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BOR - Papers in Press, published online ahead of print December 28, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.047092
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BIOLOGY OF REPRODUCTION 74, 734–743 (2006)
DOI: 10.1095/biolreprod.105.047092
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

The First Mitosis of the Mouse Embryo Is Prolonged by Transitional Metaphase Arrest1

Marta Sikora-Polaczek 3 4, Anna Hupalowska 4, Zbigniew Polanski 5, Jacek Z. Kubiak 6, and Maria A. Ciemerych 2 4

Department of Embryology,4 Institute of Zoology, Warsaw University, 02–096 Warsaw, Poland Department of Developmental Biology,5 Max-Planck-Institute of Immunobiology, D-79108 Freiburg, Germany UMR 6061 CNRS,6 University of Rennes 1, IFR 140 GFAS, Faculty of Medicine, 35043 Rennes Cedex, France

ABSTRACT

The first mitosis of the mouse embryo is almost twice as long as the second. The mechanism of the prolongation of the first mitosis remains unknown, and it is not clear whether prometaphase or metaphase or both are prolonged. Prometaphase is characterized by dynamic chromosome movements and spindle assembly checkpoint activity, which prevents anaphase until establishment of stable kinetochore-microtubule connections. The end of prometaphase is correlated with checkpoint inactivation and disappearance of MAD2L1 (MAD2) and RSN (CLIP-170) proteins from kinetochores. Spindle assembly checkpoint operates during the early mouse mitoses, but it is not clear whether it influences their duration. Here, we determine the length of prometaphases and metaphases during the first two embryonic mitoses by time-lapse video recording of chromosomes and by immunolocalization of MAD2L1 and RSN proteins. We show that the duration of the two prometaphases does not differ and that MAD2L1 and RSN disappear from kinetochores very early during each mitosis. The first metaphase is significantly longer than the second one. Therefore, the prolongation of the first embryonic mitosis is due to a prolonged metaphase, and the spindle assembly checkpoint cannot be involved in this process. We show also that MAD2L1 staining disappears gradually from kinetochores of oocytes arrested at metaphase of the second meiotic division. This shows a striking similarity between the first embryonic mitosis and metaphase arrest in oocytes. We postulate that the first embryonic mitosis is prolonged by a transient metaphase arrest that is independent of the spindle assembly checkpoint and is similar to metaphase II arrest. The molecular mechanism of this transient arrest remains to be elucidated.

developmental biology, early development, embryo, meiosis

INTRODUCTION

During cell division the correct distribution of chromosomes is a fundamental condition preventing aneuploidy. It has been well documented that successful execution of mitosis relies on the proper assembly of both the metaphase plate and the mitotic spindle. The crucial role in this process is played by the mechanisms regulating correct formation of stable connections between kinetochores of chromosomes and spindle microtubules [1–4].

In mammalian somatic cells, mitosis takes approximately 1 h. However, this timing differs significantly among various cell lines. For example, in rat kangaroo kidney epithelial cell line (PtK1), the duration of mitosis ranges between 23 and 198 min (average 50 min) [2]. In rat basophilic leukemia cells, mitosis takes approximately 32 min [5] and in HeLa cells approximately 42 min but varies from 24 to 90 min [6, 7]. The duration of different stages of mitoses can be determined by analyzing the chromosome movements or localization of prometaphase markers at the kinetochores. Continuous chromosome movements are observed during prometaphase (i.e., when microtubules are captured at kinetochores), and as a consequence of action of unbalanced pulling forces, chromosomes are rapidly displaced. Finally, these dynamic movements in and out of the chromosome plate (see, e.g., Meraldi et al. [6]) terminate because of the establishment of stable microtubule-kinetochore attachments and the stabilization of forces within the spindle, leading to the metaphase plate formation. This allows the metaphase/anaphase transition and proper separation of sister chromatids [8]. In the presence of improper chromosome-kinetochore connections, damaged spindle, or misaligned chromosomes, the spindle assembly checkpoint (SAC) is triggered, preventing the onset of the anaphase. Some of the proteins involved in generating the checkpoint signal localize to kinetochores only during prometaphase. Among these are MAD1L (also known as MAD1) [9], MAD2L1 (also known as MAD2) [10, 11], BUB1 [12], BUB1B (also known as BUBR1) [13], and BUB3 [14]. MAD2L1 has been shown to be the key player in SAC function. Its binding to CDC20 inhibits the anaphase-promoting complex (APC/cyclosome, or APC/C) by preventing degradation of multiple targets such as anaphase inhibitor PTTG1 (securin) and cyclin B [15, 16]. MAD2L1 disappearance from kinetochores delineates the inactivation of SAC and determines the end of the prometaphase [10, 17].

RSN, also known as CLIP-170, regulates dynamics of microtubules, mediates their interaction with endocytic vesicles [18], and recently was shown to play a role in spermatogenesis during spermatid differentiation and sperm head shaping [19]. Importantly, it is also a molecule that is lost from kinetochores stably attached to microtubules, both during somatic cell mitosis [20] as well as in oocytes undergoing meiotic divisions [21]. Since in these cells RSN disappears from kinetochores at the same time as MAD2L1, its presence also can be used as a prometaphase marker.

The duration of the stages of mitosis, determined by the observation of chromosome movements and/or localization of SAC markers, varies depending on the cell line studied. For example, in HeLa cells prometaphase takes approximately 25 min (it varies from 10 to 60 min), while average metaphase duration is 8 min (it varies from 2 to 15 min)[6]. In PtK1 cells the loss of MAD2L1 from all kinetochores precedes anaphase by 10 min; that is, metaphase takes 10 min [22]. Therefore, in somatic mammalian cells that were studied so far, the prometaphase is relatively long, while the metaphase sensu stricto is generally very short.

In contrast to somatic mitoses, during meiosis of mouse oocytes the first meiotic division is exceptionally long and takes approximately 9 h. During this period, MPF (M-phase promoting factor or CDC2A/cyclin B, also known as CDK1/cyclin B) activity remains high (measured by histone H1 kinase activity). Similarly to mitotic prometaphase, meiotic prometaphase is characterized by dynamic chromosome movements [21] and is also regulated by SAC [23–28]. Both the movements of chromosomes and kinetochore localization of MAD2L1 are observed for approximately 8 h [21, 24]. The metaphase of the first meiotic division (metaphase I) itself is relatively short since the onset of anaphase occurs only 1 h after the establishment of stable kinetochore-microtubule interactions marked by the cessation of the changes in chromosome localization and by MAD2L1 disappearance [21, 24]. After the accomplishment of the first meiotic division, the metaphase plate and spindle of the second meiotic division (metaphase II) are immediately formed. Since during the metaphase II arrest chromosome movements are not observed, it seems that it is the metaphase rather than prometaphase that is prolonged [21]. Metaphase II oocytes are stably arrested because of the action of cytostatic activity (CSF) [29, 30]. CSF prevents cyclin B destruction and MPF inactivation, probably through APC/cyclosome inhibition [31]. SAC seems not to be involved in CSF in mouse oocytes [27], although it participates in Xenopus CSF [32, 33].

Little is known about the regulation of the first cleavage divisions of the mouse embryo. We have shown previously that the first embryonic mitosis lasts 120 min, that is, almost twice as long as the second one, which takes approximately 70 min [34]. The difference in the duration of mitoses is reflected by the plateau of MPF activity that is observed exclusively during the first mitosis [34]. We hypothesize that this transient MPF stabilization could proceed because of the presence of factors that are also involved in meiotic cell cycle regulation and persist within the cytoplasm of the one-cell embryo. However, this hypothesis has not been carefully tested, and the molecular mechanisms underlying the observed differences remain obscure.

It also cannot be excluded that SAC is involved in the regulation of the timing of the first embryonic mitosis. It has been shown previously that SAC could be activated at very early stages of mouse embryo development upon microtubule disruption leading to the mitotic arrest of one-cell embryos [35–37], two-cell embryos [37, 38], as well as of embryos in more advanced stages of preimplantation development [39, 40]. However, there is no evidence demonstrating changes in SAC proteins localization during these embryonic divisions.

Taken together, the question whether the prolongation of the first embryonic mitosis occurs because of the longer prometaphase (as during the first meiotic division) or metaphase (as during the second meiotic division) or because of the extension of both these phases remained unanswered. To distinguish between these possibilities, we performed analyses of chromosome movements and examined the timing of disappearance from kinetochores of SAC protein MAD2L1 and RSN, during both mitoses. In addition, we focused our attention on MAD2L1 localization in metaphase II-arrested oocytes, which are characterized by prolongation of this stage of cell division due to CSF action.

MATERIALS AND METHODS

All animal investigations presented in these study were approved by Local Ethic Committee No. 1 in Warsaw, Poland, and Regierungspraesidium in Freiburg, Germany, according to European Union Council Directive 86/609/EEC of 24 November 1986 on the approximation of laws, regulations, and administrative provisions of the member states regarding the protection of animals used for experimental and other scientific purposes [41, 42]. All animals were raised on the premises.

Collection and Culture of Primary Oocytes, Ovulated Oocytes, and One- and Two-Cell Embryos

Ovarian follicle growth was stimulated by injecting F1 (CBA/HxC57Bl/10) females with 10 IU of eCG (Intervet). Primary oocytes were isolated from the ovaries 46–54 h after eCG injection and were freed of follicular cells by vigorous pipetting.

F1 female mice were superovulated by injection of 10 IU of eCG and 10 IU of hCG, (Intervet) 48–52 h apart. Ovulated, metaphase II oocytes were collected from oviducts 17–18 h after hCG injection. Cumulus cells surrounding oocytes were removed by treatment with hyaluronidase (Sigma Aldrich; 300 U/ml of PBS), and subsequently zonae pellucidae were dissolved with 0.5% pronase (Sigma Aldrich) in PBS. Fertilized one-cell embryos were obtained from oviducts of females mated with F1 males 29 h after hCG. Two-cell embryos were obtained from oviducts of mated females 49 h after hCG. All oocytes and embryos were collected and cultured in medium M2 (M16 medium supplemented with 5 mg/ml of HEPES [Sigma Aldrich] and 4 mg/ml BSA [Sigma Aldrich]) [43] under mineral oil (Sigma Aldrich) at 37°C.

Parthenogenetic Activation

One-cell parthenogenetic embryos (parthenogenotes) were obtained by exposing metaphase II (17–18 h after hCG) oocytes to 8% ethanol (POCh) in M2 for 8 min at room temperature [44] or by treating metaphase II oocytes with strontium as described previously [45] with the exception that cytochalasin B treatment was omitted. Oocytes were then washed carefully and cultured in M2 for the next 13–14 h. Only those oocytes that extruded second polar bodies and formed haploid pronuclei were used for further analyses.

Time-Lapse Recording of Chromosome Dynamics

One-cell parthenogenotes and one- and two-cell embryos obtained after in vivo fertilization were used for the analyses of chromosome dynamics. To vitally label DNA, embryos were transferred to M2 medium containing 1 µg/ml bisbenzimide (H 33258; Sigma Aldrich) for 30–60 min before starting time-lapse recording.

For time-lapse recording, the culture dish (glass-bottom dish; WillCo Wells BV) was placed in a plastic chamber incubator XL (Zeiss; Carl Zeiss Jena) mounted on a Zeiss Axiovert M200 inverted microscope (Zeiss, Carl Zeiss Jena). The temperature in the incubator was maintained at 37°C by a Tempcontrol 37–2 digital (Zeiss, Carl Zeiss Jena). The microscope was connected to AxioCam Mrm camera (Zeiss, Carl Zeiss Jena). Image acquisition was performed using DIC (30–400 ms illumination) and epifluorescence (2-sec illumination with the light intensity reduced by 70% using neutral-density filter) every 9 min under the control of AxioVision 3.4 software (Zeiss, Carl Zeiss Jena). Figures were assembled using Adobe Photoshop 7.0.

Nocodazole Treatment

One-cell parthenogenotes at different time points after nuclear envelope breakdown were subjected to 60 min of incubation in 0.25 µM nocodazole (Sigma Aldrich) in M2 medium and then fixed and proceeded for the immunodetection of MAD2L1 or RSN.

Immunofluorescence

In vitro cultured primary oocytes that resumed meiosis and reached metaphase II stage were fixed at different time points after the completion of first meiotic division. One-cell parthenogenotes and one- and two-cell embryos obtained after in vivo fertilization were fixed at different time points after nuclear envelope breakdown. All oocytes and embryos were processed for immunofluorescence as previously described [46]. For tubulin staining, mouse monoclonal antibody against {alpha}-tubulin was used (Sigma Aldrich) followed by rhodamine labeled anti-mouse antibody (Jackson ImmunoResearach). For MAD2L1 staining, rabbit polyclonal antibody (gift from Dr. Katja Wassmann, Université Pierre, and Marie Curie, Paris, France) and for RSN staining rabbit polyclonal antibody (gift from Dr. Eric Karsenti, EMBL, Heidelberg, Germany) were used followed by FITC labeled anti-rabbit antibody (Jackson ImmunoResearach). For staining control, one-cell embryos were fixed and processed for immunocytochemistry as described previously with the exception that incubation in the primary antibody (anti-{alpha}-tubulin, anti-MAD2L1, or anti-RSN) was omitted. Chromatin was visualized either with chromomycin A3 (Sigma Aldrich) or by propidium iodide (Vector Laboratories). The samples were mounted in Citifluor (Citifluor Ltd) and scanned using LSM 510 ZEISS laser scanning confocal microscope (Carl Zeiss Jena). Then the most representative, single optical sections were collected. Figures were assembled using Adobe Photoshop 7.0.

RESULTS

The length of mitoses, delineated by nuclear envelope breakdown (NEBD) and the onset of the anaphase, was analyzed in dividing one- and two-cell mouse embryos. In addition to the one-cell embryos obtained by in vivo fertilization, we also analyzed one-cell parthenogenotes. In contrast to fertilization, the parthenogenetic activation results in synchronous first embryonic division, facilitating analysis. We have shown previously that the timing of MPF activation, mitosis duration, the dynamics of chromosome condensation, and the spindle formation are identical during the first mitosis of these two types of embryos [34]. Furthermore, multiple studies have validated that one-cell parthenogenotes can serve as a model in the analyses of different aspects of the mouse embryo preimplantation development [34, 47–53]. To study the progression of the second mitosis, we used two-cell embryos obtained exclusively by in vivo fertilization.

Chromosome Dynamics During First and Second Embryonic Mitosis

We used the time-lapse recording system that enabled us to follow the behavior of chromosomes stained with vital dye Hoechst 33342 using UV light. Embryos were photographed every 9 min, and typical one-cell embryos and blastomeres of two-cell embryos are shown in Figure 1, A and B. We also monitored the progression of the mitoses in control embryos that were not recorded and thus not exposed to UV light. We followed one-cell parthenogenotes (n = 20), one-cell embryos obtained after in vivo fertilization (n = 19), and blastomeres of two-cell embryos obtained after in vivo fertilization (n = 53). Embryos were observed from the G2/M transition, that is, NEBD (Fig. 1A: NEBD; Fig. 1B: NEBD), until the onset of anaphase (Fig. 1A: NEBD + 108 min; Fig. 1B: NEBD + 90 min). Control one-cell embryos obtained by parthenogenetic activation (n = 20) or by in vitro fertilization (n = 10) reached anaphase within 120 min after NEBD (SD = 5 min), while experimental one-cell embryos obtained by in vitro fertilization did so within 111 min (n = 9; SD = 8 min). Blastomeres of control, two-cell embryos reached anaphase within 70 min after NEBD (n = 40; SD = 5 min) and experimental blastomeres within 76 min after NEBD (n = 13; SD = 9 min). These results are consistent with our previous observations [34]. Following NEBD in one-cell embryos, two haploid groups of chromatin (i.e., paternal and maternal) were observed (Fig. 1A: NEBD, NEBD + 9 min, NEBD + 18 min). Next, the common metaphase plate begins to form (Fig. 1A: NEBD + 27 min). After NEBD, in blastomeres of two-cell embryos, a single diploid group of chromosomes was present (Fig. 1B: NEBD, NEBD + 9 min, NEBD + 18 min). During the initial periods of both mitoses (i.e., between NEBD and the formation of metaphase plate), chromosomes continuously changed their positions migrating centripetally and progressively forming the chromosome plate. The most clearly distinguishable event selected as prometaphase/metaphase transition was the alignment of all chromosomes within the metaphase plate. This event occurred approximately at the same time after NEBD during the first (mean value 37 min; n = 9; SD = 7 min; Fig. 1A: NEBD + 36 min; Fig. 2) and the second mitosis (mean value 33 min; n = 13; SD = 5 min; Fig. 1B: NEBD+36 min; Fig. 2). Staring from this time point, neither shape nor position of the plate changed except that some chromosome arms altered their localization. Therefore, durations of metaphase plate formation did not differ significantly between first and the second mitosis (Student t-test, P > 0.10). Although some translocations were still observed because of the chromosome arm movements, probably because most mouse chromosomes are telocentric, no case of chromosome leaving the metaphase plate was observed (as seen in HeLa cells [6]). Positions of some particularly long chromosomes remained unchanged for longer periods within the plates, also indicating metaphase status (Fig. 1A: NEBD + 45–63 min, and Fig. 1B: NEBD + 54–63 min, arrows). The duration of the phase starting from the moment of metaphase plate formation until the onset of the anaphase was clearly longer during the first mitosis (mean value = 65 min; n = 9; SD = 8 min; Fig. 2) than during the second one (mean value = 35 min; n = 13; SD = 11 min; Fig. 2), and the observed difference was significant (Student t-test, P < 0.0005).


Figure 1
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  		FIG. 1. Chromosome movements during mitoses of one- and two-cell mouse embryos. Time-lapse video microscopy. Pictures were taken every 9 min. Arrows indicate examples of the particularly long chromosomes. A) One-cell embryo. Two sets of condensing chromosomes are visible until 18 min post-nuclear envelope breakdown (NEBD). Common chromosome plate starts to form approximately at 36 min after NEBD. B) Single blastomere of two-cell embryo. Chromosome plate starts to form approximately at 36 min after NEBD. Bar = 20 µm.


Figure 2
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  		FIG. 2. Comparison of the duration of the metaphase plate formation (prometaphase) and metaphase during the first and the second mitosis. Error bars represent standard deviation values (±SD).

These data suggest that the mechanisms responsible for the prolongation of the first mitosis operate after the formation of the metaphase plate, that is, during the metaphase stage rather than during prometaphase. However, using time-lapse recording, an unequivocal determination of the timing of the prometaphase/metaphase transition was not possible because of the wavy movements of long chromosome arms of the mouse. Therefore, we decided to correlate these observations with the localization of MAD2L1 checkpoint protein, a generally accepted prometaphase marker, by measuring the timing of its association with kinetochores.

MAD2L1 Localization During First and Second Embryonic Mitosis

MAD2L1 localization was examined in one- and two-cell embryos. We localized MAD2L1 with rabbit polyclonal antibody that was previously shown in mouse oocytes to detect MAD2L1 and colocalizes with the kinetochore protein CENPE [24].

The embryos were analyzed during G2 phase, at NEBD, as well as at 5, 10, 15, 20, 30, 40, and 60 min following NEBD. The total number of examined one-cell embryos was 238 (approximately 30 per time point). In the majority of experiments, we analyzed MAD2L1 localization in one-cell parthenogenetic mouse embryos. We verified that the dynamics of MAD2L1 localization in parthenogenotes and one-cell embryos obtained by in vivo fertilization does not differ (data not shown). MAD2L1 localization was also examined in 90 blastomeres of two-cell embryos obtained by in vivo fertilization at G2, NEBD, and at 10, 20, 30, and 40 min following NEBD (approximately 13 blastomeres per time point).

Incubation of one-cell embryos collected in G2 phase with the secondary goat anti-rabbit antibody conjugated with FITC that were used for the MAD2L1 rabbit polyclonal antibody detection showed no nonspecific staining (Fig. 3A). As a positive control, MAD2L1 was localized in one-cell embryos collected 30 min after NEBD and subjected to 60 min of incubation in the nocodazole, causing mitotic checkpoint activation and resulting in the MAD2L1 localization in kinetochores (Fig. 3B).


Figure 3
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  		FIG. 3. MAD2L1 localization in one- and two-cell embryo during mitosis. Embryos were fixed at G2 phase and at different time points after nuclear envelope breakdown (NEBD). Left panel shows MAD2L1 staining (green), and middle panel shows chromatin staining only (blue). Right panel shows the overlay of MAD2L1 (green) and chromatin (blue) staining. A) One-cell embryo in G2 phase. During the immunostaining procedure, incubation in anti-MAD2L1 antibody was omitted. B) One-cell embryo fixed 90 min after NEBD that was blocked in mitosis via incubation in the microtubule depolimerizing drug nocodazole. Because of the spindle assembly checkpoint (SAC) activation, MAD2L1 become stably localized in the kinetochores of this metaphase-arrested embryo. C) One-cell embryo. In G2 phase, MAD2L1 is localized within female and male pronuclei, at NEBD it surrounds condensing chromosomes, and at NEBD + 10 min it is visible at kinetochores. Twenty minutes after NEBD, MAD2L1 is absent from kinetochores. D) Single blastomere of two-cell embryo. G2 phase MAD2L1 is localized within blastomere nucleus, at NEBD it surrounds condensing chromosomes, and at NEBD + 10 min and NEBD + 30 min it is visible at kinetochores. Forty minutes after NEBD, MAD2L1 is absent from kinetochores. Bar = 20 µm.

During G2 phase, in both one- and two-cell embryos, MAD2L1 localization was exclusively nuclear but excluded from nucleoli (Fig. 3C: G2; Fig. 3D: G2). On NEBD, MAD2L1 remained in the vicinity of condensing chromatin (Fig. 3C: NEBD; Fig. 3D: NEBD). Ten minutes after NEBD, MAD2L1 staining was visible at kinetochores of chromosomes of one- and two-cell embryos (Fig. 3C: NEBD + 10 min; Fig. 3D: NEBD + 10min). In one-cell embryos, the kinetochore-associated signal was no longer detectable 20 min after NEBD (Fig. 3C: NEBD + 20 min, NEBD + 30 min), whereas in two-cell embryos it was still detectable at 30 min post-NEBD (Fig. 3D: NEBD + 30 min) and was lost from kinetochores by 40 min after NEBD (Fig. 3D: NEBD + 40 min).

Dynamics of MAD2L1 localization to chromosomes was correlated with the stages of mitotic spindle formation using double detection of MAD2L1 and {alpha}-tubulin. During the first mitosis, the strongest MAD2L1-positive kinetochore signal was detected when microtubules formed a radial structure or began to form a bipolar spindle, that is, 10–15 min. after NEBD (Fig. 4A: NEBD + 10 min, NEBD + 15 min), and disappeared when the spindle became bipolar, that is, 20 min after NEBD (Fig. 4A: NEBD + 20 min, NEBD + 30 min). Therefore, since our time-lapse recording experiments revealed that during the first mitosis metaphase plate formation took place approximately at 37 min after NEBD (n = 9, SD = 7 min), MAD2L1 disappearance from kinetochores occurred before the metaphase plate and spindle were formed. In contrast, during the second mitosis, the presence of MAD2L1 was still detectable on kinetochores when the bipolar spindle was formed, that is, between 20 and 40 min post-NEBD (Fig. 4B: NEBD + 20 min, NEBD + 40 min). At this point, MAD2L1 staining again became more apparent within the spindle area (Fig. 4B: NEBD + 40 min). Time-lapse recordings showed that metaphase plate formation occurred on average at 33 min after NEBD (n = 13, SD = 5 min), that is, simultaneously with the displacement of MAD2L1 from kinetochores.


Figure 4
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  		FIG. 4. MAD2L1 localization during the formation of the first and the second mitotic spindle. One- and two-cell embryos were fixed at different time points after nuclear envelope breakdown (NEBD), and MAD2L1 as well as microtubules were localized. Left panel shows MAD2L1 staining (green), and two middle panels show separate {alpha}-tubulin (red) and chromatin staining (blue). Right panel shows the overlay of MAD2L1 (green), tubulin (red), and chromatin (blue) staining. A) One-cell embryo. B) Single blastomere of two-cell embryo. Bar = 20 µm.

The length of the prometaphase, measured by the presence of MAD2L1 on kinetochores, does not differ significantly between the first and the second embryonic mitosis, although MAD2L1 kinetochore staining disappearance slightly preceded metaphase plate formation in the first mitosis but occurred at the same time in second mitosis. To confirm this, we reasoned that if MAD2L1 disappears when microtubule-kinetochore attachments are stably formed, as happens in somatic cells and mouse oocytes, we should observe simultaneous disappearance of RSN.

RSN Localization on Kinetochores During First and Second Mitosis

Fifty-two one-cell parthenogenetic embryos and 39 blastomeres of two-cell embryos were therefore analyzed for RSN localization at the same time points as for MAD2L1 (approximately seven and six embryos analyzed per time point, respectively; Fig. 5). Positive and negative controls were performed as described for the MAD2L1 analysis (Fig. 5, A and B).


Figure 5
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  		FIG. 5. RSN localization in one- and two-cell embryo during mitosis. Embryos were fixed at different time points after nuclear envelope breakdown (NEBD). Left panel shows RSN staining (green), and middle panel shows chromatin staining only (blue). Right panel shows the overlay of RSN (green) and chromatin (blue) staining. A) One-cell embryo fixed at 30 min after NEBD. During the immunostaining procedure, incubation in anti-RSN antibody was omitted. B) One-cell embryo fixed 90 min after NEBD, blocked in mitosis via incubation in the microtubule depolimerizing drug nocodazole. Because of a lack of stable microtubule-kinetochore connections, RSN becomes stably localized to the kinetochores of this metaphase-arrested embryo. C) One-cell embryo. At 10 and 20 min after NEBD, diffuse staining of RSN is visible surrounding chromatin and also on kinetochores. Forty minutes after NEBD, RSN is absent from kinetochores and visible at spindle poles. D) Single blastomere of two-cell embryo. Thirty minutes after NEBD, RSN is visible on kinetochores and absent at 40 min after NEBD. Bar = 20 µm.

During G2 phase of the first and the second cell cycle, RSN was never detectable within nuclei (data not shown). Following NEBD in one-cell embryos, RSN localized as a diffused sphere around the chromosomes in the region where the spindle was forming. However, until 20 min after NEBD, it was clearly more concentrated as a clear punctuate staining within the group of chromosomes and at the forming spindle poles (Fig. 5C: NEBD + 10 min, NEBD + 20 min). Later on, RSN was no longer detectable on the kinetochores (Fig. 5C: NEBD + 40 min). In two-cell stage embryos, RSN had the same localization as in one-cell embryos; however, it was detected on the chromosomes until 30 min after NEBD (Fig. 5D: NEBD + 30 min, NEBD + 40 min). Thus, the timing of the delocalization of RSN and MAD2L1 from the kinetochores is similar. Moreover, as a microtubule-associated protein, RSN was clearly visible at spindle microtubules during both mitoses. These observations show that RSN, similarly to MAD2L1, is lost from kinetochores of one-cell embryos before the metaphase plate is formed. In dividing two-cell embryos, RSN disappearance from the kinetochores correlates with the formation of metaphase plate. This shows that microtubule-kinetochore stable attachments are indeed formed, particularly early during the first embryonic mitosis.

MAD2L1 Localization on Kinetochores of Metaphase II-Arrested Mouse Oocytes

Our analysis of MAD2L1 localization during the first and the second mitosis strongly suggests that a mechanism responsible for the prolongation of first mitotic mitosis operates not during prometaphase but during metaphase. This bears a resemblance to maturing mouse oocytes in that metaphase of the second meiotic division is prolonged by the action of cytostatic factor (CSF) [29, 30, 54]. Therefore, we analyzed the localization of MAD2L1 at different time points of metaphase of the second meiotic division.

One hour after the first meiotic division and the formation of the metaphase plate of the second meiotic division, MAD2L1 was present at the kinetochores of 91% (21/23) oocytes. However, at the later stages of metaphase II (i.e., 6 and 10 h after the completion of the first meiotic division), only 16% (15/94) and 5% (5/111) of oocytes showed detectable kinetochore MAD2L1 staining, respectively (Fig. 6A). At this time point, the majority of oocytes lacked MAD2L1 at the kinetochores (Fig. 6B). These results, combined with our observation that the first embryonic mitosis is prolonged because of the lengthening of the metaphase, also imply that related mechanisms might cause the prolongation of metaphase of the second meiotic division and the first embryonic mitosis.


Figure 6
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  		FIG. 6. MAD2L1 localization in oocytes blocked at metaphase of the second meiotic division 6 h after completion of the first meiotic division. Left panel shows MAD2L1 staining (green), and two middle panels show separate {alpha}-tubulin (red) and chromatin staining (blue). Right panel shows the overlay of MAD2L1 (green), tubulin (red), and chromatin (blue) staining. A) Oocyte with kinetochores labeled with MAD2L1. B) Oocyte from the same time group showing that MAD2L1 is absent from kinetochores. Bar = 20 µm.

DISCUSSION

SAC Is Not Involved in the Prolongation of First Embryonic Mitosis

Using time-lapse video recording of chromosomes in living embryos and the immunolocalization of MAD2L1 and RSN proteins, we explored whether SAC could be involved in the process of the prolongation of the first mitosis during mouse embryo development. Our analyses revealed that, in contrast to somatic cells and oocytes, during the first embryonic mitosis the timing of MAD2L1 disappearance from the kinetochores is not strictly synchronized with the cessation of prometaphase chromosome movements and mitotic spindle formation [22, 23, 55]. During the first embryonic mitosis, MAD2L1 and RSN disappear from kinetochores before the metaphase plate formation, defined both by mitotic spindle formation and by chromosome alignment within the metaphase plate (Fig. 7). However, during the second mitosis, both proteins disappear from kinetochores at the same time as metaphase plate formation. Prometaphase is similar in length during these two mitoses (20–30 min), while metaphase takes 65 min during the first mitosis vs. 35 min during the second mitosis. Therefore, the prolongation of the first mitotic division depends entirely on the prolongation of the metaphase stage. Nevertheless, the proportions in the duration of prometaphase vs. metaphase observed during the first two embryonic mitoses differ significantly from that observed in dividing somatic cells. In HeLa cells, prometaphase is much longer than metaphase and takes about 25 min, while metaphase takes only 8 min [6]. This might reflect an important difference in regulatory cell cycle mechanisms between embryonic and somatic mitoses.


Figure 7
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  		FIG. 7. Schematic representation of the differences in the timing of events during the first and second embryonic mitoses in the mouse.

The loss of MAD2L1 from kinetochores before the metaphase plate formation indicates that SAC is involved neither in prolongation of the first mitosis nor in maintenance of stable MPF activity [34]. Clearly, another unknown mechanism must stabilize MPF and preserve metaphase conditions. Recently, Meraldi and colleagues have shown that besides the kinetochore-associated pool of MAD2L1 active in SAC, the cytoplasmic pool of MAD2L1, which is not involved in SAC, also regulates the timing of mitotic progression, namely, anaphase onset [6]. It was also shown in Xenopus embryo mitotic extract that the depletion of cytoplasmic pool of MAD2L1 shortens the first mitosis [56], which is also longer than the second embryonic mitosis in Xenopus [57]. We cannot, therefore, exclude that the cytoplasmic pool of MAD2L1 (that does not participate in SAC) could take part in the regulation of the duration of the first two embryonic mitoses in the mouse.

SAC Plays a Role in the Initial Stages of Metaphase II Arrest

Maturing mouse oocytes represent another type of cells in which divisions are prolonged. Fertilization or parthenogenetic activation of metaphase II-arrested oocytes triggers progressive changes from the meiotic to mitotic control of the cell cycle [58]. A part of the meiotic cell cycle controlling machinery could, therefore, operate also during early embryo development. Indeed, maturing mouse oocytes and one-cell embryos show similarities between the regulation of meiotic divisions and first embryonic mitosis. For example, the assembly of the first mitotic spindle resembles the way the first meiotic spindle is formed despite the fact that during the first meiotic division the spindle formation takes as long as 6–7 h [34, 59]. However, the vast majority of this period is in fact a prometaphase prolonged because of SAC activity, as documented by the presence of MAD2L1 and RSN, and also chromosome movements in metaphase I oocytes [21, 23, 24]. Interestingly, it has been shown recently that the second meiotic metaphase is maintained via a SAC-independent mechanism [27]. Several previous analyses of MAD2L1 and BUB1 proteins localization gave, however, contradictory results. In metaphase II-arrested mouse oocytes, the kinetochore localization of BUB1 [23] and MAD2L1 [25] suggested active SAC, while the study of rat metaphase II-arrested oocytes showed that MAD2L1 is absent from kinetochores [60]. Recently, a gradual reduction of kinetochore-bound MAD2L1 during aging of metaphase II-arrested pig oocytes has been shown [61]. The latter resembles our observation of MAD2L1 in mouse oocytes (this paper) and supports the hypothesis that the second metaphase arrest does not require SAC activity [27]. Thus, the metaphase II arrest is sustained exclusively by the cytostatic activity that involves factors such as the MOS-MAP2K1 (also known as MEK1)-MAPK1/MAPK3 (ERK2/ERK1) pathway [62]. SAC might be, therefore, required only at the very early stage of the second meiotic metaphase to prevent premature oocyte activation before the functional spindle is formed.

CSF Components During First Mitosis

The similarity of the changes of MAD2L1 localization in metaphase II oocytes and in mitotic one-cell embryos strongly suggests that also during the two first embryonic mitoses, the initial role of SAC could be rapidly replaced by other mechanism(s) stabilizing the MPF activity. This could involve a CSF-like activity, however transient in nature. The MAPK1/MAPK3 (ERK2/ERK1) pathway is necessary for the CSF activity in metaphase II-arrested mouse oocytes [62], but it does not seem to be active during the first mitosis in the mouse embryo [51, 62]. On the other hand, a partial phosphorylation of a downstream substrate of MAP kinase signaling pathway RPS6KA1 (also known as p90rsk1) during the first mitosis is very puzzling [51]. It suggests that some modifications of MAPK pathways might be involved in MPF stabilization during the first embryonic mitosis. It has been recently shown that neither RPS6KA1 nor RPS6KA2 and RPS6KA3 are involved in the CSF activity in mouse oocyte [63]. Yet it cannot be excluded that some of these factors could play a role in a pathway operating during the first embryonic mitosis.

APC/C Function May Influence the Timing of the First Mitosis

It has been postulated that CSF may act through the inhibition of APC/C [31], which has been shown to regulate the degradation of multiple cellular targets including PTTG1 (securin) and cyclin B, a regulatory subunit of MPF [15, 16]. Degradation of cyclin B is necessary for the completion of the cell division. PTTG1, on the other hand, acts as inhibitor of separase that is responsible for the cleavage of cohesins such as REC8 that mediate cohesion of meiotic chromosomes [64, 65]. Therefore, degradation of PTTG1 is a prerequisite step for the proper chromosome separation at the M-phase exit. It has been shown previously that preventing securin degradation inhibits the completion of the first meiotic division and arrests oocytes in metaphase I [66]. The prolongation of the first mitosis may be, therefore, achieved via specific regulation of APC/C activity that does not involve CSF activity. This in turn might influence the timing of MPF inactivation and/or initiation of the chromosome separation. Such an alternative mechanism might involve FBXO5 (also known as early mitotic inhibitor 1 [Emi1]), which inhibits the ability of CDC20 to activate APC/C and has been postulated recently to have the CSF activity in Xenopus oocytes [67]. In mouse oocytes, FBXO5 was also shown to be phosphorylated by RPS6KA3. Phosphorylated form of FBXO5 has an increased ability to bind CDC20 [68]. Since RPS6KA3 seems not to be involved in the metaphase arrest of mouse oocytes [63], the exact roles of these two proteins in M-phase arrest remains to be clarified. Analyses of the cyclin B and/or securin degradation during the first two mitoses of mouse embryo, as well as other factors influencing APC/C, might solve the issue of the prolongation of the first embryonic mitosis.

Another mechanism involved may act via cyclin A2, which, with cyclin B, serves as a regulatory subunit of CDC2A (CDK1, MPF). On exit from the first mitosis of the mouse embryo, an important quantity of cyclin A2 remains nondegraded. This suggests that cyclin A2 is stabilized during this particular mitosis and therefore may influence the MPF activity [69]. It is, however, unclear how this cyclin escapes degradation and how its stability could be related to the prolongation of the first embryonic mitosis [70]. The molecular basis of the modifications of the first embryonic mitosis remains to be identified.

ACKNOWLEDGMENTS

We thank Katja Wassmann for MAD2L1 antibody and Eric Karsenti for the RSN (CLIP-170) antibody. We are also grateful to Marek Maleszewski and Daniel Fisher for the critical reading of the manuscript.

FOOTNOTES

1 Supported by ARC (grant 4298) to J.Z.K., Polish-French POLONIUM Program 2004 to M.A.C. and J.Z.K., and Deutsche Forschungsgemeinschaft Special Program (Schwerpunktprogramme) 1109 to Z.P. Back

2 Correspondence: Maria A. Ciemerych, Department of Embryology, Institute of Zoology, Warsaw University, Miecznikowa 1, Room 214A, 02–132 Warsaw, Poland. FAX: 48 22 55 41 210; ciemerych{at}biol.uw.edu.pl Back

3 Current address: Department of Genetics and Evolution, Institute of Zoology, Jagiellonian University, Ingardena 6, 30–060 Krakow, Poland. Back

Received: 1 September 2005.

First decision: 16 September 2005.

Accepted: 22 December 2005.

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A. Hupalowska, I. Kalaszczynska, S. Hoffmann, C. Tsurumi, J. Z. Kubiak, Z. Polanski, and M. A. Ciemerych
Metaphase I Arrest in LT/Sv Mouse Oocytes Involves the Spindle Assembly Checkpoint
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