Using sea urchin gametes and zygotes to investigate centrosome duplication

Abstract

Centriole structure and function in the sea urchin zygote parallel those in mammalian somatic cells. Here, I briefly introduce the properties and attributes of the sea urchin system that make it an attractive platform for the study of centrosome and centriole duplication. These attributes apply to all echinoderms readily available from commercial suppliers: sea urchins, sand dollars, and starfish. I list some of the practical aspects of the system that make it a cost- and time-effective system for experimental work and then list properties that are a "tool kit" that can be used to conduct studies that would not be practical, or in some cases not possible, with mammalian somatic cells. Since centrioles organize and localize the pericentriolar material that nucleates the astral arrays of microtubules (Bobinnec et al. in J Cell Biol 143(6):1575-1589, 1998), the pattern of aster duplication over several cell cycles can be used as a reliable measure for centriole duplication (Sluder and Rieder in J Cell Biol 100(3):887-896, 1985). Descriptions of the methods my laboratory has used to handle and image echinoderm zygotes are reviewed in Sluder et al. (Methods Cell Biol 61:439-472, 1999). Also included is a bibliography of papers that describe additional methods.

Keywords: Centriole; Centrosome; Duplication; Echinoderm; Egg; Sea urchin; Sperm; Zygote.

Fig. 1

Ultrastructure of sea urchin sperm basal bodies and distal centrioles. a Longitudinal section of a sperm head. At the base of the head one can see the basal body—flagellar apparatus, the mitochondrion, and the short distal centriole lying between the mitochondrion and the nucleus. Here, the distal centriole in tangential section appears as an electron-dense patch to the left of the basal body (arrow). Scale bar 1 μm. b Longitudinal section of the head of a sperm treated with gluconate-glycine buffer. The dense cap on the proximal end of the basal body is located where it is mechanically attached to the hof in the base of the nucleus. The mitochondrion has moved to the side of the nucleus along with the distal centriole seen in longitudinal section (arrow). Scale bar 1 μm. c Cross-section of an isolated distal centriole. Scale bar 0.1 μm

Fig. 2

a Enucleated sea urchin zygote followed in vivo to characterize centrosome duplication. Each duplicated centrosome organizes a birefringent aster as visualized with a polarization microscope. The birefringence and appearance of the asters indicate that this enucleated zygote was in mitosis when the photograph was taken. The refractile sphere, slightly out of focus in the center of the cell, is a drop of the mineral oil used to cap the micropipette employed to enucleate the zygote. Scale divisions are 10 μm apart. b This particular zygote was recovered from the preparation, fixed, and serial semi-thick sectioned. Shown is a cross-section of a centriole in one of the centrosomes. The other centriole was found in a different section. Scale bar 0.1 μm

Fig. 3

Second mitosis in a Lytechinus pictus zygote. This image shows use of the polarizing microscope to image spindles and asters in living zygotes of a sea urchin that has optically clear eggs. The spindles are negatively compensated and appear dark. Quadrants of the asters are bright because the microtubules in those quadrants are oriented at right angles to those of the central spindle. Scale divisions are 10 μm apart

Fig. 4

Electron micrograph of a section through a pellet of distal centrioles isolated from sea urchin sperm. Scale bar 0.5 μm