Role of cyclic AMP in the maturation of Ciona intestinalis oocytes.

Immature oocytes are arrested at prophase I of the meiotic process and maturation onset is indicated by oocyte nuclear disassembly (germinal vesicle breakdown or GVBD). Signaling pathways that elevate intracellular cyclic AMP (cAMP) may either prevent or induce oocyte maturation depending on the species. In some marine invertebrates and, in particular, in ascidian oocytes, cAMP triggers GVBD rather than blocking it. In this paper, we tested different cAMP elevators in fully grown oocytes at the germinal vesicle stage (GV) of the ascidian Ciona intestinalis. We demonstrated that through the activation of adenylate cyclase or the inhibition and phosphodiesterases the oocyte remained at the GV stage. This effect was reversible as the GV-arrested oocytes, rinsed and incubated in sea water, are able to undergo spontaneous maturation and extrusion of follicle cells. In addition, oocytes acquire the ability to be fertilized and start early development. However, morphology of follicle cells, embryos and larvae from in vitro matured oocytes showed different morphology from those derived from in vivo mature oocytes. The role and the transduction mechanism of cAMP in the regulation of oocyte maturation were discussed. Finally, we indicated a variation of biological mechanisms present in the ascidian species; moreover, we sustain evidence proving that tunicates share some biological mechanisms with vertebrates. This information provided new hints on the importance of ascidians in the evolution of chordates.

Ion current activity and molecules modulating maturation and growth stages of ascidian (Ciona intestinalis) oocytes.

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.

Ca2+ and Na+ current patterns during oocyte maturation, fertilization, and early developmental stages of Ciona intestinalis.

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.

Calcium currents correlate with oocyte maturation during the reproductive cycle in Octopus vulgaris.

Using the whole-cell voltage clamp technique, we have studied the Ca2+ currents and the steady-state conductance during different oocyte growth stages and during the reproductive cycle of the female of Octopus vulgaris. Evidence is presented that L-type Ca2+ currents are high in small pre-vitellogenic oocytes (80-150 microm diameter) and significantly lower in early vitellogenic oocytes (180-300 microm diameter). Similarly, a significant decrease of the steady-state conductance occurred from the pre to early- vitellogenic oocytes. Octopus oocytes showed larger Ca2+ currents in the reproductive rather than non-reproductive periods. These data indicates that ion and L-type Ca2+ currents play a role in oocyte growth and cytoplasmic maturation, and possibly in preparing the plasma membrane to the interaction with the spermatozoon. By using fluorescent microscopy, we show that oocytes from 80 to 400 microm diameter have the large germinal vesicle characteristic of the immature oocytes. In subsequent stages of growth (up to 1000 microm diameter) the nucleus is no more visible and the metaphase spindle appears. These data demonstrate that Octopus vulgaris oocytes are arrested in the first meiotic prophase up to the early-vitellogenic stage and resume meiosis at this stage up to a second block presumably in metaphase I. We discuss a possible role for progesterone as the hormonal stimulus for the first prophase-metaphase meiotic transition.

Developmental potential in bovine oocytes is related to cumulus-oocyte complex grade, calcium current activity, and calcium stores.

A morphological classification of the immature cumulus-oocyte complex (COC), which grossly resembled the atresia grade of its follicle source, was used in bovine oocytes to determine 1) the developmental potential by either in vitro fertilization or parthenogenetic activation, 2) the calcium current activity by whole-cell voltage clamp technique, and 3) the intracytoplasmic calcium stores by microfluorimetric evaluation. The COC classification took into account some cumulus and ooplasm features, designated as follows: A) presence of a clear and compact cumulus and translucent ooplasm, B) dark and compact cumulus and dark ooplasm, and C) dark and expanded cumulus and dark ooplasm. We found no difference between in vitro fertilization and parthenogenetically activated oocytes in terms of cleavage rate and blastocyst production. Both protocols indicated a significant variability between the three compared COC categories. The B-COCs showed the highest embryo production efficiency as well as the greatest Ca(2+) current activity, whereas A-COCs showed an opposite pattern. The C-COCs, mostly attributed to atretic and heavily atretic follicles, showed morphological characteristics between those of A- and B-COCs. Stores of Ca(2+) were significantly greater in A-COCs than in B- and C-COCs in the case of immature oocytes, and greater in B-COCs than in C-and A-COCs in the case of in vitro-matured oocytes. These results demonstrate that in the bovine 1) the considered morphological criteria for oocyte classification are related to developmental competence, 2) plasma membrane Ca(2+) current in the immature oocyte is related to developmental potential, and 3) calcium stores are related to morphological quality in immature oocytes and to developmental competence in mature oocytes.

Bioactive aldehydes from diatoms block the fertilization current in ascidian oocytes.

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.

Biochemical and functional characterization of protein kinase CK2 in ascidian Ciona intestinalis oocytes at fertilization. Cloning and sequence analysis of cDNA for alpha and beta subunits.

The ubiquitous and pleiotropic dual specificity protein kinase CK2 has been studied and characterized in many organisms, from yeast to mammals. Generally, the enzyme is composed of two catalytic (alpha and/or alpha') and two regulatory (beta) subunits, forming a differently assembled tetramer. Although prone to controversial interpretation, the function of CK2 has been associated with fundamental biological processes such as signal transduction, cell cycle progression, cell growth, apoptosis, and transcription. Less known is the role of CK2 during meiosis and the early phase of embryogenesis. In this work, we studied CK2 activity during oocyte activation, a process occurring at the end of oocyte maturation and triggered by fertilization. In ascidian Ciona intestinalis, an organism whose complete genome has been published recently, CK2 was constitutively active in unfertilized and fertilized oocytes. The enzymatic activity oscillated through meiosis showing three major peaks: soon after fertilization (metaphase I exit), before metaphase II, and at the exit from metaphase II. Biochemical analysis of CK2 subunit composition in activated oocytes indicated that CK2-alpha was catalytically active as a monomer, independently from its regulatory subunit beta; however, CK2-beta was only detectable in unfertilized oocytes where it was associated with a bona fide identified ascidian mitogen-activated protein kinase. After fertilization, CK2-beta was undetectable, suggesting its rapid degradation. Protein sequence analysis of CK2-alpha and -beta cDNA indicated a high identity compared with vertebrate homologs. In addition, the absence of putative phosphorylation sites for Cdc2 kinase on both alpha and beta subunits suggested an important role for CK2 in regulating meiotic cell cycle in C. intestinalis oocytes.