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.

Unscrambling the oocyte and the egg: clarifying terminology of the female gamete in mammals.

Most reproductive biologists who study female gametes will agree with the 16th century anatomist William Harvey's doctrine: 'Ex Ovo Omnia'. This phrase, which literally translates to 'everything from the egg', recognizes the centrality of the egg in animal development. Eggs are most impressive cells, capable of supporting development of an entirely new organism following fertilization or parthenogenetic activation. Not so uniformly embraced in the field of reproductive biology is the nomenclature used to refer to the female germ cell. What is an oocyte? What is an egg? Are these terms the same, different, interchangeable? Here we provide functional definitions of the oocyte and egg, and how they can be used in the context of mammalian gamete biology and beyond.

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.

Oocyte aging in a marine protostome worm: The roles of maturation-promoting factor and extracellular signal regulated kinase form of mitogen-activated protein kinase.

The roles of maturation-promoting factor (MPF) and an extracellular signal regulated kinase form of mitogen-activated protein kinase (ERK MAPK) are analyzed during oocyte aging in the marine protostome worm Cerebratulus. About a day after removal from the ovary, unfertilized metaphase-I-arrested oocytes of Cerebratulus begin to flatten and swell before eventually lysing, thereby exhibiting characteristics of a necroptotic mode of regulated cell death. Based on immunoblots probed with phospho-specific antibodies, MPF and ERK are initially active in freshly mature specimens. However, as oocytes age, both kinase activities decline, with ERK deactivation occurring well before MPF downregulation. Experiments using pharmacological modulators indicate that oocyte degradation is promoted by the maturation-initiated activation of ERK as well as by the deactivation of MPF that occurs in extensively aged specimens. The potential significance of these findings is discussed relative to previously published results for apoptotic eggs and oocytes of echinoderm and vertebrate deuterostomes.

Calcium signals and oocyte maturation in marine invertebrates.

In various oocytes and eggs of animals, transient elevations in cytoplasmic calcium ion concentrations are known to regulate key processes during fertilization and the completion of meiosis. However, whether or not calcium transients also help to reinitiate meiotic progression at the onset of oocyte maturation remains controversial. This article summarizes reports of calcium signals playing essential roles during maturation onset (=germinal vesicle breakdown, GVBD) in several kinds of marine invertebrate oocytes. Conversely, other data from the literature, as well as previously unpublished findings for jellyfish oocytes, fail to support the view that calcium signals are required for GVBD. In addition to assessing the effects of calcium transients on GVBD in marine invertebrate oocytes, the ability of maturing oocytes to enhance their calcium-releasing capabilities after GVBD is also reviewed. Furthermore, possible explanations are proposed for the contradictory results that have been obtained regarding calcium signals during oocyte maturation in marine invertebrates.

Calcium signaling and endoplasmic reticulum dynamics during fertilization in marine protostome worms belonging to the phylum Nemertea.

Metaphase-I-arrested eggs of marine protostome worms in the phylum Nemertea generate a series of point-source calcium waves during fertilization. Such calcium oscillations depend on inositol-1,4,5-trisphosphate-mediated calcium release from endoplasmic reticulum (ER) stores that undergo structural reorganizations prior to and after fertilization. This article reviews fertilization-induced calcium transients and ER dynamics in nemertean eggs and compares these topics to what has been reported for other animals in order to identify unifying characteristics and distinguishing features of calcium responses during fertilization across the animal kingdom.

Immunoblotting analyses of changes in protein phosphorylations during oocyte maturation in marine nemertean worms.

Immunoblotting analyses combined with phospho-specific antibodies can provide a powerful means for assessing protein activity states in various cellular extracts. This chapter describes a traditional, film-based immunoblotting method for monitoring the phosphorylation status of proteins in marine nemertean oocytes undergoing maturation. Similarly, with minor modifications, the protocol could potentially be applied to a wider variety of cellular processes and extract types that might be analyzed in other investigations of marine invertebrate development.

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.

Comparative biology of sperm factors and fertilization-induced calcium signals across the animal kingdom.

Fertilization causes mature oocytes or eggs to increase their concentrations of intracellular calcium ions (Ca²âº) in all animals that have been examined, and such Ca²âº elevations, in turn, provide key activating signals that are required for non-parthenogenetic development. Several lines of evidence indicate that the Ca²âº transients produced during fertilization in mammals and other taxa are triggered by soluble factors that sperm deliver into oocytes after gamete fusion. Thus, for a broad-based analysis of Ca²âº dynamics during fertilization in animals, this article begins by summarizing data on soluble sperm factors in non-mammalian species, and subsequently reviews various topics related to a sperm-specific phospholipase C, called PLCζ, which is believed to be the predominant activator of mammalian oocytes. After characterizing initiation processes that involve sperm factors or alternative triggering mechanisms, the spatiotemporal patterns of Ca²âº signals in fertilized oocytes or eggs are compared in a taxon-by-taxon manner, and broadly classified as either a single major transient or a series of repetitive oscillations. Both solitary and oscillatory types of fertilization-induced Ca²âº signals are typically propagated as global waves that depend on Ca²âº release from the endoplasmic reticulum in response to increased concentrations of inositol 1,4,5-trisphosphate (IP3). Thus, for taxa where relevant data are available, upstream pathways that elevate intraoocytic IP3 levels during fertilization are described, while other less-common modes of producing Ca²âº transients are also examined. In addition, the importance of fertilization-induced Ca²âº signals for activating development is underscored by noting some major downstream effects of these signals in various animals.

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.

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.

Roles of Src family kinase signaling during fertilization and the first cell cycle in the marine protostome worm Cerebratulus.

For eggs to generate a calcium response during fertilization, the sperm of many deuterostome animals must first activate a group of egg kinases, called Src family kinases (SFKs). However, whether SFK activation is also required for fertilization-induced calcium signals in eggs of protostomes remains unknown. Thus, in this study, unfertilized oocytes of the marine protostome worm Cerebratulus were treated with either PP2 to inhibit SFKs or with U73122 to block phospholipase C activity downstream of SFK. Compared with control fertilizations, the inhibitors significantly reduced post-insemination levels of polar body formation and cleavage, but apparently did so via different mechanisms, based on the variable effects of these drugs on sperm incorporations and pronuclear differentiation. Moreover, confocal calcium imaging revealed that repetitive calcium waves (=oscillations) were blocked by U73122, but not by PP2, even though immunoblots indicated SFK activity was inhibited by PP2. Such findings fail to support the view that SFKs are required for initiating fertilization-induced calcium oscillations in Cerebratulus, and alternative mechanisms for the observed inhibition of polar body formation and cleavage in drug-treated specimens are discussed.

Stimulators of AMP-activated kinase (AMPK) inhibit seawater- but not cAMP-induced oocyte maturation in a marine worm: Implications for interactions between cAMP and AMPK signaling.

Previous studies have shown that elevations in intraoocytic cAMP prevent mammalian oocytes from maturing, whereas cAMP degradation allows these oocytes to begin maturation, as evidenced by the onset of oocyte nuclear disassembly (="germinal vesicle breakdown", GVBD). Moreover, such cAMP degradation not only reduces cAMP levels but also generates AMP, which in turn can stimulate AMP-activated kinase (AMPK), a well-documented inducer of GVBD in mice. Alternatively, in some marine invertebrates, intraoocytic cAMP triggers, rather than blocks, GVBD, and whether AMPK up- or downregulates maturation in these species has not been tested. Thus, AMPK was monitored in the nemertean worm Cerebratulus during GVBD stimulated by seawater (SW) or cAMP elevators. In oocytes lacking surrounding follicle cells, AMPK activity was initially elevated in immature oocytes but subsequently reduced during SW- or cAMP-induced GVBD, given that the catalytic alpha-subunit of AMPK in maturing oocytes displayed a decreased stimulatory phosphorylation at T172 and an increased inhibitory phosphorylation at S485/491. Accordingly, AMPK-mediated phosphorylation of acetyl-CoA carboxylase, a known target of active AMPK, also declined during maturation. Moreover, treatments with either ice-cold calcium-free seawater (CaFSW) or AMPK agonists dissolved in SW maintained AMPK activity and inhibited GVBD. Conversely, adding cAMP elevators to CaFSW- or SW-solutions of AMPK activators restored GVBD while promoting S485/491 phosphorylation and AMPK deactivation. Collectively, such findings not only demonstrate for the first time that intraoocytic AMPK can block GVBD in the absence of surrounding follicle cells, but these results also provide evidence for a novel GVBD-regulating mechanism involving AMPK deactivation by cAMP-mediated S485/491 phosphorylation.

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.