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.
Gamete cells are electrogenic, i.e. capable of responding to electrical stimuli and modifying their electrical properties during the crucial periods of maturation and fertilization. Ion channels have been widely demonstrated on the plasma membrane of the oocyte and spermatozoon in all animals studied, and electrical modifications in gametes are due to ion currents that are modulated via these ion channels. The modification of intracellular calcium levels in gametes has been extensively studied, and these modifications are recognized to be a second messenger system for gamete maturation and fertilization. Other ions also move through the plasma membrane, either in association with or independent of calcium, and these generate typical features such as fertilization currents and oscillation of resting potential. These modifications were first studied in marine invertebrates, and the observations subsequently compared with mammalian systems, including human. The precise role played by these currents in the processes of maturation and fertilization is still poorly understood; however, recent research opens new frontiers for their clinical and technological application.
Purpose To determine the level of expression for mRNAs that regulate DNA repair activity in oocytes at the germinal vesicle (GV) stage. Reactive oxygen species (ROS) have been shown to play a major role in the appearance of deleterious DNA decays, and this study focuses on the repair of damage linked to decay caused by the action of ROS. The oocyte needs a mechanism for repairing DNA decays in the early preimplantation embryo before the onset of genomic activation, since in the absence of repair, residual DNA damage would lead to either apoptosis or tolerance. Tolerance of DNA damage is a source of potential mutations. Method GV oocytes were selected for this study, both for the ethical reason that they are unsuitable for patient treatment, and because no transcription takes place during the period from GV to MII and then prior to genomic activation. The GV oocyte is therefore a good model for looking at DNA during the first cleavages of early preimplantation development. Six cohorts of GV oocytes were pooled for extraction of mRNA; the DNA was analysed using Affimetrix HG-UG133 Plus 2, containing 54,675 probe sets; spike and housekeeping genes were also added as internal controls. Results In GV oocytes, DNA repair pathways for oxidized bases are redundant. One step repair procedure (OSR), BER (base excision repair), MMR (mismatch repair) and NER (Nucleotide excision repair) are present. All the recognition proteins are also present. The chromatin assembly factors necessary for the maintenance of genomic stability are highly expressed. Conclusion Gene expression analysis shows that the oocyte does not allow a high level of tolerance for DNA decays. This regulatory mechanism should avoid transmitting mutations into the next generation.
During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed. Review Oocyte maturationOogenesis is characterized by a unique process of cell division occurring only in gametes, called meiosis; whose goal is the production of haploid cells highly specialized for fertilization. In the majority of species the oocyte arrests in different stages of meiotic division, in particular, the block occurring in the first meiotic prophase (PI) marks the state of immature oocyte characterized by a prominent nucleus called the germinal vesicle (GV), which contains de-condensed transcriptionally active chromatin [[1] for a review]. As a general scheme, in response to a stimulus, meiosis is resumed and manifested by a germinal vesicle breakdown (GVBD), it then progresses to metaphase I (MI) or II (MII) where it undergoes a second arrest that is removed after successful fertilization.Oocyte maturation is usually defined as the period of progression from the first to the second meiotic arrest and involves coordinated nuclear and cytoplasmic modifications [2]. These are highly complex processes and their interplay is regulated by a series of sequential molecular events. Nuclear maturation starts with the GVBD, ends at the meiosis exit, and is marked by the presence of the two polar bodies. Cytoplasmic maturation is a more obscure process and involves both morphological and functional alterations related to: i) changes in the expression profile of cell cycle control proteins responsible for driving the oocyte towards developmental competencies [3][4][5]; ii) relocation of organelles [6][7][8]; iii) transcriptional modifications of mRNA [9], modification of the plasma membrane permeability [10,11]; iv) the differentiation of the calcium signalling machinery [12].Although the arrest at the PI stage seems to be strictly correlated with the oocyte growth, the meiotic stage correlated with fertilizable oocyte is species-specific. In some animals, oocytes are fertilized at the PI stage
The effects of bioactive aldehydes from diatoms, unicellular algae at the base of the marine food web, were studied on fertilization and early development processes of the ascidian Ciona intestinalis. Using whole-cell voltage clamp techniques, we show that 2-trans-4-trans-decadienal (DD) and 2-trans-4-cis-7-cis-decatrienal (DT) inhibited the fertilization current which is generated in oocytes upon interaction with the spermatozoon. This inhibition was dose-dependent and was accompanied by inhibition of the voltage-gated calcium current activity of the plasma membrane. DD and DT did not inhibit the subsequent contraction of the cortex. Moreover, DD specifically acted as a fertilization channel inhibitor since it did not affect the steady state conductance of the plasma membrane or gap junctional (GJ) communication within blastomeres of the embryo. On the other hand, DD did affect actin reorganization even though the mechanism of action on actin filaments differed from that of other actin blockers. Possibly this effect on actin reorganization was responsible for the subsequent teratogenic action on larval development. The effect of DD was reversible if oocytes were washed soon after fertilization indicating that DD may specifically target certain fertilization mechanisms. Thus, diatom reactive aldehydes such as DD may have a dual effect on reproductive processes, influencing primary fertilization events such as gating of fertilization channels and secondary processes such as actin reorganization which is responsible for the segregation of cell lineages. These findings add to a growing body of evidence on the antiproliferative effects of diatom-derived aldehydes. Our results also report, for the first time, on the action of a fertilization channel blocker in marine invertebrates.
By using the whole cell voltage-clamp technique, we studied changes in plasma membrane permeability at different meiotic stages of bovine oocytes. Follicular oocytes were matured in vitro and activated by Ca(2+) ionophore. Oocytes at germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), and meiosis exit were used for electrophysiological recording. By clamping the oocytes at -30 mV, we found that the L-type voltage-dependent Ca(2+) channels were active at the GV stage and that their activity decreased after the GVBD stage. Furthermore, the resting potential decreased from the GV to the MI stage and increased again at MII. A significant decrease of the steady-state conductance occurred from the GV to the MI stage, followed by a sharp increase at the MII stage. With the addition of organic L-type Ca(2+) channel blockers (nifedipine and verapamil), we inhibited the Ca(2+) currents. However, only in the case of verapamil was there a decrease of in vitro maturation efficiency. Our results suggest that, in addition to the cumulus-oocyte junctions, the plasma membrane channels provide another mode of Ca(2+) entry into bovine oocytes during meiosis.
In vivo bovine embryos were obtained by nonsurgical flushing of uterine horns of cows submitted to superovulatory treatment, while in vitro embryos were generated from oocytes collected from slaughtered donors. Lucifer Yellow injected into single blastomeres did not diffuse into neighboring cells until the morula stage in in vivo embryos and the blastocyst stage in in vitro embryos. In both cases diffusion was limited to a few cells. In contrast, diffusion was extensive in microsurgically isolated inner cell mass (ICM) but absent in the trophectoderm (TE). At the blastocyst stage, diffusion was always more extensive in in vivo than in in vitro embryos. Ultrastructural analyses confirmed these functional observations, and gap junction-like structures were observed at the blastocyst stage. These structures were diffuse in the ICM of in vivo embryos, scarce in the ICM of in vitro embryos and in the TE of in vivo embryos, and not observed in the TE of in vitro embryos. Blastomeres at all stages of development from the 2-cell stage to the blastocyst stage in in vitro embryos and at the morula and blastocyst stage in in vivo embryos were electrically coupled, and the junctional conductance (Gj) decreased in in vitro embryos from 4.18 +/- 1.70 nS (2-cell stage) to 0.37 +/- 0.12 nS (blastocyst stage). At each developmental stage, in vivo embryos showed a significantly (P < 0. 05) higher Gj than in vitro-produced embryos. Moreover, a significantly (P < 0.01) higher Gj was found in isolated ICM than in the respective blastocyst in both in vivo- and in vitro-produced embryos (3.5 +/- 1.4 vs. 0.7 +/- 0.3 and 2.6 +/- 1.6 vs. 0.37 +/- 0. 12 nS, respectively). The electrical coupling in absence of dye coupling in the early bovine embryo agrees with observations for embryos from other phyla. The late and reduced expression of intercellular communicative devices in in vitro-produced embryos may be one of the factors explaining their developmental low efficiency.
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.
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