Microtubule distribution and reorganization in the first cell cycle of fertilized eggs of Lytechinus pictus

Abstract

We present a detailed immunofluorescence study of the distribution of microtubules in Lytechinus pictus from fertilization until first cleavage, using an improved technique for extraction and fixation of sea urchin eggs. Eggs were prepared for fixation by brief treatment with a buffer selected for its ability to maintain mitotic spindle birefringence while extracting opaque cytoplasm. Subsequent glutaraldehyde fixation and borohydride treatment provided reliable preservation of microtubule arrays with very low background fluorescence. 4'-6-Diamino-2-phenylindole (DAPI) staining of the chromosomes allowed events of the chromosomal cycle to be related to those of the microtubule cycle. By sampling cells frequently between fertilization and first cleavage, we obtained good images of transitional stages between monaster, interphase asters, pause asters, and the mitotic spindle, as well as the changes in spindle structure during mitosis. These showed that: During the growth of the sperm aster, microtubules are present elsewhere in the cell. The monaster does not persist into interphase and divide, but rather breaks down simultaneously with the formation of the bipolar interphase array. During mitotic spindle formation fibers from the pause asters extend around the nuclear envelope, on the surface of which chromosomes occupy discrete sites. Upon nuclear envelope breakdown, these fibers penetrate the nuclear region as the chromosomes move to the metaphase plate, consistent with chromosomal capture by the forming spindle. During anaphase, the mitotic poles become hollow and elongated perpendicular to the long axis of the spindle, consistent with recent studies on centrosomal shape changes during mitosis.