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Cortical granule translocation is microfilament mediated and linked to meiotic maturation in the sea urchin oocyte

Department of Molecular and Cell Biology & Biochemistry, 69 Brown Street, Box G, Brown University, Providence, RI 02912, USA

View larger version (78K): [in a new window]   Fig. 1. Oocyte maturation in vitro in sea urchins begins with movement of the germinal vesicle to an asymmetric (future animal pole) position (A), followed by germinal vesicle breakdown and release of first one polar body (B), then a second polar body (C). Frequent aberrations in maturation in vitro include generation of two pronuclei (D, egg matured in vivo; E, egg matured in vitro – note two pronuclei, each transcriptionally active for cleavage stage histone genes), and formation of enlarged pronucleus (F, compare enlarged pronucleus * to normal-sized pronuclei, arrows; all matured in vitro). Scale bars: in A, 25 µm for A-E; in F to 25 µm in F. gv, germinal vesicle; nco, nucleolus; pb(s), polar body(s); pn(s) pronucleus.

View larger version (42K): [in a new window]   Fig. 2. Regulation of oocyte maturation in vitro. Oocytes were cultured in vitro in the presence of reagents to examine the mechanisms of maturation (A). Reagents that increase the level of cAMP in a cell inhibit germinal vesicle breakdown (GVBD) (100 µg/ml dibutyrl cAMP, 125 µm IBMX; 100 µg/ml theophylline). Cells noted in recovery were treated for 12 hours with IBMX (125 µm) and dcAMP (100 µg/ml), and then washed into ASW so that in the 24 and 36 hour time points, 12 hours of this time was in the inhibitor. (B) Roscovitine at 0.1, 0.5, 2 µM; olomoucine at 10, 30 and 90 µM; LiCl, at 30 µM; and CF, coelomic fluid at 50%. Asterisk indicates significantly different from control values at P>0.01 confidence level determined by Student’s t-test analysis. (C) Control oocytes matured into eggs containing pronuclei (arrow), within 24 hours of incubation. (D) Sibling oocytes cultured for 24 hours in the presence of 100 µg/ml dibutyrl cAMP; note the distinct and centrally located germinal vesicle, arrow. (E) Sibling oocytes cultured for 24 hours in the presence of 0.1 µM roscovitine, note distinct but mis-shapened germinal vesicle, arrow. Scale bar: 50 µM.

View larger version (66K): [in a new window]   Fig. 3. Cortical granule translocation is linked to meiotic maturation. Immunolabeling cortical granules of a control cell (A) that has matured in vitro shows that nearly all of its cortical granules have translocated to the cortex. Its pronucleus, which is indicative of meiotic maturation, is evident as a uniform, 12 µm sphere, labeled with Hoechst (B). Cells inhibited from maturation by cAMP (C) or roscovitine (E) show prominent germinal vesicles with dispersed chromatin (D,F, respectively) and no translocation of cortical granules. A cell that did mature in the presence of dbcAMP (even though only a small percentage do, see Fig. 2) shows a normal looking cortical granule translocation (G) and a pronucleus (H). Scale bar: 25 µm. (I) Cortical granule translocation was quantitated by measuring immunolabeled cortical granules in eggs and oocytes cultured in vitro. Even though during some treatments a small percentage of the oocytes mature (see Fig. 2), those that do mature show translocation indistinguishable from control, whereas oocytes that have not matured by these same treatments also have no demonstrable translocation, indistinguishable from an oocyte freshly isolated from the ovary. None of the experimental treatments differ from the control at the P>0.05 confidence level by Student’s t-test analysis.

View larger version (33K): [in a new window]   Fig. 4. Cortical granule translocation is microfilament dependent. Quantitation of cortical granule location following germinal vesicle breakdown shows that disruption of microfilaments severely reduces translocation. (A) Quantitation of cortical granule translocation (%) in the presence of various inhibitors. Error bars indicate ±1 s.d. and the asterisks indicate significantly different from control values at P>0.01 (confidence level determined by Student’s t-test analysis). Treatment with either of two different microtubule inhibitors (nocodozole, C; colchicine, D) shows no significant difference in translocation from control cells (B), whereas treatment with any of three different microfilament inhibitors [cytochalasin D (E), cytochalasin B (data not shown), latrunculin A (F)] has a dramatic inhibitory effect. Recovery from treatment with cytochalasin D suggests that inhibition of translocation does not cause irreparable damage to the cell. Scale bar: 50 µm.

View larger version (99K): [in a new window]   Fig. 5. Actin filament formation during cortical granule translocation. Actin filaments were visualized and inferred by phalloidin staining followed by immunolabeling for cortical granules. Shown are early (A), mid- (B) and full- (C) sized oocytes, which have predominantly cortical actin labeling (oocyte in C is slightly compressed to better reveal the germinal vesicle). As oocytes begin meiotic maturation (D,E), the cortical granules move toward the cortex, the germinal vesicle becomes decidedly asymmetrical and phalloidin positive. The germinal vesicle then breaks down and matures to a haploid nucleus, while the cortical granules complete formation of the monolayer at the cortex. Although we can not detect individual microfilament bundles, cortical granule translocation coincides with microfilament formation in the germinal vesicle. Scale bar: 25 µm.

View larger version (64K): [in a new window]   Fig. 6. Microfilaments are functional in oocytes prior to GVBD and cortical granule translocation. Shown are oocytes treated for 30 minutes with the given reagent and then 10 minutes with FM1-43 (top) and DIC (bottom). Images were recorded by confocal microscopy. The plasma membrane is intensely labeled in all cells because of exposed surface area. Endocytosis is inhibited reversibly in latrunculin A and cytochalasin B, respectively, but no inhibition is seen in cells treated to disrupt microtubules (C). Scale bar: 25 µm. The endocytic vesicles are approximately 0.5 µm in diameter.

View larger version (55K): [in a new window]   Fig. 7. Cortical granules are not associated with structural microfilaments prior to translocation. Oocytes were subjected to isopycnic sucrose centrifugation to stratify organelles. (A) Immunolabeled cortical granules of a control cell (B) after high-speed stratification (5000 g). Note refractile band of cortical granules in B, corresponding to immunolabeled cortical granules in A. Same scheme for C,D, except that prior to stratification, cells were treated with cytochalasin D, resulting in a slightly bulged centrifugal end, sometimes exaggerated for many µm (data not shown). We interpret this disfiguration to reflect the loss of cortical microfilaments. (E) Immunolabel and (F) DIC image of low-speed spin (1500 g) in control (E,F), or (G,H) cytochalasin conditions. Conditions were sought that would maximize resolution of the cortical granule displacement, but no difference was seen. (I-L) Cells allowed to recover in control conditions after low-speed spin (I,K, recovered for 30 minutes) or from a high-speed spin (J,L, recovered for 2 hours). (Below) Graphical representation of cells represented above, error bars represent ±1 s.d.; scale bar: 50 µm. At least 10 cells were assessed for each condition. None of the experimental conditions produce result that are significantly different from control values at P>0.10 (confidence level determined by Student’s t-test analysis), but each of the recovery conditions from stratification is significantly different from the stratification alone (*), even in the presence of cytochalasin D.

View larger version (39K): [in a new window]   Fig. 8. Cortical granules associate with microfilaments at GVBD. Oocytes having undergone GVBD in vitro were subjected to stratification either in the absence (A-C) or presence (D-F) of cytochalasin D. Cortical granules were then immunolabeled and their position quantified. (A,D) immunolabeled cortical granules; (B,E) Hoechst label; (C,F) DIC images. Control oocytes are as in previous figures, prior to GVBD. Meiotic oocytes have undergone GVBD, although the precise stage of meiosis is not known. (G) Quantitation of multiple experiments; asterisks indicate changes that are significantly different from control oocytes at P>0.01 (confidence level determined by Student’s t-test analysis). Scale bar: 25 µm.