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.

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.

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.

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.

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.

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.

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 (Ca²⁺) in all animals that have been examined, and such Ca²⁺ elevations, in turn, provide key activating signals that are required for non-parthenogenetic development. Several lines of evidence indicate that the Ca²⁺ 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 Ca²⁺ 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Structural reorganizations of the endoplasmic reticulum during egg maturation and fertilization.

The endoplasmic reticulum (ER) of eggs is a major internal store of calcium ions that must be properly mobilized at fertilization for development to proceed. In most species, the ER develops distinct clusters in the cortical ooplasm as the oocyte matures into a fertilizable egg. Following fertilization, the structure of the ER rapidly reorganizes in eggs that produce a single fertilization-induced calcium wave, whereas ER clusters persist for relatively long periods in eggs that generate multiple calcium oscillations.

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.

The Cytoskeleton and Nuclear Disassembly during Germinal Vesicle Breakdown in Starfish Oocytes: (1-methyladenine/cytochalasin B/microfilaments/microtubules/oocyte maturation).

In response to maturation-inducing hormone, prophase-arrested oocytes of the starfish Pisaster ochraceus resume meiosis and undergo nuclear disassembly during a process referred to as germinal vesicle breakdown (GVBD). Time-lapse video recordings of maturing oocytes reveal that the nucleus lengthens along the animal-vegetal axis of the oocyte directly prior to GVBD. Neither taxol (10 μM) nor microtubule-depolymerizing agents [colcemid (50 μM), colchicine (250 μM), or nocodazole (1 μM)] prevent the pre-GVBD changes in nuclear shape from occurring, although correlative microscopical studies demonstrate that microtubules are nucleated (taxol) or depolymerized (colcemid, colchicine, nocodazole) at the concentrations listed above.