1 Laboratory of Genetics, Wageningen Institute of Animal Sciences, Wageningen University, 6701BH Wageningen, The Netherlands, 2 Department of Obstetrics and Gynaecology, University Medical Centre St Radboud, 6500HB Nijmegen, The Netherlands, 3 Experimental Animal Centre, 6703HD Wageningen University, The Netherlands and 4 Institute of Reproductive Medicine of the University of Münster, D-48129 Münster, Germany.
5 Current address: Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, Erasmus Medical Center, 3015 GD Rotterdam, The Netherlands.
6 Current address: Department of Obstetrics and Gynaecology, University Medical Centre St Radboud, 6500HB Nijmegen, The Netherlands. To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, UMCN St Radboud, 415 OBGYN, PO Box 9101, 6500HB Nijmegen, The Netherlands. e-mail: P.deBoer{at}Obgyn.UMCN.nl
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Abstract |
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Key words: DNA damage/ICSI/male sterility/mouse/zygote
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Introduction |
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Classically, the assessment of human sperm is carried out by WHO criteria for concentration, motility and morphology (WHO, 1999). When using sperm for ICSI, the concentration is low and there are strong indications that motility and morphology are not reliable criteria for the absence of DNA damage (human: Lopes et al., 1998
; Twigg et al., 1998
; mouse: Ahmadi and Ng, 1999
). If DNA damage is present in the spermatozoon, this may lead to de novo mutations and structural chromosome abnormalities in the developing embryo. Cytogenetic proof of this principle in human sperm at first cleavage division in heterologous fertilization systems has been obtained in a number of studies (Martin et al., 1994
; Lee et al., 1996
; Rybouchkin, 1997
). For studying the genesis of chromosome mutations from sperm of men with oligoasthenoteratozoospermia (OAT), mouse models are needed.
To this purpose, we have investigated whether mice with chromosomal male sterility and an OAT phenotype produce sperm suitable for ICSI. The majority of males heterozygous for the two semi-identical reciprocal translocations T(1;13)70H and T(1;13)1Wa, are sterile (T/T', De Boer et al., 1986; Peters et al., 1997
). During first meiotic prophase in primary spermatocytes at the time of homologous chromosome pairing, two heteromorphic bivalents (a large one between the 131H, 131Wa and a small one between the 113H, 113Wa translocation chromosomes) are produced by synaptonemal complex formation. Subsequently, in the large 131 bivalent, an adaptive structurally normal synaptonemal complex is always formed, not revealing the interruption in homology. In the majority of primary spermatocytes of around 80% of T/T' males, this process fails for the small 113 heteromorphic bivalent. In these infertile males, spermatocyte death commences during the second half of pachytene with a peak at first meiotic division, as described for other sterile reciprocal translocation heterozygotes (De Boer et al., 1986
). Thereafter, spermiogenesis results in additional cell death and a large variation in sperm morphology and motility.
When synaptic adaptation occurs for the small 113 heteromorphic bivalent as well, and a structurally normal synaptonemal complex is formed in over half of the pachytene spermatocytes, fertility is restored (De Boer et al., 1986; Peters et al., 1997
). This is in keeping with the notion that for these spermatocytes, meiotic cell cycle checkpoints no longer result into apoptosis (Odorisio et al., 1998
; De Vries et al., 1999
). The obstructive effect of aberrant synaptic behaviour of unspecified chromosomes and chromosome segments during first meiotic prophase on gametogenesis, especially spermatogenesis, has been thoroughly documented in both man (Speed, 1989
) and mouse (De Boer and De Jong, 1989
).
We have used both epididymal and testicular spermatozoa from these infertile males for injection into mouse oocytes and compared fertilization rates by pronucleus formation and rates of cleavage to the two-cell stage to those of fertile controls carrying the same genetic background with or without the translocations.
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Materials and methods |
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For oocyte collection and -injection MEM-alpha was used (Cat no. 22571, GIBCO Life Tech, Gaithersburg, MD), 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), hereafter referred to as MEM-alpha medium. After injection, oocytes were cultured in G1 (Barnes et al., 1995) under silicone oil (Aldrich, 14,615-3). During the course of the experiments however, the culture medium was changed to human tubal fluid HTF (BioWhittaker Europe, Verviers, Belgium), containing 10% GPO (pasteurized blood plasm-protein solution, CLB, Amsterdam, The Netherlands) under mineral oil (International Medical, Zutphen, The Netherlands). This did not influence fertilization or cleavage efficiencies (data not shown).
Mice
Four to eleven-month old T(1;13)70H/T(1;13)1Wa double heterozygous males (T/T') on a random bred Swiss genetic background (HsdCpb:SE), served as sperm donors. They were generated in the experimental animal centre, Wageningen University, by crossing homozygous T(1;13)70H females with homozygous T(1;13)1Wa males (De Boer et al., 1986). T/T' males were tested for infertility by mating them twice with high-littersize NMRI (HsdWin:NMRI) virgin females (De Boer et al., 1986
). Absence of decidual reactions at day 13 of pregnancy indicated sterility. The spermiogram from the caput epididymis of infertile T/T' males resembles that of OAT Ts(113)70H tertiary trisomic males (De Boer et al., 1976
): sperm counts are low, and both motility and morphology are poor. As controls, fertile T/T' males were used, next to Swiss random bred wild-type males and NMRI random bred males.
B6D2 F1 females (Iffa-Credo, Someren, The Netherlands) were used as oocyte donors and were kept in a 10 h dark/14 h light schedule, with lights on at 11 am. Superovulation was induced by i.p. injection of 7.5 IU pregnant mares serum gonadotrophin (PMSG) (Intervet, Boxmeer, The Netherlands) around 9 pm, followed by 7.5 IU hCG (Intervet, Boxmeer, The Netherlands) 48 h later.
Morphology of the epididymis of T/T' males
From four T/T' males of proven sterility, one testis with attached caput, corpus, cauda epididymidis and vas deferens was fixed for 24 h in 3.7% formaldehyde in a phosphate buffer containing 46 mM Na2HPO4 and 33 mM NaH2PO4, washed and stored in 100% ethanol until histological investigation.
Preparation of epididymal and testicular sperm
Testes weights were recorded and sperm counts taken from the caput epididymidi (Searle and Beechey, 1974). Each cauda epididymidis was placed in silicone oil and the contents squeezed into the oil with forceps. For all fertile males, contents were transferred to HTF Hepes medium and left to disperse for 30 min at 37°C. The caudal contents from infertile T/T' males were placed in either Spermatocyte Isolation Medium (SIM) (Heyting and Dietrich, 1991
) or Nucleus Isolation Medium (NIM) buffer (Kuretake et al., 1996
) without phenylmethylsulphonyl fluride (PMSF), supplemented with 0.4% BSA at 37°C for 30 min. Samples were stored at room temperature (RT) and viability was checked with a livedead sperm kit (Molecular Probes, Leiden, The Netherlands).
To obtain testicular sperm, tubuli seminiferi were isolated in NIM buffer, cut into small pieces and squeezed. Clumps of spermatogenic cells were dissolved in 100 µl NIM buffer, supplemented with 25 µg/ml DNase I and 2.6 mg/ml Collagenase IV. Cells were centrifuged for 5 min at 170 g, washed twice with NIM buffer and then resuspended in 50 µl NIM buffer at RT.
Collection of MII oocytes and intracytoplasmic sperm injection
Oocytes were freed from the oviducts 13 h after hCG, and stored without cumulus cells at 37°C for up to 4 h. Microinjection at 18°C was performed as previously described (Kimura and Yanagimachi, 1995), selecting normal looking and motile spermatozoa. A fresh sperm sample was transferred from medium to polyvinyl pyrrolidone (PVP) (360 kDa) for each injection round of eight oocytes (
30 min). Epididymal spermatozoa from infertile T/T' quickly lost motility once in 12% PVP (<10 min), so it cannot be excluded that initially immotile spermatozoa were used as well. Therefore, in one set of experiments, sperm from sterile T/T' males was specifically selected for motility or immotility directly after dispersion of the contents of two cauda epididymidi in 200 µl HTF Hepes medium with 3% BSA (Fraser and Quinn, 1981
). Motile or immotile, normal looking sperm were transferred by holding pipette to the 12% PVP and injected. Testicular spermatozoa could only be selected by morphological appearance, because of inherent lack of motion.
After injection, oocytes were kept on the cool stage for 5 min, then gradually warmed to 37°C and placed in culture medium at 37°C, 5% CO2 in air. Fertilization was determined 79 h after injection by the presence of a second polar body and two visible pronuclei. After 1920 h, zygotes were scored for first cleavage. At this point, oocytes injected with spermatozoa from infertile T/T' males not showing signs of fertilization were given hypotonic treatment and chromosome preparations were made (Tarkowski, 1966). After staining with 4,6-diamidino-2-phenylindole (DAPI) (125 ng/ml), chromosomes were examined under a Zeiss Axiophot2 fluorescence microscope.
Treatment of zygotes from infertile T/T' males with bromo-deoxyuridine or okadaic acid
In three independent injection sessions, all oocytes injected with epididymal spermatozoa from infertile T/T' males were cultured in medium containing 5 µg/ml 5-bromo-2'-deoxyuridine (BrdU) to demonstrate DNA replication. After 20 h of culture, fertilized oocytes that had failed to cleave to the two-cell stage and still possessed visible pronuclei, were fixed (Tarkowski, 1966). BrdU incorporation was detected with the RPN202 kit (Amersham Life Science, Alrington Heights, IL) in combination with a goat anti-mouse IgG conjugated with FITC (Jackson Immuno Research laboratories, PA, USA), as described previously (Baart et al., 2000
) and DNA was counterstained using DAPI.
Okadaic acid (OA), as a specific inhibitor of serine/threonine phosphoprotein phosphatases (PP) 1 and 2A was used to induce premature chromosome condensation (PCC), in a pattern dependent on the cell cycle stage of the pronucleus. (zygote G1-, S- or G2-phase; Dyban et al., 1993). In three injection sessions with sperm of sterile T/T' males, oocytes with visible pronuclei 20 h after injection, were incubated for 1 h in 10 µM OA, subsequently transferred to fresh medium and cultured for another 12 h, after which chromosome preparations were made and inspected with DAPI.
Statistics
The MannWhitney U test has been used to compare fertilization rates and first cleavage rates between males of the different experimental groups
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Results |
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Zygote development after injection of epididymal sperm
Oocytes were injected with epididymal sperm from four different types of donors and zygote development was monitored (Table I). Injection of sperm heads from NMRI males led to high fertilization rates: 95% showed two pronuclei and a second polar body 7 h after injection. Of fertilized oocytes, 96% showed normal cleavage to two-cell embryos after 20 h of culture. These results are comparable to those of others (Kimura and Yanagimachi, 1995) after injection of B6D2 F1 oocytes with B6D2 F1 sperm (98% and 99%, respectively).
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The influence of sperm motility in sterile males
Viability staining in sterile males (n = 3) showed 2240% of live spermatozoa from the cauda epididymis directly after isolation, and this decreased to 1831% after 5 h of incubation at RT. Table II gives the results of separating motile from non-motile spermatozoa prior to PVP incubation. In these small series, no effect of selection for motility or immotility was apparent when males were oligospermic.
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Discussion |
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Origin of the DNA damage in epididymal sperm from sterile T/T' males
During first meiotic prophase in these males, an indication for the persistence of recombinational double-strand DNA breaks is found in those pachytene spermatocytes, that do not form a structurally normal small translocation chromosome 113 synaptonemal complex (Mahadevaiah et al., 2001). In these primary spermatocytes, death starts during the pachytene stage, as in comparable male sterile mouse models (De Boer et al., 1986
). Spermatocytes that fail to meet the prerequisites for meiotic recombination are removed from the germ line. Therefore, it is less likely that unrepaired meiotic DNA damage persists post meiosis in the epididymal spermatozoa of these males. Thus, the chromosome damage observed at the zygote stage more likely is of a post meiotic nature, when during sperm differentiation, DNA repair becomes inactive (Sega and Sotomayor, 1982
).
In the mouse and rat, an increase in the presence of endogenous nicks in the DNA of spermatids undergoing elongation was demonstrated by in situ nick translation (McPherson and Longo, 1992, 1993; Sakkas et al., 1995
). These authors propose that the creation and ligation of DNA nicks in testicular spermatids play an important role in chromatin remodelling prior to protamination. It is not known whether in a situation of OAT the ligation step is defective, thus leading to impaired chromatin stability and the presence of DNA damage.
Sperm transport through the epididymis has been timed in the mouse using radioisotopic labelling at premeiotic S-phase. Thus, in a normal male, transport takes 23 days each for respectively caputcorpus, corpuscauda and caudavas deferens (Sega and Sotomayor, 1982). These experiments have to our knowledge never been repeated in mice with oligospermia. In humans, a reduced production of spermatozoa is highly correlated with longer transit times through the epididymis (Johnson and Varner, 1988
). If we assume the same to hold true for mice, spermatozoa from our infertile translocation carriers would start to age in the epididymis. The improvement in first cleavage rate when using testicular sperm from sterile males (Table III) also implicates epididymal storage to play a role in the reduction of two-cell embryo yield with infertile sperm.
One other mouse ICSI study used sperm from mice deficient in the protein phosphatase 1c gene that only expresses itself as male sterility. When malformed testicular sperm from Pp1c
/ males was used for ICSI in B6D2 F1 oocytes, a detrimental effect on development in vitro between the 8-cell and blastocyst stage emerged. However, in contrast to the T/T' model, no effect was found up to the two-cell stage (Davies and Varmuza, 2003
). Possibly, the testicular origin of sperm in Pp1c
/ males favours two-cell development. Furthermore, also in contrast to our T/T' model, Varmuza et al. (1999
) report that in their male mutants, meiotic prophase was unaffected (by histology).
Reduced oocyte activation by epididymal sperm from sterile males
Injections with epididymal sperm of sterile T/T' males resulted into: a) elevated levels non-decondensed sperm nuclei; and b) premature chromosome condensation (PCC) in the presence of female second meiotic chromosomes: failure of oocyte activation by the sperm. PCC is known from the IVF clinic (Schmiady et al., 1996), and has often been attributed to precocious fertilization of an unripe oocyte (Zenzes et al., 1990
). This interpretation is strengthened by IVF results in the mouse (Kubiac, 1989
). However, sperm decondensation problems were noted in the majority of activation failures after human ICSI (Lopes et al., 1998
) and a link is evident with deviant sperm chromatin compaction, as determined with CMA3 fluorescence and positive in situ nick translation (Sakkas et al., 1998
). Thus, activation of the oocyte by the sperm is likely hampered when chromatin compaction is incomplete. However, research into the heterogeneity of chromatin compaction in sterile T/T' males is needed to determine if this contributes to the observed reduction of oocyte activation. In the related OAT tertiary trisomic Ts(113)70H mouse model, no evidence for underprotamination has been found as indicated by normal total thiol levels in the sperm head (De Boer et al., 1990
).
DNA damage checkpoints and repair
In Figures 2 and 4, male chromosome rearrangements are visible, which is evidence of double-strand DNA repair. Ligation of double-strand DNA breaks leading to chromosome abnormalities at PCC (Figure 2) must have occurred before condensation of the chromatin, likely shortly after sperm penetration. With artificially inactivated mouse sperm to induce PCC after ICSI, an identical result was obtained (Perry et al., 2000).
Zygotes which did not progress timely to the first cleavage after injection of T/T' sperm consistently showed BrdU incorporation, indicating DNA synthesis to have started. Therefore, development could either be delayed during S-phase or at the subsequent G2-phase. OA stimulation of arrested pronuclear zygotes after ICSI showed the majority to have two pronuclei at G2-phase, followed by one pronucleus at S-phase and the other at G2. It was demonstrated earlier by heterologous ICSI using necrotic human sperm and mouse oocytes (Rybouchkin et al., 1997), that the human paternal pronucleus was delayed in development. So the male pronucleus most likely is the one at S-phase in this study. The presence of a checkpoint in the mouse during the first mitotic cell cycle, after the pronuclei have reached competence to replicate DNA, has been described previously (Fulka et al., 1999
). Also, a p53-dependent S-phase damage checkpoint was found recently in mouse oocytes, using irradiated sperm and genetic manipulation of p53 status (Shimura et al., 2002
). Our results, combined with data on the processing by mouse zygotes of irradiation-induced DNA breaks in spermatozoa (Matsuda and Tobari, 1989
), indicate that the zygotic cell cycle is not characterized by a G1S checkpoint for DNA damage, so typical of somatic cells (Hartwell and Kastan, 1994
). Nevertheless, commencement of DNA synthesis in zygotes is dependent on full male pronucleus development in mammals (Comizolli et al., 2003
; and references therein).
Mouse ICSI as a model system
The results obtained with OAT epididymal sperm in this study show a delay and/or interrupted development for the majority of fertilized embryos at the zygote stage, a phenomenon not observed with ejaculated OAT sperm in the human (Nagy et al., 1995; Tarlatzis en Bili, 2000
). A direct comparison between the propagating capacity of epididymal and testicular sperm within an oligospermic individual is not available. In the human, comparisons have been made between epididymal sperm in obstructive azoospermia (OA) vs. testicular sperm in non-obstructive azoospermia (NOA, Wennerholm et al., 2000
; Friedler et al., 2002
) and between testicular sperm in OA vs. testicular sperm in NOA (de Croo et al., 2000
; Vernaeve et al., 2003
). Summarizing these data, testicular sperm from NOA patients were on average somewhat less effective in fertilization and possibly cleavage rate, an effect smaller but similar to the one observed in Table III for sterile T/T'.
Using the alkaline Comet assay to assess DNA fragmentation, a difference in DNA integrity between testicular and epididymal sperm in human obstructive azoospermia was also noted (Steele et al., 1999; OConnell et al., 2002
). In agreement with our results, more damage was observed in epididymal spermatozoa. Thus, it appears that the use of poor quality semen with possible DNA damage negatively affects embryo development, but the response of the human embryo to the presence of DNA damage may be different in comparison to the mouse.
In conclusion, oligospermia in the T/T' mouse model affects fertilization and first cleavage rates. This is most pronounced for epididymal sperm, but the same tendency is observed for testicular sperm. In normospermic controls, a difference between testicular and epididymal sperm was not apparent. Epididymal sperm from fertile T/T' males did not differ from chromosomally normal fertile controls. Although epididymal sperm from sterile T/T' males is not completely representative for ejaculated human gametes used for ICSI, this study offers interesting observations on chromosome instability when PCC is observed and when first cleavage is delayed. Also, it demonstrates a role for zygotic S-phase in detecting chromosomal instability or other aspects of chromatin structure. The T/T' mouse model is a good candidate for a follow-up study of the fate of two-cell mouse embryos after TESE.
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Acknowledgements |
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Submitted on November 12, 2003; accepted on January 15, 2004.