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How do male germ cells handle DNA damage?
Ann-Karin Olsen, Birgitte Lindeman, Richard Wiger, Nur Duale, Gunnar Brunborg*
Norwegian Institute of Public Health, Department of Chemical Toxicology, P.O. Box 4404 Nydalen, N-0403 Oslo, Norway
Received 12 January 2005; revised 13 January 2005; accepted 21 January 2005
Available online 27 July 2005
Abstract
Male reproductive health has received considerable attention in recent years. In addition to declining sperm quality, fertility problems and
increased incidence of testicular cancer, there is accumulating evidence that genetic damage, in the form of unrepaired DNA lesions or de
novo mutations, may be transmitted via sperm to the offspring. Such genetic damage may arise from environmental exposure or via
endogenously formed reactive species, in stem cells or during spermatogenesis. Damaged testicular cells not removed by apoptosis rely on
DNA repair for their genomic integrity to be preserved. To identify factors with potentially harmful effects on testicular cells and to
characterise associated risk, a thorough understanding of repair mechanisms in these cells is of particular importance. Based on results from
our own and other laboratories, we discuss the current knowledge of different pathways of excision repair in rodent and human testicular
cells. It has become evident that, in human spermatogenic cells, some repair functions are indeed non-functional.
D2005 Elsevier Inc. All rights reserved.
Keywords: Testis; DNA repair; Male germ cells; Sperm; Chemical toxicity
Contents
DNA damage, fertility and reproduction ............................................. S521
Spermatogenesis ......................................................... S522
DNA repair............................................................ S522
DNA excision repair in germ cells ................................................ S522
Nucleotide Excision Repair (NER)................................................ S524
Base Excision Repair (BER) ................................................... S527
What happens with the germ cells containing unrepaired DNA lesions? ............................ S529
Concluding remarks ....................................................... S529
Epilogue ............................................................. S530
Acknowledgments ........................................................ S530
References ............................................................ S530
DNA damage, fertility and reproduction
Evidence from both animal studies and from human
epidemiology is now accumulating, indicating that environ-
mental agents can interact with the male genome, causing
specific types of genetic change in sperm that affect fertility.
Human spermatozoa frequently possess high levels of
nuclear DNA damage (Sun et al., 1997; Irvine et al.,
2000; Evenson et al., 2002). Even with significant levels of
DNA damage, sperm retain the ability to fertilise oocytes;
however, the subsequent development may be altered or
lead to zygote arrest. Further implications are early
0041-008X/$ - see front matter D2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.taap.2005.01.060
* Corresponding author. Fax: +47 2204 2686.
E-mail address: gunnar.brunborg@fhi.no (G. Brunborg).
Toxicology and Applied Pharmacology 207 (2005) S521 – S531
www.elsevier.com/locate/ytaap
abortions, congenital malformations and disease including
cancer in the offspring (Ahmadi and Ng, 1999; Zenzes et al.,
1999b; Brinkworth, 2000). The importance of DNA damage
is further illustrated by experiences with assisted reproduc-
tion (Zitzmann et al., 2003; Sun et al., 1997; Twigg et al.,
1998; Morris et al., 2002) as recently reviewed by Sharma et
al., 2004.
In addition to the data discussed by Diana Anderson in
this symposium, there are other cases of paternally trans-
mitted malformations via mechanisms not yet understood. A
cluster of malformations was discovered (e.g. club foot)
among children of fathers previously working on a marine
vessel in Norway (RR = 4.6, CI 2.6 – 8.4; Professor Bente E.
Moen, personal communication). The cause of this apparent
increased risk is however not known.
The genetic constitution of the offspring depends
logically on the integrity of the sperm and the egg DNA,
both expected to be of equal importance. Traditionally,
concern about possible developmental toxic effects from
environmental agents has focussed on the maternal con-
tribution, whereas paternal aspects have been generally
ignored. Correspondingly, chemicals have been examined
for developmental toxicity using maternal and not paternal
test systems.
Among environmental factors known to mediate DNA
damage in sperm, smoking is well-documented. Heavy
smokers exhibit increased levels of strand breaks in their
spermatozoa (Fraga et al., 1996; Potts et al., 1999), and it
has now been shown that smoking causes more abnormal
sperm, increased levels of oxidative DNA lesions such as
7,8-dihydro-8-oxoguanine (8-oxoG) and reduced fecundity
(Zitzmann et al., 2003). Furthermore, children of smoking
fathers may have an increased risk of developing childhood
cancer (Ji et al., 1997).
Spermatogenesis
Spermatogenesis is the process during which sperm is
produced from progenitor spermatogonia and is initiated
when the male has attained sexual maturity. It comprises
three main phases: the spermatogonial stage, the meiotic
stage and the spermiogenesis stage; in general, the
duration of each of these stages is approximately equal.
Some more details are given in Fig. 1 and its legend; the
reader is otherwise referred to recent reviews (Holstein et
al., 2003).
Gametes may be susceptible to environmental genoto-
xicants at different stages of spermatogenesis. Unrepaired
DNA lesions in spermatogonia may be fixed as mutations
during replication giving rise to clones of mutated sperm.
Cells in later spermatogenic stages do not replicate, and
fixation of premutagenic DNA lesions as mutations depends
on replication-independent DNA synthesis associated with
recombination or DNA repair. Alternatively, mutations may
be fixed during repair in the zygote.
DNA repair
There are several lines of defense against the induction
and persistence of DNA damage in cells. Firstly, there
are agents that prevent the formation of DNA damage,
such as detoxifying peptides and proteins, and oxyradical
scavengers, such as vitamins E and C. Secondly, DNA
damage already present in the genome may be sensed by
DNA damage response pathways, and the damage may
be removed by an array of DNA repair pathways to
reduce the possibility for inducing mutations. Thirdly, the
damaged cell itself may be eliminated by spontaneous
death or apoptosis.
DNA damage can have a variety of biological
ramifications including inhibition of transcription and/or
replication, ultimately causing cell death, mutations and
cancer. Constant excision and replacement of damaged
nucleotide residues by DNA repair are required to
counteract the occurrence of such events. The manner
in which a DNA lesion is removed depends on the nature
of the DNA lesion itself; for one specific type of DNA
lesion, there is often a redundancy of pathways that
function as back-ups for each other. Most of the repair
mechanisms are not absolutely exact and error-free,
whereas commonly occurring DNA lesions clearly must
be removed with high accuracy.
In this report, the focus is on excision repair, in which
an altered nucleotide residue is replaced following
removal of a segment of the damaged strand followed
by synthesis of intact DNA (Fig. 2). One pathway,
Nucleotide Excision Repair (NER), removes predomi-
nantly helix-distorting lesions that mostly originate via
environmental agents (Fig. 2). A second pathway, Base
Excision Repair (BER), removes mainly common mod-
ifications such as oxidations, methylations, deaminations
and base losses (Fig. 3). These types of lesions are
caused by both exogenous agents and endogenous
processes.
DNA damage can also be repaired by other mechanisms
not discussed here such as direct reversal, mismatch repair
(MMR), homologous recombination (HR) and non-homol-
ogous end joining (NHEJ) (recently reviewed in germ cells
by Baarends et al., 2001).
DNA excision repair in germ cells
There is limited information on repair functions in
male germ cells. In early studies, this was probably
related to a lack of appropriate methodologies, a situation
which has improved during recent years. Traditionally,
DNA repair in male germ cells has mostly been studied
using assays such as unscheduled DNA synthesis (UDS),
alkaline elution, the comet assay, and immunochemical
methods. Furthermore, the testes represent a challenge
with respect to the isolation and cultivation of the
A.-K. Olsen et al. / Toxicology and Applied Pharmacology 207 (2005) S521– S531S522
different male germ cell types. DNA single-strand breaks
(SSB) in male germ cells of the earlier stages of
spermatogenesis from both hamsters and rats are repaired
as efficiently as in several somatic cell types, whereas
elongated spermatids (see Fig. 1) exhibited no repair (van
Loon et al., 1991, 1993). More recently, our laboratory
Fig. 1. A schematic presentation of spermatogenesis. The general organisation of spermatogenesis is essentially the same in all mammals and can be divided
into three phases, the spermatogonial, the meiotic and the spermiogenesis stages. The spermatogonial or proliferative stage starts with a division of
spermatogonial stem cells (originating from primordial germ cells during development) into two daughter cells, one of which enters the process of
spermatogenesis, while the other remains as a stem cell. This is the period of active replicative DNA synthesis producing different types of spermatogonia. The
number of cell divisions during this stage varies with species, but ultimately type B spermatogonia give rise to tetraploid primary spermatocytes in the
preleptotene stage. During the first part (prophase) of the meiotic stage, genetic recombination takes place after which a first reduction division gives rise to
secondary 2n spermatocytes, and subsequently the second reduction division results in haploid round spermatids. During spermiogenesis, extensive changes
occur in spermatids, including nuclear condensation, leading to spermatozoa. Some additional details and the duration of the stages are indicated. Further
details may be found in the comprehensive review by Holstein et al. (2003).
A.-K. Olsen et al. / Toxicology and Applied Pharmacology 207 (2005) S521 – S531 S523
made similar observations with human (Olsen et al.,
2003), mouse and rat (Wiger et al., unpublished) male
germ cells.
Nucleotide Excision Repair (NER)
NER is a major DNA repair pathway that acts on a wide
variety of helix-distorting DNA lesions (bulky adducts) such
as those caused by UV light (cyclobutane pyrimidine dimer,
(CPD) and 6-4 photoproduct (6-4PP)) and by a range of
exogenous chemicals including environmental agents such as
benzo(a)pyrene (B[a]P) and aflatoxin B
1
and chemother-
apeutic agents such as cisplatin. A defect in one of the repair
proteins results in recessive syndromes such as xeroderma
pigmentosum (XP), associated with very high (1000-fold
increased) cancer risk (Cleaver, 1989).
NER involves recognition of the DNA lesion, incision of
the DNA strand containing the lesion, followed by DNA
synthesis and ligation to replace an excised oligonucleotide
(there are several excellent reviews including Wood, 1997;
de Laat et al., 1999; Mitchell et al., 2003). There are two
NER subpathways (Fig. 2), the global genomic repair
(GGR) that repairs DNA lesions in the entire genome and
the transcription-coupled repair (TCR) that removes DNA
lesions that block RNA synthesis, i.e. in transcribed genes
(Tornaletti and Hanawalt, 1999).
In total, 25 or more proteins are involved in NER (Fig. 2,
abbreviations for each enzyme are given in the figure
legend). GGR is initiated by binding of XPC-hHR23B to
Fig. 2. An outline of the Nucleotide Excision Repair (NER) pathway. See text for description of the pathway. Abbreviations in chronological order: XPA–G,
xeroderma pigmentosum complementation group A–G; hHR23B, human homologue of yeast RAD23B; RNA Pol II, RNA polymerase II; CSA and CSB,
Cockayne syndrome factors A and B; TFIIH, general transcription factor IIH; ERCC1, excision repair cross complementing group 1 protein; RPA, replication
protein A; PCNA, proliferating cell nuclear antigen; RFC, replication factor C; Poly/E, DNA polymerase delta/epsilon; Lig1, DNA ligase 1.
A.-K. Olsen et al. / Toxicology and Applied Pharmacology 207 (2005) S521– S531S524
disrupted base pairs. During TCR, lesions that block the
RNA polymerase are detected, and the polymerase is
displaced, making the DNA lesion accessible for repair;
this requires at least two TCR-specific factors: CSA and
CSB. During both GGR and TCR, XPG associates and the
general transcription factor TFIIH (a multi-protein complex
that includes the two helicases XPB and XPD) unwinds
about 30 base pairs surrounding the DNA lesion. The
subsequent steps of GGR and TCR are believed to be
identical. XPA associates with the single-strand binding
protein RPA to form a complex that confirms the presence
of DNA damage by sensing a simultaneous backbone and
base pair distortion of DNA (Missura et al., 2001). The
ERCC1-XPF endonuclease complex and the XPG endonu-
clease cleave the damaged strand 5Vand 3Vrelative to the
DNA lesion, generating a 24- to 32-base oligonucleotide
containing the lesion. New DNA is synthesised, and the gap
is ligated.
A third and interesting subpathway of NER has recently
been proposed and given the name differentiation-associated
repair (DAR). As opposed to TCR, in which repair is rapid in
the transcribed strand of expressed genes, during DAR in
terminally differentiated cells, both the non-transcribed and
the transcribed strands are efficiently repaired, with DAR
taking care of the non-transcribed strand which is otherwise
repaired by GGR in undifferentiated cells (Nouspikel and
Hanawalt, 2002). The significance and general existence of
DAR are however still debated.
Fig. 3. An outline of the Base Excision Pathway (BER) pathway. See text for description of the pathway. Abbreviations in chronological order: HAP1, human AP-
endonuclease 1; Polh, DNA polymerase h; XRCC1, X-ray cross complementing protein 1; LigIII, DNA ligase III; PCNA, proliferating cell nuclear antigen; RFC,
replication factor C; Poly–(, DNA polymerase y–(; FEN1, Flap endonuclease; Lig1, DNA ligase 1. The figure is modified from Ide and Kotera (2004).
A.-K. Olsen et al. / Toxicology and Applied Pharmacology 207 (2005) S521 – S531 S525
The efficiency of repair of different kinds of lesions varies
by several orders of magnitude. The rate of repair for GGR is
strongly dependent on the type of lesion, i.e. 6-4PPs are
removed much faster from the genome than CPDs (¨1h
compared to ¨6 h in Chinese hamster ovary cells, respec-
tively; Nairn et al., 1989).
About 10 years ago, we observed indications of very
low repair in rat testicular cells exposed to UV-C
(Brunborg et al., 1995). This was based on a lack of
accumulated DNA single-strand breaks (SSBs) in the
presence of repair inhibitors, using the alkaline elution
assay or the alkaline comet assay. The rationale behind
Fig. 4. Limited repair of UV-C induced DNA lesion in rat male germ cells. To measure repair, the cells are treated with UV-C to introduce bulky DNA adducts
such as cyclobutane pyrimidine dimers (CPDs) and incubated to allow repair in the absence (open bars) or presence (closed bars) of repair inhibitors
arabinofuranoside C (AraC, 0.1 mM) plus hydroxyurea (HU, 2 mM) and analysed by the comet assay (A – D). Accumulation of DNA single-strand breaks
(ssb) measured as tail moment indicates repair. The results show that crude cell suspensions (A) of rat testicular cells exhibit only limited repair following high
doses of UV-C. Populations of different rat male germ cell types (spermatocytes (C) and round spermatids (D)) did not accumulate ssb following low doses of
UV-C, while mononuclear blood cells (MNC) from rats as positive control cells (B) showed significant accumulation of ssb. NER in rat testicular cells was
alternatively analysed with a modified comet method in which the bacterial T4endoV enzyme was used to cleave CPDs in DNA to quantify their presence in
DNA in the form of ssb. Neither crude cell suspensions of rat testicular cells (E) nor isolated spermatocytes (F) removed significant numbers of CPDs within
24 h (open bars, no repair; closed bars, 24 h repair incubation), indicating very limited repair via NER in these cells. Furthermore, the results in panels (E) and
(F) show that CPDs are indeed present since they are cleaved by T4endoV. In other experiments not shown here (Jansen et al., 2001), rat male germ cells were
analysed while within their natural environment, the seminiferous tubule. Fragments of seminiferous tubules were exposed to the tissue-penetrating UV-B and
incubated for repair within the tubules. The cells were subsequently squeezed out for comet analysis; results demonstrated a similar lack of incision as the
results presented in panels A, C, and D. This also shows that lack of repair was not due to the cell isolation procedure or the absence of close contact with the
nurturing and supporting Sertoli cells.
A.-K. Olsen et al. / Toxicology and Applied Pharmacology 207 (2005) S521– S531S526
using repair inhibitors is as follows: UV-C induces bulky
lesions repaired by NER; such lesions are normally
recognised and incised by the cells’ own repair machi-
nery and are thus converted into strand breaks. These
breaks stay open and accumulate when cells are
incubated to allow repair in a medium containing repair
inhibitors (blocking DNA polymerase and deoxyribonu-
cleotide synthesis; see legend to Fig. 4). This is a
sensitive way of measuring excision repair activities in
live cells. We have subsequently studied NER in rat,
mouse and human testicular cells to get a better under-
standing of how environmental agents that cause bulky
lesions in DNA are handled in these cells. Removal of
CPDs was poor in rat testicular cells in suspension (Fig.
4), as well as in cells in their normal environment within
intact seminiferous tubules, using low doses of UV. In
enriched cell samples of rat spermatocytes of zygotene/
leptotene, mid-pachytene and diplotene stages exposed to
30 J/m
2
UV-C, TCR was also low (measured in the
transcribed strand of the meiosis specific gene SCP1)
(Jansen et al., 2001). Incision activities of cell extracts
were measured using an oligonucleotide containing a
defined lesion; excision was high with early- and mid-
pachytene spermatocytes as opposed to extracts from
other stages (diplotene spermatocytes and round sperma-
tids). It is not unexpected that protein extracts exhibit
proficient repair activities (as recently also shown by
Gospodinov et al., 2003), while in the live cell repair
may still be absent since it depends on the distribution,
spatial translocation and association of repair proteins. We
hypothesised that NER proteins are sequestered by
mispaired regions in DNA involved in synapsis and
recombination and that this explains the lack of NER
activity in premeiotic cells (Jansen et al., 2001). Later
experiments have indicated that some NER-associated
proteins may be expressed in low amounts in human
male germ cells (unpublished). At the level of mRNA,
however, transcripts of most NER-related enzymes are
expressed in high amounts (Li et al., 1996; van der Spek
et al., 1996; Cheng et al., 1999; Shannon et al., 1999).
More recently, Nouspikel and Hanawalt (2002) have
discussed observations that NER activities are highly differ-
ent among various cell types; GGR is generally switched off
in terminally differentiated cells and is replaced by DAR. In
this context, our results with rat male germ cells (Jansen et al.,
2001) are paradoxical since they apparently exhibit no TCR
(and hence no DAR).
Low NER is in compliance with the observations
recently reviewed by Sotomayor and Sega (2000).In
studies of UDS in male germ cells exposed to different
agents, as many as 59 chemicals plus UV and X-rays
were tested in spermatogenic cells of humans, rabbits,
rats and mice. Although these aspects were not discussed
in the review, in general, agents inducing DNA lesions that
are removable by NER did not show UDS (2-AAF,
aflatoxin B
1
,B[a]P, N-OH-AAF).
Base Excision Repair (BER)
BER is initiated with the release of altered bases by DNA
glycosylases (Fig. 3), as first reported by Thomas Lindahl
30 years ago (Lindahl, 1974). DNA glycosylases recognise
and excise aberrant bases that cause minor structural
changes in DNA, and each DNA glycosylase recognises a
specific set of DNA substrates. The excision of the base
generates apurinic/apyrimidinic sites (AP sites) followed by
endonuclease cleavage, re-synthesis and ligation. Some
DNA glycosylases are bi-functional, that is, besides their
DNA N-glycosylase activity, they have the ability to cleave
at AP sites (AP-lyase). At least eleven human DNA
glycosylases are known to date. BER is subdivided into
short-patch repair (SPR, 1 nucleotide (nt)) and long-patch
repair (LPR, 2 – 10 nt) (Fig. 3). During SPR, the major AP
endonuclease HAP1, or the 5V-terminal deoxyribose phos-
phatase (5V-dRPase) activity of Polh, trims the DNA ends
into suitable substrates for new DNA synthesis. Polh
incorporates one nucleotide, and the nick is sealed by
DNA ligase III/XRCC1. During LPR, Polyor (incorporates
several nucleotides in association with RPA and PCNA,
generating a 5V-dRP flap structure. This structure is nicked
by FEN1, and the gap is sealed by DNA ligase I. Similar to
NER, DNA lesions that inhibit or block transcription, such
as 8-oxoG, are probably removed by BER in a transcription-
coupled manner (Leadon and Cooper, 1993; Cooper et al.,
1997; Le Page et al., 2000), partly using the same enzymes
as in NER-TCR.
Many altered bases are repaired by BER; we have
studied some of them and their associated DNA glycosy-
lases and downstream activities. Uracil residues may lead to
mutations and occur in the genome either as a result of
erroneous incorporation via replication or by deamination of
thymine. We found that the most important uracil-DNA
glycosylase (UDG) was present in human and rat testicular
cells in amounts equal to somatic tissues (Olsen et al.,
2001). This is based upon measuring enzymatic activities
and by immunochemical detection of the enzyme. UDG has
one nuclear and one mitochondrial isoform, and both were
detected in our analyses. The data indicate that uracil is
removed from the male germ cell genome; this is
corroborated by Intano et al. (2001), who reported UDG
also in mouse testicular cells. Furthermore, Grippo et al.
(1982) detected human uracil-DNA glycosylase (UNG)
activity in the DNA-synthesising male germ cells. The
presence of at least five different and partly overlapping
DNA glycosylases for removal of uracil suggests that this
repair is important. In cases of diets low in folic acid, uracil
is incorporated into the genome in higher amounts, and
therefore pregnant women are recommended to add folic
acid to their diet to prevent the embryo from developing
spina bifida. Mice deficient in UDG have been constructed
and exhibit a 50% decreased ability to excise uracil (Nilsen
et al., 2000). These mice retain the ability to produce
offspring, indicating that UDG is not essential for repro-
A.-K. Olsen et al. / Toxicology and Applied Pharmacology 207 (2005) S521 – S531 S527
duction. The adverse effects detected in these mice include
impaired immunoglobulin production (Imai et al., 2003),
higher morbidity and a higher chance of developing B-cell
lymphomas (Nilsen et al., 2003). It has been suggested that
the reason for this apparently mild phenotype is the
redundancy of other uracil-DNA glycosylases, such as
SMUG (single-strand selective monofunctional uracil-
DNA glycosylase), that act as back-ups. Alkylations
represent a major class of DNA lesions; several types are
repaired via the methylpurine DNA glycosylase (MPG).
Mpg mRNA is abundant in mice testes (Engelward et al.,
1993; Kim et al., 2000). Other proteins that repair
alkylations are the direct acting ‘‘suicide protein’’ O
6
-
Methylguanine-DNA-glycosylase (MGMT) and the recently
identified AlkB (alkylation repair) human homologue
(ABH) proteins (Duncan et al., 2002; Aas et al., 2003),
each with their specific DNA substrates. We found that the
MGMT protein was expressed in normal amounts in human
and rat male germ cells (unpublished), similar to other
investigators’ findings in mice (Thompson et al., 2000). In
our studies, MPG is present in greater amounts in human
and rat male germ cells compared to somatic cells (Olsen et
al., 2001). Minor differences were observed between
different cellular stages of rat spermatogenesis and spermio-
genesis. DNA lesions induced by exposure to the mutagen
methyl-methane sulfonate (MMS) were 5-fold higher in rat
compared to human male germ cells, indicating major
differences in sensitivity between the two species. Repair of
methylated DNA studied at the cellular level was efficient in
both human and rat male germ cells, in primary spermato-
cytes as well as round spermatids, compared to primary
somatic cell types. This proficient repair is consistent with
the lack of increased mutation rates in male germ cells of
Big Blue transgenic mice treated with MMS (Ashby et al.,
1997); large insertions and deletions are however not scored
in the latter assay. Hence, male germ cells seem well
protected against DNA methylations such as those inflicted
by MMS; such lesions arise spontaneously or via the
environment. Other mouse germ cell alkylating mutagens,
such as ethyl nitrosourea (ENU) and isopropyl methane-
sulphonate (iPMS), attack other base positions and do
induce mutations (Ashby et al., 1997). Alkylating agents
also attack other components of a cell besides DNA bases,
and it is now believed that some of these disturb chromatin
packaging during spermiogenesis.
Oxidised lesions and their repair in male germ cells are of
special interest. One prevalent and important lesion is 8-
oxoG arising from oxidised guanine or misincorporated
oxidised dGTP. 8-oxoG is mutagenic and is an inevitable
consequence of oxidative metabolism but is also induced by
many environmental mutagens including cigarette smoke
and exhaust (Shen et al., 1997). Other important lesions
include oxidised pyrimidines such as thymine glycols (TG)
and 5-hydroxycytosine (5-OHC). We have studied the repair
Fig. 5. Limited repair of some oxidative DNA lesions in human testicular cells. Repair of oxidative DNA bases is measured using the bacterial enzyme
Formamidopyrimidine DNA N-glycosylase (Fpg) that is specific for the excision of oxidised pyrimidines such as 8-hydroxyguanine (8-oxoG). Oxidised base
lesions are introduced into the cellular DNA using the photo-activated oxidising agent Ro 12-9786 (6 AM) plus cold visible light (5 min). The number of Fpg-
sensitive DNA lesions is measured as single-strand breaks (ssb) in a modified alkaline elution assay. The damage levels are calculated from elution profiles and
are expressed as Fnormalised area above curve_(NAAC) (Brunborg et al., 1996). In these assays, genomic DNA is treated with an excess amount of Fpg crude
extract to transform Fpg-sensitive DNA lesions into ssb. (A) Repair of Fpg-sensitive DNA lesions by normal primary human fibroblasts. Cell samples were
treated with Ro 12-9786 (6 AM) plus light (5 min) to introduce oxidative base damage and treated with (diamonds) or without Fpg (triangles), respectively, after
being allowed to repair. Solid and broken lines show their mean values. Circles and squares represent control cell samples treated with or without Fpg,
respectively. (B– C) Repair of Fpg-sensitive DNA lesions by human testicular cells (B) from one individual testis biopsy and rat testicular cells (C, 3 AMRo
12-9786). For symbols, see panel A and box in panel A. The results demonstrate that human testicular cells exhibit only limited repair of Fpg-sensitive DNA
lesions as opposed to rapid repair by both rat testicular cells and human fibroblasts. On the other hand, we have observed that the repair of other oxidised DNA
lesions, such as some oxidised purines, is efficient in both human and rat testicular cells (data not shown here; Olsen et al., 2003).
A.-K. Olsen et al. / Toxicology and Applied Pharmacology 207 (2005) S521– S531S528
of these lesions in rodent and human male germ cells using
cellular extracts, identifying the relevant proteins and by
measuring active repair in live cells (Fig. 5). Oxidised
purines such as 8-oxoG were efficiently repaired in rat (Olsen
et al., 2003) and mouse (unpublished) spermatogenic cells.
However, human testicular cells from several individuals
showed no repair of these lesions, a result which we found to
be most unexpected. On the other hand, oxidised pyrimidines
such as TG were efficiently repaired in both the human and
the rat. In addition to the cellular assays, enzymatic activities
and protein levels of the relevant repair enzymes were
measured and were in accordance with the efficiency of
cellular repair. A very high level of Ogg1 mRNA was
reported in mouse testis (Rosenquist et al., 1997), whereas in
human tissues including the testis, OGG1 mRNA is
ubiquitously expressed (Radicella et al., 1997; Nishioka et
al., 1999). In conclusion, it appears that human male germ
cells may be particularly sensitive to DNA oxidation.
Mice and rats are by far the preferred species for
mutagenicity testing and reproductive toxicity studies. Since
it has been shown that humans may be different with respect
to the repair of important pre-mutagenic lesions, the results
from toxicological tests using laboratory species should be
interpreted with great care. A transgenic model has been
constructed in which the main DNA glycosylase (mOGG1)
for removal of 8-oxoG has been knocked out (Klungland et
al., 1999). Nuclear testicular extracts showed no incision of
8-oxoG (Klungland et al., 1999), also suggesting that the
testis has little back-up activity; indeed, we detected no
cellular repair of oxidative lesions in the male germ cells
from these mice (unpublished observations). We are
currently evaluating the mOGG1 KO mouse as a model
for human male germ cell toxicity testing. BER-related
enzymes involved in the later stages of the pathway seem to
be present at levels sufficient for functional repair. A number
of studies support this view. Nuclear extracts from mouse
spermatogenic germ cells contain high amounts of Ligase I,
Ape (mouse homologue of the human HAP1), Ligase III,
Xrcc1 and Polh, compared to levels in somatic cells (Intano
et al., 2001). Polhis highly expressed in bovine pachytene
spermatocytes (Hirose et al., 1989). Both Polhand DNA
ligase I are present in the rat (Prasad et al., 1996). DNA
ligase activities in the mouse were higher in premeiotic
spermatogonia and spermatocytes than in liver and bone
marrow cells (Higashitani et al., 1990). High levels of DNA
ligases I and III were also found in bovine testis (Husain et
al., 1995). Human DNA ligase III is ubiquitously expressed
at low levels, except in the testis, where the steady state
levels of DNA ligase III mRNA are at least 10-fold higher
than those detected in other tissues and cells (Chen et al.,
1995). The DNA ligase I mRNA expression in the testes
correlated with the proportion of proliferative spermatogo-
nia, in agreement with the previously defined role of this
enzyme in DNA replication. In contrast, elevated levels of
DNA ligase III mRNA were observed in primary sperma-
tocytes undergoing recombination prior to the first meiotic
division. This suggests that DNA ligase III seals DNA strand
breaks that arise during the process of meiotic recombination
in germ cells (Chen et al., 1995).
What happens with the germ cells containing unrepaired
DNA lesions?
The poor removal of bulky DNA adducts and oxidised
purines in human testicular cells are reflected in an
accumulation of DNA damage such as 8-oxoG and
benzo(a)pyrene adducts in human sperm (Sun et al., 1997;
Irvine et al., 2000; Evenson et al., 2002; Zenzes et al.,
1999b). These lesions are clearly environmentally related,
with smoking as a good (or bad) example (Shen et al., 1997;
Zenzes et al., 1999b).
Apoptosis then represents an additional mechanism for
removing cells with DNA lesions. Spontaneous apoptosis
does occur in all cell types of the testis during normal
spermatogenesis in man (Oldereid et al., 2001), and
testicular cells are very sensitive to apoptotic stimuli such
as high-dose chemotherapy (Spierings et al., 2003). It is
likely that the total level of DNA lesions is of decisive
importance for the fate of the spermatogenic cell.
On the other hand, there is evidence that B(a)P adducts
can indeed accumulate without eliciting apoptosis in
spermatogenic cells since ejaculated sperm from smokers
contain more B(a)P adducts than from non-smokers (Zenzes
et al., 1999a). Furthermore, such adducts derived from the
father do not prevent fertilisation and are persistent even at
the blastocyst level (Zenzes et al., 1999b), also indicating
that they are not always completely repaired in the fertilised
oocyte. A recent publication (Zitzmann et al., 2003) showed
that assisted fertilisation was 40% less likely to produce
a live offspring if the father was a smoker, compared to
non-smokers.
Concluding remarks
A question that emerges from these results is whether the
shutting off of some repair functions has a purpose. Several
possibilities may be envisaged. One might speculate that,
along with other functions in spermatogenic cells, energy
consumption is minimised (with priority directed to
spermatogenesis-associated processes such as meiotic
recombination and repackaging of DNA). A further
possibility is the need for a finely tuned balance (controlled
by the presence of some error-prone repair) between
preservation of the genetic integrity vs. the need for some
de novo germ-line mutations to drive evolution. Thirdly, the
presence of DNA lesions in a sperm that has reached the
oocyte may serve as a direct indicator for the oocyte as to
the genotoxic load that the sperm has been subjected to; the
oocyte may then either attempt repair or terminate the
process.
A.-K. Olsen et al. / Toxicology and Applied Pharmacology 207 (2005) S521 – S531 S529
Epilogue
In the event that sperm cell DNA has not been repaired and
apoptosis was not initiated, then – like all other problems –
we leave it up to the mother to solve the problem!
Acknowledgments
We thank Professor Bente E. Moen (Department of
Public Health and Primary Health Care, University of
Bergen, Norway) for the permission to use unpublished
data on malformations among naval serviceman. This
work has been supported by the European Union (ENV4-
CT95-0204 and QLK4-CT-2002-02198), and by The
Norwegian Research Council (Grants Nos 129614/310
and 148703/310).
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