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Hunter College, New York, NY Marine Biological Laboratory, Woods Hole, MA
* Corresponding author: cohen{at}genectr.hunter.cuny.edu
Polar body formation is of interest both as a fundamental process in sexual reproduction and as an extreme example of unequal cytokinesis in cell biology. Eggs of the surf clam Spisula solidissima, released in the germinal vesicle stage, are readily induced to form polar bodies by activation with KCl or by fertilization. However, although Spisula eggs are utilized in current studies of centrosomes and the cell cycle (e.g., 1), polar body formation in this model system is described only in older literature (2, 3). We have examined changes occurring in major cytoskeletal elements—microtubules and F-actin—in the stages immediately preceding formation of the first polar body. These stages are thought to be critical for docking of the meiotic spindle with the cell cortex, and for meiotic apparatus-cortex signaling that mediates the positioning and generation of the contractile ring. These processes occur by as yet unknown mechanisms.
In this study, confocal fluorescence microscopy was used to localize actin and tubulin relative to meiotic chromosomal stage. Ripe Spisula were obtained from the Aquatic Resources Division of the Marine Biological Laboratory (MBL) and maintained at 11–13 °C until used. Clams were opened by dissection, the gonad was removed, and the eggs were filtered through cheesecloth. Before use, the eggs were washed 3 times in 0.2-µm filtered seawater (FSW), and resuspended to a concentration of 1:10 (Veggs/VFSW). The eggs were activated, either by adding excess KCl to the seawater or by fertilization with freshly obtained sperm, according to standard methods for this species (4, 5). Time of KCl or sperm addition was recorded as t = 0. After germinal vesicle breakdown (GVBD), the eggs were resuspended to a concentration of 1:100 (Veggs/VFSW). Time-course samples, taken approximately every 2 min after GVBD, were prepared by simultaneous lysis and fixation of eggs in a medium consisting of 0.6% Brij-58, 4% formaldehyde in PEM (100 mM PIPES, 5 mM EGTA, 1 mM MgCl2, pH 6.8 using NaOH). After incubation for 1 h, the samples were washed in PEM and then stained. For F-actin, the eggs were stained with rhodamine or Alexa Fluor 568-phalloidin. Microtubules were stained with a 1:1 mass mixture of mouse monoclonal anti-
and anti-ß tubulins (Sigma T9026, T-4026) pre-labeled with Alexa fluor 488-labeled anti-mouse Fab fragments (ZenonTM, Molecular Probes), and chromosomes and nuclei were stained with DAPI. These procedures followed protocols developed in previous work on microtubule localization in a variety of cell types (6), and our measurements showed that normal Spisula egg diameters (
50–55 µm) were retained after such treatment. Confocal fluorescence microscopy of stained samples was performed using the Zeiss Laser Scanning System LSM 5 PASCAL, and images were processed with Zeiss LSM Image Examiner software.
Using the KCl-activation method at 23 °C, GVBD occurred in about 7 min, and was followed at approximately 13 min post-activation by the appearance of the first metaphase meiotic spindle. At first, the metaphase spindle was slightly eccentric (Fig. 1a). Subsequently, it moved toward the cell surface and approached the cortex while remaining in metaphase (Fig. 1b; 18 min). Microtubules of the peripheral aster, initially straight, were now observed to curve outward along the cell cortex, symmetrically away from a central microtubule-poor region (Fig. 1b, b' arrow). At 20 min the spindle entered anaphase (Fig. 1c), and a ring of thickened F-actin, 17–20 µm in diameter, then appeared in the cortex (Fig. 1d, d'). This ring surrounded a small circular cortical area, 7–9 µm in diameter, in which the F-actin was considerably thinner, creating a "bulls-eye" appearance in 3-D computer-generated rotated images (Fig. 1d', and online supplemental animation at www.mbl.edu/BiologicalBulletin/VIDEO/BB.video.html). The peripheral aster was now greatly diminished in size, and no longer visible along the cortex. Subsequently, it became apparent that the bulls-eye center of the F-actin ring (Fig. 1d, d') was the region through which the polar body nucleus and associated remaining centrosomal material passed (22–24 min post-activation; Fig. 1e). The first polar body appeared fully formed at about 26 min; it was enclosed by an actin-containing cortex that followed an outwardly and inwardly bulging contour (Fig. 1f). Results similar to those shown in Figure 1 were obtained with KCl-activated eggs from several different clams, and also with sperm-activated eggs. These observations are consistent with, and extend, older electron microscopic work on polar body formation in eggs of Spisula and Tubifex (2, 7) and are of value for comparison with current findings on unequal cell division in Saccharomyces cerevisiae and Caenorhabditis elegans (8, 9).
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It is evident that the sequence involves a mechanism for peripheral aster disassembly (Fig. 1d, inset), but such disassembly is delayed until after astral spreading along the cortex. In addition, the earlier pattern of contact between peripheral aster microtubules and the cortex, with its central microtubule-poor area (diagram b), is a close match to the "bulls-eye" pattern of thickened F-actin, with its central thinner area, which appears later (diagram d). The dimensions of the later pattern also correspond to those of the earlier aster (16–20-µm outer diameter, 7–9-µm inner diameter). Taken together, these observations strongly suggest that the signaling mechanism for contractile ring generation involves the astral microtubules.
We thank Kyeng-Gea Lee, Dr. David Burgess, Dr. Robert Palazzo, Dr. Shirley Raps, Ruben Pinkhasov, and Alex Braun, for assistance and helpful discussion. Support by the Howard Hughes Medical Institute Undergraduate Science Education Program in Biology (grant 52002679), and by PSC-CUNY64249 and NSF9726771, is gratefully acknowledged.
Literature Cited
This article has been cited by other articles:
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  R. M. Pielak, C. Hawkins, A. Pyie, J. Bautista, K.-G. Lee, and W. D. Cohen Polar Body Formation in Spisula Oocytes: Function of the Peripheral Aster Biol. Bull., August 1, 2005; 209(1): 21 - 30. [Abstract] [Full Text] [PDF]
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16 min post-activation. (b) Metaphase spindle at cell cortex with astral microtubules curving outward from central microtubule-poor region;