SEARCH

SEARCH BY CITATION

Keywords:

  • ascidian;
  • cyclic AMP;
  • follicle cell;
  • germinal vesicle;
  • oocyte

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Many ascidian oocytes undergo ‘spontaneous’ germinal vesicle breakdown (GVBD) when transferred from the ovary to normal pH 8.2 sea water (SW); however, low pH inhibits GVBD, which can then be stimulated while remaining in the low pH SW. Oocytes of Boltenia villosa blocked from GVBD by pH 4 SW undergo GVBD in response to permeant cyclic AMP (8-bromo-cyclic AMP), phosphodiesterase inhibitors (isobutylmethylxanthine and theophylline) or the adenylyl cyclase activator forskolin. This suggests that cAMP increases during GVBD. Removal of the follicle cells or addition of a protease inhibitor inhibits GVBD in response to raised pH but not to forskolin, theophylline or 8 bromo-cAMP. Isolated follicle cells in low pH SW release protease activity in response to an increase in pH. These studies imply that the follicle cells release protease activity, which either itself stimulates an increase in oocyte cAMP level or reacts with other molecules to stimulate this process. Studies with the mitogen-activated protein (MAP) kinase inhibitors U0126 and CI 1040 suggest that MAP kinase is not involved in GVBD. The Cdc25 inhibitor NSC 95397 inhibits GVBD at 200 nm in a reversible manner.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Oocytes have a large diploid nucleus, the germinal vesicle. Maturation, beginning with dissolution of the nuclear envelope (referred to as germinal vesicle breakdown [GVBD]) results in a cell capable of fertilization. In many invertebrates including ascidians, meiosis proceeds to first metaphase and maturation is arrested here until fertilization results in the completion of meiosis and the onset of mitosis. Ascidians, although invertebrates as adults, are considered stem chordates. Most chordate oocytes release the first polar body at ovulation and the eggs are fertilized at the second metaphase. Oocytes of vertebrates and asteroid echinoderms are stimulated to undergo GVBD by a hormone released by the follicle cells, maturation inducing substance (MIS). Vertebrate MIS is a steroid that is specific for each group (Byskov et al. 1999); in asteroids the MIS is 1-methyladenine. In most chordates GVBD is initiated by the synthesis of cyclin, which complexes with a cyclin-dependent kinase to form the active maturation promoting factor (MPF) (MPF operates in the interior of the cell, MIS at the cell surface). However, in some chordates and many echinoderms this is triggered by the dephosphorylation of a pre-existing MPF comprised of cyclin and a cyclin dependent kinase (Kishimoto 1999; Yamashita et al. 2000).

Vertebrate and asteroid oocytes are prevented from GVBD by an increase in intracellular cyclic AMP levels (Stricker & Smythe 2001 for review). In several invertebrates including ophiuroids (Yamashita 1988), hydrozoans (Freeman & Ridgway 1988; Takeda et al. 2006), nemerteans (Stricker & Smythe 2001) and bivalve mollusks (Yi et al. 2002), cAMP has the opposite effect: it stimulates rather than inhibits meiosis. Ascidians are invertebrates but also chordates and their oocytes are surrounded by follicle cells like those of most other chordates. Given the importance of ascidians in the evolution of the chordates and other deuterostomes and the contrary role of cAMP in many invertebrates compared with vertebrates, it is crucial to determine the role of follicle cells and cAMP in the maturation process in ascidian oocytes.

Boltenia villosa (Stimpson 1864) is a solitary pyurid ascidian abundant in the fouling community of San Juan Island, Washington. Like other ascidians, the oocytes form and grow within two layers of follicle cells, the outer and inner, and a single layer of test cells. Ovulation involves contraction of the outer follicle cells to free the oocytes from them (Burighel & Cloney 1997). The oocyte then has inner follicle cells (simply called follicle cells) outside the vitelline coat (VC) and a layer of test cells within the VC. Ovulation results in the accumulation of hundreds of oocytes within the ovaries. These oocytes all possess a large diploid nucleus: the germinal vesicle. Dissection of oocytes into normal pH 8.2 sea water (SW) results in rapid GVBD similar to many other ascidians (Hirai 1941; Sawada & Schatten 1989; Sakairi & Shirai 1991; Lambert 2005). Sakairi and Shirai (1991) have suggested that the ascidian MIS is a protease similar to trypsin, produced by the follicle cells in response to normal SW. They find that lower pH inhibits GVBD; Lambert (2005) reported that while pH 5 SW blocks GVBD in Halocynthia, pH 4 SW is needed for Herdmania oocytes. Although low pH blocks GVBD, it can still be activated by appropriate reagents even though oocytes are held at low pH (Sakairi & Shirai 1991; Lambert 2005).

This study investigates GVBD in Boltenia villosa oocytes to establish the importance of the follicle cells to GVBD and to determine the roles of cAMP, mitogen-activated protein (MAP) kinase and protein dephosphorylation in this process.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Animals and oocytes

Boltenia villosa were collected from floating pontoons in Friday Harbor, Washington and held in running sea water under constant illumination until use. A single individual was bisected through the siphons and each half was laid out in a Petri dish containing 0.01 M citrate pH 4.0 SW, and the branchial basket removed to expose the gonads. The gonads were then removed and chopped with scissors and the oocytes and sperm separated from tissue fragments and other debris by filtration through a 330-µm Nytex filter. Sperm and small oocytes were removed by washing five times in 100 mL of the low pH SW using gentle aspiration through a 100-µm Nytex screen to retain the oocytes. Self fertilization does not occur in Boltenia or other pyurid ascidians (Satoh 1994). Inhibitor studies included a 30-min incubation in the inhibitor before stimulating by adding the agonist or raising the pH. One hour after activation the oocytes were placed on microscope slides in 10-µL drops and gently flattened under a 12 × 12 mm #1.5 cover slip. This mild flattening allowed visualization of the germinal vesicle (GV) in these moderately opaque oocytes. GVs were not visible in fixed oocytes, so all experiments were carried out on living cells. At least 100 oocytes were analyzed. Each experiment was repeated at least six times using oocytes from three individuals. All experimental data were subjected to statistical evaluation using the Mann–Whitney non-parametric test (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, CA, USA).

Follicle cell isolation and protease assay

Removal of follicle cells was accomplished by a modification of a method by Fuke (1983): oocytes were placed in a 15-mL centrifuge tube of 0.56 M NaCl 1% ethylenediaminetetraacetic acid (EDTA) (4Na) pH 4 and shaken periodically for 30 min followed by 20 passages through a 163-µm Nytex filter. Follicle cells were then separated from oocytes by passage through a 100-µm Nytex filter and pelleted in a clinical centrifuge for 10 min and then re-suspended in pH 4 buffered SW. Half the follicle cells were then raised to pH 8.2 and both they and the untreated pH 4 SW oocytes were pelleted at 14 000 g for 4 min an hour later. A 50-µL aliquot of the supernatant was analyzed for protease activity using t-butoxycarbonyl (Boc)-Phe-Ser-Arg-MCA, a fluorogenic trypsin substrate. The buffer consisted of 100 mm Tris, 370 mm NaCl and 10 mm CaCl2 · 2H2O. The reaction volume was 0.5 mL of buffer containing 30 µg/mL substrate. After a 1-h incubation the reaction was stopped by the addition of 2 mL stop buffer (0.2 M Na2CO3). Fluorescence intensity was then determined with a Hoefer TKO 100 fluorometer (San Francisco, CA, USA) with an excitation of 365 nm and emission at 460 nm. Oocytes from a single individual were divided in half and one half defolliculated, the other half left intact and the experiments carried out in exactly the same way.

Chemicals

NCS 95397, theophylline, isobutylmethylxanthine (IBMX), Na citrate, soy bean trypsin inhibitor (SBTI), and protease substrate were from Sigma (St. Louis, MO, USA). Forskolin, U0126 and 8-bromo cAMP were from Tocris Bioscience (Ballwin, MO, USA). Stock solutions of hydrophilic molecules were made as a 10-mm solution in distilled water; for hydrophobic molecules, dimethylformamide or dimethylsulfoxide was used as a solvent. Neither solvent had an effect on oocytes at the concentrations used.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

GVBD is stimulated by direct application of permeant cAMP, inhibition of phosphodiesterases or stimulation of adenylyl cyclase

Boltenia oocytes normally undergo rapid GVBD when transferred from the ovary to normal pH 8.2 SW (Fig. 1). In early experiments oocytes were incubated in pH 4 SW containing increasing amounts of 8-bromo-cAMP and stimulated with mastoparan, A-23187 and dimethylbenzanthracene (Lambert 2005) in an attempt to inhibit GVBD, only to find that GVBD was activated by increasing the cAMP levels rather than inhibited, as with other chordates. Accordingly, pH 4 Boltenia oocytes were incubated in the permeant cAMP analog 8-bromo cAMP (4 mm) and the phosphodiesterase inhibitors theophylline (10 µm) and IBMX (50 µm) and all three treatments stimulated GVBD. Forskolin (100 µm) was used to activate adenylyl cyclase; this also stimulates GVBD in a dose-dependent manner. These data are all consistent with cAMP activating rather than inhibiting GVBD. In other experiments pH 8.2 SW, progesterone (2.5 µg/mL) and serotonin (5-hydroxytryptamine) (10 µm) all stimulated GVBD (Table 1). The working concentrations were determined in preliminary dose response curves for all agonists used (data not shown).

image

Figure 1. Germinal vesicle breakdown in Boltenia villosa oocytes dissected directly into normal pH 8.2 sea water (SW) at 13°C. Results are means and standard errors of six replicates using oocytes from three individuals. Oocytes were dissected into pH 8 SW and 10-µL aliquots counted at the designated times.

Download figure to PowerPoint

Table 1.  Activation of germinal vesicle breakdown (GVBD) in Boltenia villosa oocytes blocked in meiotic prophase by pH 4 sea water (SW)
AgonistpH 4 controlActivatedIncrease (%)nSignificance
  1. To 1-mL aliquots of oocytes the following agonists were added: 8 Br-cAMP (4 mm), theophylline (The) (10 µm), isobutylmethylxanthine (IBMX) (50 µm), forskolin (For) (100 µm), progesterone (Pro) (2.5 µg/mL), serotonin (Ser) (5 hydroxytryptamine) (10 µm), pH 8.2 (Tris added to bring pH to pH 8.2). After 1 h, 10-µL aliquots were withdrawn and the percentage of GVBD was determined (mean percentage GVBD ± SEM). n = number of replicates, Sig = Significance: *P < 0.05, **P < 0.01, ***P < 0.001 from Mann–Whitney U-test.

8 Br-cAMP29.57 ± 2.1855.78 ± 4.8988.614***
The28.75 ± 2.1945.5 ± 3.0658.212**
IBMX32.25 ± 3.4852.75 ± 4.4963.6 8*
For28.93 ± 1.3054.04 ± 2.5386.846***
Pro32.25 ± 1.7656.71 ± 2.6875.814**
Ser30.13 ± 2.2445.75 ± 1.2531.2 8*
pH 8.226.5 ± 1.5849.92 ± 3.4888.413***

Protease inhibitor blocks GVBD stimulated by increased pH but not by direct cAMP increases

First reported by Sakairi and Shirai (1991) and subsequently confirmed by Lambert (2005), the protease inhibitor SBTI blocks GVBD in response to increased pH. However, stimulation of an increase in cAMP by forskolin induced GVBD even in the presence of inhibitory concentrations of SBTI. This indicates that protease activity is important to the part of the pathway stimulated by increased pH but not that activated by increased cAMP levels (Fig. 2).

image

Figure 2. The effect of soybean trypsin inhibitor (SBTI) on germinal vesicle breakdown (GVBD) of Boltenia villosa oocytes. Oocytes were incubated in 0.2 mg/mL SBTI for 30 min followed by adding 100 µm forskolin or Tris to bring the pH to 8. After 1 h GVBD was determined in a 10-µL aliquot. Clear bars indicate forskolin (100 µm) or pH 8 activated pH 4 oocytes; Shaded bars indicate +0.2 mg/mL SBTI. *P < 0.05. Results are from six replicate experiments using oocytes from three individuals. The pH 4 bar is the control.

Download figure to PowerPoint

Removal of the follicle cells blocks GVBD in response to increased pH but not after direct stimulation by forskolin

Removing the follicle cells of Halocynthia roretzi oocytes inhibits GVBD in response to normal pH 8.2 SW (Sakairi & Shirai 1991) but not in another study where it had no effect. However, it is possible that the follicle cells had been removed after GVBD had already been triggered in the latter (Bates & Nishida 1998). The follicle cells were removed in the present study from freshly dissected Boltenia oocytes and GVBD stimulated by various methods; only pH-induced GVBD was inhibited in follicle-free oocytes (Fig. 3). The untreated pH 4 control values have been subtracted from the treated samples. In Halocynthia oocytes a cell-free supernatant of SW-stimulated oocytes was capable of inducing GVBD in other oocytes, but not after boiling or the addition of protease inhibitors (Sakairi & Shirai 1991). These findings suggested to them that a protease was produced from the follicle cells that induced GVBD.

image

Figure 3. The effect of follicle cell removal on germinal vesicle breakdown (GVBD) of Boltenia villosa oocytes. One-milliliter aliquots of oocytes were incubated for 1 h in the indicated agonists and GVBD determined in a 10-µL aliquot. Follicle cells were removed as stated in Materials and Methods. Each experiment used oocytes from the same individual for intact and follicle-free oocytes. 8 Br, 2 mm 8-bromo-cAMP; For, 100 µm forskolin; pH 8, tris added to bring pH to pH 8.2; Ser, 10 µm serotonin (5-hydroxytryptamine); The, 10 µm theophylline. Clear bars indicate intact oocytes, shaded bars indicate follicle cell-free oocytes. Removal of follicle cells resulted in a statistically significant (*P < 0.05) inhibition of GVBD only when triggered by pH 8.2 sea water (SW). The difference between intact and follicle-free oocytes stimulated with theophylline is not statistically significant. Results are from six replicate experiments using oocytes from three individuals; pH 4 control values subtracted.

Download figure to PowerPoint

Isolated follicle cells release protease activity in response to an increase in pH

In order to test the hypothesis that ascidian follicle cells release protease activity by an increase in pH, as would occur in transfer from the ovary to SW during spawning, Boltenia follicle cells were isolated in pH 4 SW, divided into two equal portions and one raised to pH 8.2. Protease activity in the supernatant was then measured using a fluorogenic substrate (Lambert et al. 2002). There was measurable protease activity in the supernatant of pH 4 follicle cells but an up to sevenfold increase in protease activity in a supernatant of the pH 8.2-treated cells (Fig. 4). The finding of appreciable protease release by pH 4 oocytes suggests that this may be responsible for the high levels of GVBD in inactivated, control oocytes.

image

Figure 4. Protease activity released from isolated follicle cells from Boltenia villosa oocytes. Follicle cells were isolated in pH 4 buffered sea water (SW) as stated in Materials and Methods. The follicle cells were then divided into two 0.5-mL aliquots and tris added to bring the pH to 8.2 in one of the samples. After 1 h the cells were removed by centrifugation and protease activity measured in a 50-µL sample added to 0.5 mL buffer/substrate. One hour later 2 mL stop buffer (0.2 M Na2CO3) was added. Fluorescence intensity was then determined with a Hoefer TKO 100 fluorometer. Results are from six replicate experiments using oocytes from three individuals (**P < 0.01). Protease activity in arbitrary fluorescence units from cleavage of t-butoxycarbonyl (Boc)-Phe-Ser-Arg-MCA, a fluorogenic trypsin substrate.

Download figure to PowerPoint

MAP kinase not required for GVBD

The MAP kinase cascade is involved in GVBD in frog oocytes (Haccard et al. 1995), but not those from starfish, nemerteans or the polychaete Chaetopterus (Eckberg 1997; Sadler & Ruderman 1998; Stricker & Smythe 2006a). In the present study, GVBD was activated with forskolin, and two MAP kinase inhibitors (U0126 and CI 1040) were tested at concentrations that inhibit other oocytes from completing GVBD (20 µm and 10 µm, respectively) with no inhibition of GVBD (data not shown).

Protein phosphatase inhibitor blocks GVBD in a reversible manner

The phosphatase Cdc25 dephosphorylates an inhibitory tyrosine phosphate (Y15) residue on Cdc2 to activate the MPF in organisms with pre-MPF (Borgne & Meijer 1996). Previously vitamin K3 was used to inhibit phosphatase activity in ascidian oocytes. This inhibitor blocked GVBD in Herdmania pallida and Cnemidocarpa irene oocytes but was unfortunately irreversible (Lambert 2005). It also caused cytoskeletal abnormalities as recently noted in nemertean oocytes (Stricker & Smythe 2006b). These results suggested that protein dephosphorylation was involved, but the irreversible nature of this inhibitor was troubling. NSC 95397 inhibits the phosphatase Cdc 25 rather specifically (Lazo et al. 2002) but also causes cytoskeletal abnormalities in nemertean oocytes while inhibiting GVBD at 20 µm (Stricker & Smythe 2006b). Concentrations as low as 200 nm inhibited GVBD reversibly in Boltenia oocytes even if they were not re-stimulated (Fig. 5). Furthermore, the low concentration of NSC 95397 did not cause any visible cytoskeletal abnormalities.

image

Figure 5. The effect of NSC 95397, a Cdc25 inhibitor, on germinal vesicle breakdown (GVBD) in Boltenia villosa oocytes. Results are from six replicate experiments using oocytes from three individuals. One-milliliter aliquots of oocytes were incubated in 200 nm NSC 95397 for 30 min and then forskolin was added to 100 µm. One hour later GVBD was determined in a 10-µL sample. The difference between forskolin induced GVBD is not statistically significantly different from that achieved after NSC 95397 washout. **P < 0.01. The pH 4 bar is the control.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Although direct evidence of increased cAMP levels in ascidian oocytes is lacking, activation by phosphodiesterase inhibitors or adenylyl cyclase activators, as well as cell permeant cAMP strongly suggests that increased cAMP stimulates GVBD in these oocytes as it does in several other invertebrate species including cnidarians (Freeman & Ridgway 1988; Takeda et al. 2006), brittlestars (Yamashita 1988), nemerteans (Stricker & Smythe 2001) and bivalve mollusks (Yi et al. 2002) rather than inhibiting the process as occurs in starfish, amphibians and many mammals (Stricker & Smythe 2001 for review). This is only the second record of a stimulatory function for cAMP in GVBD in any deuterostome, but the sixth record in any animal oocyte. More cases of cAMP stimulation of GVBD will surely be discovered if more marine invertebrate oocytes are examined. It has been suggested that oocytes that are released without follicle cells may be activated by cAMP, while those with a tight coupling to follicle cells might be inhibited by it (Stricker & Smythe 2006a). The finding that cAMP appears to induce GVBD in follicle enclosed ascidian oocytes is not contrary to this hypothesis because although follicle cells are present, they are outside the vitelline coat and test cells in ascidian oocytes and therefore, are not in close contact with the oocyte surface. The fact that deuterostome-protostome boundaries are not sacrosanct with regard to cAMP and its control of GVBD demonstrates that we still have a great deal to learn about the diversity of processes involved in egg maturation.

Follicle cells stimulate GVBD in starfish and vertebrate eggs by releasing a meiosis inducing substance (MIS) in response to other hormones. Removal of ascidian follicle cells only inhibits GVBD in response to increased pH but has no effect on GVBD directly stimulated by forskolin. Similar data are generated by incubation in SBTI supporting the Sakairi and Hirai (1991) hypothesis that the MIS is released by follicle cells and is a trypsin-like protease. They found that follicle cell-free oocytes failed to undergo GVBD in response to pH 8.2 SW, and a supernatant from oocytes undergoing meiosis in pH 8.2 SW stimulated GVBD in untreated pH 5 oocytes. Further, they found that boiling abolished the activity as did the addition of SBTI. In the present study it was found that isolated follicle cells released protease activity when the pH was raised from 4.0 to 8.2. This demonstrated directly the release of protease activity from follicle cells. The protease is the second enzyme released from ascidian oocyte follicle cells as they also release a glycosidase in response to sperm (Lambert et al. 1997; Lambert 2000). Eggs of the ascidian Halocynthia roretzi release a protease at fertilization that is involved with elevation of the egg coat; however, the source of the enzyme has yet to be determined (Yokosawa et al. 1989).

Although GVBD is considered to be the result of protease activity in the polychaetes Sabellaria (Peaucellier 1977) and Chaetopterus (Ikegami et al. 1976) and ascidian follicle cells release a protease, this does not necessarily verify that the protease is the ascidian MIS, as it could bind to the natural MIS receptor and modify it to activate. It could also activate a peptide that is the true MIS. Another possibility is the destruction of an ovarian inhibitor (Numakunai 2001). The studies reported here definitely support the follicle cells as the source of the MIS and it has been shown that they release protease activity. They could also release other proteases not detected in the present study's assay, and other substances could also be released in addition to the protease, as several other molecules including serotonin (5-hydroxytryptamine) and progesterone can also induce GVBD at very low concentrations. Serotonin induces GVBD in many invertebrate oocytes (Stricker & Smythe 2001 for review) and is produced by ascidians (Stach 2005). Progesterone is released by amphibian follicle cells to stimulate GVBD (Yamashita 1998) and ascidian gonads also produce progesterone (D’Aniello et al. 2003), but they lack classical nuclear steroid receptors (Sherwood et al. 2005). Therefore the true nature of ascidian MIS has not yet been fully elucidated.

Previous studies implicate a G protein and protein phosphorylation and dephosphorylation in GVBD of ascidian oocytes (Lambert 2005). In several systems the MAP kinase cascade is involved in GVBD (Haccard et al. 1995). The MAP kinase inhibitors U0126 and CI 1040 do not inhibit ascidian GVBD. In this regard the oocytes of ascidians resemble starfish (Sadler & Ruderman 1998) and nemerteans (Stricker & Smythe 2006a).

In animal oocytes GVBD is initiated by a complex kinase comprised of the cyclin dependent kinase (CDK) p34cdc2, which binds and is activated by cyclin B. Phosphorylation state controls its activity. In many organisms such as some amphibians cyclin binds to the kinase at the re-initiation of meiosis and in others such as starfish the cyclin has already bound the kinase before GVBD but remains inactive because of inhibitory phosphate groups (Yamashita et al. 2000 for review). Active p34cdc2/cyclin B induces maturation of the egg and is termed maturation promoting factor (MPF) (Eckberg 1988; Yamashita et al. 2000 for review). MPF is activated by MIS produced by follicle cells surrounding the oocyte.

Germinal vesicle breakdown occurs very rapidly after placing oocytes of the stolidobranch ascidians Halocynthia roretzi (Hirai 1941), Molgula manhattensis (Sawada & Schatten 1989), Herdmania pallida and Boltenia villosa in pH 8.2 SW (Lambert 2005 and the present study) and protein synthesis inhibitors do not inhibit it (Sakairi & Shirai 1991). This suggests that pre-MPF becomes activated rather than requiring synthesis of the cyclin. GVBD is a more leisurely process in the phlebobranch ascidian Ciona intestinalis, requiring over 1.5 h to complete (Marino et al. 1999; Cuomo et al. 2006; Prodon et al. 2006) rather than the few minutes required for the stolidobranchs. This implies that the phlebobranchs must synthesize cyclin before GVBD can begin. In vertebrates, Rana and many other amphibians require cyclin synthesis for GVBD, but Xenopus does not (Tanaka & Yamashita 1995). Thus even such closely related animals as anural amphibians can express both strategies for MPF activation. In organisms with pre-MPF, phosphatases such as Cdc25 are envisioned to dephosphorylate select phosphate residues activating the CDK (Borgne & Meijer 1996; Borgne et al. 1999). The phosphatase inhibitor vitamin K3 blocks Cdc25 activity and GVBD in starfish oocytes (Borgne & Meijer 1996) and also blocks GVBD in ascidian (Lambert 2005) and nemertean (Stricker & Smythe 2006b) oocytes. However, the effect of vitamin K3 is irreversible. The more specific vitamin K3 derivative NSC 95397 blocks GVBD at very low concentrations (200 nm) but after it is washed out GVBD still occurs. In in vitro studies NSC 95397 was irreversible (Lazo et al. 2002); however, in in vivo experiments it is reversible when used at very low concentrations. Therefore it is possible that some process other than dephosphorylation is being blocked and is also important in GVBD. Future studies will address this issue.

Here I have shown the importance of follicle cells in GVBD of ascidian oocytes. The follicle cells release protease activity, which has been speculated to be the ascidian MIS. Reagents that directly or indirectly increase cAMP levels in the oocyte stimulate GVBD without input from follicle cells. Activation of meiosis does not involve MAPK activity but does require protein phosphatase activity.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The director and staff of the Friday Harbor Laboratories have my thanks for providing laboratory space and valuable suggestions. I thank Steve Stricker for valuable suggestions on experiment design and the work in progress and manuscript and also the gift of several reagents. I am grateful to Kathy Foltz at the University of California Santa Barbara for providing laboratory space where some of the studies originated. Gretchen Lambert helped in the preparation of the manuscript. A grant from Cal ERFA helped to finance most of the supplies. Two anonymous reviewers are sincerely thanked for greatly improving the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • Bates, W. R. & Nishida, H. 1998. Developmental roles of nuclear complex factors released during oocyte maturation in the ascidians Halocynthia roretzi and Boltenia villosa. Zool. Sci. 15, 6976.
  • Borgne, A. & Meijer, L. 1996. Sequential dephosphorylation of p34cdc2 on Thr-14 and Tyr-15 at the prophase/metaphase transition. J. Biol. Chem. 271, 27 84727 854.
  • Borgne, A., Ostvold, A. C., Flament, S. & Meijer, L. 1999. Intra-M phase-promoting factor phosphorylation of cyclin B at the prophase/metaphase transition. J. Biol. Chem. 274, 11 97711 986.
  • Burighel, P. & Cloney, R. A. 1997. Urochordata: Ascidiacea. In Microscopic Anatomy of Invertebrates (eds F. W.Harrison & E. E.Ruppert), pp. 221347. Wiley-Liss Inc., New York.
  • Byskov, A. G., Andersen, C. Y., Leonardsen, L. & Baltsen, M. 1999. Meiosis activating sterols (MAS) and fertility in mammals and man. J. Exp. Zool. 285, 237242.
  • Cuomo, A., Silvestre, F., De Santis, R. & Tosti, E. 2006. Ca2+ and Na+ current patterns during oocyte maturation, fertilization, and early developmental stages of Ciona intestinalis. Mol. Reprod. Dev. 75, 501511.
  • D’Aniello, A., Spinelli, P., De Simone, A. et al . 2003. Occurrence and neuroendocrine role of d-aspartic acid and N-methyl-D-aspartic acid in Ciona intestinalis. FEBS Lett. 552, 193198.
  • Eckberg, W. R. 1988. Intracellular signal transduction and amplification mechanisms in the regulation of oocyte maturation. Biol. Bull. 174, 95108.
  • Eckberg, W. R. 1997. MAP and cdc2 kinase activities at germinal vesicle breakdown in Chaetopterus. Dev. Biol. 191, 182190.
  • Freeman, G. & Ridgway, E. B. 1988. The role of cAMP in oocyte maturation and the role of the germinal vesicle contents in mediating maturation and subsequent developmental events in hydrozoans. Roux Arch. Dev. Biol. 197, 197211.
  • Fuke, M. 1983. Self and non-self recognition between gametes of the ascidian, Halocynthia roretzi. Roux Arch. Dev. Biol. 192, 347352.
  • Haccard, O., Lewellyn, A., Hartley, R. S., Erikson, E. & Maller, J. L. 1995. Induction of Xenopus oocyte meiotic maturation by MAP kinase. Dev. Biol. 168, 677682.
  • Hirai, E. 1941. An outline of the development of Cynthia roretzi Drasche. Sci. Rep. Tohoku Imp. Univ., Biol. 16, 257261.
  • Ikegami, S., Okada, T. S. & Koide, S. S. 1976. On the role of calcium ions in oocyte maturation of the polychaete Chaetopterus pergametaceus. Dev. Growth Differ. 18, 3344.
  • Kishimoto, T. 1999. Activation of MPF at meiosis re-initiation in starfish oocytes. Dev. Biol. 214, 18.
  • Lambert, C. C. 2000. Germ-cell warfare in ascidians: sperm from one species can interfere with the fertilization of a second species. Biol. Bull. 198, 2225.
  • Lambert, C. C. 2005. Signaling pathways in ascidian oocyte maturation: effects of various inhibitors and activators on germinal vesicle breakdown. Dev. Growth Differ. 47, 265272.
  • Lambert, C. C., Goudeau, H., Franchet, C., Lambert, G. & Goudeau, M. 1997. Ascidian eggs block polyspermy by two independent mechanisms: one at the plasma membrane, the other involving the follicle cells. Mol. Reprod. Dev. 48, 137143.
  • Lambert, C. C., Someno, T. & Sawada, H. 2002. Sperm surface proteases in ascidian fertilization. J. Exp. Zool. 292, 8895.
  • Lazo, J. S., Nemoto, K., Pestell, K. E. et al . 2002. Identification of a potent and selective pharmacophore for Cdc25 dual specificity phosphatase inhibitors. Mol. Pharmacol. 61, 720728.
  • Marino, R., De Santis, R., Giuliano, P. & Pinto, M. R. 1999. Follicle cell proteasome activity and acid extract from the egg vitelline coat prompt the onset of self-sterility in Ciona intestinalis oocytes. Proc. Natl Acad. Sci. USA 96, 96339636.
  • Numakunai, T. 2001. Oocyte maturation and self-sterility by treatment with ovary extracts of the ascidian, Halocynthia roretzi. In The Biology of Ascidians (eds H.Sawada, H.Yokosawa & C. C.Lambert), pp. 117124. Springer-Verlag, Tokyo.
  • Peaucellier, G. 1977. Initiation of meiotic maturation by specific proteases in oocytes of the polychaete annelid Sabellaria alveolata. Exp. Cell Res. 106, 114.
  • Prodon, F., Chenevert, J. & Sardet, C. 2006. Establishment of animal-vegetal polarity during maturation in ascidian oocytes. Dev. Biol. 290, 297311.
  • Sadler, K. C. & Ruderman, J. V. 1998. Components of the signaling pathway linking the 1-methyl adenine receptor to MPF activation and maturation in starfish oocytes. Dev. Biol. 197, 2538.
  • Sakairi, K. & Shirai, H. 1991. Possible MIS production by follicle cells in spontaneous oocyte maturation of the ascidian, Halocynthia roretzi. Dev. Growth Differ. 33, 155162.
  • Satoh, N. 1994. Developmental Biology of Ascidians. Cambridge University Press, Cambridge, UK.
  • Sawada, T.-O. & Schatten, G. 1989. Effects of cytoskeletal inhibitors on ooplasmic segregation and microtubule organization during fertilization and early development in the ascidian Molgula occidentalis. Dev. Biol. 132, 331342.
  • Sherwood, N. M., Adams, B. A. & Tello, J. A. 2005. Endocrinology of protochordates. Can. J. Zool. 83, 225255.
  • Stach, T. 2005. Comparison of the serotonergic nervous system among Tunicata: implications for its evolution within Chordata. Org. Divers. Evol. 5, 1524.
  • Stimpson, W. 1864. Description of new species of marine Invertebrata from Puget Sound, collected by the naturalists of the North-west Boundary Commission. Proc. Acad. Nat. Sci. Philadelphia 16, 153161.
  • Stricker, S. A. & Smythe, T. L. 2001. 5-HT causes an increase in cAMP that stimulates, rather than inhibits, oocyte maturation in marine nemertean worms. Development 128, 14151427.
  • Stricker, S. A. & Smythe, T. L. 2006a. Differing mechanisms of cAMP-versus seawater-induced oocyte maturation in marine nemertean worms. I. The roles of serine/threonine kinase and phosphatases. Mol. Reprod. Dev. 73, 15781590.
  • Stricker, S. A. & Smythe, T. L. 2006b. Differing mechanisms of cAMP-versus seawater-induced oocyte maturation in marine nemertean worms. II. The roles of tyrosine kinases and phosphatases. Mol. Reprod. Dev. 73, 15641577.
  • Takeda, N., Kyozuka, K. & Deguchi, R. 2006. Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae. Dev. Biol. 298, 248258.
  • Tanaka, T. & Yamashita, M. 1995. Pre-MPF is absent in immature oocytes of fishes and amphibians except Xenopus. Dev. Growth Differ. 37, 387393.
  • Yamashita, M. 1988. Involvement of cAMP in initiating maturation of the brittle star Amphipholis kochii oocytes: induction of oocyte maturation by inhibitors of cyclic nucleotide phosphodiesterases and activators of adenylate cyclase. Dev. Biol. 125, 109114.
  • Yamashita, M. 1998. Molecular mechanisms of meiotic maturation and arrest in fish and amphibian oocytes. Seminars Cell Dev. Biol. 9, 569579.
  • Yamashita, M., Mita, K., Yoshida, N. & Kondo, T. 2000. Molecular mechanisms of the initiation of oocyte maturation: general and species-specific aspects. Prog. Cell Cycle Res. 4, 115129.
  • Yi, J. H., Lefevre, L., Gagnon, C., Anctil, M. & Dube, F. 2002. Increase of cAMP upon release from prophase arrest in surf clam oocytes. J. Cell Sci. 115, 311320.
  • Yokosawa, H., Toratani, S., Inadome, Y. & Ishii, S.-I. 1989. Phorbol ester induces elevation of the vitelline coat of eggs of the ascidian Halocynthia roretzi. Dev. Growth Differ. 31, 543548.