lopes et al., 1998; Manicardi
Origin of DNA damage in human spermatozoa 33

et al., 1998). Baccetti et al. (1996) described certain ultra-
structural features indicative of apoptosis in human spermato-
zoa, while Hadziselimovic et al. (1997) showed that there
was increased apoptosis in the testes of patients with testicular
torsion.
In our studies, we have examined whether ejaculated
human spermatozoa express Fas. Spermatozoa from a series of
patients were labelled with anti-Fas conjugated to fluoroscein
isothiocyanate (FITC) and 10
4
spermatozoa were passed
through a fluorescence-activated cell sorter. A clear increase in
Fas positivity has been found in many men who have sperm
parameters lower than normal (Fig. 2). However, why do ejacu-
lated sperm exhibit these apoptotic features when they should
be eliminated? In mice, in which apoptosis occurs via a Fas-
mediated system (Rodriguez et al., 1997), mature spermatozoa
rarely show any signs of DNA damage (Sakkas et al., 1995) or
Fas positivity (D. Sakkas, unpublished). In men with normal
sperm parameters, the percentage of Fas-positive spermatozoa
is low. However, in men with reduced sperm parameters, the
percentage of Fas-positive spermatozoa can be as high as 50%
(Fig. 2). When the percentage of spermatozoa positive for Fas is
plotted against sperm concentration, it becomes evident that
men with decreased sperm concentrations in their ejaculate
have a greater chance of expressing high concentrations of Fas-
positive spermatozoa.
These results indicate that, in these subfertile men, sper-
matozoa that have been earmarked to undergo apoptosis es-
cape this process. The above evidence suggests that the correct
clearance of spermatozoa via apoptosis is not occurring.
Therefore, production of ejaculated spermatozoa that possess
apoptotic markers (such as Fas positivity and DNA damage)
indicate that, in some men with abnormal sperm parameters,
an ‘abortive apoptosis’ has taken place. This finding suggests
that spermatozoa showing for example abnormal morphologi-
cal forms, irregular biochemical function (Huszar et al., 1997) or
nuclear DNA damage have failed to be eliminated owing to an
abortive apoptotic mechanism (Fig. 3).
The failure to clear Fas-positive spermatozoa may be due to
misfunction at various levels. First, as has been reported in
other animal models, apoptosis during spermatogenesis limits
any excess in the number of germ cells developing so that the
supportive capacity of the Sertoli cells is not inhibited. Since
it has been shown that the Sertoli cells can limit this over-
proliferation via synthesis of FasL, it could be postulated that,
in oligozoospermic men, in whom spermatogenesis is reduced,
sufficient spermatozoa may not be produced to trigger this
action by the Sertoli cells. Thus, even though a spermatogo-
nium may be Fas-positive, it may escape activation to undergo
apoptosis. Fas-positive spermatozoa may also occur because of
problems in activating Fas-mediated apoptosis. These problems
could be inherent to a particular patient or may be due to lack
of synchronization between apoptosis and spermatogenesis, in
which case, even though apoptosis has been initiated, the sper-
matozoa will go through spermiogenesis and fail to complete
apoptosis (Fig. 3). This hypothesis may explain why patients
with abnormal sperm parameters also possess a higher percent-
age of spermatozoa containing DNA fragmentation and ab-
normal spermatozoa that display signs of apoptosis.
Whereas the presence of DNA fragmentation in ejaculated
human spermatozoa may be explained by apoptosis taking
place during spermatogenesis, the initial theory that the pres-
ence of endogenous nicks indicates that incomplete endogenous
nuclease activity creates and ligates nicks during spermiogen-
esis (McPherson and Longo, 1992) may still hold. As stated
above, a spermatid that has initiated apoptosis may fail to acti-
vate its endogenous nuclease activity completely or default in
the ligation of nicks in the DNA. The evidence points to an
abortive apoptosis taking place in many males that exhibit
sperm parameters that are below normal. We hypothesize that,
in certain males, this abortive apoptosis appears to fail in the
34 D. Sakkas et al.
100
80
60
40
20
013 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55
Fig. 2. The relationship between the concentration of spermatozoa in the ejaculate of a patient ( ; 106spermatozoa ml–1) and the percentage
of ejaculated spermatozoa that are Fas positive ( ). The concentration of spermatozoa in the first five patients was >100 ×106ml–1. The
y-axis represents both the number of spermatozoa ml–1 and the percentage of Fas-positive spermatozoa, while the x-axis represents different
semen samples.

total clearance of spermatozoa that are earmarked for elimi-
nation by apoptosis. Therefore, the subsequent population of
ejaculated spermatozoa present an array of anomalies represen-
tative of the characteristics observed in cells that are in the
process of apoptosis.
Consequences of DNA damage in human spermatozoa
It has been established that there are some anomalies in the
DNA of ejaculated spermatozoa. However, the consequences of
this DNA damage during fertilization and embryo develop-
ment are unknown. The increased presence of these anomalies
in males with abnormal sperm parameters puts the population
of patients being treated by assisted reproductive technologies,
in particular ICSI, at the greatest risk.
Whether DNA-damaged spermatozoa can impair the pro-
cess of fertilization or embryo development is not clear. Studies
by Robaire and co-workers have indicated that damage to
sperm DNA may be linked to an increase in early embryo
death. They showed that treatment of male rats with cyclo-
phosphamide had little effect on the male reproductive system,
but resulted in single-strand DNA breaks in the cauda epidi-
dymal spermatozoa and altered the decondensation potential
of spermatozoa (Qiu et al., 1995a,b). Similar treatment protocols
using cyclophosphamide produced an increase in postimplan-
tation loss and malformations (Trasler et al., 1985, 1986, 1987)
and were transmissible to the next generation (Hales et al.,
1992).
In humans, failed fertilized oocytes injected with spermato-
zoa from patients with a large number of endogenous DNA
nicks in their sperm population contain more condensed sper-
matozoa (Sakkas et al., 1996). This finding indicates that DNA-
damaged spermatozoa selected for ICSI may impede the
completion or initiation of decondensation, leading to a failure
of fertilization. Lopes et al. (1998) have shown that men with a
sperm population containing > 25% DNA damage are more
likely to experience a fertilization rate of < 20% after ICSI. In
addition, in humans, ICSI patients have a lower percentage of
embryos that form blastocysts when compared with patients
undergoing routine IVF (Shoukir et al., 1998).
The use of ICSI has heightened the risk that spermatozoa
containing damaged DNA may participate in the development
of an infant. Whether spermatozoa possessing damaged DNA
will fail in their project to contribute to a viable offspring at the
time of fertilization, embryo development or fetal development
is not clear.
Origin of DNA damage in human spermatozoa 35
SpermatogenesisSpermiogenesis
Type A
spermatogonium
(a) (b)
Type B
spermatogonium
Primary
spermatocyte
Secondary
spermatocyte
Spermatids
Spermatozoa
Fas-positive
apoptopic cell Fas-positive
apoptopic cell
Fas-positive
apoptopis
rescued cell
‘Abortive apoptosis’ sperm
associated with:
abnormal morphology
abnormal function
nuclear DNA damage
Fas-positive
rescued cell
Fig. 3. The role of Fas during spermatogenesis in men with (a) normal and (b) abnormal (oligospermic, asthenospermic and teratospermic)
sperm parameters.

Conclusions
The observation that ejaculated human spermatozoa possess
DNA damage raises numerous problems relating to why and
how these spermatozoa arise in the ejaculate of some men and
what consequences they have if they succeed in their genetic
project. The chance of such spermatozoa contributing their
damaged DNA to subsequent generations has been heightened
with the advent of ICSI, although our understanding of what
ensuing problems may arise is poor. The present aim is to fur-
ther improve our understanding of the molecular basis of male
infertility. In doing so, we will need to broaden our knowledge
of the effect abnormal spermatozoa have on fertilization and
embryo development and avoid the inappropriate use of ICSI.
This work was supported by the Fonds National Suisse and
Consiglio Nazionale delle Richerche (CNR, Italy).
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