Marine Nemertean Worms Research Articles

Marine Nemertean Worms for Immunoblotting Studies of Oocyte Aging.

Immunoblotting analyses employing phospho-specific antibodies can help elucidate potential roles played by protein kinases as oocytes age and lose their ability to undergo normal fertilization. This chapter updates a previously published protocol for conducting immunoblotting analyses of oocyte maturation in marine nemertean worms by adding general methods for obtaining adult worms and for handling their gametes in experiments assessing oocyte aging.

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.

The potential roles of c-Jun N-terminal kinase (JNK) during the maturation and aging of oocytes produced by a marine protostome worm.

Previous investigations have indicated that c-Jun N-terminal kinase (JNK) regulates the maturation and aging of oocytes produced by deuterostome animals. In order to assess the roles of this kinase in a protostome, oocytes of the marine nemertean worm Cerebratulus were stimulated to mature and subsequently aged before being probed with phospho-specific antibodies against active forms of JNK and maturation-promoting factor (MPF). Based on blots of maturing oocytes, a 40-kD putative JNK is normally activated during germinal vesicle breakdown (GVBD), which begins at 30 min post-stimulation with seawater, whereas treating immature oocytes with JNK inhibitors downregulates both the 40-kD JNK signal and GVBD, collectively suggesting a 40-kD JNK may facilitate oocyte maturation. Along with this JNK activity, mature oocytes also exhibit high levels of MPF at 2 h post-stimulation. However, by ~6-8 h post-GVBD, mature oocytes lose the 40-kD JNK signal, and at ~20-30 h of aging, an ~48-kD phospho-JNK band arises as oocytes deactivate MPF and begin to lyse during a necroptotic-like mode of death. Accordingly, JNK inhibitors reduce the aging-related 48-kD JNK phosphorylation while maintaining MPF activity and retarding oocyte degradation. Such findings suggest that a 48-kD JNK may help deactivate MPF and trigger death. Possible mechanisms by which JNK activation either together with, or independently of, protein neosynthesis might stimulate oocyte degradation are discussed.

High Rates of Self-Fertilization in a Marine Ribbon Worm (Nemertea).

Organisms capable of self-fertilization ("selfing") typically exhibit two evolutionary syndromes: uniting high inbreeding depression with low levels of selfing, or low inbreeding depression with high levels of selfing. We examined the effect of inbreeding on fecundity and time to first reproduction in an apparently self-compatible, simultaneously hermaphroditic marine nemertean worm Prosorhochmus americanus. Adult and juvenile worms were raised in isolation or in pairs. Isolated worms produced significantly more offspring than paired worms (in the adult experiment), and did not exhibit inbreeding avoidance (in the juvenile experiment). The selfing rate of six natural populations was evaluated using 17 species-specific, microsatellite markers, and was consistent with preferential selfing (mean: 0.843, SD: 0.027). Our results showed that P. americanus exhibited an interesting suite of life-history traits, uniting high colonization potential through self-fertilization and high fecundity, with no dispersive larval stage, and with moderate levels of gene flow. We believe that P. americanus is an ideal model system for studies of mating system evolution, inbreeding, and sex allocation.

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.

Pharmacological Analyses of Protein Kinases Regulating Egg Maturation in Marine Nemertean Worms: A Review and Comparison with Mammalian Eggs

For development to proceed normally, animal eggs must undergo a maturation process that ultimately depends on phosphorylations of key regulatory proteins. To analyze the kinases that mediate these phosphorylations, eggs of marine nemertean worms have been treated with pharmacological modulators of intracellular signaling pathways and subsequently probed with immunoblots employing phospho-specific antibodies. This article both reviews such analyses and compares them with those conducted on mammals, while focusing on how egg maturation in nemerteans is affected by signaling pathways involving cAMP, mitogen-activated protein kinases, Src-family kinases, protein kinase C isotypes, AMP-activated kinase, and the Cdc2 kinase of maturation-promoting factor.

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 I. The roles of serine/threonine kinases and phosphatases

Unlike in most animals, oocytes of marine nemertean worms initiate maturation (=germinal vesicle breakdown, GVBD) following an increase, rather than a decrease, in intraoocytic cAMP. To analyze how serine/threonine (Ser/Thr) kinase cascades involving mitogen-activated protein kinase (MAPK), maturation-promoting factor (MPF), cAMP-dependent protein kinase (PKA), and phosphatidylinositol 3-kinase (PI3K) regulate nemertean GVBD, oocytes of Cerebratulus sp. were treated with pharmacological modulators and stimulated with cAMP-elevating drugs or seawater (SW) alone. Both cAMP elevators and SW triggered GVBD while activating MAPK, its target p90Rsk, and MPF. Similarly, neither cAMP- nor SW-induced GVBD was affected by several Ser/Thr phosphatase inhibitors, and both stimuli apparently accelerated GVBD via a MAPK-independent, PI3K-dependent mechanism. However, inhibitors of Raf-1, a kinase that activates MAPK kinase, blocked GVBD and MAPK activation during SW-, but not cAMP-induced maturation. In addition, MPF blockers more effectively reduced GVBD and MAPK activity in SW versus in cAMP-elevating treatments. Moreover, the two maturation-inducing stimuli yielded disparate patterns of PKA-related MAPK activations and phosphorylations of putative PKA substrates. Collectively, such findings suggest that in maturing oocytes of Cerebratulus sp., Ser/Thr kinase cascades differ during cAMP- versus SW-induced GVBD in several ways, including MAPK activation modes, MPF-feedback loops, and PKA-related signaling pathways. Additional differences in cAMP- versus SW-induced oocyte maturation are also described in the accompanying study that deals with the roles of tyrosine kinase signaling during GVBD.

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.

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.

Injections of Porcine Sperm Extracts Trigger Fertilization-like Calcium Oscillations in Oocytes of a Marine Worm

The precise mechanisms by which sperm trigger calcium transients in eggs or oocytes during fertilization remain unknown. Based on time-lapse confocal microscopy, we show that intracellular injections of porcine sperm extracts cause the oocytes of a marine nemertean worm to undergo repetitive calcium oscillations resembling those obtained during normal fertilizations. Such findings are consistent with the view that fertilization involves a soluble sperm factor (SF) which is capable of eliciting calcium transients without binding to externally situated receptors on the oocyte plasmalemma. This study also describes for the first time the wave-like propagation patterns of SF-induced calcium transients that are generated in a heterologous combination of gametes obtained from different phyla of animals. Such cross-reactivity between distantly related taxa suggests that the intracellular signaling pathways triggered by sperm factors can be well conserved.

THE LUMINESCENCE OF A NEMERTEAN, EMPLECTONEMA KANDAI, KATO

The luminescence of a marine nemertean worm, Emplectonema kandai, living on Chelyosoma, is described. Light appears from the whole of the body, except a small region at the head end, in response to mechanical, chemical, thermal or osmotic stimulation. The effect of salts has been studied.The photogenic cells are in the epithelium, stain with eosin, and appear to have openings in the cuticle, but no extracellular luminous secretion could be demonstrated. Histological sections are figured.Luciferin and luciferase could not be demonstrated, but since KCN does not inhibit luminescence, the origin of the light is thought to be the gland cells of the worm and not symbiotic bacteria.