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a Department of Ob/Gyn, Women & Infants Hospital, Brown University, Providence, Rhode Island 02905
b Laboratory for Reproductive Medicine, Marine Biological Laboratory, Woods Hole, Massachusetts 02543
ABSTRACT
The newly developed Pol-Scope allows imaging of spindle retardance, which is an optical property of organized macromolecular structures that can be observed in living cells without fixation or staining. Experiments were undertaken to examine changes in meiotic spindles during the initial stages of activation of living mouse oocytes using the Pol-Scope. Parthenogenetic activation of oocytes treated with calcium ionophore evoked a dynamic increase in meiotic spindle retardance, particularly of the midregion, before spindle rotation and second polar body extrusion. The pronounced increase in spindle retardance, which could, for the first time to our knowledge, be quantified in living oocytes, was maintained during polar body extrusion. Spindle retardance of newly in vivo fertilized oocytes was significantly higher than that of ovulated, metaphase II oocytes. Pol-Scope imaging of fertilized oocytes did not affect subsequent development. These results establish that increased spindle retardance precedes polar body extrusion and pronuclear formation. The increased birefringence in the spindle provides an early indicator of oocyte activation. Thus, noninvasive, quantitative imaging of the onset of activation in living oocytes might improve the efficiency of assisted fertilization and other embryo technologies.
developmental biology, fertilization, IVF/ART, oocyte development, ovum
INTRODUCTION
Identifying whether an oocyte is in metaphase II (MII) arrest or the early phases of activation would be valuable for in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and cloning, because preactivated oocytes cannot be fertilized normally [1–4]. Mammalian ovulated oocytes are arrested in second meiotic metaphase, and release of meiosis is orchestrated by a biochemical cascade triggered by either fertilization or parthenogenetic activation [5–12]. Metaphase-II-arrested oocytes are characterized by active maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and cytostatic factor [5, 10–12]. Sperm penetration triggers intracellular calcium transients that initiate the events of oocyte activation [13, 14]. Initially, cyclin B is destroyed, and MPF is inactivated coincident with the transition of MII to anaphase II. Subsequently, the spindle rotates, and the second polar body is extruded. Finally, MAPK is inactivated, and this is associated with microtubular network formation, chromatin decondensation, and pronuclear development. Although these molecular events chart the progression from metaphase to interphase, such molecular indicators at present require fixation, staining, and fluorescence microscopy; therefore, they cannot be used to visualize the progression of activation in living, individual oocytes.
Oocyte activation is associated with several morphological changes, including cortical granule exocytosis, reorganization of the fine structure of the zona pellucida, extrusion of second polar body, and pronuclear formation [1, 5, 14]. During the transition from metaphase II to anaphase and telophase II, the spindle elongates, and the chromosomes separate. However, most of these morphological changes are difficult to observe without fixation, immunostaining, and fluorescence microscopy. Therefore, second polar body extrusion and/or pronuclear formation observed with conventional light microscopy have served as the criteria for successful oocyte activation in living oocytes. Unfortunately, these morphological events occur late in the progression of activation. Movements or subtle changes in the meiotic spindle, if they could be quantitatively imaged in living oocytes, would better reflect meiotic release and provide an early, vital indicator of activation.
In addition to experimentally induced activation, oocytes also can be activated "spontaneously" or by handling, temperature variation, hyaluronidase treatment, and aging [14]. A method for identifying whether an oocyte is in MII arrest or transitioning to anaphase or telophase II would allow for prompt decisions regarding whether artificial activation of oocytes was required after ICSI. Failure of oocyte activation has been the main cause of fertilization failure after ICSI, and subsequent or combined activation treatment has improved fertilization [15, 16]. In animal cloning using somatic cells, nonactivated MII oocyte cytoplasts have been used successfully for nuclear reprogramming [17–20], whereas preactivated cytoplasts have been inefficient for nuclear transfer [21, 22]. Noninvasive methods for oocyte imaging are especially important for clinicians working with limited, valuable material such as human eggs. Lastly, studies investigating the developmental physiology of activation also would benefit from a noninvasive, quantitative assay of activation that could be applied to living oocytes.
Polarized light microscopes observe birefringence, which is an optical property that results from molecular order and allows visualization of biological structures such as spindles [23, 24]. The Pol-Scope (www.cri-inc.com) measures birefringence as birefringent retardation, which is also called retardance. The working principles and application of birefringence imaging with the Pol-Scope have been recently described in detail elsewhere [25–27]. The Pol-Scope has two notable advantages for embryologists and developmental biologists. First, details of meiotic spindles can be visualized in living oocytes without the need to fix, stain, or label them. Second, unlike previous orientation-dependent, nonquantitative, polarizing light microscopes, the Pol-Scope allows orientation-independent, quantitative measurement of the birefringent properties of a biological specimen [25–27]. Therefore, the Pol-Scope is ideally suited to quantify dynamic biological events in living cells, such as the dynamic changes occurring in meiotic spindles during oocyte activation. The Pol-Scope already has been employed to image the zona pellucida and meiotic spindles of hamster oocytes [28, 29], in which it identified a birefringent, laminar structure of the zona pellucida and a variable location of the meiotic spindle relative to the first polar body. We sought to determine whether specific morphological changes in the spindle, before the second polar body extrusion and pronuclear formation, could be viewed quantitatively with the new Pol-Scope, and also whether these changes, if any, might provide an early indicator of meiotic release and oocyte activation. In this study, we employed the Pol-Scope to visualize spindle dynamics during parthenogenetic activation of mouse oocytes and, for the first time to our knowledge, to quantify the changes in birefringence of the spindle that could serve as an early indicator of activation in living oocytes.
MATERIALS AND METHODS
Reagents, Animals, Egg Recovery, and Culture
All reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless stated otherwise. Pregnant mare's serum gonadotropin (PMSG), which was used for superovulation, was purchased from Calbiochem (La Jolla, CA). B6C3F1, CF1, or SAM mice were used in the experiments. B6C3F1 or CF1 female mice at 6 wk of age were purchased from Charles River Laboratory (Boston, MA). SAM mice were provided by Dr. S. Suzuki (Kagoshima University, Japan). Mice were subjected to a 14L:10D cycle for at least 1 wk before use. Animals were cared for according to the procedures approved by the Marine Biological Laboratory and Women & Infants Hospital Animal Care Committees. Female mice were superovulated by i.p. injection of 7.5 IU of PMSG, which was followed 46–48 h later by injection of 7.5 IU of human chorionic gonadotrophin (hCG). Females were killed by cervical dislocation 14 h after hCG injection to collect young oocytes. Female mice were mated individually with fertile males when in vivo fertilized oocytes were needed. Aged oocytes were collected from female oviducts at 21 h after hCG injection. The ovulated oocytes enclosed in cumulus mass were released from the oviductal ampullae into the modified HEPES-buffered potassium simplex optimized medium (HKSOM) containing 14 mM HEPES and 4 mM NaHCO3 [30]. The cumulus cells were removed by gentle pipetting in HKSOM containing 0.03% (w/v) hyaluronidase. Cumulus-free oocytes were washed in HKSOM three times, washed again in pre-equilibrated modified KSOM three times, and then cultured in vitro in KSOM [31, 32], at 37°C in 7% CO2 and humidified air. Oocytes were activated by exposure to 5 µM calcium ionophore A23187 (A23187) in HKSOM for 3 min. After being washed, they were imaged using the Pol-Scope. Parthenogenetic activation of oocytes induced by artificial means allows for precise control of the timing of activation.
Pol-Scope Imaging and Quantitative Analysis
Metaphase II spindles of mouse oocytes were imaged using a Zeiss Axiovert 100 inverted microscope equipped with a Cohu analogue video camera and Pol-Scope hardware consisting of liquid crystals and electro-optical controller (Cambridge Research & Instrumentation, Boston, MA). Settings of the liquid crystals were computer controlled through MetaMorph Pol-Scope imaging software (Universal Imaging Corp., Boston, MA). Oocytes were imaged at 37°C in HKSOM in a plastic Petri dish with a cover glass bottom (MatTek Corp., Ashland, MA) or in a custom-fabricated cover-glass-bottom chamber. The chambers and microscope were enclosed in a custom-made, insulated, heated box for optimal thermal control. To compare quantitatively the differences in spindle retardance between oocytes at the MII stage (14.5 h after hCG injection) and those at the early fertilization stage (19 h after hCG injection and mating, when the second polar body and pronuclei were not observed) in SAM mice, spindle retardance was determined by thresholding the images to 1.25 nm, based on average background retardance, and then averaging over the entire spindle area or over a line scan across the middle of the spindle as calculated with MetaMorph Pol-Scope imaging software. In some experiments, nuclear DNA and meiotic spindles were confirmed by immunostaining and fluorescent microscopy as described elsewhere [11, 33].
Developmental Assessment of the Pol-Scope Imaged Eggs
We further tested whether development of oocytes or embryos was compromised by Pol-Scope imaging in a condition that included exposure to the HKSOM, 37°C temperature chamber, and atmospheric gas pressures for as long as 1.8 h in addition to the circular polarized light used for imaging. B6C3F1 mouse zygotes (Day 1) at 28–30 h after hCG injection and mating were imaged with the Pol-Scope under the same conditions as oocytes. Thirty-five percent of the zygotes were in the mitotic phase as determined by the appearance of a mitotic spindle. Zygotes were then cultured in vitro in KSOM and checked for cleavage to the two-cell stage at Day 2 and for development to blastocysts at Day 4. Zygotes not submitted to imaging served as developmental controls. In most cases, the total and apoptotic cell numbers in blastocysts were determined by staining with propidium iodide (50 g/ml; Molecular Probes, Eugene, OR) and the TUNEL assay with In Situ Cell Death Detection Kit (Boehringer Mannheim, Indianapolis, IN), as described elsewhere [34].
Statistical Analysis
Comparisons of treatment means were analyzed by ANOVA using the StatView software (SAS Institute, Cary, NC).
RESULTS
Comparison of Images Obtained with the Pol-Scope in Living Oocytes and Fluorescence Microscopy of Fixed and Stained Oocytes
The Pol-Scope allowed routine visualization of spindles in living oocytes without need for fixation or staining. As depicted in Figure 1A, the Pol-Scope retardance image showed details of the birefringent metaphase spindle fibers oriented parallel to the cortical membrane. The chromosomes were not birefringent and, therefore, appeared as dark regions amongst the light spindle fibers in the retardance image. With fluorescence microscopic imaging, the chromosomes were seen to be arranged over the middle of the MII spindle (Fig. 1B). More details of the spindle could be seen with fluorescence microscope, but the orientation of the spindles was probably altered during the mounting and pressing process. Although MII spindles of oocytes were observed with both the Pol-Scope and fluorescence microscopy, the oocyte was killed for fluorescence microscopy, whereas the oocyte depicted using the Pol-Scope was alive.
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Spindles Increase in Birefringence on Meiotic Release
To determine whether morphological features of the spindle, as viewed with the Pol-Scope, were indicative of meiotic release, oocytes collected from CF1 or B6C3F1 mice were activated with calcium ionophore A23187, washed, and then imaged with the Pol-Scope. Twenty-two oocytes were imaged 15–19 h after hCG injection in three independent batches. The dynamics of spindle birefringence appeared to be similar in both CF1 and B6C3F1 MII oocytes. We observed, for the first time to our knowledge, quantitative changes in spindle birefringence at the onset of activation in living oocytes. A consistent, evident increase in spindle midregion birefringence was seen within 20–30 min after calcium ionophore treatment. Representative images are shown in Figures 2 and 3, and quantification of these images is illustrated in Figure 4. The increased birefringence of the spindle midregion (i.e., the whitening in the image) could be quantitatively detected 10 min after ionophore treatment and before any other morphological features of activation (Fig. 4, A and B). Visually evident at 26 min, the increase in spindle birefringence continued to increase through 50 min. During anaphase, retardance of the midregion of spindles increased more than three fold, at a rate of 0.1 ± 0.004 nm/min (Figs. 2–4). The increased birefringence in the midregion of spindles was so dramatic (Fig. 2) that it required recompensation of the Pol-Scope retardance images to provide more finely detailed analysis of the changes in individual spindle fiber bundles (Fig. 3). The spindle coalesced from a structure with distributed birefringence (Figs. 3 and 4A) into highly birefringent fiber bundles that were seen as peaks in the quantified retardance (Figs. 3 and 4A). At 35 and 50 min, two cytoplasmic protrusions (indicated by arrows in the figures) were evident at both spindle poles. The spindle rotated to a position perpendicular to the membrane, and the polar body formed at 1 h and 35 min. Birefringence of the spindle remained elevated throughout spindle rotation, then began to decline after polar body formation (Figs. 2, 3, and 4B). The dramatic increase in spindle birefringence was observed during every case of calcium ionophore-induced oocyte activation.
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Using the Pol-Scope to observe activation in living oocytes, we correlated the increase in spindle birefringence with other cytological events. Separation of the chromosomes occurred coincident with the increase in spindle midregion birefringence (Figs. 3 and 4), and birefringence of the spindle midregion became more pronounced as the chromosomes separated (Figs 3–5). The chromosomes began to separate 7 min after ionophore treatment, and they had markedly separated by 26 min (Fig. 5). Although chromosomal separation could be seen in the previously described CF1 mouse oocyte, we could not detect similar dynamics of chromosomes in B6C3F1 mouse oocytes because of the optical interference from cytoplasmic granules. However, the Pol-Scope allowed clear observation of spindle dynamics in living oocytes from both mouse strains.
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Spindle rotation and polar body extrusion occurred after the increased spindle birefringence. Initially, the spindle was oriented tangential to the plasma membrane, and during the increase in birefringence, it appeared to jostle laterally between two small, cytoplasmic protrusions (Figs. 2 and 5) before rotating nearly 90° to an orientation perpendicular to the plasma membrane (Figs. 2–4). Then, the spindle rotated toward one growing protrusion as the other protrusion regressed. The increased spindle birefringence had almost reached its maximum before the onset of spindle rotation (Fig. 4, B and C). The spindle rotated at a speed of 1.1 ± 0.5°/min (n = 3). Finally, a decline in spindle birefringence was associated with cytokinesis and polar body extrusion (Figs. 2F and 4, B and C).
Increases in Spindle Birefringence Precede Pronuclear Formation
Although we observed an increased spindle birefringence before polar body extrusion on parthenogenetic activation induced by calcium ionophore, we sought to determine whether oocytes fertilized in vivo also exhibited spindles with elevated birefringence. Oocytes from both unmated and mated SAM mice were collected, imaged using the Pol-Scope, and allowed to develop in individual microdrops. Pol-Scope images of spindles were then retrospectively divided into two groups: those associated with oocytes that subsequently exhibited two pronuclei, and those associated with oocytes that subsequently remained as MII oocytes. Spindles of early, in vivo fertilized oocytes exhibited greater birefringence than the unfertilized oocytes (P < 0.001, Table 1 and Fig. 6). Oocytes with spindles exhibiting elevated birefringence developed pronuclei, whereas oocytes with spindles exhibiting low levels of birefringence remained as MII oocytes. The average retardance over the spindle of fertilized oocytes was significantly greater than that of unfertilized oocytes. Moreover, retardance of the spindle midregion of fertilized oocytes was more than twice that of unfertilized oocytes (P < 0.001). Retrospectively, all fertilized oocytes that subsequently developed pronuclei initially exhibited spindles with midregion birefringences above 2.5 nm, expressed as retardance, whereas unfertilized oocytes that remained at MII after 6 h of culture initially exhibited spindles with midregion birefringences below 1.6 nm (Fig. 6). Therefore, elevated spindle birefringence provided an early indicator of oocyte activation, as evidenced by subsequent pronuclear formation.
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Some Aged Oocytes Exhibit Abnormal Spindles
Aged oocytes exhibited disrupted spindles and abnormal birefringent structures that could be visualized with the Pol-Scope. Spontaneously activated, aged oocytes (20%) contained spindles with elevated birefringence (Fig. 7A). In some aged oocytes (16%), one or several crystal-like inclusions with high birefringence were also noticed (Fig. 7B). These inclusions did not appear to be microtubule aggregates, but their composition is unknown. In a few oocytes, the spindle either appeared to have partially disassembled or even disappeared without formation of pronuclei (Fig. 7, C and D).
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Imaging with the Pol-Scope Does Not Compromise Development
The Pol-Scope illuminates the preparation with 50–70 W of green (546 nm) circularly polarized light, whereas differential interference contrast (DIC) illuminates the preparation with 30–65 W of linearly polarized, broad-spectrum light containing potentially harmful red and infrared wavelengths. Because the light intensity is similar between Pol-Scope and DIC illumination and the Pol-Scope illumination excludes the short and long wavelengths used during DIC imaging, we anticipated the Pol-Scope would not compromise development of the preparation to be imaged.
Imaging either oocytes or cleaving zygotes did not compromise their subsequent development. Pol-Scope imaging of the mitotic spindle of cleaving zygotes did not alter the frequency of cleavage to the two-cell stage or of blastocyst development (P > 0.05; Fig. 8A). The number of total cells and of apoptotic nuclei did not differ between blastocysts developed from Pol-Scope-imaged cleaving zygotes and nonimaged control zygotes (P > 0.05; Fig. 8B). Similarly, oocytes imaged with the Pol-Scope cleaved and developed to blastocysts, and to term, with a frequency to that of the nonimaged control oocytes after IVF and embryo transfer [35]. Therefore, the Pol-Scope is truly benign, and imaging oocytes and embryos with this device does not compromise their subsequent development.
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DISCUSSION
Using the new Pol-Scope, we imaged and quantified the dynamics of meiotic spindles in living oocytes at near real-time rates. On activation, we observed increased birefringence of the spindle's midregion that preceded rotation of the spindle to a position perpendicular to the plasma membrane and polar body extrusion. In addition to calcium ionophore treatment and in vivo fertilization, we also used another effective agent, strontium, to activate MII mouse oocytes [36] and observed that birefringence of spindles also increased dramatically during the initial phases of strontium-induced activation (data not shown). Thus, the increase in spindle birefringence is universally associated with the initial phases of oocyte activation. Changes in meiotic spindle morphology have been reported previously based on fluorescent microscope imaging of sets of oocytes fixed at various time-points after activation and labeled immunocytochemically [11, 37–42]; however, such oocytes could not be recovered for subsequent development. Pol-Scope imaging avoids fixation, immunostaining, and fluorescent microscopy and, therefore, allows inspection of the spindle in unperturbed, individual eggs [35]. We determined that Pol-Scope imaging did not affect subsequent development, thus providing a safe and effective method for imaging meiotic and mitotic events in living oocytes and embryos. By employing the Pol-Scope to image both parthenogenetically activated and in vivo-fertilized mouse oocytes, we observed a novel feature of the spindle, an increase of retardance in the spindle's midregion, that can serve as an early indicator of oocyte activation.
Although conventional, orientation-dependent polarization light microscopes have been used for decades to image spindles [23, 26], quantification of the dynamics of birefringent structures has been limited, because the relationship between the orientation of the specimen and the plane of polarized light inevitably changes, thus nullifying the previous calibration and preventing quantitative comparison of sequential images [25, 43]. This is especially true during activation, when the orientation of the spindle changes both rapidly and dramatically. We report here the first use, to our knowledge, of an orientation-independent polarizing microscope, the Pol-Scope, to image the dynamics of the mammalian meiotic spindle in a quantitative fashion. Indeed, other investigators have described many of the same dynamics of oocyte activation described herein, including the cytoplasmic protrusions appearing at either spindle pole, the regression of one of these protrusions, and the rotation of the spindle toward the growing protrusion [37–39, 44, 45]. However, we identified an increased retardance of the spindle midregion before, during, and after spindle rotation, because the Pol-Scope allowed quantitative assessment of the spindle birefringence, regardless of the orientation of the living specimen.
The mechanism underlying the increased birefringence of the spindle midregion is not yet clear. Multiple factors could contribute to increased spindle birefringence. First, it may reflect changes in the microtubular structure of the spindle and arise either from an increased density of microtubules and/or alignment of elongating polar microtubules [46]. Acetylated microtubules accumulate in the anaphase spindle [37, 42, 45, 47] and, thus, also could contribute to the increased spindle retardance. The measured retardance (expressed as nm) is equal to the coefficient of birefringence multiplied by the thickness of the object; in turn, the coefficient of birefringence is directly proportional to the concentration of the birefringent material [43]. Before the thickness of the spindle remains constant within the relevant dimensions, changes in retardance perhaps represent changes in concentration of the microtubules. By visualizing dynamics in living oocytes, we observed that the onset of increased spindle birefringence coincided with the separation of the chromosomes. Treadmilling microtubules generate a pulling (or "contractile") force, and polymerizing or assembling microtubules generate a pushing (or "extensive") force [24]. Both forces might move the chromosomes and would be expected to increase spindle retardance. The increased birefringence also might arise from the actin filament ring, because actin filament bundles are birefringent [27] and because actin filament ring surrounds the spindle midregion during initial oocyte activation and, subsequently, forms the cleavage furrow during polar body extrusion [38]. Lastly, the increased birefringence might arise from edge birefringence of lipid vesicles translocated to the midregion in preparation for formation of the cleavage furrow [44, 46]. We propose that the initial increase of spindle birefringence in the midregion results from overlapping polar microtubules as well as from an increase in microtubule concentration as the chromosomes prepare for separation and spindle rotation. The later, elevated spindle birefringence may involve the microfilament ring and lipid vesicles, forming midbody structures, that contribute to polar body extrusion.
Using the Pol-Scope, we have identified abnormalities in spindle morphology and unusual, highly birefringent inclusions in aging and degenerative oocytes. Because aged oocytes exhibit a decline in MPF, which is indicative of "spontaneous" activation [11, 48], the increased spindle birefringence in some aged oocytes might relate to decreased MPF. Indeed, the onset of elevated spindle birefringence and chromosomal separation both coincide with the metaphase-anaphase transition, when MPF is inactivated [9, 10, 12]. Integrity of the metaphase spindles in oocytes is important for subsequent normal fertilization and embryo development. Interestingly, the spindle from different mouse strains varies in the frequency of disruption [49]. Pol-Scope imaging of the meiotic spindle may provide an efficient marker for selecting high-quality oocytes for IVF, ICSI, and cloning. Studies investigating the developmental physiology of activation also may benefit from this noninvasive, quantitative assay of activation applicable to living oocytes.
ACKNOWLEDGMENTS
We thank Dr. Shinya Inoue for his critical reading and evaluation of the manuscript and Mr. Douglas Bowman from UIC and Mr. Cliff Hoyt from CRI for technical assistance.
FOOTNOTES
First decision: 21 December 1999.
1 Supported in part by the National Institute of Health (NIH K081099) and Women & Infants Hospital/Brown Faculty Research Fund. 
2 Correspondence: David Keefe, Department of Ob/Gyn, Women & Infants Hospital, 101 Dudley St., Providence, RI 02905. FAX: 401 453 7500; dkeefe{at}smtp.wihri.org
Accepted: February 29, 2000.
Received: November 19, 1999.
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