Hum. Reprod. Advance Access originally published online on June 18, 2007
Human Reproduction 2007 22(9):2368-2376; doi:10.1093/humrep/dem166
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Motile human normozoospermic and oligozoospermic semen samples show a difference in double-strand DNA break incidence
1 Department of Obstetrics and Gynaecology, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands
5 Correspondence address. Tel: +31-243610869; Fax: +31-24-3668597; E-mail: p.deboer{at}obgyn.umcn.nl
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Abstract |
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BACKGROUND: Among ICSI children de novo structural chromosome aberrations of male descent are increased. Misrepair of double-strand DNA breaks (DSBs) is a prerequisite for such aberrations to occur. To date, no absolute assessment of the number of DSBs in human sperm nuclei after gamete fusion has been described.
METHODS: Using man-mouse heterologous ICSI and 
H2AX immunofluorescent staining, capable of detecting a single DSB, the number of lesions in ICSI selected sperm from normozoospermic men (n = 2) and oligozoospermic patients (n = 3) was quantified. A comparison with a subfertile male mouse model (n = 5) has been made. In addition, the fate of morphologically normal ejaculated immotile sperm after ICSI was examined.
RESULTS: A significant increase in the fraction of sperm cells bearing DSBs was found in oligozoospermic semen compared with that from normozoospermic men (P < 0.01). The majority of morphologically normal immotile human sperm showed excess H2AX staining and nuclear disintegration. However, some had a non-deviant DSB pattern.
CONCLUSIONS: The increased fraction of DSB-positive sperm in both human and mouse oligozoospermic semen is adding to the surmise that semen from oligozoospermic patients has a reduced chromatin quality, causally related to reduced preimplantation embryo development. The use of ejaculated immotile sperm for in vitro reproduction is debatable due to sperm DNA degradation.
Key words: ICSI/double-strand DNA break/sperm/chromatin/DNA degradation
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Introduction |
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With the introduction of assisted reproduction techniques (ART), infertility no longer poses an absolute hurdle for couples to parent a child. In biological terms, ICSI is a less physiological form of ART, as this technique circumvents biological selection. As sperm selection is a poorly defined concept (Cohen and McNaughton, 1974
) and ICSI is the sole option for many cases of male factor infertility, this poses a degree of concern. Sperm from such men usually meet several or all of the World Health Organization (WHO) criteria for oligo-astheno-teratozoospermia (OAT). One major concern pertains to an increased risk of de novo mutation at both the level of the gene and chromosome. An increased risk of de novo mutation at the chromosomal level has been indicated. In sperm of OAT males, the normally low incidence of numerical chromosomal abnormalities is about doubled implying meiotic defects (Marchetti and Wyrobek, 2005), possibly illustrated by lower recombination rates (Gonsalves et al., 2004), giving rise to an increased number of aneuploid offspring (Bonduelle et al., 2002).
The aetiology of structural chromosome aberrations, that are also more frequent among ICSI descendants (Bonduelle et al., 2002), remains unclear. For a long time, this class of mutation has been known to be largely of male descent (Olson and Magenis, 1988). Misrepair of double-strand DNA breaks (DSBs) is a prerequisite for structural chromosome aberrations to occur (Richardson and Jasin, 2000). Indeed, radiation-induced DSBs in mouse and human sperm lead to chromosome abnormalities, detectable in the paternal chromosomes at the first cleavage division (Matsuda et al., 1985; Kamiguchi and Tateno, 2002).
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DSBs are a characteristic of living cells in that they do occur spontaneously in the cell cycle during DNA replication and are instrumental in the generation of antibody diversity (Hoeijmakers, 2001
) and meiotic recombination. If left unrepaired, DSBs would inhibit the cell cycle or alternatively would lead to gross structural alteration of the chromosome complement, most likely by a high frequency of deletions. Therefore a DSB is considered to be a most serious DNA lesion. Hence, special DNA repair pathways, notably homologous recombination (HR) repair and non-homologous end joining (NHEJ) evolved in the course of evolution to take care of such damage (see for review Wyman and Kanaar, 2006). Structural chromosome abnormalities such as dicentric chromosomes, reciprocal translocations and acentric fragments are conceptualized as failures of both NHEJ and HR, for which the word misrepair is coined.
DSBs are also used to rearrange chromatin, a most prominent example being the elongation of round spermatids when the enzyme Topoisomerase II (Laberge and Boissonneault, 2005) both creates and religates these lesions. This breakage activity is implicated in the loss of the nucleosomal chromatin structure on the way to transition protein and protamine occupation of DNA in sperm.
Sperm present a special group of nuclei with respect to DNA damage as nuclei are very compact. Yet, several methods have yielded indications for the presence of DNA damage at a larger scale than in somatic cell systems that rely on active DNA repair, that is not available to the sperm nucleus (Kofman-Alfaro and Chandley, 1971; Sega et al., 1978).
For the past decades, the DNA integrity of the sperm nucleus has been measured by numerous techniques, i.e. in situ nick translation, terminal deoxynucleotidyl transferase dUTP end labelling (TUNEL), single-cell electrophoresis (SCE, or comet assay in alkaline and ‘neutral’ variants), sperm chromatin dispersion test and sperm chromatin structure assay (SCSA) (Fernandez et al., 2005; Evenson and Wixon, 2006). Apart form the neutral comet assay (Van Kooij et al., 2004), these methods do not specifically sense DSBs. Moreover, the neutral comet assay does not generate absolute numbers of breaks.
For the cytogenetic analysis of human sperm after heterologous insemination, hamster (Rudak et al., 1978) and mouse (Lee et al., 1996) secondary oocytes have been used in the past. Heterologous ICSI of mouse oocytes is a proven method for the assessment of oocyte activating power and chromosomal constitution of human sperm, mimicking the ICSI-involved sperm selection procedure (Rybouchkin et al., 1995; Lee et al., 1996). Frequencies of 1.3 and 6.9% for, respectively, numerical and structural chromosome aberrations have been found in morphologically normal semen using these methods (Lee et al., 1996). However, from the data on structural chromosome abnormalities, the absolute number of breaks per sperm nucleus cannot be deduced, due to an absence of knowledge regarding the reliability of repair mechanisms, i.e. the amount of misrepair of HR and NHEJ, in the zygote (Fiorenza et al., 2001). Because of the fact that DNA repair is not error free, knowledge about the absolute number of DNBs in sperm is required.
As every living cell has to cope with DNA damage, highly sensitive signalling and repair mechanisms have evolved (Hoeijmakers, 2001). The surveillance and repair machinery that protects the cell from DSBs uses enzymes that recognize the DSB and phosphorylate proteins surrounding the break, notably Histone H2AX, denoted as H2AX (Rogakou et al., 1998).
This reaction, appearing within minutes after the insult in most cellular systems, is pivotal to genome stability and conserved from yeast to human (Rogakou et al., 1998; Fernandez-Capetillo et al., 2004). As a prerequisite for the present investigation, we have investigated whether H2AX signalling was operative in the early mouse zygote, which was the case for both paternal and maternal chromatin (Derijck et al., 2006).
Upon gamete fusion followed by activation of the secondary oocyte, the second meiotic division is completed. Simultaneously, a speedy transition from sperm nucleus to male pronucleus occurs, (van der Heijden et al., 2005) during which paternal chromatin undergoes remodelling from a protamine-rich sperm chromatin configuration, to the histone-based nucleosomal configuration. H2AX can be detected after the onset of chromatin remodelling.
During temporary recondensation of the unfolded sperm nucleus, histone H3 is increasingly phosphoryated at serine 10. With uniform staining for this epitope, H2AX foci are distinctly present and large foci representing DSBs can be positively identified (Derijck et al., 2006).
Here, we quantify DSBs present in human sperm after chromatin remodelling using the mouse heterologous ICSI and H2AX staining (HIGH) assay. Sperm from normozoospermic men and oligozoospermic patients, selected by WHO criteria, was analysed. A comparison with a subfertile male mouse model has been made. For the first time, absolute numbers of sperm DSBs could be determined.
Sperm motility is a crucial factor that influences the outcome of ICSI. Both pregnancy rate and embryo quality are reduced after ICSI with immotile sperm (Stalf et al., 2005). DNA damage assays have been shown to give lower readings in sperm selected for motility, both after swim up (Van Kooij et al., 2004) and manual selection (Ramos and Wetzels, 2001). By theory and in practice, immotile sperm is composed of a viable and non-viable fraction with a large variation between samples. Hence, the use of ejaculated immotile sperm for ICSI is still under debate. Therefore, we also examined ejaculated immotile but morphologically normal sperm by the HIGH assay under the assumption that this dichotomy could be clarified from zygote chromatin behaviour, which was the case.
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Materials and Methods |
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Reagents
For oocyte storage, injection and culture, minimum essential medium alpha medium was used (Cat no. 22571, GIBCO Life Tech, Gaithersburg, MD, USA), supplemented per 500 ml with 2.5 g HEPES, 684 mg 50% sodium lactate solution, 55 mg sodium pyruvate, 65 mg penicillin G (1596 U/mg) and 6% fetal calf serum (BioWhittaker Europe, Verviers, Belgium).
Semen donors
Semen samples from three OAT men [by WHO criteria (World Health Organization, 1999)] attending our fertility clinic for diagnosis were cryopreserved for this study. Cryopreserved semen samples of two normozoospermic men of proven fertility served as controls. The percentage of severely DNA-damaged sperm of each sample was assessed by TUNEL assay (Ramos and Wetzels, 2001) after thawing. All patients gave written informed consent before inclusion in this study.
Preparation of cryopreserved human sperm
Sperm samples were diluted 1:1 (drop wise) with test yolk buffer (Irvine Scientific, Santa Ana, CA, USA) and equilibrated for 10 min at room temperature. The sperm/cryoprotectant mixture was aspirated in 0.5 ml straws (CBS, France), sealed, placed in a cooling chamber and rapidly frozen in liquid nitrogen vapour for 10 min (Tournaye et al., 1999; Friedler et al., 2002). Straws were stored in liquid nitrogen. Thawing occurred over 10 min at room temperature followed by dilution of cryoprotectant with 1 ml human tubal fluid (HTF)-HEPES. Suspension was centrifuged for 5 min at 500g and the pellet was gently resuspended in HTF-HEPES. This was repeated once and the samples were kept at room temperature until ICSI.
Preparation of mouse sperm
Male mice, heterozygous for two semi-identical reciprocal translocations T(1;13)70H and T(1;13)1Wa (abbreviated T/T') usually are sterile by OAT (de Boer et al., 1986). Sterility is caused by reduced chromosome synapsis at first meiotic prophase for translocation chromosomes. This mouse model is maintained on a Swiss random bred background. Zygotes derived by ICSI with cauda epididymal sperm show a severely reduced cleavage rate and developmental delay/arrest during the zygotic S-/G2-phases (Baart et al., 2004). Sperm of five OAT mice (8–10 weeks, testes 50–70 mg, Baart et al., 2004) was obtained by dispersion of the contents of the two cauda epididymidi in 200 µl HTF-HEPES 3% bovine serum albumin (Sigma, A-4503). Samples were kept at room temperature.
Preparation of mouse oocytes
B6D2 F1 females (4–10 weeks) (Charles River, Sulzfeld, Germany) were used as oocyte donors and kept at a 9.00 am–9.00 pm light schedule. Superovulation was induced by i.p. injection of 7.5 IU pregnant mare's serum gonadotrophin (Intervet, Boxmeer, The Netherlands) around 9 pm, followed by 7.5 IU HCG (Intervet) 48 h later. Oocytes were freed from the oviducts 13 h after HCG, and stored without cumulus cells at 37°C for up to 5 h.
Heterologous and homologous ICSI
Microinjection was performed as described (Kimura and Yanagimachi, 1995) with some adaptations. The injection medium was kept at 24°C. Both human and mouse sperm were selected for normal morphology and motility at 400 x magnification. Morphologically normal immotile sperm of OAT men were used as well. The sperm tail of mouse sperm was removed using the piezo driven injection needle. After injection, oocytes were kept on the microscope stage for 5 min, were then gradually warmed to 37°C and placed in culture medium at 37°C, 5% CO2 in air.
Fixation and staining of zygotes
For immunofluorescent detection of H2AX chromatin domains and status of paternal chromatin remodelling (by anti-H3S10ph) zygotes were processed two hours after ICSI as described (van der Heijden et al., 2005). The following antibodies were used: H2AX mouse monoclonal (Upstate #05–636, clone JBW301, 1:10,000) and rabbit anti-H3S10ph (Upstate #06–570, 1:1,000). Secondary antibodies were: Molecular Probes, Oregon, USA: A11001 fluor 488 goat anti-mouse immunoglobulin (Ig)G (H + L), A11012
[GenBank]
fluor 594 goat anti-rabbit IgG (H + L), both in a 1:500 dilution. DNA was stained with 4',6-diamidino-2-phenylindole (DAPI) in phosphate-buffered saline (0.33 mg/l) and fading was counteracted with Vectashield (Vector Laboratories).
Analysis
Microscopic observations were made from coded samples by one observer (A.A.H.A.D.). Oocyte activation status was assessed, as was the degree of decondensation and chromatin remodelling of the sperm nucleus. Large DSB related H2AX foci of fully remodelled paternal nuclei (judged by histone H3 serine10 phosphorylation and DAPI morphology) were counted. Unlike small foci, large foci can be induced by sperm irradiation and treatment of the early zygote with proven inducers of DSBs (Derijck et al., 2006).
Non-parametric statistical analysis was performed using Prism (Graphpad) and Statistical Package for the Social Sciences (Appache software foundation) software.
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Results |
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Semen quality and heterologous ICSI efficiency
Semen samples were categorized using the hallmark parameters of fertility; concentration, motility and morphology. The TUNEL staining provides a further reference to semen quality. A TUNEL score > 14% is indicative for poor DNA integrity and correlates with reduced fertility (Ramos and Wetzels, 2001
; Chohan et al., 2006). As expected, the DNA integrity of OAT sperm was reduced (higher TUNEL scores, Table 1). Heterologous ICSI was performed on 274 mouse oocytes, resulting in 225 analysable zygotes (82.1% survival). Zygotes were classified in three classes: (i) normal zygotes with maternal chromosomes in anaphase II–telophase II and fully remodelled sperm (Fig. 1A); (ii) activated secondary oocytes without sperm decondensation (iii) sperm nucleus expansion without oocyte activation. Between patient samples, no significant differences were observed for class distributions (Table 2). However, morphologically normal immotile sperm (pooled from OAT semen, all patients) showed a significant shift in ICSI characteristics, with almost half of the sperm lacking oocyte activating capacity (Table 2).
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DSB analysis of human sperm using
H2AXAll normal developing zygotes were analysed for
H2AX signalling. The mouse oocyte remodels the human sperm nucleus to a nucleosomal chromatin structure (van der Heijden et al., 2005,2006). The DNA damage signalling mechanism of the oocyte marks DSBs via H2AX which show as large foci in paternal chromatin (Fig. 1A). These can be counted provided the remodelling process has advanced far enough, which is indicated by uniform H3S10ph staining (Derijck et al., 2006, Fig. 1A). Background staining and small H2AX foci, intrinsic to the sperm remodelling stage are not DSB related (Derijck et al., 2006). Occasionally a non-focal pattern, often covering the larger part of the nucleus, is observed (Fig. 1B and C). This type of H2AX staining was mostly found in morphologically normal immotile sperm (see below).
When the ranges and averages of DSB related H2AX foci from all five men were compared, no significant differences could be detected (Fig. 2A).
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Zygotes were divided into three groups on the basis of paternal
H2AX staining (Table 3): without DSB foci (negative), with foci and with an aberrant H2AX pattern. The fraction of sperm with DSB-related foci is increased in OAT men, as shown in the graphical representation (Fig. 2B).
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Chromatin from morphologically normal ejaculated immotile sperm displays an increased incidence of aberrant
H2AX stainingEjaculated immotile sperm are prone to lack oocyte activating activity (Table 2). In addition, both immotile sperm which do activate oocytes and those which do not, show a significant increase in aberrant
H2AX staining patterns (Table 3 and Fig. 2B). Larger positive nuclear domains up to heavily stained paternal chromatin prevail (Fig. 1B and 1C). Of the sperm nuclei lacking oocyte activating capacity, 4 out of 9 showed chromatin disintegration within the oocyte cytoplasm, as indicated by chromatin fragments double stained for H2AX and H3S10ph (Fig. 1D). Although ICSI with immotile sperm resulted in 81% (17/21) abnormal zygotes, 28.6% (6/21) was able to produce normal remodelled paternal chromatin (2 non-activating and 4 activating sperm). The level of DSB related foci of these nuclei, showed no significant difference with motile sperm (average 1.8).
Maternal chromatin reacts to abnormal paternal chromatin from immotile sperm
The maternal chromatin arrested at meiotic metaphase II shows a clear punctuate non-DSB-related H2AX staining, similar to that found in normal mitosis (McManus and Hendzel, 2005). After resumption of meiosis II this meiotic H2AX diminishes rapidly (Fig. 1A, Derijck et al., 2006). However, zygotes containing aberrantly H2AX stained paternal chromatin from immotile sperm (Fig. 1B and C) often showed an abnormal intense labelling of the maternal anaphase II (Fig. 1C). Table 4 shows the status of parental chromatin of all oocytes/zygotes from injected immotile sperm. Abnormal H2AX intensities/patterns often correlated between parental chromatin complements.
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ICSI with epididymal sperm of a sterile mouse model
Morphologically normal motile sperm of T/T' mice was used for ICSI. A total of 46 analysable zygotes was obtained (90.2% survival). Zygote development is given in Table 2. DSB-related
H2AX foci were counted in normal zygotes and compared with sperm from normozoospermic males (Table 5, Fig. 2). The average amount of DSBs was doubled in OAT mouse sperm. However, no statistical significant difference was obtained (Fig. 2A). A significant increase of the fraction of H2AX positive remodelled nuclei was also observed in OAT mouse sperm (Fig. 2B).
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Discussion |
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In situ DSB detection in sperm via
H2AX at chromatin remodelling post gamete fusionThe chromatin composition of sperm nuclei differs from somatic nuclei by the presence of protamines. Although human sperm chromatin remodelling during spermiogenesis is incomplete when compared with mouse (residual nucleosomes: mouse ~1%, human ~15% Bench et al., 1996
), no H2AX signal can be detected in in vitro decondensed sperm of both species (L. Ramos personal communication). The analysis of H2AX phosphorylation in human sperm after heterologous ICSI, indicates that human sperm is comparable to mouse sperm (Fig. 1, Derijck et al., 2006).
It was already known that mouse oocytes can efficiently remodel human sperm chromatin to a somatic state, capable of chromosome condensation during first mitosis (Lee et al., 1996). Both studies demonstrate the suitability of the heterologous zygote system for the study of human sperm chromatin and chromosomes.
Sperm quality measurements compared
Sperm quality assessments based on the basic WHO sperm parameters are often supported by DNA integrity measurements like TUNEL, SCE and SCSA (Chohan et al., 2006). The fraction of presumably apoptotic (i.e. TUNEL positive) sperm within an ejaculate correlates well between assays and is ~11% in normozoospermics (Aravindan et al., 1997; Chohan et al., 2006, see also Table 1). Selection of individual morphologically normal progressively motile sperm reduces the TUNEL-positive fraction to < 1% (Ramos and Wetzels, 2001) in line with the general notion that more heavily DNA-compromised sperm nuclei are found in the immotile fraction (Van Kooij et al., 2004).
The HIGH assay measures the absolute amount of DSBs (Derijck et al., 2006) after sperm selection by ICSI criteria and sperm chromatin remodelling in the oocyte. The DSB-‘positive’ fraction in the HIGH assay was 36% (fertile donor average) and 57% (OAT average), showing discriminative power for motile sperm (in contrast to TUNEL, Ramos and Wetzels, 2001) and demonstrating the improved sensitivity of the HIGH assay. Hence, both at the level of the total sperm population (DNA integrity assays) and in stringent ‘ICSI’-selected sperm (HIGH assay) a difference between normozoospermic and infertile semen was found, adding to the evidence that sperm chromatin and DNA integrity is altered in male infertility syndromes. It is reassuring that for these small samples the average absolute number of DSBs in semen from normozoospermic donors was not significantly different from that of OAT patients. We have to stress, however, that the OAT donors used, were not at the extreme end of the spectrum. Assuming the incidence of DSBs to be applicable to ICSI candidates in general, the increase of de novo structural chromosome abnormalities after ICSI, by at least 4-fold (Bonduelle et al., 2002), can not be attributed to an elevated number of DSBs in OAT semen per se (Fig. 2) and could therefore arise early in embryonic development (zygote and cleavage stage) or during spermatogenesis. Indeed, inspection of synaptonemal complexes in pachytene spermatocytes of infertile men has yielded indications for quadrivalents representing reciprocal translocations (Vendrell et al., 1999).
The minor increase of DSBs in OAT sperm could relate to a hampered preimplantation development
Zygotes derived from cauda epididymal sperm of T/T' OAT mice showed a reduced cleavage rate (33 versus 87–96%) due to developmental delay and arrest during zygotic S- or G2-phase (Baart et al., 2004). In sperm of these mice, the measured number of DSBs was similar to 1.5–2 Gy sperm irradiation (Derijck et al., 2006). Irradiation-induced damage that leads to chromosome abnormalities at first cleavage division, including reciprocal translocations, does not block the progression of the zygotic cell cycle (Matsuda et al., 1985). Therefore, the marginal increase of DSBs in conjunction with the developmental block at S/G2 phase found after insemination with T/T' OAT sperm (Baart et al., 2004) indicates that the HIGH assay does not measure the total burden of aberrant chromatin. Cauda epididymal sperm from Tnp1–/–, Tnp2 +/– mice that have an abnormal chromatin compaction due to a defect in the nucleosome to protamine transition, produced lower implantation rates and yields of live born offspring (Suganuma et al., 2005). Therefore, depending on the aetiology of these OAT mouse models, an early or late onset phenotype of preimplantation development has been described.
In human reproduction, a link between a paternal factor and poor embryo quality (Shoukir et al., 1998) resulting in reduced pregnancy rates, has been observed (Loutradi et al., 2006). Poor sperm quality as judged by the conventional DNA integrity assays is often found to be linked to reduced cleavage/blastocyst rates (Sun et al., 1997; Morris et al., 2002; Seli et al., 2004; Virro et al., 2004; Zini et al., 2005), reduced in vivo fertility and ART outcome (Evenson et al., 1999; Tomlinson et al., 2001; Duran et al., 2002; Larson-Cook et al., 2003; Tesarik et al., 2004; Virro et al., 2004) (for reviews see Lewis and Aitken, 2005; Spano et al., 2005). Early onset paternal effects on zygote development (Tesarik et al., 2002) and early cleavage (Lewis and Aitken, 2005) have also been described. Thus, also for the human, the increased fraction of sperm with DSBs (Fig. 2B) could indicate a pathology related to aberrant chromatin, i.e. expressed during preimplantation development. However, this burden does not originate from the absolute number of DSBs that were either present in the sperm before penetration or were induced at chromatin remodelling before pronucleus formation (Bizzaro et al., 2000; Derijck et al., 2006).
Immotile sperm and DNA fragmentation
Protamine dominated sperm chromatin is generally regarded as highly stable. In contrast, chromatin from immotile human sperm often disintegrated after heterologous ICSI. In line with our results, Rybouchkin and co-workers (1997) found a strong correlation between zygote arrest and the proportion of non-viable sperm among the immotile ones, also using man-mouse heterologous ICSI. H2AX staining revealed male nucleus disintegration during the chromatin remodelling phase before pronucleus formation (Fig. 1).
Recently, mammalian sperm including human sperm was found to contain endonucleases which become activated after membrane permeabilization in the presence of divalent cations (Sotolongo et al., 2005). DNA degradation by these endonucleases was noticed already after 15 min and eventually digested the DNA to 50 kb loop domain fragments (Sotolongo et al., 2005), suggestive of an apoptosis-like pathway, represented by the fraction of TUNEL-positive spermatozoa (Ward and Ward, 2004). The H2AX pattern of 70% of nuclei from immotile sperm is indicative for high DNA damage (Fig. 1B–D). We have to stress that nuclear fragmentation after gamete fusion (Fig. 1D) was not found after ICSI with motile sperm that indeed are TUNEL negative (Ramos and Wetzels, 2001). In 71% (10/14) of the cases, highly damaged non-motile sperm triggered a signalling response in the maternal chromatin, as deduced from the increase in H2AX phosphorylation (Table 4, Fig. 1C). Normally, the condensed maternal second meiotic chromosomes loose H2AX upon oocyte activation (Fig. 1A). A functional role for the oocyte chromatin in supporting preimplantation development of zygotes with damaged paternal chromatin has been shown in the mouse (Suganuma et al., 2005). The nature of this maternal role remains elusive and needs further investigation. The maternal reaction found by us supports the existence of a role of maternal chromatin in determining paternal chromatin integrity in the zygote.
In ~20% of immotile ejaculated sperm injections (4/21, Table 3), we found a normal H2AX pattern in paternal and maternal chromatin. This assessment may well be in line with clinical results obtained with immotile ejaculated sperm, notwithstanding the multifactorial background of this condition.
Conclusively, by use of man-mouse heterologous ICSI combined with the examination of the most pronounced chromatin marker for DSBs (H2AX) we have for the first time obtained an objective assessment for the absolute number of these most serious DNA lesions in sperm. The incidence of DSBs was comparable in mouse and man (Fig. 2). For both species, an indication was found for a decrease of the fraction of DSB-free sperm in OAT. This observation could be one aspect of data in the current literature, that hypothesize a paternal chromatin-linked factor to negatively influence preimplantation embryonic development. In addition, this approach has unequivocally demonstrated the nuclear disintegration of the larger part of morphologically normal ejaculated immotile human sperm and supports further deliberation on the use of such sperm in assisted reproduction.
In this project, we for practical reasons made an exclusive use of cryopreserved samples, which in case of an interaction between resistance to cryopreservation and donor status (i.e. fertile or subfertile) could influence the interpretation of our data. On the other hand, survival after cryopreservation could lead to a general improvement of DNA integrity as measured by a modified alkaline Comet assay (Donnelly et al., 2001) no mention being made of any relation between sperm quantity and direction of change with this assay.
The HIGH assay could also be an asset in the analysis of DNA integrity of testicular sperm in non-obstructive azoospermia and of immature testicular spermatids, in both cases capable of addressing possible relations with altered sperm chromatin remodelling. Due to the delicacy of mouse ICSI and time-consuming analysis, the HIGH assay will not be a suitable system for clinical sperm DNA integrity testing. From a biological point of view, however, this system provides a highly sensitive single-sperm analysis, to study principal questions on sperm DNA integrity and the oocyte response in man and mouse.
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Acknowledgements |
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The Dutch ministry of Health, Welfare and Sport financed this research.
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Footnotes |
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2 Present address: University Medical Center Utrecht, Department of Pharmacology and Anatomy, Rudolf Magnus Institute of Neuroscience, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands
3 Present address: Carnegie Institution of Washington, Department of Embryology, 3520 San Martin Drive, Baltimore, MD 21218, USA
4 Present address: Orthopedic Research Laboratory, Department of Orthopedics, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
* Both authors contributed equally.
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Submitted on November 13, 2006; resubmitted on April 16, 2007; accepted on May 15, 2007.
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