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Oocyte maturation and fertilization in marine nemertean worms: using similar sorts of signaling pathways as in mammals, but often with differing results.

Abstract. 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 1,45-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.


For animals to develop normally. oocytes must resume meiotic maturation and undergo a process of nuclear disassembly, referred to as germinal vesicle breakdown (GVBD) (Voronina and Wessel. 2003). In most oocytes. GVBD occurs before fertilization and is required for proper activation by sperm (Whitaker and Swann, 1993; Stricker. 1999; Runft et al., 2002). Given the fundamental roles played by oocyte maturation and fertilization, it is not surprising that a database search for such topics can yield over one hundred thousand references. The majority of these articles cover deuterostome animals (e.g., chordates and echinoderms) rather than protostomes (e.g., arthropods and molluscs), and most recent reviews of such topics have concentrated on the vast literature that has been compiled for mammals (e.g., Evans and Gadella, 2011; Kang and Han. 2011; Silvestre et al., 2011; Wakai et al., 2011; Yamada and Isaji, 2011; Ito and Kashiwazaki, 2012; Miao and Williams, 2012; Nogueira et al., 2012; Sobinoff et al., 2012; Swann and Lai, 2013).

In addition to various studies of mammals, aspects of gamete biology have also been examined in such marine invertebrates as jellyfish. annelids, molluscs, echinoderms, and ascidians, owing in part to the ease with which eggs and sperm can be obtained (e.g., Deguchi et al., 1996, 2005; Yi et al., 2002; Chausson et al., 2004; Dupont and Dumollard, 2004; Takeda et al., 2006; Deguchi, 2007; Lambert, 2008; Vacquier, 2011; McDougall et al., 2012; Santella et al., 2012). However, in spite of their benefits, commonly used marine invertebrate oocytes may also present some drawbacks. For example, jellyfish and sea urchin eggs complete meiosis in the ovary (Freeman and Ridgway, 1988: Wessel et al., 2002; Miyata et al., 2006), which can complicate analyses of prophase-I arrest and meiotic resumption. Similarly, the solitary calcium wave generated at fertilization in starfish and sea urchins (Stricker et al., 1992; Stricker, 1995) differs from repetitive calcium transients in other oocytes such as those of mammals, which, unlike starfish oocytes and sea urchin eggs, are fertilized at a metaphase-arrest point during maturation (Jones, 1998; Sardet et al., 1998; Stricker, 1999; Swann and Yu, 2008).

Thus, to assess alternative types of oocytesu1/4 analyses began to be conducted about 15 years ago on marine nemertean worms, which are sometimes confused with alike-sounding nematodes. However, nemerteans, or "ribbon worms," belong to their own phylum of about 1300 species in the lophotrochozoan clade of protostomes that includes molluscs and annelids, whereas nematodes and related invertebrates such as arthropods constitute separate phyla of ecdysozoan protostomes (Kajihara et al., 2008; Struck and Fisse 2008). Prior to the 1990s, much of what was known about nemertean oocytes was derived from classical studies (e.g., Coe, 1899, Wilson. 1900), and except in a few cases (e.g., Freeman, 1978; Jaffe et al., 1985), the mechanisms of oocyte maturation or fertilization in nemerteans had not been analyzed. However, more recent investigations have begun to show that nemerteans can utilize similar sorts of intraoocytic signaling pathways as in mammals, and yet, as reviewed further in the following sections, such pathways often mediate different responses in nemertean than in mammalian oocytes.

Materials and Methods

Ripe specimens of the nemerteans Cerebratulus sp., Emplectonema gracile (Stimpson, 1837), Micrura ala.skensis Coe, 1901, and Tubulanus polymorphus Renier, 1804 were collected at low tides on San Juan Island, Washington. USA. whereas C. lacteus (Leidy. 1851) adults were purchased from the Marine Biological Laboratory, Woods Hole, Massachusetts. Immature oocytes obtained from cut females were pre-incubated in ice-cold calcium-free seawater (CaFSW) (Schroeder and Stricker. 1983) for 1.5-2 h to reduce spontaneous GVBD. Dejellied oocytes were then transferred to seawater (SW), artificial seawater (ASW), or CaFSW and kept at ambient SW temperatures (-12-14 [degrees]C) with or without pharmacological modulators. Drug dosages were adopted on the basis of literature reports or dose-response curves that identified minimum effective concentrations. Western blots of total cell lysates were performed as described elsewhere (Stricker, 2011; Escalona and Stricker, 2013). Similarly, histological methods and confocal imaging of oocytes injected with probes for tracking endoplasmic reticulum (ER) dynamics, calcium ions, or cyclic adenosine monophosphate (cAMP) levels were carried out as detailed previously (Stricker, 1996; Stricker et al., 1998, 2001; Stricker and Whitaker, 1999; Stricker and Smythe, 2001). Nearly all images and graphs are comparable to previously published figures in cited papers, which provide full protocol descriptions. However, Figures 2E and 3A document some as-of-yet unreported data and are thus supplemented with methodological details in the figure legends. Given that mammalian gamete biology has been thoroughly described in recent reviews, coverage here of mammals is restricted to topics that can be directly compared to what has been reported for nemerteans.

Results and Discussion

Oogenesis and the production of follicle-free oocytes in nemerteans

In most marine nemerteans, sexes are separate, and ripe females spawn their oocytes prior to undergoing external fertilizations (Stricker, 1982, 1985, 1986, 1987; Stricker and Folsom, 1998) (Fig. 1A, B). Follicle cells are characteristically lacking during nemertean oogenesis, and in species such as Cerebratulus lacteus, Micrura alaskensis, and Par-borlasia corrugatus, numerous ovaries develop along the body, with each gonad producing dozens to hundreds of microlecithal oocytes that average 70-130 p.m in diameter (Stricker et al., 2001; Norenburg and Stricker, 2002; Grange et al., 2011). Thus, nemerteans can supply copious amounts of oocytes for analyses that are not subject to the potentially confounding effects of follicle cells (Fig. 1B-G).

Similarly, mammals are normally dioecious and generally produce oocytes similar in size to those of microlecithal nemertean specimens (Hartman, 1929). However, compared to most nemerteans, mammals ovulate fewer oocytes (Zuckerman, 1951) prior to internal fertilizations in the female reproductive tract. Moreover, unlike in nemerteans, mammalian oocytes develop in a follicle that can exhibit some species-specific differences (Griffin et al., 2006) but invariably promotes complex interactions between oocytes and their surrounding follicle cells (Matzuk et al., 2002; Rodrigues et al., 2008; Su et al., 2010; Gilchrist, 2011). In fully grown mammalian follicles, GVBD is prevented by inhibitory cues that are produced by the follicle itself. Maturation can then be triggered by the pituitary-derived gonadotropin, luteinizing hormone, and by signals of follicle cell origin, such as epidermal growth factor-like ligands (Park et al., 2004). Alternatively, after removal from their follicles, cumulus-enclosed or denuded oocytes of mammals are prevented from maturing by exogenously supplied GVBD antagonists. Compared to intact follicles, such preparations may provide more straightforward assessments of experimental results, but neither responds to the same spectrum of maturation-inducing cues that elicit GVBD in whole follicles (Downs, 2010).

Stimulation of nemertean GVBD by seawater or cAMP-elevating drugs vs. the blockage of GVBD by calcium-free seawater

At 10-14[degrees]C. fully grown nemertean oocytes can initiate GVBD within about 15-45 min after being stimulated by natural seawater (SW) (Stricker and Smythe, 2000). Conversely, nemertean oocytes remain immature with an intact germinal vesicle when treated with calcium-free seawater (CaFSW) (Fig. 2A-D), and such inhibition is not due to morbidity, since oocytes that are removed from CaFSW after a day of incubation are capable of undergoing normal maturation and fertilization (Stricker and Smythe, 2000).

The differential effects of SW versus CaFSW on GVBD are not solely mediated by calcium ions, given that CaFSW supplemented with 10 mmol [l.sup.-1] calcium such as found in SW elicits less GVBD than in SW controls (Stricker and Smythe, 2000). Moreover, treatments with calcium channel blockers and the calcium chelator BAPTA inhibit GVBD in calcium-containing artificial seawater (ASW) but fail to prevent GVBD in SW-stimulated oocytes (Stricker and Smythe, 2000). Furthermore, conditioning ASWs for several weeks with marine sediments transforms such solutions into as robust stimulators of GVBD as SW (Stricker and Smythe. 2000). Thus, SW apparently contains maturation-inducing substance(s) other than just calcium itself, and although such substances have yet to be identified, experiments using boiled SW or SW that had been fractionated by MW-cutoff filters suggest the presence in SW of maturation-inducing stimuli that are heat-stable and less than 30 kD (Fig. 2E). Accordingly, the molecular weight and apparent non-proteinaceous nature of active component or components in SW are at least consistent with some GVBD inducers identified for other oocytes, including maturation-inducing sterols (Byskov et al., 2002).

In addition to SW's stimulatory effects, cAMP-elevating drugs also trigger GVBD and can do so by alternative modes of action, given that nemertean oocytes mature in response to (1) 8-bromo cAMP (Fig. 2F); (2) the adenylate cyclase (AC) activator forskolin; (3) inhibitors of phosphodiesterases (PDEs), including the broadly acting PDE antagonist IBMX and other PDE blockers (etazolate, Ro-20-1724. and rolipram) that are more specific for type 4 PDEs; and (4) serotonin (=5HT), which may act as both an AC stimulator and a PDE inhibitor (Stricker and Smythe, 2001). Such drugs induce a marked pre-GVBD increase in intraoocytic cAMP at physiologically relevant doses (Fig. 2G) (Stricker and Smythe, 2001). Moreover, cAMP elevators cause maturation in calcium-free ASW and can trigger GVBD without markedly affecting intraoocytic calcium ion levels (Stricker and Smythe, 2000, 2001), collectively suggesting that cAMP-elevating drugs elicit GVBD Oct their shared ability to increase cAMP rather than by off-target effects, such as calcium-mediated signals. Owing to technical limitations of the experimental setup used for such microinjection and imaging analyses, it has yet to be determined if immature nemertean oocytes also generate either a cAMP or a calcium transient in response to SW stimulations. However, as discussed further in the following sections. several lines of evidence suggest that even if SW elicits such transients, the amplitudes or spatiotemporal properties of these signals do not fully mimic the intraoocytic responses mediated by exogenously applied cAMP elevators.

Protein kinase A (PKA) isotypes are fundamental mediators of cAMP signaling in cells (Taylor et al., 2004). Based on studies of mammalian follicles where dibutryl-cAMP (db-cAMP)-induced cAMP rises are thought to target type I PKAs, and 8-bromo-cAMF's actions are apparently associated with type H PICA signaling (Downs and Hunzicker-Dunn, 1995), the differing effectiveness of 8-bromoversus db-cAMP (Fig. 2F) in nemertean oocytes could indicate that a type II PKA mediates cAMP signaling. In any case, regardless of which specific PKA functions in nemertean oocytes. GVBD in response to cAMP elevators is blocked by PKA inhibitors (Sticker and Smythe, 2001). Accordingly, in blots probed with an antibody to sites phosphorylated by active PKAs, maturing oocytes display increased phosphorylations of several unidentified putative PKA targets, including a major band at about 130 kD, and such responses are reduced to varying degrees by the PKA blocker H89 in oocytes that had been stimulated by SW versus cAMP elevators (Stricker and Smythe, 2001, 2006A).

The alternative effects of H89 during SW versus cAMP stimulations suggest that cAMP signaling does not fully overlap the pathways that mediate SW-induced GVBD. This conclusion is also supported by the findings that (1) some oocyte batches respond fully to one, but not the other, kind of stimulation; (2) sensitivities to SW versus cAMP elevators decline at different rates during oocyte aging; and (3) as discussed further in the section "Inhibition of SW-induced GVBD in nemerteans by various pharmacological modulators ... ", other pharmacological modulators block SW-induced GVBD, whereas co-adding cAMP elevators along with those modulators restores GVBD (Stricker and Smythe, 2000; Stricker et al., 2010). In any event, although it remains unknown if oocytes in the field are initially stimulated by intraovarian cAMP signaling before spawning or if spawned oocytes begin to mature only after contacting SW, laboratory analyses reveal that nemertean oocytes can mature in response to either SW or cAMP elevators.

When compared to nemertean GVBD that can start as early as 15 min post-stimulation, the lag between maturation stimulation and GVBD onset is longer in mammals, even at the higher temperatures used for such oocytes. Thus, although GVBD timing varies widely among species and is affected by culture conditions, mammalian oocytes typically do not begin GVBD until 2-24 h post-stimulation (Hegele-Hartung et al., 1999; Lu et al., 2001; Fan and Sun, 2004).

Furthermore, as noted for nemertean oocytes, some studies of mammals have also indicated that GVBD does not require intraoocytic calcium transients (Paleas and Powers, 1981; Carroll and Swami. 1992; Tombes et al., 1992; Mehlmann et al., 2006). However, other analyses have shown that mammalian GVBD is calcium-dependent (Homa, 1995; Su et al., 1999; Tosti, 2006) and that external calcium influx can play an important role in meiotic resumption (Bae and Channing, 1985; Goren et al., 1990; Zhou and Jin, 2007). Such conflicting results could be due to species-specific differences or to variations in experimental procedures such as the type of maturation inducer that is used and whether or not oocytes are associated with follicle cells (Homa, 1995; Fan et al., 2004).

By contrast, a well-unified literature has demonstrated that prolonged elevations in intraoocytic cAMP prevent GVBD in mammalian oocytes and that meiotic resumption is associated with a decrease in intraoocytic cAMP (Mehlmann, 2005; Downs, 2010; Jaffe and Norris, 2010; Sole et al., 2010; Tripathi et al., 2010; Tsafriri and Dekel, 2010). Accordingly. intraoocytic PKA inhibits GVBD in mammals, whereas the relief of PKA's effects in response to decreased cAMP levels promotes GVBD (Kovo et al., 2006; Zhang et al., 2008; Pirino et al., 2009; Oh et al., 2010). Alternatively, in a few cases, mammalian GVBD has been shown to involve an initial transient increase in intraoocytic cAMP (Salustri et al., 1985; Yoshimura et al., 1992; Mattioli et al., 1994), and pulsed applications of cAMP elevators can trigger maturation (Chen et al., 2009). Nevertheless, the generally inhibitory effects of intraoocytic cAMP/PKA on GVBD in mammals differ from the stimulatory actions of cAMP signaling in nemerteans and other marine invertebrates that undergo cAMP-induced GVBD (Deguchi et al., 2011).

Roles of maturation-promoting factor, mitogen-activated protein kinases, and protein neosynthesis during nemertean GVBD

Two key cell cycle regulators in eggs are maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) subtypes, called extracellular signal regulated kinases 1/2 (=ERK1/2, "MAPKs 3/1," or hereafter, simply MAPKs) (Kishimoto, 2003; Sun et al., 2009). In nemerteans, MPF is activated before GVBD, and such activation does not appear to depend on the neosynthesis of MPF's regulatory subunit, cyclin B, given that cyclin B1 is present in immature oocytes and does not markedly increase before GVBD (Fig. 3A). Moreover, although such results do not preclude the possibility that an alternative isoform of cyclin affects MPF activity, protein neosynthesis in general appears to be nonessential for nemertean oocyte maturation, since several protein synthesis inhibitors fail to block GVBD (Stricker and Smythe, 2000; Smythe and Stricker, 2005). Alternatively, MPF apparently involves phosphorylation changes in MPF's kinase moiety (=Cdc2 or Cdk1), as maturing oocytes exhibit both decreased inhibitory phosphorylation at the Y15 site of Cdc2 and increased stimulatory phosphorylation at T161 on Cdc2 (Stricker and Smythe, 2003, 2006a) (Fig. 3B). Furthermore, MPF activation is necessary for GVBD, given that MPF inhibitors consistently block GVBD, although, for undetermined reasons, higher doses of MPF antagonists are needed to inhibit GVBD in oocytes that are stimulated by cAMP elevators versus SW (Fig. 3C) (Stricker and Smythe, 2003. 2006a).

As is the case with MPF, MAPK is activated before GVBD (Smythe and Stricker, 2005; Stricker and Smythe, 2003, 2006a). However, oocytes incubated with the MAPK kinase (MAPKK) inhibitors CI1040 (ci) and PD98059 still complete GVBD without marked MAPK activity (Smythe and Stricker, 2005; Snicker and Smythe, 2006a; Stricker, 2009b) (Fig. 3B). Moreover, although some oocyte batches fail to mature in SW solutions of the MAPKK inhibitor U0126 (uo) (Smythe and Stricker, 2005), such inhibition is apparently due to off-target effects rather than to MAPK deactivation per se. This conclusion is based on the findings that co-adding cAMP elevators along with uo restores GVBD without activating MAPK and that combining uo with ci prevents the onset of GVBD that normally occurs when ci alone is used to deactivate MAPK (Fig. 3B) (Stricker. 2009a. 2011).

As noted for nemerteans, mammalian GVBD typically involves changes in Cdc2 phosphorylations that convert inactive pre-MPF into active MPF (Choi et al., 1991: Kishimoto, 2003; Jones, 2004). Thus, MPF activation in nemerteans and mammals contrasts with the de novo synthesis of cyclin that activates MPF either in fish and non-Xenopus amphibians that lack pre-MPF (Yamashita, 2000: Suwa and Yamashita, 2007) or in Xenopus, where cyclin neosynthesis during progesterone stimulation can help to transform pre-MPF into MPF (Gaffre et al., 2011). Moreover, contrary to the view that the cyclin-dependent kinase activating kinase (CAK) responsible for phosphorylating 1161 of MPF is constitutively active throughout maturation of Xenopus oocytes and is thus not involved in regulating MPF activity during GVBD (Brown et al., 1994), T161 phosphorylation increases in maturing pig oocytes (Fujii et al., 2011), as has also been noted for nemertean oocytes.

As opposed to its pre-GVBD onset in nemerteans, MAPK activation can

occur either before or after GVBD in mammals (Fan and Sun, 2004; Liang et al., 2007; Sun et al., 2009). Nevertheless, regardless of the timing of MAPK activation, most studies indicate that intraoocytic ERK MAPK activity is not required for mammalian GVBD (Sun et al., 2009). Conversely, such activation must occur in mammalian follicle cells for GVBD to proceed (Fan et al., 2009), and active ERK MAPK is needed within post-GVBD oocytes of mammals for the successful completion of meiosis (Fan and Sun, 2004; Liang et al., 2007; Sun et al., 2009).

Unlike in nemerteans, in ungulate mammals protein synthesis inhibitors block GVBD (Fulka et al., 1986; Kastrop et al., 1991; LeGal et al., 1992; Alm and Hinrichs, 1996). Conversely, such treatments fail to prevent GVBD in rodents (Schultz and Wassarman, 1977; Fulka et al., 1986; Plancha and Albertini, 1992). Aside from species-specific differences, such varying responses may depend on the timing of inhibitor application or on the presence versus absence of follicle cells (Ekholm and Magnusson, 1979; Motlik et al., 1989).

Inhibitory effects of AMP-activated kinase signaling on SW-induced GVBD in nemerteans

In addition to modulating energy states (Hardie, 2007), AMP-activated kinases (AMPKs) can also regulate cell cycling (Alessi et al., 2006; Motoshima et al., 2006). Accordingly, blots probed with antibodies to active AMPK, inactive AMPK, or a phosphorylated substrate of active AMPK indicate that AMPK is active in prophase-arrested nemertean oocytes and deactivated during GVBD stimulated by SW or cAMP elevators (Fig. 4A) (Stricker et al., 2010a, b). Similarly, various AMPK agonists, including the AMPK mimetic AICAR, block AMPK deactivation and prevent SW-induced GVBD in nemerteans, whereas co-adding cAMP elevators overrides such blockage and restores GVBD (Fig. 4B-D). Moreover, AMPK activation in immature oocytes apparently downregulates two MPF stimulators--target of rapamycin (TOR) and nitric oxide synthase (NOS) (Stricker, 2011, 2012)--while upregulating the MPF inhibitor p27 Kip1 (Stricker, 2011), collectively indicating that AMPK signaling serves to inhibit GVBD in nemerteans (Stricker et al., 2010a, b; Stricker, 2011).

Conversely, intraoocytic AMPK is a potent trigger of GVBD in mice (Downs et al., 2002; Chen et al., 2006; LaRosa and Downs, 2006, 2007; Chen and Downs, 2008), and to a lesser degree, in rats (Downs, 2011). Alternatively, AMPK agonists block GVBD in cumulus-enclosed cow and pig oocytes (Bilodeau-Goeseels et al., 2007; Mayes et al., 2007; Tosca et al., 2007), although such inhibition is apparently due either to AMPK's actions on follicle cells rather than on the oocyte itself, or to ectopic effects on AMPK-independent pathways (Mayes et al., 2007; Tosca et al., 2007; Bilodeau-Goeseels et al., 2011). Moreover, although numerous studies have analyzed the effects of NO-related pathways on mammalian GVBD. potential interactions between intraoocytic AMPK levels and either NOS or TOR signaling have apparently not been reported for mammals.

Inhibition of SW-induced GVBD in nemerteans by various pharmacological modulators, and the overriding of such blockage by co-addition of cAMP elevators

As noted for AMPK activators, other drugs also block SW-induced GVBD in nemerteans, whereas such blockage is readily reversed by co-adding a cAMP elevator. For example, GVBD is prevented during stimulation in SW--but not in the presence of cAMP-elevators--following treatments of nemertean oocytes with blockers of Raf1, protein tyrosine kinases (Fig. 5A, B), protein tyrosine phosphatases, and the dual-specificity phosphatase Cdc25 (Stricker and Smythe, 2006a, b; Stricker et al., 2010a).

Similarly, SW-induced GVBD is inhibited by broad-spectrum protein kinase C (PKC) blockers and by more specific inhibitors of the calcium- and diacylglycerol-independent atypical PKCs (aPKCs) (Stricker, 2009a, b). Conversely, combining cAMP elevators with these PKC inhibitors restores GVBD (Stricker, 2009a, b) (Fig. 5C, D). Accordingly, blots also indicate that aPKCs, rather than conventional or novel PKCs, are activated during nemertean GVBD, and that the blockage of PKC activity induced by broad-spectrum PKC inhibitors is restored by co-adding cAMP-elevating drugs (Stricker, 2009a).

In addition, antioxidants and other commonly used antagonists of nitric oxide (NO)/cGMP signaling, including NOS inhibitors, a NO scavenger, and soluble guanylate cyclase (sGC) blockers, prevent GVBD induced by SW, but not by cAMP elevators (Stricker, 2012) (Fig. 5E-H). Moreover, NOS activity normally induced by SW stimulation is blocked by the AMPK activator AICAR, whereas co-addition of cAMP elevators reverses such blockage while restoring GVBD (Stricker, 2012).

One interpretation of these results is that SW and cAMP elevators trigger a single signaling cascade and that all of the tested inhibitors simply disrupt steps upstream to a cAMP rise. However, a solitary GVBD-promoting cascade is difficult to reconcile with the fact that some of the drugs exhibiting differential effects on SW versus cAMP-induced GVBD alter targets downstream to cAMP such as PKA, the PKA substrate Cdc25, or even MPF itself (Fig. 3C). Instead, then, these findings along with other data suggest that nemertean GVBD can be elicited by multiple signaling pathways (Stricker and Smythe, 2000, 2001, 2006a, b) and that, compared to GVBD triggered by cAMP elevators, SW-induced GVBD may be more dependent upon Raf1, tyrosine kinases, protein tyrosine phosphatases, Cdc25, atypical PKCs, NO or cGMP signaling. Regardless of how such differential drug effects are interpreted, the ability of cAMP elevators to restore maturation provides a means of confirming that GVBD blockages by drugs in SW alone are not simply due to oocyte morbidity.

Several studies have suggested that, as proposed for nemerteans, multiple signaling cascades mediate mammalian GVBD (Faerge et al., 2001; Lu et al., 2001; Zhang et al., 2005). However, some of the support for this view comes from comparing denuded oocytes to cumulus-enclosed ones and may reflect the effects of a "cAMP paradox" (Tsafriri and Dekel, 2010), wherein signaling cascades in mammalian follicles allow cAMP-elevating pathways in follicle cells to promote GVBD, whereas intraoocytic cAMP elevations serve an opposite, inhibitory role (Tsafriri et al., 1972; Dekel et al., 1988; Xia et al., 1994; Tsafriri et al., 1996). In any case, there appears to be no wide-ranging collection of modulators that can block or not block mammalian GVBD, simply depending on the presence or absence of cAMP elevators. Moreover, most pharmacological analyses of rodent oocytes indicate that inhibiting intraoocytic PKC promotes GVBD, and that, unlike the stimulatory aPKCs of nemertean oocytes, conventional PKCs block mammalian GVBD (Bornslaeger et al., 1986; Alexandre and Mulnard, 1988; Lefevre et al., 1992; Luria et al., 2000; Downs et al., 2001; Lu et al., 2001; Quan et al., 2003; Fan et al.. 2004; Denys et al., 2007). Similarly, although conflicting data have been reported for mammalian species, several studies of rat oocytes show that, unlike in nemerteans, NO/cGMP blocks GVBD (Nakamura et al., 2002; Yamagata et al., 2002; Sela-Abramovich et al., 2008; Tripathi et al., 2009).

Post-GVBD processes induced by fertilization in nemerteans

After GVBD, nemertean oocytes normally arrest at metaphase I by about 60-90 min post-stimulation. Fertilization of metaphase-I oocytes is followed by sperm incorporation, pronuclear reorganization, polar body production, and cleavage by about 2-6 h post-insemination (Stricker, 1987) (Fig. 6A). In the presence of PKC or MAPK inhibitors, polar body formation continues, whereas such antagonists usually block cleavage (Stricker, 2009b). Moreover, although inhibitors of phospholipase C (PLC) and Src-family kinases (SFKs) fail to block GVBD, both types of inhibitors prevent polar body formation and cleavage (Stricker et al., 2010c). However, such blockages apparently occur via alternative mechanisms, since the SFK antagonist PP2 promotes polyspermy (Fig. 6B) while disrupting pronuclear decondensation, whereas the PLC blocker U73122 (U7) reduces sperm incorporations and pronuclear migrations (Stricker et al., 2010c). Moreover, U7 prevents fertilization-induced calcium oscillations (see next section), whereas repetitive calcium transients continue to occur during fertilizations in the presence of PP2 (Stricker et al., 2010c).

Unlike those in nemerteans, mammalian oocytes characteristically arrest at metaphase II before fertilization, and even at the higher culture temperatures used for mammals, cleavage onset occurs later than in nemerteans. Thus, mice begin cleaving at about 25 h (Edwards and Gates, 1959) or about 17.5 h (Kaufman, 1973) post-fertilization during in vivo or in vitro treatments, respectively. Similarly, rat zygotes can take 21-23 h to cleave (Shalgi et al., 1985). Moreover, mammalian oocytes differ from those of nemerteans in that MAPK or PKC inhibitors can prevent polar body formation (Gallicano et al., 1993; Quan et al., 2003). Conversely, mammalian oocytes treated with PLC or SFK antagonists exhibit defects in sperm incorporations, pronuclear reorganizations, and cleavage that resemble what has been reported for nemerteans, and some of these defects appear to be associated with abnormal calcium signals (Dupont et al., 1996; Talmor-Cohen et al., 2004; Meng et al., 2006; McGinnis et al., 2011).

Fertilization-induced calcium dynamics

In all animals examined, fertilizing sperm cause the levels of intracellular free calcium ions in eggs to rise (Stricker, 1999; Miyazaki, 2006). Accordingly, fertilization of metaphase-I-arrested nemertean oocytes generates repetitive calcium increases that begin with a more-or-less simultaneous rise, or "cortical flash" (Parrington et al., 2007), around the oocyte perimeter (Fig. 7A, B) (Stricker, 1996, 1997, 2000, 2004; Stricker et al., 1998, 2010c; Stricker and Smythe, 2003). Several lines of evidence indicate that the cortical flash involves calcium influx, helps to prevent polyspermy, and depends on proper SFK signaling (Stricker, 1996; Stricker et al., 2010c).

Within 5-10 min after cortical flash production, each nemertean zygote begins to generate a series of calcium waves that travel about 10-15 [micro]m/s. Such calcium oscillations typically arise every 2-8 min for 20-60 min post-fertilization and end before polar body formation is completed (Stricker, 1996). Thus, pronuclei, which are generated only after polar bodies are fully formed, do not appear to play a vital role in terminating the calcium signal in nemerteans, unlike in some mammalian oocytes where pronuclear formation is involved in the cessation of calcium oscillations (Marangos et al., 2003).

In C. lacteus (Stricker, 1996), the initial one to three calcium waves arise from the site of sperm entry and fail to spread across the entire ooplasm, thereby resembling incompletely propagating transients in ascidians (Yoshida et al., 1998), or what have been called "tango waves" in computer models of calcium wave propagations (Li, 2003). Thereafter, calcium transients spread across the entire ooplasm as point-source waves and usually attain lower amplitudes than are exhibited by the calcium waves of mammalian fertilizations (Stricker, 1999). In addition, even if a sperm enters the animal hemisphere of the nemertean oocyte, later waves shift vegetally toward a repeated onset site (Stricker, 1996) (Fig. 7C, D) resembling the "vegetal pacemaker" in oocytes of other animals (Dumollard et al., 2002).

Since repetitive calcium transients continue to be generated in nemertean oocytes that are transferred to CaFSW after fertilizations in SW (Stricker, 1996), internal calcium sources mediating calcium signaling have been analyzed. Such studies reveal that [IP.sub.3]-sensitive stores are present and required for calcium signaling, on the basis of the findings that photoactivations of caged [IP.sub.3] cause calcium transients in unfertilized oocytes and that heparin injections to block [IP.sub.3]-mediated calcium release prevent fertilization-induced calcium oscillations (Stricker, 1996).

Given that immature nemertean oocytes fail to generate an oscillatory calcium response upon insemination (Fig. 7E) (Stricker, 1996; Stricker and Smythe, 2003), the possible effects of the maturation-associated kinases MPF and MAPK on calcium signaling have been examined. In such tests. MAPK kinase inhibitors eliminate ERK 1/2 MAPK activation but fail to prevent fertilization-induced calcium oscillations (Fig. 7F) (Stricker and Smythe, 2003). Conversely, the MPF inhibitor roscovitine either blocks repetitive calcium waves if used on mature oocytes before fertilization (Fig. 7G) or dampens existing oscillations if added after the onset of a fertilization-induced calcium response (Stricker and Smythe, 2003). Accordingly, treatments with colchicine to maintain MPF activity prolong oscillations during fertilization (Fig. 7H), collectively indicating that fertilization-induced calcium signaling in nemerteans depends on MPF, rather than MAPK, activity (Sticker and Smythe, 2003).

As noted for nemerteans, fertilization of mammalian eggs triggers calcium oscillations (Jones, 2007; Ducibella and Fissore. 2008: Swarm and Yu, 2008; Whitaker. 2006; Wakai et al., 2011). However, as opposed to what occurs in nemerteans, the first fertilization-induced calcium transient in rodents spreads as a point-source wave rather than a distinct cortical flash (Deguchi et al., 2000). In addition, subsequent transients in mammals can continue for a few hours after second polar body formation (Jones et al., 1995) and thus, unlike the short-lived oscillations of nemerteans, may persist for more than 20 h (Wakai et al., 2011). Moreover, oscillation intervals can be as little as 3 min or up to 50 min in mice versus cows, respectively (Bedford et al., 2006), and some of the later calcium transients in mammals propagate via a non-wavelike mode (Miyazaki et al., 1993) that has yet to be observed in oscillating nemertean oocytes. Furthermore, although the initial calcium oscillations during mammalian fertilization persist in calcium-free medium as noted for nemerteans, later oscillations require external calcium influx (Sardet et al., 1998). Also, MAPK-mediated alterations in [IP.sub.3] receptor sensitivity can play a key role in mammalian oocytes (Lee et al., 2006: Ito et al., 2008; Wakai et al., 2011), whereas in nemerteans, blocking MAPK activity does not markedly affect calcium responses.

Evidence for a soluble sperm factor mediating calcium oscillations at fertilization

In the nemertean C. lacteus, injecting whole sperm or sperm extracts into metaphase-I-arrested oocytes generates fertilization-like calcium oscillations (Stricker, 1996, 1997) (Fig. 8A. B). Similarly, injections of porcine sperm extracts can also produce calcium oscillations (Stricker et al., 2000). These findings indicate that the calcium response of nemertean oocytes is not dependent upon an external oolemmal receptor; instead, they are consistent with the view that sperm introduce a soluble oscillogenic factor into the ooplasm, as has been documented for other marine invertebrates, such as ascidians (Kyozuka et al., 1998; McDougall et al., 2000; Runft and Jaffe, 2000; Levasseur et al., 2007). Although the ability of porcine sperm extracts to trigger oscillations in nemertean oocytes suggests shared features in sperm factor signaling, the putative sperm factor of nemerteans remains uncharacterized other than that its oscillogenic activity is heat labile and contained in fractions of 10 to 100 kD (Stricker, 1997). Thus, it has yet to be determined if a sperm factor in nemerteans resembles what has been reported for mammals or any other animal in which sperm factor composition or function has been assessed (Coward et al., 2005, 2011; Harada et al., 2007, 2011; Wu et al., 2007; Aarabi et al., 2010).

As reviewed in detail elsewhere (Ito et al., 2011; Wakai et al., 2011; Nomikos et al., 2012; Swann and Lai, 2013), fertilization in mammals involves a soluble sperm factor. Several lines of evidence indicate that the sperm-derived oscillogenic factor of mammals corresponds to a sperm-specific PLC isotype, called PLC[zeta] (Heytens et al., 2009; Kashir et al., 2010, 2012; Ramadan et al., 2012), although not all findings support this view (Aarabi et al., 2012). In mice, sequestration of PLC[zeta] into nascent pronuclei is thought to function in terminating oscillations (Larman et al., 2004; Yoda et al., 2004). However, even though the PLC[xi]s of other mammals contain a nuclear localization signal, such isotypes fail to translocate into pronuclei and thus argue against nuclear sequestration of PLC[zeta] being the sole mechanism of terminating oscillations (Cooney et al., 2010).

Structural reorganizations of the endoplasmic reticulum during oocyte maturation and fertilization

As a result of the interrelated findings that immature nemertean oocytes fail to generate a robust fertilization-induced calcium response (Fig. 7D) and that the endoplasmic reticulum (ER) is the major mobilizable store of calcium during fertilization (Eisen and Reynolds, 1985), morphological changes in the ER have been assessed. Maturing nemertean oocytes that had been injected with the vital ER-specific probe DiI were examined with confocal microscopy (Teraski and Jaffe, 2004). Such analyses reveal that prophase-arrested nemertean oocytes possess a relatively homogeneous ER (Fig. 9A), except for scattered DiI-positive strands that may correspond to annulate lamellae-containing subcompartments of the ER (Beckhelling et al., 2003).

However, in metaphase-arrested nemertean oocytes, the ER is reorganized into microdomains, or "clusters," of about 5 pm in diameter (Fig. 9B, C), in both the animal and vegetal hemispheres, albeit without displaying any overt animal-vegetal heterogeneity that might correspond to the vegetal pacemaker activity observed during fertilization (Stricker et al., 1998; Stricker and Smythe, 2003; Stricker, 2006). After insemination, such clusters disassemble at about 20-60 min post-fertilization, which in turn precedes second polar body formation and corresponds to the cessation of oscillations (Fig. 9C) (Stricker et al., 1998; Stricker and Smythe, 2003). Inhibition of ERK-type MAPK activation fails to affect ER reorganizations (Stricker and Smythe, 2003). Alternatively, the MPF inhibitor roscovitine promotes both ER disassembly in unfertilized specimens (Fig. 9D) and premature ER breakdown coupled with oscillation termination in fertilized oocytes (Stricker and Smythe, 2003). Conversely, treatments with colchicine to delay MPF degradation cause persistent ER clustering and prolonged oscillations (Stricker and Smythe, 2003), presumably because under such conditions, exit from meiosis is prevented by a spindle checkpoint (Kishimoto, 2003) that remains operative after microtubular disruption. In any case, such findings indicate that a robust fertilization-induced calcium response in nemertean oocytes is associated with ER clusters that depend upon MPF activity.

As in nemerteans, mammalian oocytes form ER clusters during maturation (Kline, 2000; Mann et al., 2010), and in mice, such clusters disassemble after roscovitine treatments (FitzHarris et al., 2003). However, compared to those in nemerteans, such structures in mice tend to be smaller as well as more cortically restricted and vegetally enhanced (Stricker, 2006). In addition, unlike in nemerteans, repetitive calcium transients in mouse oocytes continue for 2 h after ER clusters have disassembled (FitzHarris et al., 2003). Nevertheless, along with altered IP3 receptor sensitivities (Wakai et al., 2011), such ER reorganizations apparently contribute in fundamental ways to shaping calcium signals in both nemertean and mammalian oocytes.


Many marine invertebrates can provide an abundant source of gametes and are thus well suited for studies of oocyte maturation and fertilization. Moreover, analyses of marine invertebrates, particularly in understudied protostome lineages, help expand perspectives in gamete biology and add to our current knowledge of these topics, which is predominantly based on studies of mammals.

As reviewed above, the regulation of oocyte maturation and fertilization exhibits some common features in nemertean and mammalian oocytes. For example, GVBD in these two groups involves the conversion of pre-MPF into active MPF and does not require intraoocytic MAPK activation. Moreover, in both types of oocytes, the ER undergoes dramatic pre--and post-fertilization reorganizations, and the fertilizing sperm triggers calcium oscillations that are apparently mediated by a soluble sperm factor that elevates IP3 levels. However, although oocyte maturation and fertilization can utilize the same general kinds of intraoocytic signals in nemerteans and mammals, marked differences can also be seen in the effects of such key modulators as AMPK, cAMP, cGMP, NO, PICA, and PKC (Fig. 10).

Some of these differences may be due to the fact that nemerteans arrest at metaphase I prior to fertilization, whereas mammalian oocytes characteristically reach metaphase II before being fertilized. Alternatively, or in addition, heterogeneous subcompartments within these oocytes could differentially affect how various signals are utilized (Webb et al., 2008: Oh et al., 2010).

Similarly, the absence versus presence of follicle cells might have influenced the particular kinds of signaling molecules that have evolved in nemertean versus mammalian oocytes. Thus, as proposed for the "cAMP paradox" in which increased cAMP levels within oocytes tend to block mammalian GVBD, whereas elevating cAMP in follicle cells can promote GVBD (Downs and Hunzicker-Dunn, 1995; Tsafriri et al., 1996: Tsafriri and Dekel, 2010), varying effects of the same signal may be due to the presence of alternative isotypes of regulators and targets for that signal. For example, cAMP's inhibitory effects on GVBD depends on type 3 PDE and type I PKA in mammalian oocytes, whereas nemertean oocytes and mammalian follicle cells seem to utilize predominantly type 4 PDE and type II PKA to stimulate GVBD (Downs and Hunzicker Dunn, 1995; Stricker and Smythe, 2001; Conti et al., 2002; Sasseville et al., 2006). Considering these and other apparent similarities between follicle cells and nemertean oocytes (Stricker, 2009b; Stricker et al., 2010a), it remains possible that nemerteans evolved intraoocytic signals that resembled those used by follicle cells in an ancestral lineage. Conversely, mammalian follicle cells might have co-opted the kinds of signaling pathways that previously existed in oocytes of a more basal group with non-follicular oogenesis.

For further testing of such hypotheses, the particular isotypes and subcellular localizations of key regulatory signals in nemertean oocytes need to be analyzed by genomic and proteomic studies conducted in conjunction with experimental manipulations and imaging methods. Similarly, to analyze the potential contributions of follicle cell versus cell cycle effects, comparative analyses could be pursued, especially in sister taxa such as molluscs, where follicle cells can be present or absent, and oocytes do not universally arrest at metaphase 1 before fertilization. In any case, currently available data indicate that oocyte maturation and fertilization in nemerteans and mammals are often regulated by similar types of signaling pathways that nevertheless can lead to fundamentally different results.


Parts of these studies were conducted at Friday Harbor Laboratories of the University of Washington with financial support from NSF (#0114319) and UNM's Research Allocation Committee. We dedicate this article to the memory of C. C. Lambert, who contributed greatly to the knowledge of marine invertebrate gametes.

Received 14 January 2013; accepted 24 May 2013.

* To whom correspondence should be addressed. E-mail: Abbreviations: AC, adenylate cyclase; AMP, adenosine monophosphate; AMPK. AMP-activated kinase; ASW, artificial seawater; CaFSW, calcium-free seawater; ER, endoplasmic reticulum; GMP, guanosine monophosphate; GV, germinal vesicle: GVBD, germinal vesicle breakdown; [IP.sub.3], inositol 1.4,5-trisphosphate: MAPK, mitogen-activated protein kinase; MPF, maturation-promoting factor; NO, nitric oxide; NOS, nitric oxide synthase: PDE, phosphodiesterase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C: SFK, Src family kinase; SW. seawater.

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STEPHEN A. STRICKER*, CORY CLINE, AND DAVID GOODRICH Department of Biology. University of New Mexico, MSCO3 2020, Albuquerque, New Mexico 87131
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