1 Oregon Regional Primate Research Center, Division of Reproductive Sciences, Beaverton, Oregon, 2 Departments of Obstetrics and Gynecology, and Cell & Developmental Biology, and Center for Women's Health, Oregon Health Sciences University, Portland, Oregon, USA 3 Department of Anatomy and Cell Biology, Queens University, Kingston, Ontario, Canada, 4 Department of Molecular and Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, USA and 5 Center for Neuroscience and Cell Biology of Coimbra, Department of Zoology, University of Coimbra, 3000 Coimbra, Portugal
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Abstract |
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Key words: acrosome/fertilization/ICSI/perinuclear theca/spermatozoa
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Introduction |
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Following its initial introduction in mammals (Uehara and Yanagimachi, 1976) ICSI has been employed in many species, ultimately resulting in live human births (Palermo et al., 1992
; Van Steirteghem et al., 1993
). Although some questions have been raised regarding the technique (In't Veld et al., 1995; Bowen et al., 1998; Lamb, 1999), ICSI remains a reasonably safe and effective treatment to address many types of male factor infertility (Palermo et al., 1996
; Bonduelle et al., 1998a
,b
; Loft et al., 1999
; Schlegel, 1999
).
However, further information on the peculiar cell biology of ICSI is required. Notably, the unusual handling of sperm components not present inside the oocyte during conventional in-vitro fertilization, as well as the possible implications for the first embryonic cell cycle, should be investigated. Given the ethical dilemmas involved, information from a clinically relevant model is needed. Such a model could help expand the information that can be garnered from discarded human material, which is often limited and/or of impaired reproductive quality. Although the mouse would seem an appropriate choice, given the detailed knowledge available on the fertilization and early embryology in this system, a critical event in the first embryonic cell cycle in rodents differs completely from what takes place in primates, including humans. Namely, the primate spermatozoon provides a centriole, which, once transformed into a zygotic centrosome, is responsible for nucleating microtubules, and thus mediating pronuclear apposition during the first embryonic cell cycle. In the mouse, both sperm centrioles are dismantled during spermiogenesis (Manandhar et al., 1998), and centrosomal inheritance is strictly maternal (Schatten et al., 1985
, 1986
; Schatten, 1994
; Simerly et al., 1995
). We have therefore been actively engaged in developing the rhesus macaque (Macaca mullata) as a model for ICSI, since fertilization events in this system closely resemble those that take place in humans (Simerly et al., 1995
; Hewitson et al., 1996
, 1998
, 1999
; Sutovsky et al., 1996a
).
The success of ICSI implies that a well-choreographed disassembly of sperm components, normally lost before or during gamete fusion, must take place before the first embryonic cell cycle is successfully completed. Notable among these sperm-specific structures is the acrosome, a large cap-like secretory vesicle localized on the heads of spermatozoa from most species. Acrosomal contents mediate sperm-oocyte interactions by aiding the sperm's penetration through the mammalian oocyte's zona pellucida. Release of these contents occurs during the acrosome reaction, an exocytotic event resulting in the fusion of the acrosomal membrane and the sperm plasma membrane that takes place before the spermatozoon binds to the oolemma (Breitbart and Spungin, 1997; Wassarman, 1999
). Recent parallels have been drawn between the sperm acrosome reaction and exocytosis in somatic cells, implicating members of the `SNARE hypothesis' for intracellular membrane fusion in the release of acrosomal contents. This hypothesis postulates that a specific soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) on the membrane of a vesicular carrier (termed v-SNARE) specifically interacts with a complementary SNARE on the target membrane (t-SNARE), and that this interaction results in the recognition, docking and/or fusion of the two membranes (Rothman, 1994
; Hanson et al., 1997
; Gotte and von Mollard, 1998
). Accordingly, homologues of the v-SNARE vesicular associated membrane protein (VAMP), also known as synaptobrevin, and of the t-SNARE syntaxin were identified on the acrosome/plasma membrane of mammalian spermatozoon (Ramalho-Santos et al., 2000
). We have therefore studied the behaviour of rhesus sperm VAMP after ICSI, using this protein as a marker for the inserted acrosome.
Another important sperm structure that must be removed during ICSI is the sub- and post-acrosomal perinuclear theca. Unlike the acrosome, this cytoskeletal structure is removed from the sperm head and discarded shortly after gamete fusion (Sutovsky et al., 1997). The removal of the acrosome and perinuclear theca following ICSI might also have profound implications for nuclear remodelling and DNA synthesis during the first embryonic cell cycle, which could be related to the increased frequency of sexual chromosome abnormalities in the progeny of subfertile couples (In't Veld et al., 1995; Bonduelle et al., 1998a,b).
In the present study we address these issues by providing a cellular analysis of ICSI-fertilized rhesus oocytes, and comparing the results with zygotes obtained by traditional IVF. We extend previous reports by using only monospermic ICSI, and by providing information on the handling of male-specific molecules after sperm injection.
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Materials and methods |
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Media
Throughout this work several variations of TALP medium were used. TALP is modified Tyrode-lactate medium with pyruvate and albumin: 114 mmol/l NaCl, 3.2 mmol/l KCl, 2 mmol/l CaCl2, 0.5 mmol/l MgCl2, 25 mmol/l NaHCO3, 0.4 mmol/l NaH2PO4, 10 mmol/l sodium lactate, 6.5 IU penicillin, 25 µg/ml gentamicin, 6 mg/ml fatty acid-free bovine serum albumin, 0.2 mmol/l pyruvate. TALP-HEPES consists of TALP buffered with 10 mmol/l HEPES at pH 7.4 (Bavister et al., 1983).
Gamete collection
Females exhibiting normal menstrual cycles were hyperstimulated by a regimen of exogenous gonadotrophic hormones. Females were down-regulated by daily s.c. injections of a gonadotrophin-releasing hormone (GnRH) antagonist (Serono, Randolph, MA, USA) at 0.5 mg/kg body weight for 6 days during which recombinant human FSH (rFSH; Organon Inc., West Orange, NJ, USA) was administered twice daily (30 IU, i.m.). This was followed by 13 days of rFSH + recombinant human LH (30 IU each, i.m. twice daily). Ultrasonography was performed on day 7, and when there were at least four follicles 4 mm in diameter, a final i.m. injection of 1000 IU recombinant human chorionic gonadotrophin (HCG; Serono, Randolph, MA, USA) was administered. Follicles were aspirated by laparoscopy 27 h post HCG injection, and the collected oocytes assessed for maturity. Germinal vesicle (GV), germinal vesicle breakdown (GVBD) and mature oocytes were cultured for up to 6 h in pre-equilibrated TALP (Bavister et al., 1983), containing 3 mg/ml BSA at 37°C in 5% CO2 under mineral oil (Sigma, St Louis, MO, USA). Following this incubation, only mature, metaphase II-arrested, oocytes were used for fertilization by ICSI or IVF.
Spermatozoa from rhesus macaques were obtained by penile electroejaculation, washed in TALP-HEPES and resuspended in TALP (Bavister et al., 1983) at a final concentration of 20x106 spermatozoa/ml.
Intracytoplasmic sperm injection
Fertilization by ICSI was accomplished by injecting a single spermatozoon into a mature rhesus oocyte. A holding pipette with external and internal diameters of 100 and 20 µm, respectively, and an injection pipette with an outer diameter of 67 µm and an internal diameter of 45 µm, bevelled at 50°, were used (Hewitson et al., 1996, 1998
, 1999
). Washed spermatozoa were immobilized prior to aspiration by first diluting 1:50 in 10% polyvinylpyrrolidone (PVP; Sigma) followed by scoring across the sperm tail with the injection pipette. A single, immobilized spermatozoa was aspirated tail-first into the pipette and then injected into an oocyte after ensuring oolemma breakage by controlled cytoplasmic aspiration. Injection was carried out in TALP-HEPES, and injected oocytes were returned to culture in TALP (37°C in 5% CO2 under mineral oil). Oocytes were fixed at several time-points following sperm injection.
In some cases rhesus sperm mitochondria were prelabelled with the fluorescent dye MitoTrackerTM (Molecular Probes, Eugene, OR, USA). For this purpose sperm samples were pelleted (700 g, 10 min) and resuspended in KMT medium (100 mmol/l KCl, 2 mmol/l MgCl2, 10 mmol/l Tris-Cl, 5 mmol/l EGTA, pH 8.2). Spermatozoa were then incubated for 10 min at 37°C with MitotrackerTM (400 nmol/l), washed twice by centrifugation/resuspension in TALP-HEPES and finally resuspended in this medium prior to injection (Sutovsky et al., 1996b, 1999). Mitotracker was stored as a stock 5 µmol/l solution in dimethyl sulphoxide.
In-vitro fertilization
For control IVF purposes (Wu et al., 1996), rhesus spermatozoa were obtained as described above and cultured at 37°C for 12 h in TALP containing caffeine and dibutyryl-cAMP (1 mmol/l each) to ensure capacitation prior to use. After confirming adequate hyperactivation, IVF was carried out by incubating no more than 10 mature rhesus oocytes (obtained as described above) in 100 µl TALP drops with capacitated spermatozoa at a final concentration of 20x104 spermatozoa/ml (37°C in 5% CO2 under mineral oil). Inseminated oocytes were removed from culture at several time-points following insemination, washed in TALP-HEPES (by pipetting the oocytes up and down for 25 min) to remove unbound spermatozoa, and fixed as described below.
Immunocytochemistry
The zona pellucida of fertilized oocytes was removed with a short (15 min) incubation at 37°C in TL-HEPES (TALP-HEPES without pyruvate and albumin, pH 7.4) containing pronase (1 mg/ml). The oocytes were then carefully placed with a pipette on poly-L-lysine-coated coverslips containing a drop of calcium/magnesium-free TL-HEPES (TL-HEPES without CaCl2 and MgCl2, and with 5 µmol/l EGTA, pH 7.4) and allowed to attach for 510 min at room temperature.
For microtubule visualization (Simerly and Schatten, 1993) the coverslips were placed in cold methanol and fixed for 20 min (-20°C) and subsequently rehydrated in phosphate-buffered saline (PBS, pH 7.2) containing 0.1% of Triton X-100. Rehydration was carried out by carefully removing methanol (without ever exposing the coverslips to air), and by adding PBS with Triton. This operation was repeated at least five times, to ensure that the methanol had been replaced by the hydrophilic solution. The use of methanol as a fixative also ensures permeabilization of the oocyte plasma membrane, since the reagent partially extracts membrane lipids (Simerly and Schatten, 1993
). For labelling purposes the samples were incubated with PBS containing both the E7 anti-beta tubulin antibody (dilution 1:5) and the anti-perinuclear theca antibody (dilution 1:200) for 1 h at 37°C, and then washed twice in PBS with 0.1% Triton X-100. For secondary antibody labelling the coverslips were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (for perinuclear theca) and tetramethylrhodamine-beta-isothiocyanate (TRITC)-conjugated goat anti-mouse antibody (for E7), both from Zymed (San Francisco, CA, USA) and diluted 1:40 in PBS. Following a 40 min incubation (37°C) the samples were again washed twice in PBS with 0.1% of Triton X-100, and further incubated with the DNA stain DAPI (2 µg/ml; Molecular Probes) for 510 min at 37°C. Finally, and after an aditional washing in PBS with 0.1% of Triton X-100 to remove excess DAPI, the coverslips were carefully inverted, mounted on a slide containing a drop of VectaShield mounting medium (Vector Labs, Burlingame, CA, USA), and sealed with nail polish.
For all other procedures (VAMP, NPC/perinuclear theca) poly-L-lysine-coated coverslips with attached zygotes were placed in PBS containing 2% formaldehyde and fixed for 1 h at room temperature. Unlike methanol fixation this procedure usually preserves membrane integrity. Therefore, following fixation in formaldehyde, the samples were permeabilized by a 3060 min incubation in PBS containing 1% Triton X-100 at room temperature, and non-specific reactions were blocked by further incubation in PBS containing 2 mg/ml bovine serum albumin and 400 mmol/l glycine. For labelling, the antibodies were solubilized in this blocking solution and incubated on the coverslips at room temperature for 12 h at the appropriate dilutions (for perinuclear theca 1:200; for VAMP 1:100; for nuclear pore complexes 1:250). After two washes in PBS containing 0.1% Triton X-100, the coverslips were sequentially incubated with TRITC- or FITC-conjugated secondary antibodies (40 min at room temperature), and with DAPI (510 min at room temperature), and mounted in VectaShield as described in detail above.
Samples were examined with a Zeiss Axiophot or a Nikon Eclipse E1000 epifluorescence-equipped microscope and images were aquired using a RTE/CCD 1217 camera (Princeton Instruments Inc., Trenton, NJ, USA) or a Hamamatsu C-4742 Digital Camera (Hamamatsu Photonics K.K., Hamamatsu, Japan), both operated with Metamorph software. For VAMP imaging, samples were examined with a Leica TCS NT confocal microscope. Images were processed using Adobe Photoshop 5.0 software (Adobe Systems Inc., Mountain View, CA, USA) and printed on a Sony UP-D 8800 dye sublimation printer.
Detection of DNA synthesis using BrdU
Following fertilization by either ICSI of IVF, rhesus zygotes were incubated with 100 mmol/l of bromodeoxyuridine (BrdU; Boehringer Manheim, Germany) in TALP for 16 h. After zona removal the oocytes were attached to poly-L-lysine-coated coverslips (see previous section), fixed in 70% ethanol for 20 min at 20°C and rehydrated in PBS containing 0.1% Triton X-100 (see previous section). To visualize incorporated BrdU, coverslips were incubated in PBS containing mouse anti-BrdU monoclonal antibody (dilution 1:20) for 40 min at room temperature. Washes in PBS, incubation with the FITC-conjugated anti-mouse secondary antibody (dilution 1:40), staining with DAPI, mounting of coverslips and examination of samples by epifluorescence microscopy were carried out as described above.
Electron microscopy
For electron microscopy analysis, oocytes were fixed for 6 h after ICSI or IVF and embedded in Epon 812 as described previously (Sutovsky et al., 1996a). Briefly, oocytes were fixed for 90 min at room temperature with 2.5% glutaraldehyde and 0.6% paraformaldehyde in PBS (pH 7.3), washed in 0.1 mol/l PBS containing 0.15 mol/l sucrose, postfixed for 1 h in 1% osmium tetroxide, dehydrated by an ascending concentration (30100%) series of ethanol, perfused with a mixture of propylene oxide and PolyBed 812 (both from Polyscience, Warrington, PA, USA), and embedded in PolyBed 812. Ultrathin sections were cut on a Sorvall MT-5000 ultramicrotome (Ivan Sorvall, Inc., Norwalk, CT, USA), placed on copper grids, sequentially stained by uranyl acetate and lead citrate, and photographed on a Philips EM 300 electron microscope. Negatives were scanned with a Umax Powerlook 3000 flat bed scanner (Umax Technologies Inc., Freemont, CA, USA) and printed on a Sony UP-D 8800 dye sublimation printer using Adobe Photoshop 4.0 software.
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Results |
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Fertilization by IVF and ICSI was carried out in mature oocytes, and the fertilization rate (judged by second polar body extrusion and/or pronuclear formation) was 81% for ICSI and 75% for IVF. The results discussed here were obtained from material fixed before the first cleavage. When embryos produced by IVF or ICSI during these sessions were allowed to develop for other purposes (Hewitson et al., 1999; Chan et al., 2000
), the cleavage rate was 95% in both cases.
DNA decondensation, microtubule organization and processing of the perinuclear theca and acrosome after ICSI
Sperm DNA decondensation occurred asynchronously after ICSI, with the apical (i.e. acrosomal) portion of the sperm head remaining condensed, when compared with the basal area. The condensed (apical) male DNA was covered by a perinuclear theca `cap' (Figure 1A-C).
Centrosome-derived sperm aster formation (Figure 1B-E, asterisks), microtubule organization and pronuclear apposition after ICSI did not require complete decondensation of the sperm nucleus (Figure 1B, C
). The sperm perinuclear theca were still seen on the apex of the sperm head after pronuclear apposition 12 h after ICSI (Figure 1C
), at a time when complete male DNA decondensation had occurred in IVF embryos (Figure 1F
). Remarkably, this perinuclear accessory structure seemed to be shed virtually intact into the zygote cytoplasm 2024 h after ICSI (Figure 1D, E
), sometimes with a clearly visible equatorial segment (Figure 1D
, arrowhead).
To illustrate this peculiar male DNA decondensation at the ultrastructural level, the fate of the perinuclear theca after ICSI was also followed by transmission electron microscopy (Figures 2 and 3). As noted above, in both cases the persistence of male-derived structures on the decondensing sperm head did not affect the organization and function of the zygotic centrosome (Figures 2B and 3A
). Spermatozoa that failed to decondense and form the male pronucleus after ICSI had an intact acrosome, perinuclear theca, nuclear envelope, equatorial segment, and post-acrosomal sheath (Figure 2A
). In the spermatozoa that underwent the process of nuclear decondensation 6 h after ICSI, the postacrosomal sheath was absent from the injected sperm nucleus (Figure 2B
), while the subacrosomal perinuclear theca and acrosome were found almost intact in serial sections (Figure 2B-E
, arrowheads). As expected, sperm nuclear decondensation was partial and relatively homogeneous, except for small patches of heterochromatin found at the base of the perinuclear theca cap (Figure 2C
, arrows), and more prominently in its apical portion (Figure 2E
, arrows). A normal sperm aster and zygotic centrosome were found in such oocytes (see Figure 3 D, E
). Clusters of maternal mitochondria were found adjacent to the sperm mitochondrial sheath (Figure 2F
).
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Persistence of acrosomal VAMP on the sperm head following ICSI
During IVF the v-SNARE VAMP/synaptobrevin, although initially present on spermatozoa at the oocyte surface, was lost from the sperm head prior to oocyte penetration and could not be found associated with the male pronucleus (data not shown, Figure 4I). In contrast, VAMP was detectable on intact sperm heads a short time after sperm injection, with the same pattern that can be found in isolated spermatozoa (Figure 4A
). As the sperm nucleus asynchronously decondensed, a two-piece VAMP `collar', probably derived from the equatorial segment, clearly separated the condensed apical DNA from the decondensed posterior portion (Figure 4B-D
). IVF embryos fixed at a comparable time-point lacked this `collar', and the male pronucleus was fully formed in this case (Figure 4I
). Although DNA decondensation seemed to be retarded in the apical portion of the sperm head, complete pronuclear formation eventually occurred, and, reminiscent of what takes place for the perinuclear theca, the VAMP `collar' was shed and discarded in the vicinity of the decondensed male-derived DNA (Figure 4E, F
). VAMP remnants were no longer found at first mitosis (Figure 4G
) and were not associated with the female pronucleus (Figure 4H
) or metaphase spindles of unfertilized oocytes (not shown).
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Synthesis of DNA after ICSI (Figure 6B), as evaluated by BrdU incorporation into pronuclei, only took place if the spermaozoon had completely decondensed, and was therefore delayed when compared to traditional IVF (Figure 6A
). Although a fully formed female pronucleus was present in ICSI oocytes before complete sperm decondensation, DNA synthesis seemed to take place synchronously in both pronuclei.
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Discussion |
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We have attempted to dissect the peculiar choreography of fertilization by ICSI at the subcellular level. For this purpose we followed two distinct, but interconnected, sets of phenomena: the removal of sperm head accessory structures normally discarded during the initial stages of sperm-oocyte interactions, and the nuclear remodelling that takes place following ICSI.
Sperm head components introduced in the oocyte by ICSI include the acrosome and an intact perinuclear theca (Hewitson et al., 1996, 1999
; Sutovsky et al., 1996a
, 1997
; Sathananthan et al., 1997
; Bourgain et al., 1998
; Kupker et al., 1998
; Yanagimachi, 1998
). Using specific markers we have found that complete disassembly of the acrosome and perinuclear theca is apparently unrelated to pronuclear apposition and microtubule organization in the zygote. An acrosomal/perinuclear theca cap is present on the sperm head following ICSI, and persists on the apical portion of the asynchronously decondensing male-derived DNA. This could help explain why this area remains condensed in ICSI embryos at a time when pronuclear formation has been completed in oocytes fertilized by IVF (Figures 1, 3, 4 and 6
). However, ICSI embryos seemed to `catch up' by shedding these structures virtually intact into the oocyte cytoplasm (Figures 1 and 4
), where they were probably degraded by the oocyte's proteolytic machinery. Strikingly, a VAMP `collar' seems to separate decondensing DNA at the posterior end of the sperm head from the condensed anterior portion. This `collar' probably represents the sperm equatorial segment, a double leaf of perinuclear theca and acrosomal membranes that is not released from the sperm head following the acrosome reaction, and that is thought to mediate sperm-oolema binding and/or fusion (Wassarman, 1999
).
After traditional fertilization, nuclear pore complexes are normally detected on both male and female pronuclear envelopes, as well as in specialized storage organelles in the oocyte cytoplasm, the annulate lamellae (Sutovsky et al., 1998). Our data suggest that NPC recruitment may also occur asynchronously after ICSI. Indeed, recruitment took place at the forming female pronucleus, but not at the condensed male DNA (Figure 5B
). However, ICSI and IVF embryos were virtually indistinguishable 20 h after fertilization (Figure 5E-G
).
More importantly, complete decondensation of paternal chromatin was required for DNA replication, and thus for the successful completion of the first embryonic cell cycle (Figure 6). At a time when IVF embryos had already begun DNA replication, in ICSI embryos showing asynchronously decondensed male chromatin this process had not yet been initiated. It is possible that sperm-derived structures still present on the paternal DNA could be responsible for this delay in nuclear remodelling. This can also be noted by the selective import of the nuclear matrix protein NuMA, which can be found only in the posterior (decondensing) region of the sperm head, but is excluded from the apical (condensed) region (Hewitson et al., 1999
).
The fact that fertilization by ICSI can be arrested at intermediate stages of male pronuclear formation (Hewitson et al., 1996; Sutovsky et al., 1996a
) suggests that ICSI failures are not solely related to failures in oocyte activation (Sousa and Tesarik, 1994
), and some concerns regarding ICSI stem from the possible hazardous effect of this non-traditional processing of embryonic DNA. Transmission of congenital male chromosomal abnormalities by ICSI has been the focus of many recent studies (e.g. Bofinger et al., 1999; Kamischke et al., 1999; Page et al., 1999). However, while the transmission of such abnormalities by ICSI are to be expected (Bonduelle et al., 1999
), more worrisome is the slight possibility that `de novo' problems may arise, due to technical aspects of the procedure itself. Asynchronous decondensation of the sperm nucleus and delayed DNA replication, although overcome, could be related to the reported higher rate of sex chromosome disorders in embryos and fetuses conceived by ICSI (In't Veld et al., 1995; Bonduelle et al., 1998a, 1999). The authors of one of these studies ruled out maternal age as a possible cause for this higher incidence of sex chromosome abnormalities, and proposed that it might merely reflect increased abnormalities in the spermatozoa of infertile men (Bonduelle et al., 1999
). On the other hand, no greater incidence of abnormalities following ICSI has been noted in other reports (Palermo et al., 1996
; Loft et al., 1999
). In developmental studies it has also been suggested that children conceived by ICSI have a risk of mild delays in development compared with children conceived by parents of comparable age and background using IVF or natural conception (Bowen et al., 1998
). However, another study failed to detect any such delay, although in this case children conceived by ICSI were compared with the general population (Bonduelle et al., 1998b
). Clearly these studies underscore the need for more comprehensive clinical data in order to resolve the debate. Regardless, it is of interest to note that the X chromosome shows a preference for the apical portion of the sperm head (Luetjens et al., 1999
), precisely the region with delayed decondensation after ICSI, which could possibly result in inappropriate chromosomal duplication during first interphase and/or improper positioning at first mitosis. It has also been recently shown that inhibition (and possibly delays) in DNA replication during early embryogenesis may result in disturbances in gene expression (Memili and First, 1999
).
Removal of sperm accessory structures prior to ICSI might be considered in order to reduce the possible risks of this widely employed assisted reproductive techniques. Some studies have shown that induction of the acrosome reaction (Lee et al., 1997), or incubation of spermatozoa with detergents (Ahmadi and Ng, 1999
; Kasai et al., 1999
) prior to ICSI increases the rate of sperm nuclear decondensation, although this may not be extendable to the clinical setting (Liu et al., 1994
). However, it is not known whether, for example, such treatments can affect vital sperm structures, such as the centriole.
In summary, these results show several differences during the elaborate choreography that takes place following successful ICSI, when compared with traditional IVF. Although ultimately overcome, these differences may result in delays in nuclear remodelling and DNA replication, and may be correlated to the peculiar handling of male-derived structures that are not present on spermatozoa following IVF, but are introduced by ICSI.
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Acknowledgments |
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Notes |
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7 To whom correspondence should be addressed at: Oregon Regional Primate Research Center, Oregon Health Sciences University, 505 NW 185th Avenue, Beaverton, OR 97006, USA. E-mail: schatten{at}ohsu.edu
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References |
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Submitted on June 12, 2000; accepted on September 21, 2000.