Article Interactions of the meiotic spindle with mitotic chromosomes in GV mouse oocytes

RBMOnline - Vol 13 No 2. 2006 213-221 Reproductive BioMedicine Online; www.rbmonline.com/article/2054 on web 23 May 2006 Article Interactions of the meiotic spindle with mitotic chromosomes in GV mouse oocytes Ching-Chien Chang obtained his BSc (1994) and MSc degrees (1996) at the National Chung-Hsing University in Taiwan. He worked as an embryologist at Lee Women s Hospital in Taiwan from 1998 1999. He joined the porcine cloning project for xenotransplantation at the Animal Technology Institute, Taiwan from 1999 2001. In 2001, he joined the Centre for Regenerative Biology at the University of Connecticut to start his PhD, and his PhD degree was conferred in 2005. At present, he is an embryologist/research fellow at Reproductive Biology Associates, Atlanta, USA. His research interests are oocyte and epigenetic reprogramming. Dr Ching-Chien Chang Ching-Chien Chang 1,5, Zsolt Peter Nagy 2, Roger Abdelmassih 3, Ji-Long Liu 1, Xiangzhong Yang 1, X Cindy Tian 1,4 1 Centre for Regenerative Biology and the Department of Animal Science, University of Connecticut, Storrs, CT, USA; 2 Reproductive Biology Associates, Atlanta, GA, USA; 3 Clínica e Centro de Pesquisa em Reprodução Humana Roger Abdelmassih, São Paulo, Brazil; 4 Advanced Technology Laboratory, 1392 Storrs Road, U 4243, University of Connecticut, Storrs, CT 06269, USA; 5 Current address: Reproductive Biology Associates, Atlanta, GA, USA 4 Correspondence: Fax: +1 860 486 8809; e-mail: xiuchun.tian@uconn.edu Abstract During mitosis, a spindle checkpoint detects chromosome misalignment and halts the cell cycle progression. In meiosis of female germ cells, however, it is debatable whether such a checkpoint is present. This research employed a unique model in the mouse, mitotic chromosomes transferred to meiotic cytoplasts to investigate whether a meiotic oocyte s microtubule apparatus can effectively separate mitotic metaphase chromosomes, and whether a spindle checkpoint exists during its division. The intact germinal vesicle (GV) oocytes, enucleated GV cytoplasts, and enucleated GV cytoplasts at 15 h in-vitro maturation were transferred with a metaphase fibroblast cell. When mitotic chromosomes were transferred into enucleated or intact mouse GV oocytes, the first bipolar meiotic spindles were established and the reconstructed oocytes were able to extrude polar bodies. However, none of the reconstructed oocytes showed complete and accurate alignment of chromosomes, except the enucleated GV cytoplasts reconstructed after maturation. The spindle formation and polar body extrusion suggest that the first meiotic spindle was functional, and the chromosome misalignment did not prevent the onset of anaphase. The data indicate that a spindle checkpoint, providing surveillance of misaligned chromosomes, was overridden or compromised by the incompatibility between somatic chromosomes and meiotic spindles during the first meiotic division. Keywords: meiosis, nuclear transfer, oocyte maturation Introduction Mis-segregation of chromosomes during cell division leads to aneuploidy. It can induce tumours and birth defects when occurring in mitosis and meiosis, respectively. In mitotic cells, a surveillance mechanism, termed the metaphase anaphase checkpoint or spindle checkpoint, detects abnormalities in chromosome alignment and spindle formation, and halts the progression of metaphase to anaphase to prevent these severe consequences (Hardwick, 1998; Shah and Cleveland, 2000; Gorbsky, 2001; Hoyt, 2001; Millband et al., 2002). This checkpoint can effectively detect both minute abnormalities, such as the presence of a single unattached kinetochore as well as massive spindle disruptions (Rieder et al., 1995; Rudner and Murray, 1996; Hoffman et al., 2001). In meiosis of female germ cells, however, conflicting evidence has been reported regarding the existence of such a spindle checkpoint. On the one hand, the presence of a misaligned X chromosome in an XO mouse model did not delay the onset of anaphase at the first meiotic division (LeMaire- Adkins et al., 1997). Therefore, it had been assumed that there is no such chromosome-mediated, metaphase anaphase checkpoint mechanism in mammalian oocytes (Hunt et al., 1995; LeMaire-Adkins et al., 1997; Woods et al., 1999). Furthermore, in humans, aneuploidy is reported to occur in 10 25% of conceptuses (Hassold et al., 1996; Hassold and Hunt, 2001), a high error rate due to female meiosis (Hassold, 1996), suggesting the absence of a spindle checkpoint in oocytes (Hunt et al., 1995; LeMaire-Adkins et al., 1997). 213

214 Female meiosis appears error prone with up to 20% of human oocytes displaying chromosomal abnormalities, while less than 5% of human sperm appear abnormal. Even the presence of an unpaired centromere is not sufficient to arrest male meiosis by a tension-sensitive spindle checkpoint mechanism, implying that misattached chromosomes are normally eliminated by one or more mechanism(s) in spermatogenesis other than a spindle checkpoint (Mee et al., 2003; Ehrmann and Elliott 2005; Tesarik, 2005). This is also supported by studies in Xenopus eggs which suggest that the spindle checkpoint is deficient during meiosis I, and the anaphase-promoting complex/cyclosome (APC/C) seems to be dispensable for the first meiotic anaphase (Peter et al., 2001). On the other hand, evidence for the presence of a spindle checkpoint came from studies in which proteins active in the mitotic spindle checkpoint, i.e. mitotic arrest deficient 1 (Mad), Mad2 and budding uninhibited by benzimidazole 1 (Bub), were shown to also play important roles in meiosis of mouse oocytes (Kallio et al., 2000; Brunet et al., 2003; Wassmann et al., 2003; Zhang et al., 2004, 2005; Homer et al., 2005; Ma et al., 2005), and that the APC/C is required in mammalian female meiosis (Herbert et al., 2003; Terret at al., 2003). Although the mitotic division executes the separation of sister chromatids, the first meiotic division involves the segregation of homologues rather than sister chromatids. The unique chromosome behaviour in the first meiotic division includes: first, chiasma, which maintain the physical connections between two homologues; and second, physical constraints on the centromeres of sister chromatids (Hassold and Hunt, 2001). Therefore, the chromosome configuration in meiosis I is fundamentally divergent from that in mitosis. It is still an open question: whether those mitotic spindle checkpoint proteins, monitoring the separation of sister chromatids, also function the same way as in meiosis. In the present study, a unique model in the mouse was employed. Mitotic chromosomes transferred to meiotic cytoplasts to investigate whether a meiotic oocyte s microtubule apparatus can effectively separate mitotic metaphase chromosomes, and whether a spindle checkpoint exists during its division. This procedure is similar to somatic cell haploidization (Tesarik and Mendoza, 2003; Nagy and Chang, 2005; Takeuchi et al., 2005) in which the oocytes are used as the machinery to reduce the number of chromosomes in somatic cells by half through meiosis. If the meiotic spindle can carry out a division of somatic chromosomes, it would indicate that either there is no spindle checkpoint, while a halt or delay of the cell cycle progression would indicate that the oocytes are able to detect the incompatibility of the somatic chromosomes with the meiotic spindle, thus confirming the absence of spindle checkpoint in oocytes during division. Materials and methods Chemicals and culture media Unless otherwise indicated, all chemicals were purchased from Sigma Chemical Co (St. Louis, MO, USA). All media were prepared fresh and filter-sterilized through a 0.22 -μm filter (Acrodisc; Pall Gelman Laboratory, Ann Arbor, MI, USA). Animals and recovery of germinal vesicle (GV) oocytes The BDF1 (C57BL/6 DBA/2) and CD-1 mice were purchased from Charles River Laboratories (Wilmington, MA, USA). All animal use was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Connecticut, Storrs. Ovaries were obtained from 8 12 week old CD-1 mice 44 48 h after 5 IU of equine chorionic gonadotrophin (ecg) injection. Oocytes at the GV stage were retrieved from each ovary by puncturing the follicles with a sterile 25-gauge needle and releasing the cumulus oocyte complexes (COC). Oocytes with loosely attached cumulus cells and a diameter of more than 75 μm were regarded as fully grown GV oocytes and selected for subsequent use. The oocytes were stripped of cumulus cells by repeated aspiration through a glass pipette, the tip diameter of which was slightly larger than the diameter of an oocyte. The cumulus-denuded GV oocytes were then transferred into human tubal fluid (HTF, Specialty Media, Phillipsburg, NJ, USA) supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT, USA) and 50 μg/ml of 3-isobutyl-1-methylxanthine (IBMX) (Liu et al., 2000), and were cultured for 3 h in 5% CO 2 in air at 37 C. The 3 h exposure to IBMX was included to help oocytes develop a perivitelline space and to prevent GV breakdown during oocyte in-vitro culture. Germinal vesicle stage oocytes that had a visible perivitelline space were selected with the use of an inverted microscope (TE300, Nikon, MVI, Avon, MA, USA), and randomly assigned for either micromanipulation or in-vitro maturation. Somatic cell culture and cell cycle synchronization Ear biopsies of female BDF1 mice were cut into pieces of 1 2 mm 2, and incubated as tissue explants in Dulbecco s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal calf serum (FCS). Fibroblast monolayers, which formed around the tissue explants, were harvested following incubation in phosphatebuffered saline (PBS) containing 0.25% trypsin and 0.75 mm EDTA. For storage, confluent cells were detached, placed in DMEM with 20% FCS and 10% dimethyl sulphoxide, and frozen in liquid nitrogen. To synchronize donor cells at metaphase, cells at passages 5 10 were cultured to approximately 50 70% confluency and treated with 0.125 μg/ml nocodazole in DMEM supplemented with 10% FCS for 3 h at 37 C, which arrests cells at metaphase (Ono et al., 2001a,b). After gentle pipetting, metaphase fibroblast cells, which were floating in the medium, were collected. The synchronized cells were stained with 7.5 μm propidium iodide to confirm their cell cycle stage by examining the presence of condensed chromosomes. In the population of large (25 30 μm) floating cells, 93% (85/92) were at metaphase (Figure 1).

Micromanipulation of GV oocytes Micromanipulations were performed using an inverted microscope (TE300, Nikon, Japan) equipped with two IM- 6 microinjectors with two oil hydraulic micromanipulators (Narishige, East Meadow, NY, USA). The GV oocytes were incubated in a microdroplet of M2 containing cytochalasin B (CCB, 7.5 μg/ml) and IBMX (50 μg/ml) for 30 min at room temperature (25 C), and then a slit was made through the zona pellucida of each oocyte with a sharp needle. To remove the GV nucleus, an enucleation pipette (25 μm) was inserted into the cytoplasm through the slit on the zona pellucida and the GV was aspirated. The synchronized donor cells were screened, and larger cells (25 30 μm), presumably at metaphase, were selected for insertion into the perivitelline space of the enucleated oocytes in M2 medium containing 0.125 μg/ml nocodazole. After the insertion, cell ooplast complexes were washed thoroughly in M2 to remove CCB, IBMX, and nocodazole. For electrofusion, two microelectrodes, 100 μm in diameter, were applied to align the cell ooplast complexes (Chang et al., 2004) in electrofusion medium (0.28 M mannitol, 100 μm CaCl 2, 100 μm MgSO 4, and 0.005% bovine serum albumin, BSA). The cell cytoplast complexes were then subjected to two pulses of 2.0 kv/cm direct current with a BTX 200 Electro Cell Manipulator (BTX Inc., San Diego, CA, USA) for 15 μs. They were washed in M2 and examined for fusion 30 min after the electrical pulses. Experimental groups The experimental design is illustrated in Figure 2. The oocytes were randomly divided into one of four groups, group A: denuded GV oocytes (controls); group B: denuded GV oocytes fused with synchronized somatic cells, group C: denuded oocytes were first enucleated and then fused with synchronized somatic cells. Group D: denuded oocytes were first enucleated and the ooplasts were in-vitro matured for 15 h, which allowed them reach the MII stage. Subsequently, the ooplasts were fused with synchronized somatic cells and then cultured for 4 h. Oocyte in-vitro maturation All oocytes were incubated in in-vitro maturation medium, which is human tubal fluid supplemented with 10% FCS, in 5% CO 2 in air at 37 C. To visualize the metaphase I to anaphase I stage, oocytes in group C were fixed at 6 h post fusion. After 17 h of maturation of all three groups, the percentages of polar body (PB) extrusion were determined for each group. Oocytes with a PB were regarded as metaphase II (MII) stage, and oocytes without a PB were regarded as arrested at MI stage. Immunohistochemistry and laser-scanning confocal microscopy The control and micromanipulated oocytes were fixed at 37 C for at least 30 min in a microtubule stabilizing buffer containing 2% formaldehyde, 0.5% Triton X-100, 1 μm taxol, 10 units/ml aprotinin and 50% deuterium oxide. They were then washed three times in washing buffer (PBS containing 3 mm NaN 3, 0.01% Triton X-100, 0.2% non-fat dried milk, 2% normal goat serum, 0.1 M glycine, and 2% BSA) and left in washing buffer overnight at 4C for blocking and permeabilization (Carabatsos et al., 2000). Oocytes were then stained to visualize microtubules, microfilaments and/ or DNA. Briefly, to stain for microtubules, samples were incubated in mouse anti-α-tubulin antibody (1:200) for 4 h at 37 C or overnight at 4C, then in fluorescein isothiocyanate (FITC)-conjugated goat anti mouse immunoglobulin G (1:200) for 1 h at 37 C. To stain for microfilaments, the oocytes were subsequently incubated with Rhodamine- Phalloidin (Molecular Probes, Eugene, OR, USA) (1:200) for 1 h at 37C. Finally, the oocytes were washed, stained for DNA with 7.5 μm propidium iodide, mounted in PBS containing 50% glycerol, as an anti fading reagent, and 25 mg/ml NaN 3, and examined with a laser-scanning confocal microscope. For gauge of pole-to-pole distance the two extremes of spindle were located and spindle lengths recorded as the distance between the two poles (Sanfins et al., 2003; Roberts et al., 2005) by using the function of image scaling of confocal microscope (TCS SP2 True scanning; Leica Microsystems, Heidelberg, Germany). Statistical analyses The percentages for bipolar spindle formation and complete chromosome alignments (Table 1) were analysed using chisquared tests and pole-to-pole distance (Table 2) was analysed by Tukey s HSD test in the Statistical Analysis System (Gary, NC, USA). Results Extrusion of the first polar body during invitro maturation Following in-vitro maturation for 17 h, 90.1% (128/142) of control oocytes (group A) extruded the first PB, indicating completion of meiosis I. Of the oocytes in group B that fused successfully, 91.8% (56/61) extruded a PB. In group C, 71.6% (48/67) of fused oocytes extruded their PB. Additionally, the time of the first PB extrusion differed among these three groups of oocytes. For control GV oocytes, extrusion of the first PB occurred 9 10 h after their release from IBMX, which is consistent with prior reports. However, group B oocytes extruded their first PB 6 8 h after fusion, which was approximately 2 3 h earlier than controls (group A), while those in group C extruded their first PB 5 7 h after fusion, which was approximately 3 4 h earlier than controls. Spindle establishment and chromosome alignment during in-vitro maturation The establishment of a bipolar spindle and the chromosome alignment within the spindle were observed at the presumed MII stage (those that extruded a PB) and oocytes arrested at MI after 17 h of maturation. Ninety-four control oocytes were examined at the presumed MII stage, and all but one of these (93/94; Table 1) revealed a bipolar spindle (Figure 215

3a) and the majority (95.7%; 90/94; Table 1) displayed complete chromosome alignment (Figure 3a). Control oocytes had chromosomes positioned equidistantly between two poles of a barrel-shaped spindle at both MII (Figure 3a) and MI-arrest stages (Figure 3b). For those control oocytes that were arrested at MI, 13 out of 14 (92.9%) contained a bipolar spindle (Figure 3b and Table 1), and 85.7% (12/14) of these (Figure 3b and Table 1) had all their chromosomes completely aligned along the metaphase plate. It was then determined whether the formation of a bipolar spindle and the complete alignment of chromosomes also occurred at the presumed MII and MI-arrest stages when mitotic chromosomes interacted with GV oocytes (group B), GV ooplasts (group C), or in-vitro-matured GV ooplasts (group D). In these cases, a bipolar spindle was formed at the presumed MII stage in 94.6% (53/56) of un-enucleated GV oocytes (group B; Figure 3c and Table 1), in 64.6% (31/48) of enucleated GV oocytes (group C; Figure 3e and Table 1), and in 57.1% (8/14) of in-vitro-matured GV ooplasts (group D; Figure 4 and Table 1). Despite bipolar spindle formation, none of these oocytes achieved complete chromosome alignment, in either group B (Figure 3c; 0/56; Table 1), or group C (Figure 3e; 0/48; Table 1). In contrast, the complete chromosome alignment could be found in the group D (Figure 4; 6/14; Table 1). For oocytes at the presumed MI-arrest stage, they were capable of assembling a bipolar spindle in both group B (Figure 3d; 5/5, 100%; Table 1) and C (Figure 3f; 10/19, 52.6%; Table 1), while oocytes in neither group B (Figure 3d; 0/5; Table 1), nor C (Figure 3f; 0/19; Table 1) exhibited proper chromosome alignment. Metaphase anaphase transition of meiosis I To determine whether the newly assembled meiotic spindle containing mitotic chromosomes is functional and capable of segregating the mitotic chromosomes at the expected MI to anaphase transition stage, control oocytes were fixed at 9.5 h after release from the IBMX, which corresponds to the metaphase anaphase transition of meiosis I. In the enucleated oocytes in group C, the PB extrusion was expected at approximately 5 7 h post-fusion, consequently, the oocytes were fixed at 6 h post-fusion, the predicted metaphase anaphase transition of meiosis I. This timing was determined from the time of PB extrusion. Among 35 control MI oocytes, there were 31 that accomplished complete and accurate chromosome alignment or were undergoing metaphase anaphase transition. Although the meiosis I metaphase anaphase transition was initiated in reconstructed oocytes, in all instances, the mitotic chromosomes were not aligned at the metaphase plate (0/49, Figure 5a). At the metaphase anaphase transition of meiosis I, which is also the stage when the first PB is formed, however, oocytes were able to co-ordinately segregate chromosomes by microtubule networks and the movement of microfilaments to the cortex. Subsequently, the mitotic chromosomes were separated into two regions by a meiosis I-like asymmetrical cell division (Figure 5b), and one portion of chromosomes were relegated to the PB, while another resided in the cytoplasm (Figure 5c). The anaphase spindle midzone was also observed in reconstructed oocytes (Figure 5c). Pole-to-pole distance of MII and MI-arrest spindles The interpolar distance of a spindle is an appropriate marker for the fidelity of chromosome segregation along a metaphase spindle. Because the mitotic chromosomes did not align at the metaphase plate in oocytes in which somatic cells were inserted (groups B and C), an unbalanced pulling force between sister kinetochores could cause the expansion of spindle pole-topole distance (Goshima et al., 1999). Therefore, the interpolar spindle distance of reconstructed oocytes containing mitotic chromosomes and meiotic spindles was measured (Table 2). At the presumed MI-arrest stage, the spindles of reconstructed oocytes in groups B and C had extended interpolar distances, compared with those of controls (group A). Likewise, at the presumed MII stage, the interpolar distance of spindles in groups B and C were also significantly longer than those of controls (Table 2, P < 0.05). 216 Figure 1. Metaphase synchronization of fibroblast cells. (a) Fibroblast cells cultured to 50 70% confluency before synchronization. (b) After 3 h of 0.125 μg/ml nocodazole treatment, cells that were arrested at mitotic metaphase stage (arrows) became rounded up and became detached from the bottom of the dish. (c) Metaphase fibroblasts 25 30 μm in diameter were selected for nuclear transfer. The inset image in (c) shows a selected fibroblast cell embedded in the perivitelline space of an enucleated germinal vesicle oocyte and the metaphase status was confirmed by staining DNA with 7.5 μm propidium iodide. Scale bars represent 200 μm (a, b) and 50 μm (c and inset of c).

Figure 2. Schematic illustration of treatments in control (group A) and manipulated oocytes (groups B, C, and D). The duration of in-vitro maturation (IVM) was 15 17 h in all groups. For control oocytes (group A), this was from the release of maturation inhibitor to metaphase II (MII) arrest. The germinal vesicle (GV) oocytes in this group were denuded and no micromanipulation was performed. Oocytes in group B were denuded and metaphase somatic cells were transferred into the perivitelline space. Oocytes in group C were denuded, GV removed and metaphase somatic cells transferred. In-vitro maturation of oocytes in groups B and C was 17 h from fusion. For groups A, B, and C, oocytes that extruded a polar body (PB) were regarded as MII stage, while those that did not have a PB were regarded as MI-arrested. In group D, the GV nuclei were removed from GV oocytes firstly, and the enucleated GV cytoplasts were further in-vitro matured for 15 h. Immediately after in-vitro maturation, the ooplasts were fused with synchronized somatic cells, i.e. a metaphase somatic cell was transferred into the GV cytoplast, and advanced by 4 h of in-vitro culture (IVC). NT = nuclear transfer. MII MI arrest Figure 3. Confocal images of individual oocytes from control (group A; a and b) and manipulated oocytes (groups B and C; c f) ) collected after 17 h of in-vitro maturation. Oocytes were immunostained for α-tubulin (green) and chromosomes (red). (a) A control oocyte at metaphase II (MII) stage showing all chromosomes lining up at the metaphase plate (arrow). (b) A control oocyte at MI-arrest (no polar body, PB) showing all chromosomes positioned in the middle between two poles (arrow). (c) At the presumed MII stage (17 h post-fusion), a group B oocyte extruded a PB and had misaligned chromosomes on a bipolar spindle. (d) A group B oocyte arrested at MI stage (17 h post-fusion) showing no PB and some chromosomes aligned at the metaphase plate (arrow), while others did not congress to the middle of the bipolar spindle. (e) After 17 h of in-vitro maturation, a group C oocyte arrested at MII stage had a PB and condensed chromosomes randomly attached to the presumed MII bipolar spindle. (f) After 17 h in-vitro maturation, a group C oocyte arrested at MI stage had no PB and condensed chromosomes randomly positioned within or around the presumed MI spindle. Bar = 30 μm. 217

Table 1. Meiotic spindle formation and mitotic chromosome alignment in metaphase I (MI) and metaphase II (MII) oocytes. Group a Fusion Polar body Presumed cell No. oocytes Bipolar spindle Complete rate (%) extrusion cycle stage b examined formation (%) chromosome alignment (%) A N/A + MII 94 93 (98.9) 90 (95.7) MI-arrest 14 13 (92.9) 12 (85.7) B 61/92 (66.3) + MII 56 53 (94.6) 0 (0) c MI-arrest 5 5 (100) 0 (0) c C 67/111 (60.4) + MII 48 31 (64.6) c 0 (0) c MI-arrest 19 10 (52.6) c 0 (0) c D 14/25 (56.0) N/A MII 14 8 (57.1) c 6 (42.9) c a Group A: denuded GV oocytes (controls); group B: denuded GV oocytes fused with synchronized somatic cells, group C: denuded oocytes were first enucleated and then fused with synchronized somatic cells; group D: denuded oocytes were first enucleated and the ooplasts were then in-vitro matured (see text). b MI = metaphase I; MII = metaphase II. c Significantly different from the corresponding control (Group A) (P < 0.05, chi-squared test). Table 2. Pole-to-pole length of meiotic spindle. Group d MI-arrest spindle MII spindle No. spindles Spindle No. spindles Spindle examined length (μm) examined length (μm) A 14 29.9 ± 3.9 a 28 24.9 ± 3.7 a B 5 39.6 ± 6.0 b 44 34.3 ± 7.2 b C 8 38.1 ± 9.4 b 30 29.6 ± 7.3 c D 8 26.7 ± 1.3 a,c abc Values with same letter within a column are not significantly different (P < 0.05). d For description of groups A to D see Table 1 footnote. 218

Figure 4. A confocal image of an manipulated oocyte (group D) that was fixed at presumed metaphase II stage and was immunostained for α-tubulin (green), actin (red), and chromosomes (red). The reconstructed oocytes showing a well-organized bipolar spindle with all mitotic chromosomes aligned at the metaphase plate beneath the actin-rich domain. Bar = 30 μm. Figure 5. Confocal images of manipulated oocytes (group C; a, b, and c) collected and fixed during metaphase anaphase transition of meiosis I after 6 h of in-vitro maturation. The oocytes were immunostained for α-tubulin (green), actin (red), and chromosomes (red). (a) An oocyte at 6 h post-fusion and before polar body (PB) extrusion (presumed metaphase I) showing a haphazard distribution of mitotic chromosomes in the spindle. (b) An oocyte at the presumed early-anaphase stage showing mitotic chromosomes moving towards two discrete poles. (c) An oocyte at the presumed late-anaphase stage showing mitotic chromosomes nicely separated in the cytoplasm, and the actin-rich domain on the cortex where the PB would form. Bar = 30 μm. Discussion The present study developed and utilized a unique mouse model. Somatic chromosomes transferred into either intact or enucleated GV oocytes, to study the spindle checkpoint in the reconstructed oocytes. Because of the incompatibility of a meiotic spindle with mitotic chromosomes, the reconstructed oocyte should stall at metaphase if a checkpoint can detect this incompatibility. It was observed that reconstructed oocytes were able to form a bipolar spindle, demonstrating that the meiotic spindle can be induced to form by the presence of mitotic chromosomes. However, the incompatibility between the mitotic chromosomes and meiotic spindle prevented complete chromosome alignment at the metaphase plate in 100% of the manipulated oocytes. Despite these misalignments, PB extrusion and meiosis I-like division were accomplished when somatic chromosomes were in either intact GV oocytes (91.8%; 6 8 h) or enucleated GV ooplasts (71.6%; 5 7 h), suggesting that the progression of the cell cycle of meiosis was not blocked nor delayed by this incompatibility and misalignment. On the contrary, the transition from metaphase to anaphase was accelerated, as shown by the earlier extrusion of the first PB, possibly due to the transfer of chromosomes that had already condensed. Although in prior studies an XO mouse model was used to show that failure of congress of the X chromosome to the spindle equator was not a prerequisite to prevent anaphase onset in mouse oocytes (Hunt et al., 1995; LeMaire-Adkins et al., 1997), the possibility exists that misalignment of a single 219

220 chromosome may not be sufficient to induce anaphase delay. The results showed that even massive chromosome misalignment did not prevent anaphase progression, thus convincingly demonstrating that a spindle checkpoint does not exist when mitotic chromosomes are introduced to MI spindle of mouse oocytes. These observations may suggest that the oocyte s spindle checkpoint may be overridden or compromised during the first meiotic division. This hypothesis is also supported by the high rate of aneuploidy, which has been found in human embryos (Munné et al., 2004; Van Blerkom et al., 2004), which is mostly due to the errors of the first meiotic division in human oocytes (Hassold, 1996; Hassold and Hunt, 2001). Previously, a spindle checkpoint was proposed to exist in mouse oocytes at the first meiotic division before spindle assembly rather than after spindle formation as in the case of mitosis of somatic cells (Woods et al., 1999). In the previous study, the deletion of mlh1, which affects homologous pairing, rendered meiotic chromosomes at MI univalent, similar to chromosomes in somatic cells and those used in the present study. A majority of these chromosomes in oocytes from the mlh1 null mice, were not attached properly to the meiotic microtubules, resulting in the failure of a well assembled MI spindle and a subsequent lack of anaphase I onset. The arrest of cell cycle progression led to the conclusion that the failure of homologous pairing was detected by a cell cycle checkpoint, which prevented the formation of a functional spindle and delayed the onset of anaphase (Woods et al., 1999). The similarity between this study and the previous one is that all chromosomes appeared to be paired sister chromatids, while in this study, the mitotic chromosomes induced formation of fully assembled bipolar spindles, which were capable of initiating anaphase and separating mitotic chromosomes to two poles for extrusion of the PB and no anaphase delays were present. The discrepancy between this study and the previous one can be explained by the fact that in the prior study, the protein mlh1 was knocked out. It is possible that, in addition to its function in homologous pairing, the protein mlh1 may also be important for the formation of the first meiotic spindle, and knocking out mlh1 might hinder the formation of the spindle. In the absence of a functional spindle, there would not be enough driving force for cell division, and therefore no anaphase. This postulation is also consistent with the widely accepted observation that the onset of anaphase can be prevented, or significantly delayed, even in mitotic cells which have normal spindle checkpoints, when they are exposed to spindle disrupting drugs (e.g. nocodazole) (Eichenlaub- Ritter and Boll, 1989; Pesty et al., 1994; Eichenlaub-Ritter and Betzendahl, 1995; Can and Albertini, 1997) and fail to form a spindle and do not have the driving force for cell division. In the reconstructed oocytes, increased spindle length was observed in the presumed MI-arrest, further demonstrating the incompatible nature of the MI spindle and mitotic chromosomes. This is consistent with previous observations that expanded spindle length (pole-to-pole distance) was detected in newly established spindles of maturing ooplasts when univalent chromosomes rather than homologously paired chromosomes were present (Woods et al., 1999). Interestingly, an expanded pole-to-pole distance was also observed in this study in the MII spindle, which also had incorrect chromosome alignment after the reconstructed oocytes were matured in vitro and had extruded PB. When mitotic metaphase chromosomes were transferred directly into enucleated oocytes arrested at the MII stage, however, an organized MII spindle with well-aligned chromosomes was observed (Ono et al., 2001b), suggesting the MII spindle is quite compatible to mitotic chromosomes. Likewise, in this study, the mitotic chromosomes were well aligned on the organized MII spindle when the enucleated GV cytoplasts have been in-vitro matured to MII stage. This, however, is in clear contrast to the observations that mitotic chromosomes could not be aligned properly within the MI spindle. Taken together, these observations indicate that major differences exist between the functions and structures of the MI versus MII spindles. These differences may be caused by kinetochore configurations on chromosomes that interact with MI versus MII spindles. This should not be surprising, because the first meiotic division is unique in that during early prophase I the MI spindle binds to paired homologous chromosomes that recombine between homologues. The MII spindle, on the other hand, functions only to separate the duplicated sister chromatids, which are similar to mitotic chromosomes during mitosis. It is unclear why the MII spindles, in this study, also had extended pole-to-pole lengths. This could be due to the possibility that the MII spindles are not identical to a mitotic spindle or that the chromosomes in this study were suboptimal, in that they had an abnormal MI division. In conclusion, these results suggest that mitotic chromosomes can initiate meiotic spindle assembly in a maturing ooplast, even though the mitotic chromosomes were not well positioned because of the incompatibility between the somatic chromosomes and the meiotic MI spindle. A spindle checkpoint does not appear to exist at MI when mitotic chromosomes were introduced and differences reside between MI and MII spindles. The observations herein may provide key insights in exploring the emerging and complex biotechnology of somatic cell haploidization. As the idea of generation of artificial gametes is emerging, comprehending the mechanisms of the meiotic division are just a beginning, and the complexity of the process and the challenges involved in chromosome segregation and oocyte spindle checkpoint need to be further addressed. The effort is highly worthwhile as it promises to identify completely new avenues to overcome the challenges of producing artificial gametes. Acknowledgements The authors thank Marina Julian for her help with revising this manuscript, Dr Michele Barber for the assistance with confocal microscopy, and Dr John Riesen, Dr Hiroyuki Suzuki and Dr Brian Enright for helpful discussions. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. References Brunet S, Pahlavan G, Taylor S, Maro B 2003 Functionality of the spindle checkpoint during the first meiotic division of mammalian oocytes. Reproduction 126, 443 450. Can A, Albertini DF 1997 Stage specific effects of carbendazim (MBC) on meiotic cell cycle progression in mouse oocytes. Molecular Reproduction and Development 46, 351 362. Carabatsos MJ, Combelles CM, Messinger SM, Albertini DF 2000 Sorting and reorganization of centrosomes during oocyte maturation in the mouse. Microscopy Research and Technique 49, 435 444.

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