ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications

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1 Human Reproduction vol.15 no.12 pp , 2000 ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications João Ramalho-Santos 1,5,6, Peter Sutovsky 1, Calvin Simerly 1, Richard Oko 3, Gary M.Wessel 4, Laura Hewitson 1 and Gerald Schatten 1,2,7 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 6 Current address: Center for Neuroscience and Cell Biology of Coimbra, Department of Zoology, University of Coimbra, 3000 Coimbra, Portugal 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. schatten@ohsu.edu We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mullata). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle. Key words: acrosome/fertilization/icsi/perinuclear theca/ spermatozoa Introduction Intracytoplasmic sperm injection (ICSI) is an assisted reproduction technique used to overcome various types of male factor infertility. ICSI involves the direct injection of a single spermatozoon into a metaphase II arrested oocyte, thus bypassing many of the steps normally present in gamete interactions, both during in-vivo and in-vitro fertilization. These include the sperm acrosome reaction, sperm-zona binding and penetration, and sperm-oolemma binding and fusion (Hewitson et al., 2000; Sutovsky and Schatten, 2000). 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 2610 European Society of Human Reproduction and Embryology

2 Sperm structures and nuclear remodelling after ICSI 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. Materials and methods Antibodies The antibody against VAMP was prepared as described previously (Conner et al., 1997). This probe cross-reacts with VAMP isoforms (VAMP1 and VAMP2) involved in secretory granule exocytosis, and that are present in mammalian acrosomes (Ramalho-Santos et al., 2000). pab 427 antibody against a set of perinuclear theca proteins was obtained as described (Oko and Maravei, 1994). Nuclear pore complexes (NPC) were detected using the MAb414 antibody (BabCo, Berkely, CA, USA; Sutovsky et al., 1998). Microtubules were detected using the E7 antibody against beta-tubulin (Developmental Studies Hybridoma Bank, University of Iowa, IA, USA). 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 CaCl 2, 0.5 mmol/l MgCl 2, 25 mmol/l NaHCO 3, 0.4 mmol/l NaH 2 PO 4, 10 mmol/l sodium lactate, 6.5 IU penicillin, 25 µg/ml gentamicin, 6 mg/ml fatty acidfree 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 1 3 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 in5%co 2 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 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 6 7 µm and an internal diameter of 4 5 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 in5%co 2 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 MitoTracker TM (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 MgCl 2, 10 mmol/l Tris-Cl, 5 mmol/l EGTA, ph 8.2). Spermatozoa were then incubated for 10 min at 37 C with Mitotracker TM (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 1 2 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 spermatozoa/ml (37 Cin5%CO 2 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 2 5 min) to remove unbound spermatozoa, and fixed as described below. Immunocytochemistry The zona pellucida of fertilized oocytes was removed with a short (1 5 min) incubation at 37 C in TL-HEPES (TALP-HEPES without 2611

3 J.Ramalho-Santos et al. pyruvate and albumin, ph 7.4) containing pronase (1 mg/ml). The oocytes were then carefully placed with a pipette on poly-l-lysinecoated coverslips containing a drop of calcium/magnesium-free TL- HEPES (TL-HEPES without CaCl 2 and MgCl 2, and with 5 µmol/l EGTA, ph 7.4) and allowed to attach for 5 10 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 5 10 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-llysine-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 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 1 2 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 (5 10 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 (30 100%) 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. Results IVF and ICSI For this study, oocyte collections were done on four separate occasions. When directly compared, IVF and ICSI were done on sibling oocytes, using the same sperm sample. The results presented below were obtained from a total of 118 successfully fertilized oocytes (85 fertilized by ICSI, 33 IVF controls, see figure legends for details). Oocytes showing obvious abnormalities (lysis, DNA fragmentation, lack of oocyte activation, etc.) were not taken into account, except when noted (Figure 1A, 2A and 5A), but were 8% of the total number. Abnormal fertilization patterns after ICSI in this system have been described previously (Hewitson et al., 1996; Sutovsky et al., 1996a). However, in this study no such pattern was detected on a regular basis (i.e. more than once in any given experiment). Upon collection, 46% of the retrieved oocytes were mature (46% with one polar body; 52% GVBD; 2% GV). Following a 6 h incubation in TALP the maturation rate was 90%. 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 2612

4 Sperm structures and nuclear remodelling after ICSI Figure 1. Microtubule organization and persistence of perinuclear theca after intracytoplasmic sperm injection (ICSI). (A) Injected oocyte that failed to activate. Microtubules (MT; red) can only be detected in the female meiotic spindle, while the sperm head (arrow) maintains its typical perinuclear theca label (PT; green). DNA was labelled with DAPI (blue). For clarity the digital, non-colour encoded DNA images were also included as an insert in B-E. (B) Oocyte 30 min after injection (four oocytes examined). Female meiosis is almost complete and the sperm DNA is still mostly condensed. In this image the basal portion of the spermatozoon, where the sperm aster is forming (asterisk), is turned towards the viewer, the apical portion can be visualized by the PT label (arrow). (C) Although paternal DNA (M) remains partially condensed 12 h after ICSI (insert, arrow), the centrosome (asterisk) has promoted microtubule organization and pronuclear apposition. The female pronucleus in this image (F) is underneath the male (eight oocytes examined). (D, E) Sperm decondensation and centrosomeorchestrated (asterisk) pronuclear apposition have been completed (20 24 h after ICSI, eight oocytes examined), the perinuclear theca has been shed almost intact into the cytoplasm, and the sperm equatorial segment is clearly visible (arrowhead). (F) In contrast, 12 h following IVF (five oocytes examined) both pronuclei have completely decondensed and no traces of the perinuclear theca are found in the cytoplasm. Bar 10 µm. 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 2613

5 J.Ramalho-Santos et al. Figure 2. Perinuclear theca disassembly and sperm nucleus decondensation after intracytoplasmic sperm injection (ICSI). Oocytes fixed for transmission electron microscopy were observed 6 h after ICSI (five oocytes examined). (A) Rhesus spermatozoon that failed to form a male proncleus after ICSI displays an intact equatorial segment/post-acrosomal sheath complex (ps) and sub-acrosomal perinuclear theca/ acrosomal layer (spt). Asterisk denotes the position of the proximal centriole under the observed striated columns. Oocyte mitochondria (arrows) are adjacent to the sperm head and sperm mitochondrial sheath (ms). (B) Inside the ICSI-fertilized oocyte the post-acrosomal sheath has been removed from the partially decondensed sperm nucleus/male pronucleus (m). (C-E) Sections showing an almost intact acrosome and perinuclear theca (arrowheads); small patches of heterochromatin (arrows) are found at the base of the acrosome/perinuclear theca cap (C), and more prominently in its apical portion (E). (F) Clusters of maternal mitochondria (arrows) adjacent to the sperm mitochondrial sheath (arrowheads). Bars 1 µm. 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 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 2614 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). In other cases (Figure 3), the decondensation of apical sperm chromatin in the acrosome and perinuclear theca-covered part of the nascent male pronucleus was less even, resulting in the persistence of long cords of heterochromatin in otherwise homogeneous sperm chromatin (Figure 3A, B). In this case, part of the post-acrosomal sheath was also found loosely associated with the sperm nucleus/male pronucleus (Figure 3C). This perinuclear theca segment sheltered a distinct clump of chromatin with the appearance of a fully individualized

6 Sperm structures and nuclear remodelling after ICSI Figure 3. Abnormal male chromatin patterns during sperm disassembly 6 h after intracytoplasmic sperm injection (ICSI). (A-G) Uneven sperm chromatin decondensation 6 h after ICSI (five oocytes examined). (A) Male chromatin (m) remains partially condensed, and a clump of male chromatin/paternal karyomere appears slightly detached (asterisk). At higher magnification (C) this clump (asterisk) is shown to be sheltered by the sperm post-acrosomal sheath (arrowheads in C), which is apparently being separated from the sperm nucleus at this stage. Note the intact acrosomal vesicle and perinuclear theca (arrowheads in B and F) and the presence of large heterochromatin cords (arrows in B) in the apical part of the male chromatin. Serial sectioning revealed that, at this stage, there is already disassembly of the sperm tail connecting piece (D, E) with microtubule polymerization (E, arrowheads) from the sperm centriole (E, asterisk). Note that the centriole is still partially embedded in the remnants of sperm tail connecting piece component called capitulum. Maternal, oocyte-derived mitochondria are found adjacent to the sperm nucleus (arrows in F) and to the membranes of the sperm mitochondrial capsule (arrows in G). (H) Low magnification electron micrograph of the male pronucleus (m) in an oocyte generated by conventional IVF (five oocytes examined) that contains no remnants of the sperm perinuclear theca. Arrow delineates the position of the sperm tail on this cross-section. Bars 1 µm. chromosome. A nuclear envelope was not yet found at this early stage of pronuclear development, whereas the functional zygotic centrosome, with a microtubule sperm aster, was formed around the sperm-derived centriole (Figure 3D, E), suggesting proper disassembly of the sperm tail connecting piece. Maternal, oocyte-derived mitochondria were found adjacent to the sperm nucleus (Figure 3F), and, similar to Figure 2, to the membranes of the sperm mitochondrial capsule 2615

7 J.Ramalho-Santos et al. (Figure 3G). Such atypical features were never seen in male pronuclei from oocytes generated by conventional IVF (Figure 3H, see also Sutovsky et al., 1996a). 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). Nuclear remodelling and DNA synthesis after ICSI To further monitor pronuclear remodelling and development following ICSI, a combined study of perinuclear theca disassembly and nuclear pore complex (NPC) assembly was carried out (Figure 5). At 2 h after ICSI, a spermatozoon with an intact perinuclear theca was observed in an inactivated oocyte, as judged by the presence of metaphase-arranged female chromosomes, with no obvious assembly of NPC on either male or female chromatin (Figure 5A, see also Figure 1A). The swelling and diffusion of subacrosomal perinuclear thecae (Figure 5B, D) were observed in the oocytes with activated female chromatin. Association of NPC with female chromosomes was seen in most of these oocytes (Figure 5C). Another set of fertilized oocytes was examined 20 h after ICSI and most oocytes successfully completed both male and female pronuclear development. At this stage a great amount of NPC seemed to be actively transported to the apposed pronuclei, while the remnants of the perinuclear theca were often seen in association with one of them (Figure 5E, arrow). Control IVF oocytes at corresponding time-points (20 h post-insemination) also contained two full-size, apposed pronuclei (Figure 5F, G), and (in one case) some remnants of the perinuclear theca in the cytoplasm (Figure 5G, arrow). 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 2616 ICSI oocytes before complete sperm decondensation, DNA synthesis seemed to take place synchronously in both pronuclei. Discussion Since its clinical introduction, ICSI has challenged a number of established reproductive paradigms. Considering the fact that it bypasses many steps normally completed during traditional fertilization, the success of ICSI provokes questions as to what is necessary and/or sufficient for productive gamete interactions. Additionally, the recent discovery that ICSI can potentiate sperm-mediated delivery of exogenous DNA, and thus be successfully used as a technique to produce transgenic animals (Perry et al., 1999; Chan et al., 2000), raises concerns that it may also promote the entry into the oocyte of unwanted DNA of parasitic or infectious origin. 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

8 Sperm structures and nuclear remodelling after ICSI Figure 4. Confocal imaging of the persistence of vesicular associated membrane protein (VAMP) on the sperm head following intracytoplasmic sperm injection (ICSI). (A) Sperm with a typical intact sperm VAMP (red) pattern (30 min post-injection, four oocytes examined). (B-D) DNA decondensation at the anterior portion of the sperm head seems to be prevented or retarded by a VAMP two-piece collar (8 16 h post injection, 12 oocytes examined). Eventually, the male DNA decondenses completely, forming a pronucleus with VAMP remnants in the vicinity (E, F, 24 h post-injection, eight oocytes examined). VAMP became undetectable by first mitosis (G, first mitotic anaphase, two oocytes examined), and is not observed associated with female pronuclei, the polar body (H, PB polar body), or with the male pronucleus following IVF (I, 16 h post insemination, eight oocytes examined). Arrows denote the position of the sperm tail as observed by phase-contrast microscopy. Nucleoli are detected as round dark regions within the labelled male chromatin (DAPI: blue). Bars represent 10 µm. 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 2617

9 J.Ramalho-Santos et al. Figure 5. Assembly of nuclear pore complexes following intracytoplasmic sperm injection (ICSI). Assembly of nuclear pore complexes (NPC; green) on the nascent pronuclei was examined, concomitantly with the removal of the perinuclear theca (PT; red). DNA was labelled using DAPI (blue). (A) Two hours after ICSI (seven oocytes examined). The spermatozoon shows an intact perinuclear theca and the oocyte has not been activated. (B) In activated oocytes, swelling and diffusion of the perinuclear theca was seen. In most oocytes, NPC were already being recruited to the female chromatin (C and D are magnified details of B, showing the female and male chromatin, respectively). (E) Twenty hours after ICSI (seven oocytes examined) Successful completion of both male and female pronuclear development, with remnants of the perinuclear theca (arrow) still associated with one of the pronuclei. (F, G) Control IVF oocytes at 20 h post-insemination (five oocytes examined), containing two full size, apposed pronuclei and, in some cases, remnants of perinuclear theca in the cytoplasm (G, arrow). Bars 10 µm. Bar in A refers to A and B. Bar in G refers to E G. Figure 6. DNA replication in male and female pronuclei after IVF and intracytoplasmic sperm injection (ICSI). (A) Following traditional IVF, bromodeoxyuridine (BrdU; green) is visible in both decondensed pronuclei (DNA; blue, insert) 16 h post-insemination (10 oocytes examined). (B) Although the female pronucleus is completely formed, BrdU has not been incorporated into either pronucleus 16 h after ICSI (18 oocytes examined); the apical region of the sperm head remains condensed (insert, arrow). Bar 10 µm. Figure 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

10 Sperm structures and nuclear remodelling after ICSI 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. Acknowledgements Dr Ramalho-Santos and Dr Sutovsky are joint co-authors, as they contributed equally to this manuscript. The technical assistance of Diana Takahashi, Crista Martinovich, Bryan McVay, Michael Webb and Dr Anda Cornea (ORPRC) and the editorial and clerical assistance of Nancy Duncan, Hollie Wilson and Michelle Emme are gratefully acknowledged. This work was supported by research grants from the NIH to G.S. (NICHD, NCRR) and R.O. (R-21), from the Mellon Foundation (L.H.), and from MRC of Canada (R.O.). The ORPRC is sponsored as an NCRR Regional Primate Research Center. J.R.-S. is the recipient of a Praxis XXI postdoctoral fellowship from Fundação para a Ciência e Tecnologia (FCT, Portugal), and wishes to thank M.I.Ramalho, B.S.Santos, J.Saramago, A.L.Antunes, P.Auster, B.Katchor, B.Chatwin, L.Ferlinghetti, G.G.Márquez and J.L.Borges for continuous encouragement and inspiration. References Ahmadi, A. and Ng, S.C. (1999) Influence of sperm plasma membrane destruction on human sperm head decondensation and pronuclear formation. Arch. Androl., 42, 1 7. Bavister, B.D., Boatman, D.E., Leibfried, L. et al. (1983) Fertilization and cleavage of rhesus monkey oocytes in vitro. Biol. Reprod., 28, Bofinger, M.K., Needham, D.F., Saldana, L.R. et al. (1999) 45,X/46,X,r(Y) karyotype transmitted by father to son after intracytoplasmic sperm injection for oligospermia. A case report. J. Reprod. Med., 44, Bonduelle, M., Aytoz, A., Van Assche, E. et al. (1998a) Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum. Reprod., 13, Bonduelle, M., Joris, H., Hofmans, K. et al. (1998b) Mental development of 201 ICSI children at 2 years of age. Lancet, 351, Bonduelle, M., Camus, M., De Vos, A. et al. (1999) Seven years of intracytoplasmic sperm injection and follow-up of 1987 subsequent children. Hum. Reprod., 14 (Suppl. 1), Bourgain, C., Nagy, Z.P., De Zutter, H. et al. (1998) Ultrastructure of gametes after intracytoplasmic sperm injection. Hum. Reprod., 13 (Suppl. 1), Bowen, J.R., Gibson, F.L., Leslie, G.I. et al. (1998) Medical and developmental outcome at 1 year for children conceived by intracytoplasmic sperm injection. Lancet, 351, Breitbart, H. and Spungin, B. (1997) The biochemistry of the acrosome reaction. Mol. Hum. Reprod., 3, Chan, A.W., Luetjens, C.M., Dominko, T. et al. (2000) Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live Rhesus monkey births. Mol. Hum. Reprod., 6, Conner, S., Leaf, D. and Wessel, G. (1997) Members of the SNARE hypothesis are associated with cortical granule exocytosis in the sea urchin egg. Mol. Reprod. Dev., 48, Gotte, M. and von Mollard, G.F. (1998) A new beat for the SNARE drum. Trends Cell Biol., 8, Hanson, P.I., Heuser, J.E. and Jahn, R. (1997) Neurotransmitter release ø four years of SNARE complexes. Curr. Opin. Neurobiol., 7, Hewitson, L.C., Simerly, C.R., Tengowski, M.W. et al. (1996) Microtubule and chromatin configurations during rhesus intracytoplasmic sperm injection: successes and failures. Biol. Reprod., 55, Hewitson, L., Takahashi, D., Dominko, T. et al. (1998) Fertilization and embryo development to blastocysts after intracytoplasmic sperm injection in the rhesus monkey. Hum. Reprod., 13, Hewitson, L., Dominko, T., Takahashi, D. et al. (1999) Unique checkpoints during the first cell cycle of fertilization after intracytoplasmic sperm injection in rhesus monkeys. Nat. Med., 5, Hewitson, L., Simerly, C., Dominko, T. et al. (2000) Cellular and molecular events after in vitro fertilization and intracytoplasmic sperm injection. Theriogenology, 53, In t Veld, P., Brandenburg, H., Verhoeff, A. et al. (1995) Sex chromosomal abnormalities and intracytoplasmic sperm injection. Lancet, 346, 773. Kamischke, A., Gromoll, J., Simoni, M. et al. (1999) Transmission of a Y chromosomal deletion involving the deleted in azoospermia (DAZ) and chromodomain (CDY1) genes from father to son through intracytoplasmic sperm injection: case report. Hum. Reprod., 14, Kasai, T., Hoshi, K. and Yanagimachi, R. (1999) Effect of sperm immobilisation and demembranation on the oocyte activation rate in the mouse. Zygote, 7, Kupker, W., Diedrich, K. and Edwards, R.G. (1998) Principles of mammalian fertilization. Hum. Reprod., 13 (Suppl. 1), Lamb, D.J. (1999) Debate: is ICSI a genetic time bomb? Yes. J. Androl., 20, Lee, D.R., Lee, J.E., Yoon, H.S. et al. (1997) Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection. Fertil. Steril., 67, Liu, J., Nagy, Z., Joris, H. et al. (1994) Intracytoplasmic sperm injection does not require special treatment of the spermatozoa. Hum. Reprod., 9, Loft, A., Petersen, K., Erb, K. et al. (1999) A Danish national cohort of 730 infants born after intracytoplasmic sperm injection (ICSI) Hum. Reprod., 14, Luetjens, C.M., Payne, C. and Schatten, G. (1999) Non-random chromosome positioning in human sperm and sex chromosome anomalies following intracytoplasmic sperm injection. Lancet, 353, Manandhar, G., Sutovsky, P., Joshi, H.C. et al. (1998) Centrosome reduction during mouse spermiogenesis. Dev. Biol., 203, Memili, E. and First, N. L. (1999) Control of gene expression at the onset of bovine embryonic development. Biol. Reprod., 61, Oko, R. and Maravei, D. (1994) Protein composition of the perinuclear theca of bull spermatozoa. Biol. Reprod., 50, Page, D.C., Silber, S. and Brown, L.G. (1999) Men with infertility caused by AZFc deletion can produce sons by intracytoplasmic sperm injection, but are likely to transmit the deletion and infertility. Hum. Reprod., 14,

11 J.Ramalho-Santos et al. Palermo, G., Joris, H., Devroey, P. et al. (1992) Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 340, Palermo, G.D., Colombero, L.T., Schattman, G.L. et al. (1996) Evolution of pregnancies and initial follow-up of newborns delivered after intracytoplasmic sperm injection. J. Am. Med. Assoc., 276, Perry, A.C., Wakayama, T., Kishikawa, H. et al. (1999) Mammalian transgenesis by intracytoplasmic sperm injection. Science, 284, Ramalho-Santos, J., Moreno, R.D., Sutovsky, P. et al. (2000) SNAREs in mammalian sperm: possible implications for fertilization. Dev. Biol., 223, Rothman, J.E. (1994) Mechanisms of intracellular protein transport. Nature, 372, Sathananthan, A.H., Szell, A., Ng, S.C. et al. (1997) Is the acrosome reaction a prerequisite for sperm incorporation after intra-cytoplasmic sperm injection (ICSI)? Reprod. Fertil. Dev., 9, Schatten, G. (1994) The centrosome and its mode of inheritance: the reduction of the centrosome during gametogenesis and its restoration during fertilization. Dev. Biol., 165, Schatten, G., Simerly, C. and Schatten, H. (1985) Microtubule configurations during fertilization, mitosis, and early development in the mouse and the requirement for egg microtubule-mediated motility during mammalian fertilization. Proc. Natl. Acad. Sci. USA, 82, Schatten, H., Schatten, G., Mazia, D. et al. (1986) Behavior of centrosomes during fertilization and cell division in mouse oocytes and in sea urchin eggs. Proc. Natl. Acad. Sci. USA, 83, Schlegel, P.N. (1999) Debate: is ICSI a genetic time bomb? No: ICSI is safe and effective. J. Androl., 20, Simerly, C. and Schatten, G. (1993) Techniques for localization of specific molecules in oocytes and embryos. Methods Enzymol., 225, Simerly, C., Wu, G.J., Zoran, S. et al. (1995) The paternal inheritance of the centrosome, the cell s microtubule- organizing center, in humans, and the implications for infertility. Nat. Med., 1, Sousa, M. and Tesarik, J. (1994) Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum. Reprod., 9, Sutovsky, P. and Schatten, G. (2000) Paternal contributions to the mammalian zygote: fertilization after sperm-egg fusion. Int. Rev. Cytol., 195, Sutovsky, P., Hewitson, L., Simerly, C.R. et al. (1996a) Intracytoplasmic sperm injection for Rhesus monkey fertilization results in unusual chromatin, cytoskeletal, and membrane events, but eventually leads to pronuclear development and sperm aster assembly. Hum. Reprod., 11, Sutovsky, P., Navara, C.S. and Schatten, G. (1996b) Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization. Biol. Reprod., 55, Sutovsky, P., Oko, R., Hewitson, L. et al. (1997) The removal of the sperm perinuclear theca and its association with the bovine oocyte surface during fertilization. Dev. Biol., 188, Sutovsky, P., Simerly, C., Hewitson, L. et al. (1998) Assembly of nuclear pore complexes and annulate lamellae promotes normal pronuclear development in fertilized mammalian oocytes. J. Cell Sci., 111, Uehara, T. and Yanagimachi, R. (1976) Microsurgical injection of spermatozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. Biol. Reprod., 15, Van Steirteghem, A.C., Nagy, Z., Joris, H. et al. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, Wassarman, P.M. (1999) Mammalian fertilization: molecular aspects of gamete adhesion, exocytosis, and fusion. Cell, 96, Wu, G.J., Simerly, C., Zoran, S.S. et al. (1996) Microtubule and chromatin dynamics during fertilization and early development in rhesus monkeys, and regulation by intracellular calcium ions. Biol. Reprod., 55, Yanagimachi, R. (1998) Intracytoplasmic sperm injection experiments using the mouse as a model. Hum. Reprod., 13 (Suppl. 1), Received on June 12, 2000; accepted on September 21,