CHANGES IN THE SPATIOTEMPORAL PATTERNS OF INTRACELLULAR CALCIUM TRANSIENTS DURING STARFISH EARLY DEVELOPMENT

In order to analyze the spatiotemporal patterns and possible biologica l significance of intracellular calcium transients during early develo pment, starfish oocytes were microinjected with calcium-sensitive fluo rescent probes and subjected to time-lapse imaging using confocal lase r scanning microscopy (CLSM) or conventional microscopy equipped for f ura-ratioing studies. Based on treatments with inositol 1,4,5-trisphos phate (IP3) or caffeine in the presence or absence of low-molecular-we ight heparin (a competitive inhibitor of IP3-sensitive receptors), pro phase-arrested oocytes were capable of mobilizing internal stores of b ound calcium via IP3-sensitive or IP3-insensitive receptors. The ampli tudes of the calcium transients elicited by caffeine or IP3 were simil ar to those obtained at fertilization. However, fertilization-induced transients were significantly more prolonged than were caffeine- or IP 3-mediated transients, which suggests that the kinetics of the calcium transients can be affected by the type of stimulatory agent. In proph ase-arrested oocytes that were treated with the maturation-inducing ho rmone 1-methyladenine (1-MA), distinct calcium transients were typical ly lacking prior to germinal vesicle breakdown (GVBD), indicating that nuclear disassembly can occur without marked calcium spikes during me iotic maturation. When subjected to monospermic fertilizations, howeve r, both prophase-arrested and maturing oocytes showed a single, prolon ged increase in free calcium that travelled in a wavelike fashion thro ughout the oocyte at similar to 5 mu m/s. Compared to the calcium wave s of normally developing specimens that were fertilized after GVBD, fe rtilization waves in prophase-arrested specimens decayed more rapidly and typically lacked a well defined wave front, indicating that the pr opagation patterns of the waves can differ depending on the state of o ocyte maturation. Within 90 min after fertilization, most starfish zyg otes began to display repetitive calcium oscillations in ''dual-channe l'' ratioed images obtained using dextran-conjugated forms of the calc ium-sensitive fluorochrome Calcium Green and the calcium-insensitive d ye Rhodamine. Each transient constituting these oscillations lasted si milar to 1-3 min and tended to be enhanced in the cortical cytoplasm. Calcium oscillations often preceded nuclear envelope breakdown (neb), anaphase onset, and cleavage furrow formation during the first cell cy cle and continued through the first five cleavages in specimens that d eveloped into normal blastulae. In oocytes microinjected with colchici ne to arrest cytokinesis, calcium transients also occurred in the abse nce of cell divisions, indicating that calcium transients can be uncou pled from cytokinesis. In addition, injections of low-molecular-weight heparin -- but not the control molecule de-N-sulfated heparin -- caus ed abnormal fertilization-induced calcium dynamics in a dose-dependent fashion and typically abolished marked postfertilization calcium osci llations, neb, and normal cleavage. Collectively, these imaging analys es reveal that the spatiotemporal patterns of intracellular calcium tr ansients can change during starfish early development and suggest that IP3-mediated calcium release is involved in producing a complex reper toire of Gee calcium elevations that may in turn help regulate normal development.