Nemertean Oocytes Research Articles

Marine Nemertean Worms for Studies of Oocyte Maturation and Aging.

Many marine invertebrates are capable of providing an abundant supply of oocytes that are fertilized external to the female body, thereby making these specimens well suited for studies of development. Along with intensively analyzed model systems belonging to such groups as echinoderms, tunicates, mollusks, and annelids, various lesser-studied taxa can undergo an external mode of fertilization. For example, nemertean worms constitute a relatively small phylum of marine protostome worms whose optically clear oocytes are easily collected and fertilized in the laboratory. Thus, to help promote the use of nemertean oocytes as a potential model in embryological analyses, this chapter begins by describing general methods for obtaining adults and for handling their gametes. After presenting such protocols, this chapter concludes with some representative results obtained with these specimens by summarizing the roles played by adenosine monophosphate-activated kinase (AMPK) during oocyte maturation and by c-Jun N-terminal kinase (JNK) during oocyte aging and death.

Oocyte Maturation and Fertilization in Marine Nemertean Worms: Using Similar Sorts of Signaling Pathways as in Mammals, but Often With Differing Results

In marine worms belonging to the phylum Nemertea, oocyte maturation and fertilization are regulated by the same general kinds of signals that control such processes in mammals. However, unlike mammalian oocytes that develop within follicles, nemertean oocytes characteristically lack a surrounding sheath of follicle cells and often respond differently to maturation-related cues than do mammalian oocytes. For example, elevators of cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) levels promote the resumption of meiotic maturation (=germinal vesicle breakdown, GVBD) in nemertean oocytes, whereas increasing intraoocytic cAMP and cGMP typically blocks GVBD in mammals. Similarly, AMP-activated kinase (AMPK) signaling keeps nemertean oocytes from maturing, but in mouse oocytes, AMPK activation triggers GVBD. In addition, protein kinase C (PKC) activity is required for seawater-induced GVBD in nemerteans, whereas some PKCs have been shown to inhibit GVBD in mammals. Furthermore, although fertilization causes both types of oocytes to reorganize their endoplasmic reticulum and generate calcium oscillations that can involve soluble sperm factor activity and inositol 1,4,5-trisphosphate signaling, some discrepancies in the spatiotemporal patterns and underlying mechanisms of fertilization are also evident in nemerteans versus mammals. Thus, to characterize differences and similarities in gamete biology more fully, aspects of oocyte maturation and fertilization in marine nemertean worms are reviewed and briefly compared with related findings that have been published for mammalian oocytes. In addition, possible causes of the alternative responses displayed by oocytes in these two animal groups are addressed.

Inhibition of germinal vesicle breakdown by antioxidants and the roles of signaling pathways related to nitric oxide and cGMP during meiotic resumption in oocytes of a marine worm

In mammalian oocytes, cAMP elevations prevent the resumption of meiotic maturation and thereby block nuclear disassembly (germinal vesicle breakdown (GVBD)), whereas nitric oxide (NO) and its downstream mediator cGMP can either inhibit or induce GVBD. Alternatively, some invertebrate oocytes use cAMP to stimulate, rather than inhibit, GVBD, and in such cases, the effects of NO/cGMP signaling on GVBD remain unknown. Moreover, potential interactions between NO/cGMP and AMP-activated kinase (AMPK) have not been assessed during GVBD. Thus, this study analyzed intraoocytic signaling pathways related to NO/cGMP in a marine nemertean worm that uses cAMP to induce GVBD. For such tests, follicle-free nemertean oocytes were stimulated to mature by seawater (SW) and cAMP elevators. Based on immunoblots and NO assays of maturing oocytes, SW triggered AMPK deactivation, NO synthase (NOS) phosphorylation, and an NO elevation. Accordingly, SW-induced GVBD was blocked by treatments involving the AMPK agonist AICAR, antioxidants, the NO scavenger carboxy-PTIO, NOS inhibitors, and cGMP antagonists that target the NO-stimulated enzyme, soluble guanylate cyclase (sGC). Conversely, SW solutions combining NO/cGMP antagonists with a cAMP elevator restored GVBD. Similarly, AICAR plus a cAMP-elevating drug reestablished GVBD while deactivating AMPK and phosphorylating NOS. Furthermore, sGC stimulators and 8-Br-cGMP triggered GVBD. Such novel results indicate that NO/cGMP signaling can upregulate SW-induced GVBD and that cAMP-elevating drugs restore GVBD by overriding the inhibition of various NO/cGMP downregulators, including AMPK. Moreover, considering the opposite effects of intraoocytic cAMP in nemerteans vs mammals, these data coincide with previous reports that NO/cGMP signaling blocks GVBD in rats.

Comparative biology of cAMP‐induced germinal vesicle breakdown in marine invertebrate oocytes

During maturation, oocytes must undergo a process of nuclear disassembly, or "germinal vesicle breakdown" (GVBD), that is regulated by signaling pathways involving cyclic AMP (cAMP). In vertebrate and starfish oocytes, cAMP elevation typically prevents GVBD. Alternatively, increased concentrations of intra-oocytic cAMP trigger, rather than inhibit, GVBD in several groups of marine invertebrates. To integrate what is known about the stimulation of GVBD by intra-oocytic cAMP, this article reviews published data for ascidian, bivalve, brittle star, jellyfish, and nemertean oocytes. The bulk of the review concentrates on the three most intensively analyzed groups known to display cAMP-induced GVBD-nemerteans, ascidians, and jellyfish. In addition, this synopsis also presents some previously unpublished findings regarding the stimulatory effects of intra-oocytic cAMP on GVBD in jellyfish and the annelid worm Pseudopotamilla occelata. Finally, factors that may account for the currently known distribution of cAMP-induced GVBD across animal groups are discussed.

Potential upstream regulators and downstream targets of AMP-activated kinase signaling during oocyte maturation in a marine worm

Unlike in mice, where the onset of oocyte maturation (germinal vesicle breakdown, GVBD) is blocked by cAMP and triggered by AMP-activated kinase (AMPK), oocytes of the marine nemertean worm Cerebratulus undergo GVBD in response to cAMP elevations and AMPK deactivation. Since the pathways underlying AMPK's effects on mammalian or nemertean GVBD have not been fully defined, follicle-free nemertean oocytes were treated with pharmacological modulators and subsequently analyzed via immunoblotting methods using phospho-specific antibodies to potential regulators and targets of AMPK. Based on such phosphorylation patterns, immature oocytes possessed an active LKB1-like kinase that phosphorylated AMPK's T172 site to activate AMPK, whereas during oocyte maturation, AMPK and LKB1-like activities declined. In addition, given that MAPK can deactivate AMPK in somatic cells, oocytes were treated with inhibitors of ERK1/2 MAPK activation. However, these assays indicated that T172 dephosphorylation during maturation-associated AMPK deactivation did not require MAPK and that an observed inhibition of GVBD elicited by the MAPK kinase blocker U0126 was actually due to ectopic AMPK activation rather than MAPK inactivation. Similarly, based on tests using an inhibitor of maturation-promoting factor (MPF), T172 dephosphorylation occurred upstream to, and independently of, MPF activation. Alternatively, active MPF and MAPK were necessary for fully phosphorylating a presumably inhibitory S485/491 site on AMPK. Furthermore, in assessing signals possibly linking AMPK deactivation to MPF activation, evidence was obtained for maturing oocytes upregulating target-of-rapamycin activity and downregulating the cyclin-dependent kinase inhibitor Kip1. Collectively, these findings are discussed relative to multiple pathways potentially mediating AMPK signaling during GVBD.

Interactions between mitogen‐activated protein kinase and protein kinase C signaling during oocyte maturation and fertilization in a marine worm

In the marine nemertean worm Cerebratulus, follicle-free oocytes re-initiate meiosis and undergo nuclear disassembly (=germinal vesicle breakdown, GVBD) after being stimulated to mature by seawater (SW) or cAMP-elevating drugs. Previously, it has been shown that inhibitors of mitogen-activated protein kinase (MAPK) or protein kinase C (PKC) signaling can reduce SW-induced GVBD in nemertean oocytes without affecting cAMP-induced GVBD. Thus, SW and cAMP elevators may trigger alternative pathways that vary in their dependence on MAPK and PKC. To further characterize such signaling cascades, immunoblotting analyses of MAPK and PKC activities were conducted on oocytes treated with U0126, an inhibitor of the MAPK kinase (MAPKK) that is responsible for activating MAPK. Based on these analyses and comparisons with the MAPKK inhibitor CI1040 that inactivates MAPK without preventing GVBD, U0126 seems to block GVBD via a non-MAPK-mediated effect that involves PKC. Moreover, evidence is presented for post-GVBD oocytes establishing positive feedback between MAPK and PKC signaling. Such feedback apparently allows the activities of both kinases to be maintained before insemination and to undergo concomitant downregulation after fertilization. Furthermore, in oocytes treated with MAPKK and PKC inhibitors during fertilization, sperm incorporation and polar body formation still occur, but normal cleavage is prevented. This suggests that although GVBD and aspects of post-fertilization activation may proceed in the absence of MAPK or PKC, such kinases are apparently required for proper embryogenesis. Collectively, these results are discussed relative to previous analyses of the interactions and functions of MAPK and PKC signaling during oocyte maturation and fertilization.

Roles of protein kinase C isotypes during seawater‐versus cAMP‐induced oocyte maturation in a marine worm

Based on immunoblotting analyses using phospho-specific antibodies, follicle-free oocytes of the marine nemertean worm Cerebratulus sp. activate protein kinase C (PKC) when induced to mature by either seawater (SW) or cAMP-elevating drugs. In SW-stimulated oocytes, the onset of maturation (=germinal vesicle breakdown, "GVBD") can be inhibited by broadly acting PKC antagonists such as bisindoylmaleimide (BIM)-I or BIM-IX. Conversely, co-treatment with SW solutions of BIM-I or BIM-IX plus a cAMP elevator (forskolin, serotonin, or a phosphodiesterase inhibitor) restores GVBD, indicating that the blockage of SW-induced GVBD by PKC antagonists is not simply due to oocyte morbidity and that such inhibition is somehow reversible by cAMP signaling. In tests to determine which specific PKC may be involved in regulating GVBD, immunoblots fail to provide strong evidence for the presence of conventional or novel PKCs, which are characteristically activated by 12-O-tetradecanoylphorbol-13-acetate (TPA). Moreover, inhibitors of TPA-sensitive PKCs do not prevent SW-induced GVBD, and TPA itself serves to downregulate, rather than stimulate, GVBD. Alternatively, maturing oocytes apparently possess phosphorylated forms of TPA-insensitive isotypes, including an approximately 67-kDa atypical PKC and an approximately 130-kDa PKC-related kinase (PRK). Accordingly, inhibitors of atypical PKC signaling block SW-but not cAMP-induced GVBD, collectively suggesting that instead of depending on a conventional or novel isotype, SW-induced GVBD may require atypical PKC and/or PRK. In addition, such findings provide further support for the view that GVBD in nemertean oocytes can be achieved via multiple mechanisms, with SW triggering different signaling pathways than are stimulated in the presence of cAMP-elevating drugs.

Differing mechanisms of cAMP‐ versus seawater‐induced oocyte maturation in marine nemertean worms II. The roles of tyrosine kinases and phosphatases

Instead of blocking oocyte maturation as it does in most animals, cAMP causes oocytes of marine nemertean worms to initiate maturation (=germinal vesicle breakdown, "GVBD"). To characterize cAMP-induced GVBD in nemerteans, inhibitors of tyrosine kinase signaling were tested on Cerebratulus sp. oocytes that had been incubated in cAMP-elevating drugs versus seawater (SW) alone. Such tests yielded similar results for Src-like tyrosine kinase blockers, as the inhibitors prevented mitogen-activated protein kinase (MAPK) activation without stopping either GVBD or maturation-promoting factor (MPF) activation in both SW and cAMP-elevating treatments. Alternatively, genistein, a general tyrosine kinase antagonist, and piceatannol, an inhibitor of the tyrosine kinase Syk, reduced GVBD and MAPK/MPF activities in SW-, but not cAMP-induced maturation. Similarly, inhibitors of the human epidermal growth factor receptor-2 (HER-2) tyrosine kinase prevented GVBD and MAPK/MPF activations in oocytes treated with SW, but not with cAMP-elevating drugs. Antagonists of either protein tyrosine phosphatases (PTPs) or the dual-specificity phosphatase Cdc25 also reduced GVBD and MAPK/MPF activities in SW-treated oocytes without generally affecting cAMP-induced maturation. Collectively, these data suggest cAMP triggers GVBD via pathways that do not require MAPK activation or several components of tyrosine kinase signaling. In addition, such differences in tyrosine kinase cascades, coupled with the dissimilar patterns of Ser/Thr kinase signaling described in the accompanying study, indicate that nemertean oocytes are capable of utilizing multiple mechanisms to activate MPF during GVBD.

Germinal vesicle breakdown is not fully dependent on MAPK activation in maturing oocytes of marine nemertean worms

Previously, it has been shown that oocytes of marine nemertean worms resume meiosis and undergo germinal vesicle breakdown (GVBD) following treatment with either natural seawater (NSW), or the neurohormone serotonin (5-hydroxytryptamine or 5-HT). In this investigation of the nemerteans Cerebratulus lacteus and Cerebratulus sp., immunoblots and kinase assays were used to compare the roles of two regulatory kinases: mitogen-activated protein kinase (MAPK) and Cdc2/cyclin B (referred to as maturation promoting factor or MPF). Based on such analyses, an ERK (extracellular signal regulated kinase) type of MAPK was found to be activated concurrently with Cdc2/cyclin B during NSW- and 5-HT-induced maturation. MAPK activation occurred prior to GVBD and seemed to be controlled primarily by phosphorylation rather than de novo protein synthesis. Inhibition of MAPK signaling by U0126 was capable of delaying but not permanently blocking Cdc2/cyclin B activation and GVBD in 5-HT treated oocytes and subsets of NSW-treated oocytes. Collectively such data indicated that GVBD is not fully dependent on MAPK activation, since Cdc2/cyclin B can apparently be activated by MAPK-independent mechanism(s) in maturing nemertean oocytes.

Endoplasmic reticulum reorganizations and Ca2+ signaling in maturing and fertilized oocytes of marine protostome worms: the roles of MAPKs and MPF.

Before a proper Ca(2+) response is produced at fertilization, oocytes typically undergo a maturation process during which their endoplasmic reticulum (ER) is restructured. In marine protostome worms belonging to the phylum Nemertea, the ER of maturing oocytes forms numerous distinct clusters that are about 5 micro m in diameter. After fertilization, mature oocytes with such aggregates generate a normal series of Ca(2+) oscillations and eventually disassemble their ER clusters at around the time that the oscillations cease. Immature oocytes, however, lack prominent ER clusters and fail to exhibit repetitive Ca(2+) oscillations upon insemination, collectively suggesting that cell cycle-related changes in ER structure may play a role in Ca(2+) signaling. To assess the effects of meiotic regulators on the morphology of the ER and the type of Ca(2+) response that is produced at fertilization, nemertean oocytes were treated with pharmacological modulators of mitogen-activated protein kinases (MAPKs) or maturation-promoting factor (MPF) prior to confocal microscopic analyses. Based on such imaging studies and correlative assays of kinase activities, MAPKs of the ERK1/2 type (extracellular signal regulated kinases 1/2) do not seem to be essential for either structural reorganizations of the ER or repetitive Ca(2+) signaling at fertilization. Conversely, MPF levels appear to modulate both ER structure and the capacity to produce normal Ca(2+) oscillations. The significance of these findings is discussed with respect to other reports on ER structure, MPF cycling and Ca(2+) signaling in oocytes of deuterostome animals.

5-HT causes an increase in cAMP that stimulates, rather than inhibits, oocyte maturation in marine nemertean worms.

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.

Multiple triggers of oocyte maturation in nemertean worms: the roles of calcium and serotonin.

To analyze the process of oocyte maturation in nemertean worms, oocytes with a large nucleus (=germinal vesicle, or GV) were removed from gravid ovaries of Cerebratulus lacteus and Micrura alaskensis. Following transfer to natural seawater (NSW), fully grown oocytes spontaneously matured as indicated by their completion of germinal vesicle breakdown (GVBD), whereas GVBD was reversibly blocked if the oocytes were initially placed in calcium-free seawater (CaFSW). Similarly, calcium ionophore treatments triggered GVBD in calcium-containing artificial seawater (ASW) but not in CaFSW, suggesting that external calcium influx may facilitate maturation. However, compared to the overall levels of maturation elicited by ASW, significantly higher percentages of GVBD were achieved with NSW or with ASW that had been conditioned with marine sediment. Moreover, calcium channel blockers decreased GVBD rates in ASW but not in NSW, which is consistent with the view that substances other than external calcium ions can trigger maturation. Accordingly, oocytes underwent equally high levels of GVBD when treated with serotonin (=5-hydroxytryptamine, or 5-HT) in ASW or CaFSW. The 5-HT-induced maturation was blocked by inhibitors of 5-HT receptors but continued to occur in the presence of calcium channel blockers or the calcium chelator BAPTA. In addition, oocytes microinjected with fluorescent calcium indicators underwent GVBD in response to 5-HT without displaying marked calcium transients during confocal imaging runs. Collectively, such findings suggest that nemertean oocytes can mature via multiple pathways that may include external calcium influx or a 5-HT-induced signaling cascade that lacks prominent calcium fluctuations. J. Exp. Zool. 287:243-261, 2000.