In the nemertean worms Cerebratulus lacteus and Micrura alaskensis, 5-HT (=5-hydroxytryptamine, or serotonin) causes prophase-arrested oocytes to mature and complete germinal vesicle breakdown (GVBD). To identify the intracellular pathway that mediates 5-HT stimulation, follicle-free oocytes of nemerteans were assessed for GVBD rates in the presence or absence of 5-HT after being treated with various modulators of cAMP, a well known transducer of 5-HT signaling and an important regulator of hormone-induced maturation in general. Unlike in many animals where high levels of intra-oocytic cAMP block maturation, treatment of follicle-free nemertean oocytes with agents that elevate cAMP (8-bromo-cAMP, forskolin or inhibitors of phosphodiesterases) triggered GVBD in the absence of added 5-HT. Similarly, 5-HT caused a substantial cAMP increase prior to GVBD in nemertean oocytes that had been pre-injected with a cAMP fluorosensor. Such a rise in cAMP seemed to involve G-protein-mediated signaling and protein kinase A (PKA) stimulation, based on the inhibition of 5-HT-induced GVBD by specific antagonists of these transduction steps. Although the downstream targets of activated PKA remain unknown, neither the synthesis of new proteins nor the activation of MAPKs (mitogen-activated protein kinases) appeared to be required for GVBD after 5-HT stimulation. Alternatively, pre-incubation in roscovitine, an inhibitor of maturation-promoting factor (MPF), prevented GVBD, indicating that maturing oocytes eventually need to elevate their MPF levels, as has been documented for other animals. Collectively, this study demonstrates for the first time that 5-HT can cause immature oocytes to undergo an increase in cAMP that stimulates, rather than inhibits, meiotic maturation. The possible relationship between such a form of oocyte maturation and that observed in other animals is discussed.

During oogenesis, animal oocytes initially arrest at prophase I of meiosis and develop an enlarged nucleus (‘germinal vesicle’, GV). After remaining in prophase for a variable length of time, fully grown oocytes can be triggered by hormones to undergo a maturation process during which the oocyte completes germinal vesicle breakdown (GVBD) and advances to metaphase I or II prior to fertilization (Masui and Clarke, 1979; Sagata, 1997). Oocyte maturation is crucial for subsequent development, as prophase-arrested oocytes fail to fertilize properly (Barrios and Bedford, 1979; Ducibella and Buetow, 1994; Stricker et al., 1994; Stricker et al., 1998), except in the case of a few animals (e.g. echiuran worms and some annelids and molluscs) that normally undergo fertilization at prophase I (Austin, 1965). Thus, considerable effort has been directed towards understanding the mechanisms by which oocytes mature, and in various instances, the secondary messenger cAMP has been shown to play a key role in modulating the resumption of meiosis by prophase-arrested oocytes (Eppig, 1989).

As reviewed elsewhere (Maller, 1983; Maller, 1985; Eppig and Downs, 1984; Schultz, 1991), high levels of cAMP in oocytes typically block both spontaneously triggered GVBD and maturation that is induced by hormones. Accordingly, GVBD tends to be prevented by treating prophase-arrested oocytes with: (1) cAMP; (2) forskolin, which stimulates the cAMP-synthesizing enzyme adenylate cyclase; or (3) inhibitors of the phosphodiesterases (PDEs) that degrade cAMP (Cho et al., 1974; Magnusson and Hillensjo, 1977; Maller et al., 1979; Bornslaeger et al., 1984; Racowsky, 1985; Meijer et al., 1989; Warikoo and Bavister, 1989; Bilodeau et al., 1993; Tornell and Hillensjo, 1993; Karaseva and Khotimchenko, 1991; Karaseva and Khotimchenko, 1995; Haider and Chaube, 1996). Similarly, when oocytes are triggered to complete GVBD, their concentrations of cAMP have been shown to drop in several studies of invertebrates (Mazzei et al., 1981; Meijer and Zarutskie, 1987), fish (Finet et al., 1988; Haider and Chaube, 1995; Cerda et al., 1998), amphibians (Speaker and Butcher, 1977; Maller et al., 1979; Schorderet-Slatkine et al., 1982; Cicirelli and Smith, 1985) and mammals (Schultz et al., 1983; Vivarelli et al., 1983; Nagyova et al., 1993).

Collectively, such findings suggest that elevations in intra-oocytic cAMP and/or perhaps other inhibitory molecules (Tsafriri and Chandler, 1975; Eppig et al., 1983; Sato and Koide, 1987; Downs et al., 1989) serve to maintain oocytes in prophase-I arrest. Conversely, instead of blocking maturation, agents that increase cAMP concentrations actually trigger GVBD in follicle-free oocytes of brittle stars (Yamashita, 1988) and cnidarians (Freeman and Ridgway, 1988). Moreover, a transient increase in intra-oocytic cAMP may facilitate hormone-induced maturation in sheep (Moor and Heslop, 1981), rabbits (Yoshimura et al., 1992a; Yoshimura et al., 1992b) and pigs (Mattioli et al., 1994), although it remains possible that such findings are confounded by cumulus cell remnants that remain attached to the zona pellucida of at least some oocytes (Schultz, 1991).

In the marine worms Cerebratulus lacteus and Micrura alaskensis (Phylum Nemertea), follicle-free oocytes undergo GVBD in response to unidentified stimuli that occur in Ca2+-containing, but not Ca2+-free, seawater (Stricker, 1996; Stricker and Smythe, 2000). Such idiopathic GVBD is referred to here as ‘spontaneous maturation’ as no exogenous inducers need to be added to the seawaters in order to elicit GVBD. Alternatively, GVBD can be reliably triggered by 5-HT (=5-hydroxytryptamine, or serotonin) (Stricker and Smythe, 2000), a neurohormone that also stimulates maturation in bivalve molluscs (Guerrier et al., 1996; Ram et al., 1996). However, unlike bivalve oocytes that typically need Ca2+ fluxes for 5-HT stimulation (Kadam et al., 1990), 5-HT is capable of causing nemertean oocytes to mature in either Ca2+-containing or Ca2+-free seawater, and such maturation can proceed without marked Ca2+ transients occurring within the maturing oocyte (Stricker and Smythe, 2000). Thus, 5-HT does not seem to require Ca2+ signaling in nemerteans and instead apparently stimulates a Ca2+-independent pathway that has not yet been elucidated (Stricker and Smythe, 2000).

Given that cAMP modulates both the resumption of meiosis in many oocytes and the stimulation of somatic cells by 5-HT in general (Saudou and Hen, 1994; Saxena, 1995), the possible contribution of cAMP to oocyte maturation in nemerteans was assessed by treating prophase-arrested oocytes of C. lacteus and M. alaskensis with various modulators of cAMP signaling in the presence or absence of 5-HT. Based on these investigations, we show for the first time that a 5-HT-induced increase in cAMP can stimulate, rather than inhibit, GVBD, and we discuss the possible implications of such an apparently aberrant form of maturation relative to the patterns observed in maturing oocytes of other animals.

Animals

Adult females of Cerebratulus lacteus and Micrura alaskensis were maintained in aerated aquaria at Friday Harbor Laboratories (San Juan Island, WA) or the University of New Mexico as described previously (Stricker and Smythe, 2000). For tests of 5-HT-induced signaling pathways, oocytes were removed from cut pieces of worms and initially kept in 5-10 ml of ice-cold, EGTA-containing Ca2+-free seawater (CaFSW) (Schroeder and Stricker, 1983) for 30 minutes before being transferred to another ice-cold solution of CaFSW. At approx. 2 hours post-removal, one or two concentrated drops of oocytes were routinely placed in 24-well polystyrene culture dishes containing 1-2 ml of test solution per well, and the oocytes were then incubated at 10-14°C. Alternatively, some prophase-arrested oocytes were immersed in membrane-permeable inhibitors (RS-23597-190, melittin, mastoparan, H-89, MAPK blockers and roscovitine) for 30-60 minutes before treatment with 5-HT. For all studies, GVBD was assessed 2-3 hours after incubation in the test solutions by examining 50-250 oocytes per well with an inverted microscope, and such counting times were adopted, given that GVBD is typically completed by 1-1.5 hours after transfer to a maturation-inducing solution (Stricker, 1996; Stricker and Smythe, 2000).

Chemicals

Chemicals were purchased from: (1) Sigma, St Louis, MO (8-bromo-cAMP, cholera toxin, dibutyryl cAMP, DMSO, emetine, forskolin, guanosine 5μ-O-(2-thiodiphosphate) (GDP-β-S), mastoparan, MDL 12,330A, Ro-20-1724, SQ 22,536, 5-HT creatinine sulfate and stock solutions for artificial seawaters and injection buffers); (2) Tocris, Ballwin, MO (8-bromo-cAMP, etazolate, melittin, rolipram, 2-[1-(4-piperonyl)piperazinyl] benzothiazole (2-PPB), PD-98059, RS-23597-190, SB-203580 and U-0126); (3) LC Laboratories, Woburn, MA (H-89 and Rp-cAMP-S); (4) CalBiochem, San Diego, CA (2μ5μ dideoxyadenosine (2-DDA) and IBMX); (5) AG Scientific, San Diego, CA (roscovitine); and (6) Molecular Probes, Eugene, OR (FlCRhR). For Cerebratulus oocytes, the drugs were typically dissolved in CaFSW, rather than Ca2+-containing artificial seawater (MBL ASW; Cavanaugh, 1956), in order to minimize spontaneous maturation (Stricker and Smythe, 2000). Stock solutions of non-water-soluble chemicals were routinely made in DMSO at concentrations at least 200-1000 times the dose to be tested, and the final levels of diluted DMSO (<0.5%) did not significantly affect GVBD rates. Drug concentrations were either adopted based on previous reports in the literature or directly determined via dose-response curves generated in this study. In such cases, IC50 and EC50 values (i.e. inhibitory and excitatory concentrations that yield GVBD in 50% of the oocytes) were obtained by plotting dose-response curves and interpolating 50% points from the plots.

Microinjection

For microinjections of GDP-β-S and Rp-cAMP-S, Cerebratulus oocytes were stripped of their raised extracellular coat, or ‘chorion’ (Stricker, 1996), and the oocytes were subsequently maintained at prophase I in MBL artificial seawater by the addition of 25 μM PD-98059 or 10 μM U-0126, which serve to block spontaneous maturation (see Figs 12C, 13). Such oocytes were attached to protamine-sulfate-coated specimen dishes and injected by means of an Eppendorf injection system (Stricker, 1994) to ∼1-2% of their volume with stock solutions made in a potassium aspartate/Hepes injection buffer (Chiba et al., 1990).

Analysis

Time-lapse confocal analyses of cAMP levels were conducted on immature oocytes of Cerebratulus that had been injected under red light to 1-5% of their volume with a 20 μM phosphate-buffered solution of FlCRhR, which is a recombinant protein kinase A (PKA). Based on fluorescence resonance energy transfer (FRET) between fluorescein and rhodamine fluorophores conjugated to the PKA, FlCRhR can be used to monitor cAMP levels in vivo, since the fluorescein/rhodamine ratio increases as the regulatory subunits of the PKA bind cAMP and dissociate from the catalytic subunits (Adams et al., 1991). Thus, within 5-10 minutes after being injected with FlCRhR, the dye-loaded oocytes were examined with a BioRad MRC-600 confocal laser-scanning microscope using 488-nm excitation in the dual-channel mode (T1/T2A filter cubes, BioRad), and the emissions from the cAMP probe were processed as fluorescein/rhodamine ratioed images by means of MetaMorph software (Universal Imaging; Stricker, 1995). Graphs of uncalibrated FlCRhR runs expressed in arbitrary units of fluorescence were constructed using Excel spreadsheets (Microsoft).

Statistics

In all studies of 5-HT signaling pathways, experiments were conducted at least three times, with the exact sample size being specified with each dataset. For most investigations, oocytes were obtained from two to five females, whereas experiments utilizing forskolin, IBMX, or inhibitors of the MAPK (mitogen-activated protein kinase) pathway were conducted with oocytes from 8-11 females. Statistical analyses involved a one-way ANOVA (Christensen, 1996) or Mann-Whitney U (Sokal and Rohlf, 1973) test, for large and small datasets, respectively.

Agents that elevate cAMP levels in follicle-free nemertean oocytes mimic the maturation-inducing effects of 5-HT by triggering GVBD

As is typical for the phylum Nemertea (Stricker et al., 2001), oocytes examined in this study lacked a surrounding sheath of follicle cells throughout oogenesis and possessed an enlarged nucleus (GV) during the later stages of prophase I arrest. After removal from the ovary and transfer to Ca2+-containing seawaters, fully grown oocytes spontaneously matured by undergoing GVBD and subsequently arrested at metaphase I (Stricker, 1996; Stricker and Smythe, 2000). However, oocytes isolated directly in CaFSW typically remained in prophase I (Fig. 1A) until maturation was elicited by treatment with either natural seawater or 0.01-1 μM 5-HT that had been dissolved in Ca2+-containing or Ca2+-free artificial seawater (Stricker and Smythe, 2000).

Fig. 1.

The cAMP-elevating drug 8-Br-cAMP stimulates GVBD in nemerteans. (A) Prophase-arrested oocytes of Cerebratulus possessing a germinal vesicle (GV) at 3 hours after removal from the ovary and dechorionation in Ca2+-free seawater (CaFSW). (B) Completion of GVBD by 2 hours after addition of 5 mM 8-Br-cAMP to the CaFSW solution. Scale bar: 100 μm.

Fig. 1.

The cAMP-elevating drug 8-Br-cAMP stimulates GVBD in nemerteans. (A) Prophase-arrested oocytes of Cerebratulus possessing a germinal vesicle (GV) at 3 hours after removal from the ovary and dechorionation in Ca2+-free seawater (CaFSW). (B) Completion of GVBD by 2 hours after addition of 5 mM 8-Br-cAMP to the CaFSW solution. Scale bar: 100 μm.

As an alternative to either 5-HT-stimulated- or spontaneously triggered GVBD, prophase-arrested oocytes could also be induced to mature by 2-5 mM solutions of the membrane-permeable analog 8-bromo-cAMP dissolved in either Ca2+-containing or Ca2+-free seawater (Figs 1B, 2). Similarly, simply treating nemertean oocytes with the adenylate cyclase activator forskolin to elevate cAMP concentrations (Seamon et al., 1981) elicited maturation in a dose-dependent manner (EC50=410±240 nM; n=14; Fig. 3A), and at 10 μM concentrations, forskolin was as effective as 5-HT in stimulating GVBD (Fig. 3B). Nemertean oocytes also underwent high levels of GVBD in response to 3-isobutyl-1-methylxanthine (IBMX) (Fig. 4A,B; Table 1), a broadly acting inhibitor of the PDEs that act to lower cyclic nucleotide levels (Conti et al., 1998). To verify that the results obtained with IBMX were not simply due to nonspecific effects on Ca2+ signaling or adenosine receptors (Schultz, 1991), three inhibitors of PDE4 enzymes – etazolate, Ro-20-1724 and rolipram – were used as specific blockers of cAMP degradation (Conti et al., 1995) and were likewise found to cause GVBD (Fig. 5A,B; Table 1).

Table 1.

EC50 values for stimulation of GVBD by phosphodiesterase (PDE) inhibitors*

EC50 values for stimulation of GVBD by phosphodiesterase (PDE) inhibitors*
EC50 values for stimulation of GVBD by phosphodiesterase (PDE) inhibitors*
Fig. 2.

8-Br-cAMP triggers GVBD in either Ca2+-containing or Ca2+-free seawater. Cerebratulus (5 mM 8-Br-cAMP); Micrura (2 mM 8-Br-cAMP); *, significantly higher than controls; P<0.05.

Fig. 2.

8-Br-cAMP triggers GVBD in either Ca2+-containing or Ca2+-free seawater. Cerebratulus (5 mM 8-Br-cAMP); Micrura (2 mM 8-Br-cAMP); *, significantly higher than controls; P<0.05.

Fig. 3.

GVBD is elicited by forskolin, which stimulates adenylate cyclase and thereby elevates cAMP. (A) Cerebratulus oocytes in Ca2+-free seawater (n=14). (B) Micrura oocytes in Ca2+-containing artificial seawater (n=19). 5-HT, 1 μM 5-HT; FK, 10 μM forskolin; *, significantly lower than all three test solutions; P<0.05.

Fig. 3.

GVBD is elicited by forskolin, which stimulates adenylate cyclase and thereby elevates cAMP. (A) Cerebratulus oocytes in Ca2+-free seawater (n=14). (B) Micrura oocytes in Ca2+-containing artificial seawater (n=19). 5-HT, 1 μM 5-HT; FK, 10 μM forskolin; *, significantly lower than all three test solutions; P<0.05.

Fig. 4.

GVBD is triggered by IBMX, which can elevate cAMP by blocking phosphodiesterases. (A) Cerebratulus oocytes in Ca2+-free seawater (n=10). (B) Micrura oocytes in Ca2+-containing artificial seawater (n=15). *, significantly lower than both test solutions; P<0.05.

Fig. 4.

GVBD is triggered by IBMX, which can elevate cAMP by blocking phosphodiesterases. (A) Cerebratulus oocytes in Ca2+-free seawater (n=10). (B) Micrura oocytes in Ca2+-containing artificial seawater (n=15). *, significantly lower than both test solutions; P<0.05.

Fig. 5.

Three inhibitors of cAMP-specific (type 4) phosphodiesterases – Ro-20-1724, rolipram and etazolate – trigger GVBD. (A) Micrura oocytes in Ca2+-containing seawater. (B) Cerebratulus oocytes in Ca2+-free artificial seawater.

Fig. 5.

Three inhibitors of cAMP-specific (type 4) phosphodiesterases – Ro-20-1724, rolipram and etazolate – trigger GVBD. (A) Micrura oocytes in Ca2+-containing seawater. (B) Cerebratulus oocytes in Ca2+-free artificial seawater.

5-HT elicits a rise in cAMP as nemertean oocytes mature

To determine if 5-HT also triggers a rise in cAMP, as might be expected based on the GVBD rates elicited by cAMP-elevating agents, immature oocytes were injected with the cAMP fluorosensor FlCRhR. In such oocytes that went on to complete GVBD, the addition of 1 μM 5-HT was rapidly followed by a marked rise (32±16%; n=16) in cAMP-related fluorescence over pre-stimulation baseline values (Fig. 6A-C), whereas control oocytes not treated with 5-HT lacked fluorescence increases (Fig. 6C, inset). In 13 of the 16 cases, the 5-HT-induced increase in fluorescence was maintained above the halfway point between baseline and peak levels for at least 30 minutes after stimulation (Fig. 6B), whereas the other three responses were more ephemeral (Fig. 6C). Whether or not 5-HT-induced GVBD required a pulsatile stimulation by cAMP (i.e. a rise and subsequent drop in cAMP prior to GVBD) was not directly tested. However, the occurrence of more or less continuously elevated cAMP levels before GVBD (e.g. Fig. 6B) suggested that the cAMP concentrations did not need to decline fully to pre-stimulatory baseline levels in order for GVBD to proceed.

Fig. 6.

Cerebratulus oocytes injected with the cAMP fluorosensor FlCRhR undergo a marked rise in cAMP after addition of 1 μM 5-HT. (A) Time-lapse confocal images of fluorescein/rhodamine ratioed images taken every 2 minutes, showing a FlCRhR-injected oocyte directly before 5-HT addition (first frame) and subsequent time-points with elevated cAMP levels (arrow); blues, relatively low cAMP; yellows, higher cAMP; Scale bar, 100 μm. (B) Typical 5-HT-induced rise in cAMP-related FlCRhR fluorescence (representative of 13/16 traces) inset, lack of cAMP rise in control oocyte not treated with 5-HT (representative of 6/6 traces).

Fig. 6.

Cerebratulus oocytes injected with the cAMP fluorosensor FlCRhR undergo a marked rise in cAMP after addition of 1 μM 5-HT. (A) Time-lapse confocal images of fluorescein/rhodamine ratioed images taken every 2 minutes, showing a FlCRhR-injected oocyte directly before 5-HT addition (first frame) and subsequent time-points with elevated cAMP levels (arrow); blues, relatively low cAMP; yellows, higher cAMP; Scale bar, 100 μm. (B) Typical 5-HT-induced rise in cAMP-related FlCRhR fluorescence (representative of 13/16 traces) inset, lack of cAMP rise in control oocyte not treated with 5-HT (representative of 6/6 traces).

Blocking Gs-associated 5-HT receptors prevents 5-HT-induced GVBD

To verify that a cAMP increase is actually required for 5-HT-induced GVBD, various inhibitors of cAMP-related signaling pathways were analyzed. Initially, such studies tested 5-HT receptors that utilize μs subunits of receptor-associated G proteins to stimulate adenylate cyclase and thereby elevate cAMP (Saxena, 1995; Hamblin et al., 1998). For these tests, immature oocytes were pre-incubated in RS-23597-190 (Eglen et al., 1993), a blocker of the 5-HT4 type of receptor, which (along with the more poorly characterized 5-HT6 and 5-HT7 receptors) couples to G proteins that possess μs subunits in various somatic cells (Ford and Clarke, 1993; Eglen et al., 1995).

In the presence of the 5HT4-receptor antagonist, 5-HT-stimulated GVBD was inhibited in a dose-dependent manner (IC50=34.7±10.8 μM, n=7), and at concentrations above 50 μM, the GVBD rates declined to near the baseline levels of spontaneous maturation that occurred in the absence of 5-HT (Fig. 7). Such blockage of GVBD was not simply due to nonspecific morbidity, as prophase-arrested oocytes in 50 μM RS-23597-190 underwent high rates of GVBD when subsequently washed in natural seawater (99.8%±0.9%, n=7). Furthermore, the specificity of the response was supported by the fact that an agonist of 5-HT4 receptors – 2-PPB (Monge et al., 1994) – stimulated GVBD in the absence of 5-HT (EC50=32.8±7.9 μM, n=8; Fig. 7).

Fig. 7.

Treatment with RS-23597-190, a specific antagonist of Gs-coupled 5HT4 receptors, prevents 5-HT-induced GVBD, whereas the 5HT4R agonist 2-PPB stimulates GVBD in the absence of 5-HT. Such findings are consistent with the mediation of 5-HT stimulation by G protein μs subunits that elevate cAMP.

Fig. 7.

Treatment with RS-23597-190, a specific antagonist of Gs-coupled 5HT4 receptors, prevents 5-HT-induced GVBD, whereas the 5HT4R agonist 2-PPB stimulates GVBD in the absence of 5-HT. Such findings are consistent with the mediation of 5-HT stimulation by G protein μs subunits that elevate cAMP.

Blockers of G protein signaling can inhibit 5-HT-induced GVBD

To confirm that the results obtained with the 5HT4 receptor blocker were due to the inhibition of Gs-mediated signaling, prophase-arrested oocytes of Cerebratulus were initially injected with the G protein antagonist GDP-β-S at a 2-3 mM final concentration, as has been used to inactivate G proteins in sea urchin eggs (Turner et al., 1987). Following injection, GDP-β-S-loaded oocytes typically failed to undergo GVBD when stimulated with 1 μM 5-HT (2/15 (13%) GVBD), whereas control oocytes injected with buffer alone matured in response to 5-HT (23/25 (92%) GVBD).

Although such findings suggested that G-protein functioning is required for 5-HT-induced GVBD, they did not demonstrate that it was actually μs, rather than some other G-protein subunit, that mediated the 5-HT signal. Thus, as an alternative to highly specific antibodies that must be injected into cells to inhibit Gs functioning (Gallo et al., 1995), prophase-arrested oocytes of Cerebratulus were pre-incubated in 3.5 μM melittin, a blocker of Gs activity as well as a stimulator of μi/o subunits (Fukushima et al., 1998). In such cases, melittin-treated oocytes displayed significantly lower 5-HT-induced GVBD levels than did controls (Fig. 8A). That this inhibition was mainly due to the blockage of Gs activity rather than the stimulation of μi/o subunits was supported by the fact that mastoparan, which stimulates only Gi/Go signaling (Fukushima et al., 1998), did not significantly inhibit 5-HT-induced GVBD (i.e. in Cerebratulus controls treated with 100 nM 5-HT in CaFSW, GVBD averaged 87.8±17.4% versus 82.8±21.6% in cohort oocytes pre-treated with 2 μM mastoparan prior to stimulation with 100 nM 5-HT; P>0.05; n=8).

Fig. 8.

5-HT stimulation of GVBD in Cerebratulus oocytes is apparently transduced by Gs signaling. (A) Blockage of 5-HT-induced GVBD by pre-incubation in CaFSW containing 3.5 μM melittin, which is an inhibitor of Gs (and stimulator Gi/Go) μ subunits; *, significantly lower than 5-HT-treated controls; P<0.05 (n=7). (B) Effective triggering of GVBD in Ca2+-containing MBL artificial seawater by 5μ10–10 M cholera toxin (CT), a stimulator of Gs signaling. FK, 10 μM forskolin; IBMX, 100 μM IBMX; 5-HT, 1 μM 5-HT; n=4 for all solutions; *, significantly lower than the four test solutions; P<0.05.

Fig. 8.

5-HT stimulation of GVBD in Cerebratulus oocytes is apparently transduced by Gs signaling. (A) Blockage of 5-HT-induced GVBD by pre-incubation in CaFSW containing 3.5 μM melittin, which is an inhibitor of Gs (and stimulator Gi/Go) μ subunits; *, significantly lower than 5-HT-treated controls; P<0.05 (n=7). (B) Effective triggering of GVBD in Ca2+-containing MBL artificial seawater by 5μ10–10 M cholera toxin (CT), a stimulator of Gs signaling. FK, 10 μM forskolin; IBMX, 100 μM IBMX; 5-HT, 1 μM 5-HT; n=4 for all solutions; *, significantly lower than the four test solutions; P<0.05.

Accordingly, high levels of GVBD were also elicited in the absence of 5-HT by cholera toxin, which specifically catalyzes the ADP-ribosylation of μs subunits to lock them in an activated state and thereby keep cAMP levels elevated (Gilman, 1987). In fact, cholera toxin at concentrations ranging from 10–9 to 10–10 M was essentially as effective as supramicromolar doses of forskolin, IBMX or 5-HT in inducing GVBD (Fig. 8B).

Given that Gμs stimulates adenylate cyclase (AC), three inhibitors of AC activity – MDL-12,330A, SQ-22536 and 2-DDA – were tested for their effects on oocyte maturation. However, contrary to the data obtained with cAMP-elevating drugs or G-protein modulators, pre-incubation in 100 μM solutions of the AC inhibitors did not prevent 5-HT-induced GVBD in either Micrura or Cerebratulus oocytes (data not shown). Whether such results show that 5-HT-induced GVBD did not require AC activity or that the inhibitors simply did not block AC functioning at the concentrations tested remains unclear. However, the latter explanation coincided with the finding that pre-treatment of Micrura oocytes with 100 μM doses of the AC inhibitors before stimulation with 10 μM forskolin continued to yield GVBD rates that were not significantly lower than those displayed by forskolin-treated controls, i.e. forskolin-induced GVBD after incubation in the following AC inhibitors was: (1) MDL-123,30A, 77.1±23.1%(2) SQ-22536, 99.3±0.8%; and (3) 2-DDA, 95.4±4.6% versus (controls) (a) MBL ASW without forskolin or AC inhibitor, 33.4±29.1%; (b) MBL ASW plus forskolin but no AC inhibitor, 98.0±0.5% (n=4).

Blockers of the MAP kinase signaling pathway do not prevent 5-HT-induced GVBD

Given that PKA is a primary target of cAMP in various cells (Skalhegg and Tasken, 2000), two specific blockers of PKA activity − Rp-cAMP-S and H-89 − were used on prophasearrested Cerebratulus oocytes prior to 5-HT stimulation. In such cases, oocytes injected with 100 mM Rp-cAMP-S typically failed to undergo GVBD when subsequently treated with 1 mM 5-HT (2/19 (11%) GVBD) versus 19/19 (100%) GVBD in control oocytes that were injected with buffer alone before 5-HT treatment; Fig. 9). Similarly, pre-incubation in H-89 inhibited 5-HT-induced maturation in a dose-dependent fashion (IC50=3.3±1.9 mM; n=10; Fig. 10), and such inhibition was not simply due to oocyte morbidity, as prophase-arrested oocytes in 10-15 mM H-89 underwent high levels of GVBD (92.8±8.0%; n=6) when washed in natural seawater.

Fig. 9.

Injection of 100 μM Rp-cAMP-S, a PKA blocker, prevents 5-HT-induced GVBD in Cerebratulus oocytes (GV indicates continued prophase arrest), whereas controls injected with just injection buffer consistently complete GVBD after 5-HT-mediated stimulation. Scale bar: 100 μm.

Fig. 9.

Injection of 100 μM Rp-cAMP-S, a PKA blocker, prevents 5-HT-induced GVBD in Cerebratulus oocytes (GV indicates continued prophase arrest), whereas controls injected with just injection buffer consistently complete GVBD after 5-HT-mediated stimulation. Scale bar: 100 μm.

Fig. 10.

The protein kinase A (PKA) blocker H-89 prevents 5-HT-induced GVBD in Cerebratulus oocytes that are incubated in CaFSW (n=10).

Fig. 10.

The protein kinase A (PKA) blocker H-89 prevents 5-HT-induced GVBD in Cerebratulus oocytes that are incubated in CaFSW (n=10).

5-HT-induced GVBD does not appear to require protein synthesis

As targets of activated PKA include transcription factors such as CREB (cAMP response element-binding protein) that regulate gene expression (Habener et al., 1995; Shaywitz and Greenberg, 1999), the role of de novo protein synthesis during For various cell types, the effects of extracellular stimuli can be transduced intracellularly by the MAPK signaling system (Ferrell, 1996), and at least in a few oocytes (Gotoh et al., 1995; Fissore et al., 1996; Goudet et al., 1998), MAPK activity facilitates GVBD. Thus, the possible contribution of MAPKs to nemertean maturation was analyzed by treating prophase-arrested oocytes with: (1) PD-98059 or U-0126, which inhibit the kinase responsible for activating the erk1/erk2 (extracellular signal regulated kinase) types of MAPKs; or (2) SB-203580, which blocks p38 (HOG) MAPKs. In both cases, there was no significant reduction in the GVBD levels induced by 1 μM 5-HT (Fig. 12A,B). This lack of inhibition was not simply due to inadequate permeability, as the inhibitory drugs were highly effective in preventing spontaneous maturation in the absence of 5-HT (IC50 for PD-98059=94.8±54.2 nM, n=4; IC50 for U-0126=9.5±5.9 nM, n=14; Fig. 12C). Moreover, as further support for the dispensability of MAPK signaling during 5-HT-induced cAMP rises, forskolin and IBMX were capable of reversing the blockage of spontaneous GVBD that was caused by MAPK inhibitors (Fig. 13).

Fig. 11.

(A) 400-μM solutions of the protein synthesis blocker emetine fail to prevent 5-HT-induced GVBD in CaFSW cultures of Cerebratulus oocytes. *, significantly lower GVBD rates than both test solutions, n=33, P<0.05. (B) Such a lack of GVBD inhibition is not simply due to inadequate permeability of the protein synthesis blocker, as emetine can trigger significantly higher GVBD rates (*P<0.05) than either controls that lack emetine and 5-HT or oocytes pretreated with 10 μM of the MAPK kinase blocker U-0126 prior to emetine application (n=22).

Fig. 11.

(A) 400-μM solutions of the protein synthesis blocker emetine fail to prevent 5-HT-induced GVBD in CaFSW cultures of Cerebratulus oocytes. *, significantly lower GVBD rates than both test solutions, n=33, P<0.05. (B) Such a lack of GVBD inhibition is not simply due to inadequate permeability of the protein synthesis blocker, as emetine can trigger significantly higher GVBD rates (*P<0.05) than either controls that lack emetine and 5-HT or oocytes pretreated with 10 μM of the MAPK kinase blocker U-0126 prior to emetine application (n=22).

Fig. 12.

Blockers of the MAPK pathway do not inhibit 5-HT-induced GVBD in Cerebratulus oocytes. (A) In the presence of 10 μM U-0126, an inhibitor of MAPK signaling, GVBD rates rise with increasing concentrations of 5-HT and 1 μM 5-HT fully overcomes the inhibitory effects of U-0126 (n=6). (B) Inhibition of neither erk1/erk2 MAPKs (PD-98059 and U-0126) nor p38-like MAPKs (SB-203580) prevents GVBD induced by 1 μM 5-HT. (C) The lack of GVBD inhibition by such blockers is not due to inadequate permeability, as both U-0126 and PD-98059 effectively block spontaneous maturation in MBL artificial seawater lacking 5-HT.

Fig. 12.

Blockers of the MAPK pathway do not inhibit 5-HT-induced GVBD in Cerebratulus oocytes. (A) In the presence of 10 μM U-0126, an inhibitor of MAPK signaling, GVBD rates rise with increasing concentrations of 5-HT and 1 μM 5-HT fully overcomes the inhibitory effects of U-0126 (n=6). (B) Inhibition of neither erk1/erk2 MAPKs (PD-98059 and U-0126) nor p38-like MAPKs (SB-203580) prevents GVBD induced by 1 μM 5-HT. (C) The lack of GVBD inhibition by such blockers is not due to inadequate permeability, as both U-0126 and PD-98059 effectively block spontaneous maturation in MBL artificial seawater lacking 5-HT.

Fig. 13.

Both 10 μM forskolin (FK) and 100 μM IBMX reverse the blockage of spontaneous maturation by 10 μM U-0126 in seawater solutions that lack 5-HT, which is consistent with the view that the pathway elicited by 5-HT-induced cAMP elevations does not require MAPK signaling. MBL ASW, Ca2+-containing artificial seawater; CaFSW, Ca2+-free seawater; n=6 for all solutions; *, significantly lower than either all four pairs of test solutions or controls lacking U-0126.

Fig. 13.

Both 10 μM forskolin (FK) and 100 μM IBMX reverse the blockage of spontaneous maturation by 10 μM U-0126 in seawater solutions that lack 5-HT, which is consistent with the view that the pathway elicited by 5-HT-induced cAMP elevations does not require MAPK signaling. MBL ASW, Ca2+-containing artificial seawater; CaFSW, Ca2+-free seawater; n=6 for all solutions; *, significantly lower than either all four pairs of test solutions or controls lacking U-0126.

The MPF inhibitor roscovitine blocks 5-HT-induced GVBD

During hormone-induced maturation, animal oocytes require an increase in active MPF (maturation-promoting factor) to complete GVBD (Eckberg, 1988; Yamashita, 1998). To verify that such a requirement holds true for 5-HT-induced GVBD, prophase-arrested oocytes of Cerebratulus were pre-incubated in the MPF inhibitor roscovitine (Meijer et al., 1997) before stimulation with 1 μM 5-HT. In such tests, roscovitine blocked both 5-HT-induced GVBD (IC50=11.9±6.0 μM; n=6; Fig. 14) and spontaneous maturation in the absence of 5-HT (i.e. GVBD for Cerebratulus oocytes in MBL ASW without 5-HT or roscovitine was 64.0±29.8% versus 3.3±3.2% in 50 μM roscovitine; P<0.05, n=8). Moreover, such inhibition was not solely due to morbidity, since prophase-arrested oocytes in 50 μM roscovitine subsequently underwent high levels of GVBD (99.5±0.8%, n=3) after washes in seawater.

Fig. 14.

Roscovitine, an inhibitor of maturation-promoting factor (MPF), prevents GVBD induced by 1 μM 5-HT in a dose-dependent manner (n=6).

Fig. 14.

Roscovitine, an inhibitor of maturation-promoting factor (MPF), prevents GVBD induced by 1 μM 5-HT in a dose-dependent manner (n=6).

What role does cAMP play during 5-HT-induced GVBD in nemerteans?

Unlike the inhibitory effects of cAMP in many animals examined (Eppig and Downs, 1984; Maller, 1985; Schultz, 1991; Downs, 1993), millimolar doses of 8-bromo-cAMP cause follicle-free oocytes of the nemerteans Cerebratulus and Micrura to undergo GVBD. Although such concentrations of cAMP seem somewhat high compared with doses used on mammalian oocytes (Eppig, 1989), 10 mM cAMP solutions are needed to trigger maturation in brittle star oocytes (Yamashita, 1988), and cnidarians undergo GVBD in response to 8-bromo-cAMP concentrations ranging from 0.6-23 mM (Freeman and Ridgway, 1988). As in cnidarians (Freeman and Ridgway, 1988) and contrary to the situation in some mammals (Dekel and Beers, 1978), GVBD in nemertean oocytes is more readily promoted by 8-bromo-cAMP than another membrane-permeable analog, dibutyryl cAMP (db-cAMP) (data not shown). The enhanced efficacy of 8-bromo-cAMP in cnidarians and nemerteans could simply be due to differences in permeabilities and/or resistances to phosphodiesterases (Freeman and Ridgway, 1988; Homa, 1988). Alternatively, such a response might be related to the type of PKA that is present in nemertean oocytes (Beebe and Corbin, 1986; Downs and Hunzicker-Dunn, 1995).

In any case, the stimulatory effects of cAMP are further supported by the fact that the cAMP-elevating drugs forskolin and IBMX cause GVBD, as has been as noted for brittle stars (Yamashita, 1988). Moreover, unlike in some mammalian oocytes, where type 3 rather than type 4 PDEs predominate (Tsafriri et al., 1996), several inhibitors of type 4 PDEs trigger GVBD in nemerteans, although with marked species-specific differences in the dose-response curves. Whether such differences reflect variations in membrane permeabilities and/or alternative isoforms of PDEs remains unknown. Nevertheless, the type 4 PDE inhibitors and all other cAMP-elevating drugs were tested on follicle-free nemertean oocytes in the absence of surrounding tissues. Thus, GVBD was directly elicited by effects on the oocyte itself, rather than by an indirect excitation involving a cAMP-stimulated secretion of maturation inducers by nearby follicle cells (Iwamatsu et al., 1987; Downs et al., 1988; Andersen et al., 1999) or ovarian tissues (Freeman, 1994).

Not only can cAMP-elevating drugs mimic the maturation-inducing effects of 5-HT in nemertean oocytes, but a marked rise in cAMP also occurs in FlCRhR-injected oocytes directly after applying 5-HT. In this study, FlCRhR data are expressed in arbitrary units of cAMP-related fluorescence, owing to the prohibitive costs of the fluorophore and other modulators of cAMP levels needed for such calibrations, as has been noted in other investigations (Sammak et al., 1992). However, the magnitudes of the peak fluorescence rises obtained in nemertean oocytes are among the highest reported for FlCRhR-injected cells (Sammak et al., 1992; DeBernardi and Brooker, 1996; Kume et al., 2000) and presumably correspond to substantial increases of at least several micromoles cAMP, based on calibrations performed with other cell types (Adams et al., 1991).

Although such 5-HT-induced increases in cAMP consistently occurred prior to GVBD, it remains possible that some FlCRhR-injected oocytes were initially stimulated to mature by the injection procedure or FlCRhR molecule itself (Sammak et al., 1992), as 7/8 FlCRhR-injected oocytes underwent GVBD in the absence of 5-HT, which is higher than normal spontaneous maturation rates (data not shown). In any case, 5-HT stimulation is rapidly followed by a distinct cAMP rise prior to GVBD, as has been noted for brittle star oocytes that were triggered to mature by the cAMP-elevating drugs forskolin or theophylline (Yamashita, 1988).

Accordingly, 5-HT-induced GVBD in nemerteans is prevented by inhibiting the G-protein pathway that elevates cAMP. For example, pre-treatment with RS-23597-190, an antagonist of 5-HT4 receptors, which use μs subunits to stimulate adenylate cyclase, blocks GVBD, albeit at a relatively high IC50 that may suggest some nonspecificity. However, regardless of whether 5-HT4 receptors or some other kind of 5-HT receptors actually mediate the 5-HT signal, experiments using GDP-β-S, melittin and mastoparan also suggest that 5-HT stimulation of nemertean ooctyes requires Gs signaling. Moreover, unlike in other oocytes where cholera toxin inhibits maturation (Maller et al., 1979; Schorderet-Slatkine et al., 1982; Schultz et al., 1983), stimulation of the Gs signaling pathway by cholera toxin causes GVBD in nemertean oocytes. Conversely, 5-HT-induced GVBD continues to occur in the presence of AC inhibitors, which might argue against the necessity of Gs signaling. However, such inhibitors also fail to prevent forskolin-induced GVBD. Therefore, they may simply fail to block the AC isoforms in nemertean oocytes at the concentrations tested in this study, or cAMP could be elevated in the absence of AC activity by alternative mechanisms such as the modulation of PDEs (Sadler and Maller, 1987).

A cAMP-mediated stimulation of GVBD during 5-HT-triggered maturation is further supported by the fact that PKA inhibitors effectively block 5-HT-induced GVBD. Thus, unlike in vertebrate oocytes where PKA blockers stimulate GVBD (Maller and Krebs, 1977; Rose-Hellekant and Bavister, 1996), both Rp-cAMP-S injections and incubations in H-89 prevent hormone-induced GVBD in nemerteans. Collectively, the results from experiments using cAMP-elevating drugs, FlCRhR-based assays of cAMP levels, various modulators of G protein signaling, and inhibitors of PKA suggest that cAMP has a stimulatory effect on maturation in nemertean oocytes and that 5-HT causes a Gs-facilitated rise in cAMP which activates PKA prior to GVBD.

What are the potential downstream targets of activated PKA in maturing oocytes of nemerteans?

Based on the continued occurrence of GVBD in 5-HT-treated oocytes that were pre-incubated with emetine, the stimulation of nemertean oocytes by 5-HT does not appear to require de novo protein synthesis. Such high GVBD levels are not simply due to inadequate drug permeability, because emetine actually triggers meiotic resumption in the absence of 5-HT, as has been reported for metaphase-I-arrested oocytes in bivalves (Neant et al., 1994). Although the mechanism of emetine-induced maturation remains unknown, MAPKs are apparently involved, because inhibition of MAPK signaling prevents GVBD in the presence of emetine. Thus, emetine may eliminate short-lived proteins that, when present, promote prophase arrest by inhibiting a MAPK-stimulated pathway of non-hormone-induced GVBD. Alternatively, emetine could activate MAPK by pleiotropic effects not related to the blockage of protein synthesis. In any case, the fact that emetine fails to inhibit 5-HT-induced GVBD is consistent with the reported dispensability of protein synthesis during GVBD in some other animals (Zampetti-Bosseler, 1973; Hunt et al., 1992; Kishimoto, 1999).

In the absence of an apparent need for de novo protein synthesis during 5-HT-induced GVBD, the MAPK and MPF signaling pathways were analyzed to determine if such likely targets of activated PKA are required for nemertean GVBD. Given that inhibitors of erk1/erk2 or p38-like MAPKs eliminate spontaneous maturation in the absence of 5-HT but fail to prevent 5-HT-induced GVBD, the PKA activated by 5-HT does not seem to target the MAP kinase pathway. Such findings coincide with the dispensability of MAPK functioning that has been reported in other studies of GVBD (Eckberg, 1997; Fisher et al., 1999; Gould and Stephano, 1999; Kajiura-Kobayashi et al., 2000), but the data do not rule out the possible participation of alternative MAPK types (Ferrell, 1996) that are not affected by the particular inhibitors tested in this study.

As opposed to the results obtained with MAPK inhibition, 5-HT-induced GVBD in nemerteans is significantly reduced by roscovitine, a blocker of MPF. Coupled with the emetine data suggesting that protein synthesis is not required for 5-HTinduced GVBD, these findings indicate that 5-HT stimulation generates active MPF from an existing pool of inactive pre-MPF that is already present in prophase-arrested nemertean oocytes. Such a form of MPF activation is similar to that described for Xenopus (Yamashita, 1998) and unlike the mode employed by fish and non-Xenopus amphibians, where pre-MPF pools are lacking and cyclin B must be synthesized de novo in order to produce active MPF (Tanaka and Yamashita, 1995; Kondo et al., 1997).

In prophase-arrested oocytes of Xenopus, activated PKA can apparently convert pre-MPF into active MPF by phosphorylating an okadaic acid-sensitive phosphatase that keeps the Cdc25 phosphatase dephosphorylated and hence incapable of removing inhibitory phosphates on the Cdc2 kinase in inactive pre-MPF (Grieco et al., 1994). Alternatively, although the downstream targets of activated PKA in nemerteans remain unknown, they would presumably be opposite to those in Xenopus, where high cAMP/PKA levels inhibit GVBD, rather than stimulate it (as in nemerteans). Thus, activating PKA in nemerteans is postulated to promote pre-MPF conversion into MPF by: (1) upregulating Cdc25 and/or CAK (cyclin-dependent kinase activating kinase), both of which function to stimulate Cdc2 kinase activities; (2) downregulating inhibitory pathways involving wee-1 and myt-1; and/or (3) modulating targets that have yet to be identified (Yamashita, 1998; Kishimoto, 1999).

How does the signal transduction pathway of 5-HT-induced GVBD compare with that used by alternative modes of oocyte maturation in nemerteans and other animals?

In bivalve molluscs, 5-HT triggers GVBD in follicle-free signaling pathways were analyzed to determine if such likely targets of activated PKA are required for nemertean GVBD. Given that inhibitors of erk1/erk2 or p38-like MAPKs eliminate spontaneous maturation in the absence of 5-HT but fail to prevent 5-HT-induced GVBD, the PKA activated by 5-HT does not seem to target the MAP kinase pathway. Such findings coincide with the dispensability of MAPK functioning that has been reported in other studies of GVBD (Eckberg, 1997; Fisher et al., 1999; Gould and Stephano, 1999; Kajiura-Kobayashi et al., 2000), but the data do not rule out the possible participation of alternative MAPK types (Ferrell, 1996) that are not affected by the particular inhibitors tested in this study. As opposed to the results obtained with MAPK inhibition, 5-HT-induced GVBD in nemerteans is significantly reduced by roscovitine, a blocker of MPF. Coupled with the emetine data suggesting that protein synthesis is not required for 5-HT-induced GVBD, these findings indicate that 5-HT stimulation oocytes, and with the exception of the oyster Crassostrea (Kyozuka et al., 1997), such maturation involves Ca2+ transients arising from external Ca2+ influx and/or a phospholipase-C-mediated release from internal stores (Guerrier et al., 1996; Krantic and Rivailler, 1996). Conversely, unlike the Ca2+-dependent pathways that have been documented for bivalves or hormone-treated vertebrate oocytes (Maller, 1985; Racowsky, 1986; Homa, 1995; Mattioli and Barboni, 2000), 5-HT-stimulated GVBD in nemertean oocytes does not appear to require Ca2+ signaling (Stricker and Smythe, 2000). Moreover, in the bivalve Spisula, elevated levels of cAMP inhibit 5-HT-induced GVBD (Sato et al., 1985), whereas data presented here support a stimulatory, rather than inhibitory role for cAMP in nemertean oocytes. Although the exact types of 5-HT receptors have not been identified for bivalves (Fong et al., 1993; Krantic and Rivailler, 1996; Osada et al., 1998) or nemerteans (Stricker and Smythe, 2000), it remains possible that a fundamental difference accounting for this discrepancy is that bivalve oocytes lack the Gs-associated 5-HT receptors that presumably function in nemerteans to elevate cAMP.

Aside from the case of bivalve and nemertean oocytes, where 5-HT stimulation is sufficient to trigger GVBD, 5-HT can also modulate the effects of other maturation-inducing hormones. For example, 5-HT prevents progesterone-induced GVBD in amphibians (Buznikov et al., 1993) and increases intra-oocytic cAMP levels to inhibit GVBD in fish oocytes that had been treated with the maturation-inducing hormone 17μ, 20β DHP (Cerda et al., 1998). Conversely, 5-HT facilitates GVBD in starfish oocytes exposed to the maturation-inducing hormone 1-methyladenine (1-MA) (Buznikov et al., 1990a), although presumably by alternative mechanisms than those in nemertean oocytes, given that 5-HT by itself does not trigger GVBD in the absence of 1-MA (Buznikov et al., 1990a).

5-HT-induced GVBD in nemerteans also seems to differ from the so-called spontaneous form of maturation that occurs when nemertean oocytes are placed in Ca2+-containing seawaters lacking any exogenous inducers of maturation (e.g. added hormones, enzyme modulators, etc.). This view is based on the fact that: (1) some oocytes respond to natural seawater (NSW) but not to 5-HT and vice versa; (2) aged oocytes show a more rapid decline in sensitivity to 5-HT than to NSW; and (3) blocking 5-HT receptor function inhibits 5-HT-induced GVBD but not spontaneous maturation (Stricker and Smythe, 2000). Moreover, experiments reported here indicate that spontaneous maturation is stimulated by emetine and requires MAPK signaling, whereas emetine and MAPK inhibition do not affect 5-HT-induced GVBD. Collectively, such findings suggest 5-HT-induced GVBD and spontaneous maturation use separate pathways (Fig. 15), but whether nemertean oocytes in the field normally employ a 5-HT-stimulated mode of maturation or a spontaneous one triggered by unidentified seawater constituents remains a fundamental question to be answered.

Fig. 15.

Summary of oocyte maturation pathways either elicited spontaneously in the absence of 5-HT (left side of diagram) or induced by 5-HT (right side of diagram). Spontaneous maturation refers to idiopathic GVBD that occurs in response to unknown stimuli in seawaters without exogenous inducers such as 5-HT having to be added. AC, adenylate cyclase; CaFSW, Ca2+-free seawater; MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PDE, phosphodiesterase; PKA, protein kinase A.

Fig. 15.

Summary of oocyte maturation pathways either elicited spontaneously in the absence of 5-HT (left side of diagram) or induced by 5-HT (right side of diagram). Spontaneous maturation refers to idiopathic GVBD that occurs in response to unknown stimuli in seawaters without exogenous inducers such as 5-HT having to be added. AC, adenylate cyclase; CaFSW, Ca2+-free seawater; MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; PDE, phosphodiesterase; PKA, protein kinase A.

Regardless of which signaling pathway is used during natural reproduction, a 5-HT-induced rise in intra-oocytic cAMP initially seems to be a highly unusual trigger for oocyte maturation. However, both brittle star and cnidarian oocytes mature after increases in their cAMP levels, and a facilitation of GVBD by elevations in intra-oocytic cAMP may also occur in some mammals (Moor and Heslop, 1981; Yoshimura et al., 1992a; Yoshimura et al., 1992b; Mattioli et al., 1994). Moreover, although cAMP inhibits oocyte maturation in most starfish species (Meijer and Zarutskie, 1987; Buznikov et al., 1990b; Buznikov et al., 1992; Karaseva and Khotimchenko, 1991; Karaseva et al., 1996), db-cAMP apparently increases GVBD rates in some starfish oocytes treated with low levels of maturation-inducing hormone (Moreau and Guerrier, 1979). Thus, the stimulation, rather than inhibition, of GVBD by elevations in intra-oocytic cAMP may be more widespread than previously indicated by paradigms that are based solely on commonly studied vertebrate systems.

We thank Dr A. O. D. Willows for use of laboratory space at Friday Harbor Laboratories. This study was made possible by facilities and services of the Department of Biology’s Confocal Microscopy Facility at the University of New Mexico.

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