Hum. Reprod. Advance Access originally published online on April 28, 2005
Human Reproduction 2005 20(8):2207-2210; doi:10.1093/humrep/dei044
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Spindle positions and their distributions in in vivo and in vitro matured mouse oocytes
1 Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam-si, Kyunggi-do, 463-707 and 2 Department of Obstetrics and Gynecology, Seoul National University Hospital, 28 Yeongun-dong, Chongno-gu, Seoul 110-744, Korea
3 To whom correspondence should be addressed. Email: suhcs{at}snu.ac.kr
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Abstract |
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BACKGROUND: This study was carried out to compare spindle locations and their developmental competencies both in vivo and in vitro in matured mouse oocytes. Spindle locations were identified using a polscope. Since meiotic spindles in living oocytes are highly birefringent, their structures can be viewed non-invasively by using a polscope. METHODS: In vivo matured metaphase II oocytes were collected from the oviducts of mice. Immature oocytes were collected from mouse ovaries, and then cultured in YS medium until the first polar body (PB) extrusion. In vitro and in vivo matured oocytes were classified into four categories according to their spindle positions relative to the first PB (0°, 0–90°, 90–180° and without a spindle image), and rates of fertilization and blastocyst formation were assessed. In vivo matured oocytes with a 0° spindle disposition relative to PB were cultured in vitro for 24 h, and then their spindle positions were re-assessed. RESULTS: Most in vivo matured oocytes (89.1%) had a 0° spindle position. Only 6 and 3% of oocytes had spindle positions of 0–90° and 90–180°, respectively. No spindle image was observed in 2%. However, most in vitro matured oocytes (83.1%) had a 0–90° spindle position and, in contrast, only 6.5% of these oocytes had a 0° spindle position. The rate of fertilization and blastocyst rate were significantly higher for in vivo matured oocytes than in vitro matured oocytes (87.1 versus 64.9% and 76.1% versus 66.0%, respectively, P<0.05 for each). We also observed that 71.7% of the in vivo matured oocytes with the 0° spindle position showed a spindle position change to 0–90° after 24 h of culture. These oocytes had a poor fertilization rate (43%) and a zero blastocyst rate. CONCLUSION: In vitro matured mouse oocytes showed quite different spindle positions compared with in vivo matured oocytes. Moreover, in vivo matured oocytes cultured for 24 h had a spindle position distribution that was similar to that of in vitro matured oocytes. The different spindle positions observed in in vivo and in vitro matured oocytes may reflect differences in their cytoplasmic maturation processes. These findings have implications regarding the lower developmental competency of in vitro matured oocytes.
Key words: in vivo and in vitro matured oocytes/meiotic spindle/polscope
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Introduction |
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The intracellular bipolar barrel-shaped microtubule spindles that form during meiosis play an essential role in accurate chromosome segregation. Spindle locations and their morphology can be identified using a polscope. Since meiotic spindles in living oocytes are highly birefringent, their presence and structures can be viewed non-invasively by using a polscope. Recent reports indicate that birefringent spindle image analysis allows us to evaluate spindle positions in living oocytes and to compare these findings with developmental competency after fertilization (Wang et al., 2001a
,b). We and others have reported that human metaphase II (MII) oocytes exhibit various spindle positions with respect to the first polar body (PB), and that oocytes without a birefringent spindle image have low developmental competency (Wang et al., 2001b; Moon et al., 2003). Thus, spindle positions in MII oocytes may be used to predict successful fertilization and improve developmental competency during assisted reproduction treatment (ART). Rienzi et al. (2003) suggested that PB displacement during manipulations for cumulus removal results in various spindle relocations angled from the first PB in human oocytes. They explained that these various spindle locations are the result of the strong mechanical deformation that occurs during the pipetting process required for corona radiata removal. Furthermore, it was reported that the majority of in vitro matured oocytes exhibiting aberrant chromatin configurations were associated with morphologically viable oocytes (Racowsky and Kaufman, 1992), and all in vitro matured oocytes without birefringent spindles had abnormal microtubule organization and abnormal chromosome alignment (Wang and Keefe, 2002). In the present study, we investigated whether the in vitro maturation process could be related to their spindle positioning, subsequent fertilization and the development of mouse oocytes. A polscope was used to determine meiotic spindle positions in living mouse MII oocytes matured either in vivo or in vitro.
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Materials and methods |
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Experiment 1: comparison of spindle positions and the developmental competencies of in vivo and in vitro matured mouse oocytes
Collection of in vivo and in vitro matured mouse oocytes. B6CBAF1 female mice at 5 weeks of age were superovulated by injecting 5 IU of pregnant mare's serum gonadotrophin (PMSG). For in vivo maturation, 5 IU of HCG was injected 46–48 h later, and then cumulus-enclosed oocytes were collected from oviducts 17 h later into Ham's F-10 medium containing 10% human follicular fluid. Cumulus removal was safely performed by gentle pipetting in 0.01% hyaluronidase using a series of finely drawn glass Pasteur pipettes. For in vitro maturation, immature oocytes with cumulus were collected from oviducts 46–48 h after PMSG injection by puncturing the ovarian follicles (2–5 mm in diameter) with a 19 gauge needle and placed in Ham's F-10 medium containing 10% human follicular fluid. Cumulus removal was performed as described above, and cumulus-denuded germinal vesicle (GV) oocytes were incubated for 17 h in YS medium (Yoon et al., 2001
) containing 30% human follicular fluid, 1 IU/ml FSH (IVF-C:LG Chemicals, Seoul, Korea), 10 IU/ml HCG (IVF-C:LG Chemicals) and 10 ng/ml recombinant human epidermal growth factor (rhEGF; Sigma Chemical Co., St Louis, MO) until the first PB extrusion. The human follicular fluid was prepared as described by Chi et al. (1998). Classification of MII oocytes according to spindle positions. Spindle positions in cumulus-denuded in vivo (n=101) and in vitro (n=77) matured mouse oocytes were visualized by a polscope (CRI, Cambridge, MA) at 37°C. Oocytes were divided into four categories according to the angle formed between a spindle position and PB in both in vivo and in vitro matured mouse oocytes; i.e. 0°, 0–90°, 90–180° and those showing no spindle image (Figure 1).
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Evaluation of fertilization and the development of in vivo matured and in vitro matured oocytes. Spermatozoa were obtained from male mice at 8 weeks of age. The vas deferens and cauda epididymides were dissected, and then the spermatozoa were released into 1 µl of Ham's F10 with 10% human follicular fluid for 30 min at 37°C. After dispersion, the concentration was adjusted to a final value of 1x106 spermatozoa/ml. Then in vivo and in vitro matured MII oocytes and capacitated spermatozoa were transferred into the YS medium containing 10% human follicular fluid for fertilization. After 4 h of culture with spermatozoa, the oocytes were washed and then co-cultured with human cumulus cells in YS medium containing 10% human follicular fluid until the blastocyst stage. Oocytes were judged to be fertilized by the presence of two uniform blastomeres with two definite nuclei and a second PB at 24 h after insemination (Figure 2A). The embryos were examined to the blastocyst stages for 4 or 5 days (Figure 2B).
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Experiment 2: assessment of the spindle positions of in vivo matured mouse oocytes with a 0° spindle after in vitro culture for 24 h and their developmental competency
In vivo matured mouse oocytes, as a new batch, were obtained to test whether an additional culture in vitro is associated with a change of their spindle positions as described for experiment 1. Sixty in vivo matured oocytes with a 0° spindle position were incubated for 24 h. Their spindle positions were then re-assessed, and their fertilization and blastocyst rates were evaluated.
Statistics
Statistical analysis was performed using the 
2 test. A P-value of <0.05 was considered to be statistically significant.
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Experiment I
In the in vivo matured group, most oocytes (89.1%, 90 out of 101) had a spindle located beneath the first PB (0°). Only 6% (six out of 101) and 3% (three out of 101) of these oocytes had a spindle positioned at 0–90° or 90–180° from the first PB, respectively. A birefringent spindle image was not observed in 2% (two out of 101). In contrast, in the in vitro matured group, the majority of oocytes (83.1%, 64 out of 77) had a spindle at 0–90° from the first PB, and only 6.5% (five out of 77) of these oocytes had a 0° spindle exactly under the first PB.
The rates of fertilization and of oocytes reaching the blastocyst stage were significantly higher in the in vivo matured group that in the in vitro matured group; 87.1% (88 out of 101) versus 64.9% (50 out of 77), and 76.1% (67 out of 88) versus 66.0% (33 out of 50), respectively (P<0.05).
Experiment II
Sixty oocytes in vivo matured oocytes with a spindle at 0° were cultured for 24 h from the time of collection to the first PB extrusion. We observed that 71.7% (43 out of 60) of these oocytes exhibited a spindle shift from 0° to 0–90°. The rates of fertilization and of blastocyst development of these oocytes were 43, and 0%, respectively.
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Discussion |
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Recent investigations have shown that spindle location does not always correspond to the position of the first PB in human and hamster oocytes (Silva et al., 1999
; Wang et al., 2001a). These studies utilized a polscope that can examine birefringent spindle images in living cells and continuously monitor changes in spindle architecture. We previously reported that human MII oocytes show variously angled spindle locations with respect to the first PB, and that the highest rates of fertilization and embryonic development during ICSI were obtained by avoiding spindle damage in human MII oocytes (Moon et al., 2003). In the present study, we compared spindle locations in in vivo and in vitro matured MII mouse oocytes with first PB extrusion to investigate whether in vitro maturation alters the cytoskeletal structure related to cytoplasmic maturity and developmental competency. We observed different spindle positional distributions in in vivo and in vitro matured oocytes, which may reflect differences in their cytoplasmic maturities, even though the two were MII stage with the complete first PB extrusion. We speculate that these various spindle locations are unlikely to be the result of displacements caused by the cumulus removal process. It has been reported that a deviation of a spindle from the PB is the result of the misalignment of PB displacement during the process of cumulus removal by pipetting (Rienzi et al., 2003). In contrast, another researcher pointed out that the distance between the PB and the meiotic spindle cannot be explained completely by an angular difference caused by cumulus denudation (Hardarson et al., 2000). Our present study supports the notion that mechanical manipulation cannot account for the various spindle locations observed in MII oocytes. However, for in vitro matured mouse oocytes, which underwent mechanical pipetting at the GV stage not MII, they also had their spindles located at various degrees away from the PB. We observed that most in vivo matured oocytes (89.1%) had a spindle location exactly beneath the PB, whereas the majority of in vitro matured oocytes had a 0–90° spindle location. We postulated that the spindle displacement is a time-dependent process that involves a deficiency in GV oocyte maturation by unknown factors that cause the observed differences between in vivo and in vitro conditions. Another explanation is that spindle locations result from the dynamic movements of the oolemma after the first PB extrusion. Unfortunately, use of a polscope results in a fixed image in time. Thus, in order to study changes in dynamic processes such as those occurring in spindle morphology and position during oocyte maturation, time-lapse photography is required. If a time-lapse video recording could be made, it would provide concrete evidence of spindle movement in the cortical area during oocyte maturation following PB extrusion.
Developmental competency was higher in in vivo matured oocytes than in in vitro matured oocytes according to the present experiment. The possibility cannot be excluded that cytoplasmic maturity affects subsequent embryo development. It has been shown that oocytes or embryos of poor quality or with chromosomal abnormalities after in vitro maturation have a lower ART success rate (Gras et al., 1992; Racowsky and Kaufman, 1992), although higher success results have been reported (Chian et al., 2000; Son et al., 2001). In vitro maturation systems can induce an asynchrony in the progression of nuclear and cytoplasmic maturation, which results in embryos with reduced developmental potential (Trounson et al., 2001). In addition, it has been reported that diminished constitutive proteins of microtubule-organizing centres (MTOCs) were observed more so in in vitro matured oocytes than in in vivo matured oocytes, which suggests a fundamental distinction in the regulation of microtubules between the cytoplasm of in vivo and in vitro matured oocytes (Sanfins et al., 2003). When in vivo matured oocytes with a 0° spindle location were incubated for 24 h for the complete extrusion of the first PB, a shift of spindle location was observed from 0° to 0–90° in the majority. Moreover, subsequent embryonic development was poor in this oocyte group. This observation suggests that prolonged in vitro culture of in vivo matured oocytes causes cytoplasmic ageing which is accompanied by a change in spindle location. Interestingly, the resultant distribution of spindle locations of in vivo matured oocytes cultured in vitro was similar to those of in vitro matured oocytes, which indicates that spindle location may be a marker of cytoplasmic condition. Considering the findings of our second experiment, different spindle locations in in vivo and in vitro matured oocytes may reflect oocyte ageing. Since in vitro matured oocytes showed spindle distributions similar to 1-day-old in vivo matured oocytes, they may have experienced premature cytoplasmic ageing.
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This study was supported by the research fund of the College of Medicine, Seoul National University (Grant No. 8002002116).
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References |
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Submitted on August 11, 2004; resubmitted on March 23, 2005; accepted on April 1, 2005.
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