Metamorphosis in the ascidian Ciona intestinalis is a very complex process which converts a swimming tadpole to an adult. The process involves reorganisation of the body plan and a remarkable regression of the tail, which is controlled by caspase-dependent apoptosis. However, the endogenous signals triggering apoptosis and metamorphosis are little explored. Herein, we report evidence that nitric oxide (NO) regulates tail regression in a dose-dependent manner, acting on caspase-dependent apoptosis. An increase or decrease of NO levels resulted in a delay or acceleration of tail resorption, without affecting subsequent juvenile development. A similar hastening effect was induced by suppression of cGMP-dependent NO signalling. Inhibition of NO production resulted in an increase in caspase-3-like activity with respect to untreated larvae. Detection of endogenously activated caspase-3 and NO revealed the existence of a spatial correlation between the diminution of the NO signal and caspase-3 activation during the last phases of tail regression. Real-time PCR during development, from early larva to early juveniles, showed that during all stages examined, NO synthase (NOS) is always more expressed than arginase and it reaches the maximum value at late larva, the stage immediately preceding tail resorption. The spatial expression pattern of NOS is very dynamic, moving rapidly along the body in very few hours, from the anterior part of the trunk to central nervous system (CNS), tail and new forming juvenile digestive organs. NO detection revealed free diffusion from the production sites to other cellular districts. Overall, the results of this study provide a new important link between NO signalling and apoptosis during metamorphosis in C. intestinalis and hint at novel roles for the NO signalling system in other developmental and metamorphosis-related events preceding and following tail resorption.

Using the whole-cell voltage clamp technique, the electrical changes in oocyte and embryo plasma membrane were followed during different meiotic and developmental stages in Ciona intestinalis. We show, for the first time, an electrophysiological characterization of the plasma membrane in oocytes at the germinal vesicle (GV) stage with high L-type calcium (Ca2+) current activity that decreased through meiosis. Moreover, the absence of Ca2+ reduced germinal vesicle breakdown (GVBD), which is consistent with a role of Ca2+ currents in the prophase/metaphase transition. In mature oocytes at the metaphase I (MI) stage, Ca2+ currents decreased and then disappeared and sodium (Na+) currents first appeared remaining high up to the zygote stage. Intracellular Ca2+ release was higher in MI than in GV, indicating that Ca2+ currents in GV may contribute to fill the stores which are essential for oocyte contraction at fertilization. The fertilization current generated in Na+ free sea water was significantly lower than the control; furthermore, oocytes fertilized in the absence of Na+ showed high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, suggesting that signaling pathways that mediate first cleavage do not rely on ion current activities. At the 8-cell stage embryo, a resumption of Na+ current activity and conductance occurred, without a correlation with specific blastomeres. Taken together, these results imply: (i) an involvement of L-type Ca2+ currents in meiotic progression from the GV to MI stage; (ii) a role of Na+ currents during electrical events at fertilization and subsequent development; (iii) a major role of plasma membrane permeability and a minor function of specific currents during initial cell line segregation events.

Electrophysiological techniques were used to study ion currents in the ascidian Ciona intestinalis oocyte plasma membranes during different stages of growth and meiosis. Three stages (A, B, C) of immature oocytes were discriminated in the ovary, with the germinal vesicle (GV) showing specific different features of growth and maturation. Stage A (pre-vitellogenic) oocytes exhibited the highest L-type Ca(2+)current activity, and were incompetent for meiosis resumption. Stage B (vitellogenic) oocytes showed Na(+) currents that remained high during the maturation, up to the post-vitellogenic stage C oocytes. The latter had acquired meiotic competence, undergoing spontaneous maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation plays a specific role in embryo development. Spontaneous maturation was inhibited at low pH whereas trypsin was able to trigger germinal vesicle breakdown (GVBD) regardless of pH; in addition spontaneous maturation was not affected by removal of follicle cells or by inhibiting junctional communication between oocyte and follicle cells. Taken together these results imply: (i) Ca(2+) and Na(+) currents are involved in meiotic progression, growth, and acquisition of meiotic competence; (ii) trypsin-like molecules may have a role as candidates for providing the physiological stimulus to resume meiosis. Finally, we provide evidence that follicle cells in Ciona are not involved in triggering GVBD as it occurs in other ascidians.

Growing oocytes are arrested at the first prophase of meiosis which is morphologically identified by the presence of a large and vesicular nucleus, called the germinal vesicle. The dissolution of the germinal vesicle marks the resumption of meiosis during which the oocyte undergoes massive modifications up to the second meiotic block, which is removed at fertilization. The interval between the first and the second meiotic block is defined as maturation and the events occurring during this period are crucial for ovulation, fertilization, and embryo development. Oocytes are excitable cells that react to stimuli by modifying their electrical properties as a consequence of ion currents flowing through ion channels on the plasma membrane. These electrical changes have been largely described at fertilization whereas little information is available during oocyte maturation. The aim of this review is to give an overview on the involvement of ion channels and ion currents during oocyte maturation in species from invertebrates to mammals. The results summarized here point to the possible functional role of ion channels underlying oocyte growth and maturation.

SUMMARYElectrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the postvitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous ''rosette'' embryos. Current amplitudes became negligible in embryos at the 2-and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.Mol.