Eggshells are protective structures, deposited shortly after fertilization in egg-laying animals. Composition and properties vary across species; however, most eggshells include a tough outer layer that provides physical support, and semipermeable layers that participate in gas exchange and the maintenance of osmotic balance (Cavaliere et al.,2008; Cree,1996; Margaritis et al.,1980; Nickles et al.,2002; Rose and Hincke,2009; Wharton,1980). Recent studies in invertebrates have elucidated additional roles for the eggshell in directing the pivotal events in embryonic development. For example, Drosophila Toll pathway-dependent dorsal-ventral polarity is dependent on an instructive cue embedded in the eggshell vitelline membrane layer (Cho et al.,2010; Moussian and Roth,2005; Zhang et al.,2009). As well, patterning of Drosophila embryonic termini requires Torso-like accumulation in the anterior and posterior vitelline layer (VL) for localized activation of the Torso receptor tyrosine kinase (LeMosy,2003; Stevens et al.,2003; Ventura et al.,2010). In the nematode C. elegans, recent studies have revealed multiple essential roles for the eggshell during the oocyte-to-embryo transition, as discussed below.
The nematode eggshell is comprised of at least three morphologically distinct layers deposited by the zygote (Fig. 1) (Bird,1971; Foor,1967; Lee,2002; Wharton,1980). The outside layer is thin and protein-rich and is called the VL. The middle layer is thick and contains both chitin and protein and is named the chitin layer (CL). The inner layer contains lipids and proteolipids and is called the lipid-rich layer (LrL). In some species, for example Heterodera sp., the LrL is further divided into multiple sublayers and the CL appears bilamellate, although the latter conclusion has been challenged (Burgwyn et al.,2003; Perry and Trett,1986). In addition to these three layers, additional outer layers deposited by somatic cells surrounding the zygote have been reported in the pig parasite Ascaris lumbricoides and the plant parasite Xiphinema diversicaudatum (Bleve-Zacheo et al.,1993; Foor,1967). Inside the eggshell, a clear zone separates the LrL and the embryo plasma membrane. This region is variably called the perivitelline fluid/space, the perimembrane space, or the extraembryonic matrix (EEM) (Benenati et al.,2009; Gunsalus et al.,2005; Hall and Altun,2008). Here, we refer to this zone as the EEM.
Electron microscopy of the C. elegans eggshell prepared by high pressure freezing and freeze-substitution has revealed three major eggshell layers (Benenati et al.,2009; Hall and Altun,2008; Rappleye et al.,1999). The eggshell is about 0.3–0.4 μm thick, except at the anterior pole, where the first polar body is embedded, resulting in a total thickness of >2 μm. Electron density in embryos fixed and stained with osmium tetroxide, uranyl acetate, and lead citrate is consistent with the interpretation of an outer VL, a middle CL, and an inner LrL (Benenati et al.,2009; Rappleye et al.,1999). An inner LrL is also supported by studies examining the ability of the membrane dye (FM 4-64) to gain access to the embryonic plasma membrane. The dye barrier is intact in embryos treated with bleach (removes VL) or bleach plus chitinase (removes VL and CL), whereas laser pulse (breaches VL, CL and LrL) results in FM 4-64 uptake (Rappleye et al.,1999). Differences in electron density within the eggshell layers indicate further complexity in organization. Molecular details are now being analyzed using probes that mark specific eggshell constituents, including chitin and proteoglycans. Inside the eggshell proper, the embryo is surrounded by an EEM comprised of an outer clear zone and an inner filamentous embryonic layer (EmL) (Benenati et al.,2009). The EmL is about 0.1–0.4 μm thick, except around the embedded second polar body, where it is thickened to >1 μm. The size of the outer clear zone changes rapidly during embryonic cell movement, suggesting a liquid or gel-like composition.
PREPARING FOR FERTILIZATION AND EGGSHELL FORMATION
Deposition of eggshell layers begins at fertilization and depends on prior preparation of the oocyte in the gonad. The hermaphrodite gonad is comprised of germ cells surrounded by somatic cells that form a pair of U-shaped gonad arms (Hall and Altun,2008; Hall et al., 1999). Each arm is connected to a spermatheca, where sperm are stored and the mature oocyte is fertilized. Spermathecae are connected by spermathecal-uterine (sp-ut) valves to the common uterus. Germ nuclei are generated in a distal mitotic niche, dependent on LAG-2, which is produced by the somatic distal tip cell (DTC) and signals through germline GLP-1 (Notch) receptors (Kimble and Crittenden,2005). Germ nuclei are compartmentalized into cubicle-like units that share cytoplasm via a common central rachis. Continuous production of germ nuclei pushes older nuclei out of the mitotic niche, resulting in their entry into prophase of meiosis I (MI) (Lee and Schedl,2001). The first prophase stages (leptotene and zygotene) are completed in the transition zone, after which nuclei enter pachytene. The pachytene zone extends along most of the dorsal gonad arm to the bend (loop) region (Hansen and Schedl,2006; McCarter et al.,1999). At the bend, mitogen-activated protein kinase (MAPK) signaling is activated, triggering nuclei to exit pachytene, enter diplotene, grow and begin to cellularize into oocytes. Continuing toward the proximal region of the gonad, oocytes progress through diplotene to diakinesis, and cellularization is completed (Greenstein,2005).
Major Sperm Protein Signals Oocyte Growth, Maturation, and Cytoskeletal Rearrangement
Maturation of the oocyte closest to the spermatheca (“−1” oocyte) involves nuclear envelope breakdown (NEBD) and chromosome rearrangement and depends on major sperm protein (MSP) (Greenstein,2005). MSP is released from sperm in the spermatheca and diffuses into the proximal gonad, where it signals to relieve MAPK inhibition and allow meiotic progression (Han et al.,2010; Miller et al.,2003; Yang et al.,2010). Progression also requires relief of inhibition of maturation promoting factor (MPF), comprised of CDK-1 and cyclin B3. MPF is maintained in an inactive form in oocytes, possibly by WEE-1.3-dependent phosphorylation, and activation is proposed to require dephosphorylation by the phosphatase CDC-25.2, acting downstream of MSP (Burrows et al.,2006; Kim et al.,2010a). MSP signals to somatic sheath cells via multiple unidentified G-protein-coupled receptors (Govindan et al.,2006; Miller et al.,2001), and the signal is communicated indirectly to oocytes, possibly via gap junctions (Govindan et al.,2009; Whitten and Miller,2007). MSP also signals to oocytes directly through VAB-1 receptors, to modulate the sheath cell-provided signal (Govindan et al.,2009).
Accompanying maturation, MSP stimulates oocyte microtubule reorganization, coordinating meiotic spindle assembly with cell cycle progression and NEBD (Harris et al.,2006; Nadarajan et al.,2009). MSP also promotes cytoplasmic flow from the rachis into developing oocytes (Govindan et al.,2009; Nadarajan et al.,2009), and together with RME-2-dependent endocytosis of yolk by the oocyte (Grant and Hirsh,1999), ensures the oocyte is large enough with sufficient nutrient stores to carry the embryo through to hatching. Yolk provides lipids essential for synthesis of the eggshell LrL, as well as polyunsaturated fatty acids used in the synthesis of F-series prostaglandins that attract sperm into the spermatheca (Edmonds et al.,2010; Kubagawa et al.,2006). A finite number of sperm (∼ 150) is made by each gonad arm of the L4 larval hermaphrodite, and deposited into the spermatheca. As such, the concentration of MSP that diffuses into the gonad is likely high in young adults and declines as more oocytes are fertilized. GLP-1 signaling from the DTC opposes MSP activity, limiting oocyte growth, possibly serving to control oocyte size in an environment in which the supply of MSP can be variable (Govindan et al.,2009; Nadarajan et al.,2009).
The Oocyte Surface and Cortex Are Remodeled
As the oocyte grows and matures, its surface, plasma membrane and cortex are prepared for fertilization and rapid eggshell deposition. On the surface, a thin basal lamina becomes visible (Hall and Altun,2008) and contains laminin, hemicentin, and the chitin-binding protein CBD-1 (Huang et al.,2003; Johnston et al.,2010; Xu and Vogel,2011). The basal lamina may be a diminutive C. elegans equivalent of the zona pellucida (Hall and Altun,2008), which in mammals is essential for sperm recognition and monospermic fertilization (Wassarman,1987). In the oocyte plasma membrane, the LDL receptor-like molecules EGG-1 and EGG-2 become localized (Kadandale et al.,2005), dependent on CBD-1 (Johnston et al.,2010). CBD-1 is also essential for the normal distribution of oocyte membrane CHS-1, which may prepare for rapid and even secretion of eggshell chitin at fertilization (Johnston et al.,2010). Fluorescence recovery after photobleaching (FRAP) experiments indicate CBD-1 immobilizes EGG-1 in the oocyte membrane (Johnston et al.,2010), suggesting a model in which multiple oocyte plasma membrane proteins are tethered, either directly or indirectly, by CBD-1 in the basal lamina. CBD-1 is a lectin with four predicted mucin-like domains and 12 chitin-binding domains, which may enable interaction with different types of membrane and ECM glycoconjugates.
Underneath the plasma membrane, a complex is assembled in the oocyte cortex. The complex contains the phosphatase-related proteins EGG-3, EGG-4/5, and the DYRK-family kinase MBK-2 and depends on CHS-1 for localization (Cheng et al.,2009; Heighington and Kipreos,2009; Johnston et al.,2006; Parry et al.,2009). CHS-1, EGG-3, and MBK-2 are subsequently cointernalized in zygotes, indicating membrane CHS-1 may physically associate with the EGG-3-5/MBK-2 cortical complex (Maruyama et al.,2007). In vitro, EGG-3 and EGG-4/5 bind MBK-2, while in vivo EGG-3-dependent cortical localization of MBK-2 requires EGG-4/5, indicating EGG-3 may serve as an adaptor to localize EGG-4/5-bound MBK-2 to the cortex (Cheng et al.,2009). In the mature oocyte, CDK-1 is proposed to phosphorylate and activate MBK-2, and localization of MBK-2 at the oocyte cortex may be a mechanism to sequester active MBK-2 to prevent premature phosphorylation of its cytoplasmic targets (Cheng et al.,2009). Membrane EGG-1 and EGG-2 are essential to maintain the normal homogeneous distribution of CHS-1, EGG-3, and MBK-2 (Johnston et al.,2010), consistent with a hierarchical model in which CBD-1 in the oocyte basal lamina supports tethering of plasma membrane molecules, including EGG-1/2 and CHS-1, to prevent clumping and premature internalization of CHS-1 and the cortical EGG-3-5/MBK-2 complex (Fig. 2b,c and Fig. 3).
Just prior to ovulation, the “−1” oocyte undergoes actomyosin rearrangement and reshapes into an oval, and the nucleus relocates to the “back” (distal part) of the cell (McCarter et al.,1999; Harris et al.,2006). During ovulation, the “front” end of the oocyte enters the spermatheca first, with the result that the fertilizing sperm usually contacts the pole opposite the maternal DNA. In cases where the sperm is at a lateral position, the sperm pronucleus/centrosomal complex (sp/centrosome) is repositioned during the subsequent phase of cortical contact and initiation of polarization, implying a requirement for a polar localization of the sp/centrosome (Goldstein and Hird,1996). In addition to positioning the male nucleus opposite to the maternal DNA, an oval oocyte also provides the template for deposition of an oval eggshell, which then constrains the shape of the zygote (McCarter et al.,1999). Experiments in Drosophila have shown that cell shape determines myosin distribution and cleavage furrow positioning during cytokinesis (Higgins and Goldstein,2010; Rappaport and Rappaport,1994). Therefore, the oval shape may ensure optimal arrangement of the actomyosin and microtubule cytoskeletons during AP polarization and mitosis.
Eggshell Transcripts Are Released for Translation
In the germline, many maternal messages are transcribed from leptotene through pachytene, followed by a transcriptionally quiescent state during diakinesis (Lee and Schedl,2001). Beginning at meiotic entry and continuing through pachytene, the tumor suppressor GLD-1 binds and represses translation of multiple maternal mRNA transcripts essential for oocyte and zygote development (Lee and Schedl,2001; Wright et al.,2011). As nuclei progress to diplotene, GLD-1 levels drop and transcripts are released and translated, beginning around the bend region and continuing into the proximal gonad (Lee and Schedl,2001; Lee and Schedl,2004). A number of GLD-1-bound transcripts encode proteins important for eggshell chitin synthesis, including the chitin biosynthetic enzyme, CHS-1 (Zhang et al.,2005), as well as glucosamine fructose 6-phosphate aminotransferase (F22B3.4 and F07A11.2), glucosamine 6-phosphate N-acetyltransferase (GNA-2), and UDP N-acetylglucosamine pyrophosphorylase (C36A4.4) in the hexosamine pathway (Fig. 4). gna-2 transcript abundance is further regulated by SMG-2-dependent nonsense-mediated decay, confining expression to the proximal few oocytes in the gonad (Lee and Schedl,2004). Chitin is a linear polymer of N-acetylglucosamine (GlcNAc), and the hexosamine pathway is essential for synthesis of the activated sugar substrate, UDP-GlcNAc.
In addition to transcripts encoding CHS-1 and hexosamine pathway enzymes, GLD-1 binds mRNA encoding the eggshell chitin-binding proteins CBD-1, CEJ-1 (also known as CPG-1) and CPG-2, and the chondroitin pathway enzymes SQV-4,5,7,8 (Lee and Schedl,2001; Wright et al.,2011), which are necessary for chondroitin modification of CEJ-1/CPG-1 and CPG-2 (Olson et al.,2006). GLD-1 also binds mRNA encoding the LDL-like receptors RME-2, EGG-1, and EGG-2, the pumilio RNA-binding protein PUF-5, the microtubule regulator RMD-1, the novel protein H02I12.5, and the phosphatase-related EGG-4 and EGG-5 (Lee and Schedl,2001; Wright et al.,2011). Depletion of F22B3.4, F07A11.2, GNA-2, C36A4.4, CHS-1, SQV-4, RME-2, EGG-1, EGG-2, PUF-5, RMD-1, CBD-1, EGG-4, EGG-5, or CEJ-1/CPG-1 (in combination with CPG-2 depletion) results in osmotically sensitive embryos, implying a role for the proteins in the eggshell (Hwang and Horvitz,2002; Hwang et al.,2003; Johnston et al.,2006; Johnston et al.,2010; Lublin and Evans,2007; Oishi et al.,2007; Parry et al.,2009; Sonnichsen et al.,2005) (Fig. 2a).
A subset of the GLD-1-regulated transcripts that are important for the eggshell are also regulated by the poly(A) polymerase/RNA-binding protein complex, GLD-2/RNP-8 (Kim et al.,2009; Kim et al.,2010b). The coregulated group includes chs-1, egg-1,2, egg-4,5, sqv-5,7,8, H021125 and mRNAs. In addition, GLD-2/RNP-8 binds mRNA encoding the cytochrome P450, POD-7(CYP-31A2), which contributes to the LrL osmotic barrier of the eggshell (Kim et al.,2009; Kim et al.,2010b) (Fig. 2a). RNP-8 levels are high in most of the proximal gonad, but drop in the “−1” oocyte, which may limit translation of eggshell mRNA to the period of eggshell assembly (Kim et al.,2010b).
The layers of the C. elegans eggshell and EEM appear to be generated sequentially by the zygote, beginning with the VL, and followed by the CL, the LrL, and the EEM. Shortly after fertilization, a thin, crenellated covering appears on the surface of the zygote (Hall and Altun,2008). The covering resembles the fertilization envelope of other animals, a modified basal lamina that prevents polyspermy and protects the developing embryo (Foor,1967; Wong and Wessel,2008; Wessel and Wong,2009). CBD-1 is found in the oocyte basal lamina and is required to prevent polyspermy (Johnston et al.,2010). CBD-1 is also present in the zygote eggshell and is essential for structural integrity during exit from the spermatheca (Johnston et al.,2010). The localization and functions of CBD-1 support the interpretation that it is part of an oocyte basal lamina that is first transformed into a fertilization envelope, and subsequently becomes part of the outer VL of the eggshell (Johnston et al.,2010).
The first evidence of the middle CL is a continuous ring of chitin surrounding the newly fertilized zygote in the spermatheca. Deficiency of the sperm-provided protein SPE-11 restricts eggshell chitin to a small region overlying the male nucleus, indicating the signal for deposition likely initiates at the site of sperm contact or entry and depends on SPE-11 for propagation around the zygote (Johnston et al.,2010). SPE-11 is a novel protein that is functional when provided either paternally or maternally, implying it requires zygotic activation (Browning and Strome,1996; Hill et al.,1989). Chitin deposition occurs before cell cycle progression past MI metaphase and is not dependent on genes required for the metaphase-to-anaphase transition, such as mat-1, which encodes the CDC27/APC3 subunit of APC/C (Golden et al.,2000; Johnston et al.,2010). However, chitin deposition depends on EGG-3 to localize CHS-1 to the membrane, as well as GLD-1 release of transcripts for de novo synthesis of the UDP-GlcNAc substrate. Interestingly, EGG-4/5 is not required for CHS-1 localization to the membrane but is essential for chitin deposition (Parry et al.,2009), indicating a possible role in CHS-1 activation at fertilization.
After chitin deposition, and during MI anaphase, zygotic cortical granules are released in a wave originating around the meiotic spindle (Bembenek et al.,2007; Sato et al.,2008). Degranulation requires the small GTPase, RAB-11.1 and the syntaxin, SYN-4, which mediate trafficking, as well as the protease SEP-1, which partially relocates from the nucleus and cytoplasmic filaments to cortical granules during anaphase (Bembenek et al.,2007; Sato et al.,2008). Depletion of SQV-4, an enzyme in the chondroitin biosynthesis pathway, or the chondroitin-modified chitin-binding proteins, CEJ-1/CPG-1 and CPG-2, results in smaller cortical granules and disrupted eggshell function (Bembenek et al.,2007). Thus, CEJ-1/CPG-1 and CPG-2 are candidate cortical granule cargo molecules, which may be incorporated into the middle CL. We have observed that normal distribution of CEJ-1/CPG-1 in the eggshell depends on chitin (Johnston and Dennis, unpublished), whereas chitin is deposited in embryos depleted for CEJ-1/CPG-1 and CPG-2, but the eggshell does not support development (Johnston et al.,2006). Therefore, chitin in the CL is deposited first and appears to organize CEJ-1/CPG-1 and CPG-2 chitin-binding proteins within the CL.
Lipid-rich Layer and EEM
Yolk is secreted by intestinal cells and taken up by the oocyte, providing lipid substrate for the LrL (Grant and Hirsh,1999; Kimble and Sharrock,1983; Sharrock,1983). Yolk granules also contain Apo-B-like lipid transfer proteins called vitellogenins. Vitellogenins are ligands for the oocyte LDL receptor RME-2, which mediates yolk uptake by receptor-mediated endocytosis (Grant and Hirsh,1999; Paupard et al.,2001). RME-2 deficiency causes osmotic sensitivity (Sonnichsen et al.,2005), suggesting yolk lipids or their derivatives generate the osmotic barrier.
Deficiency of the fatty acid synthesis enzymes, acetyl CoA carboxylase (POD-2), or fatty acid synthase-1 (FASN-1) causes osmotic sensitivity (Rappleye et al.,2003) (Fig. 5), implicating fatty acid-containing lipids in the eggshell LrL osmotic barrier. Candidate molecules include glycolipids, phospholipids, triglycerides, and myristoylated or palmitoylated proteins. In Ascaris sp., the barrier requires ascaroside glycolipids, comprised of long-chain fatty acids (C22-C37) and an ascarylose sugar (Foor,1967; Joo et al.,2009; Mei et al.,1997; Pungaliya et al.,2009; Wharton,1980). In C. elegans, ascarosides appear dispensable (Zagoriy et al.,2010). The observation that dietary supplementation of C16:0, C18:0 or C18:1 fatty acids rescues pod-2(ye60) polarity defects, but not osmotic defects, implies the relevant molecule(s) may carry a shorter (<C16) chain fatty acid (Rappleye et al.,2003). However, deficiency of FAT-6 and FAT-7, which catalyze desaturation of C18:0, or FAT-2, which desaturates C18:1Δ9, results in eggshell defects, suggesting longer-chain fatty acids may also be important (Brock et al.,2007). Experiments examining the ability of different lipid fractions to rescue osmotic sensitivity in worms mutant at multiple steps in fatty acid synthesis, desaturation, and elongation may be helpful to assess the requirement for different fatty acids.
Osmotic sensitivity and eggshell LrL defects are also seen in worms deficient in the cytochrome P450s, POD-7, POD-8, and CYP-31A5, and the cytochrome P450 reductase EMB-8 (Benenati et al.,2009; Rappleye et al.,2003). POD-7, POD-8, or EMB-8 deficiency can be rescued by feeding a hydrophilic lipid extract, but not by a more hydrophobic extract or C16:0, C18:0, or C18:1 fatty acids, consistent with a requirement for shorter chain fatty acids, and/or nonfatty acid lipids in the lipid barrier (Benenati et al.,2009; Sonnichsen et al.,2005). Deficiency of the mevalonate pathway enzymes, HMG-CoA synthase (F25B4.6) or HMG CoA reductase (F08F8.2), also results in osmotic sensitivity (Sonnichsen et al.,2005). Mevalonate is essential for the synthesis of farnesyl-pyrophosphate, which provides substrate for biosynthesis of a number of important nonfatty acid lipids, including cholesterol and isoprenoids used for protein prenylation (Casey,1992) (Fig. 5). Like other animals, C. elegans requires cholesterol, but squalene synthase is absent, preventing cholesterol synthesis from farnesyl pyrophosphate. Therefore, cholesterol is acquired from the diet (Entchev and Kurzchalia,2005; Vinci et al.,2008). As such, cholesterol deficiency is not likely to explain the eggshell defects in HMG CoA reductase deficiency, pointing instead to an alternative lipid or prenylated protein. Treatment of worms with fluvastatin inhibits protein prenylation and results in swollen embryos (Morck et al.,2009). Moreover, deficiency of M57.2 (a geranylgeranyltransferase) is embryonic lethal and partially phenocopies fluvastatin treatment (Morck et al.,2009), consistent with a role for prenylation in the eggshell osmotic barrier. Therefore, in addition to requiring fatty acids, the eggshell barrier may depend on prenylated molecules.
Following LrL synthesis, the EEM is elaborated, separating the eggshell from the zygotic plasma membrane (Benenati et al.,2009). Very little is known about the EEM, except that its secretion depends on the LrL (Benenati et al.,2009). Importantly, the EEM is the immediate “extracellular” environment for the plasma membrane of the late-stage zygote, and the apical plasma membrane of the early embryo. Proteoglycans and other glycoconjugates in the EEM may modulate signaling at early developmental stages; an exciting prospect for future research.
FUNCTIONS OF THE EGGSHELL IN ZYGOTE DEVELOPMENT
One of the earliest challenges facing a zygote is avoiding the lethality of polyspermy. In humans, dispermic fertilization is the major cause of triploidy, which occurs in ∼ 1% of conceptions and accounts for ∼ 6% of spontaneous abortions (McFadden et al.,2002; Zaragoza et al.,2000). In metazoans, a cytoplasmic Ca2+ wave is seen in the zygote immediately following sperm contact or entry, which blocks penetration by additional sperm (Runft et al.,2002; Santella et al.,2004). Studies in vertebrates, as well as echinoderms, have identified a biphasic polyspermy barrier, with a “fast” block at the level of the plasma membrane and a “slow” Ca2+-dependent block that depends on changes to the ECM (Gardner et al.,2007; Jaffe,1976; Wessel and Wong,2009; Wong and Wessel,2008). In echinoderms, as well as amphibians, the “fast” block results from rapid and transient membrane depolarization, whereas in mammals, the fast block is poorly defined, but appears to be independent of changes in membrane potential (Gardner et al.,2007). During the “slow” block the extracellular “vitelline” layer surrounding the oocyte is lifted and converted into a fertilization envelope. In starfish (echinoderm), lifting of the VL has been shown to depend on actin remodeling beginning at the site of sperm entry (Chun and Santella,2009; Chun et al.,2010; Kyozuka et al.,2008; Puppo et al.,2008).
The C. elegans oocyte is exposed to a sperm-filled spermatheca but polyspermy is very rare, indicating a robust polyspermy barrier. As in other metazoans, fertilization evokes a zygotic Ca2+ wave (Samuel et al.,2001). Actin is reorganized to form a cap at the site of sperm entry (Parry et al.,2009) and a zygote covering (the presumptive eggshell VL; pVL) is separated from the plasma membrane (Hall and Altun,2008). Polyspermy is frequent in zygotes made chitin-deficient by depletion of CHS-1, GNA-2, EGG-3, or EGG-4/5 (Johnston et al.,2010; Parry et al.,2009). In egg-4/5(RNAi), zygotic actin cap formation is defective (Parry et al.,2009) and in chs-1(RNAi), lifting of the pVL is incomplete (Johnston et al.,2010). Therefore, chitin is required to generate a robust polyspermy barrier, which may involve polarized actin remodeling and pVL lifting, radiating out from the site of sperm entry. The VL protein CBD-1 organizes membrane proteins and has 12 predicted chitin binding domains. cbd-1(RNAi) results in polyspermy (Johnston et al.,2010), suggesting chitin secreted at fertilization may bind to CBD-1 to contribute to pVL lifting. Alternatively or additionally, chitin and CBD-1 might form a scaffold to organize a sperm-impermeant matrix, analogous to the role of chitin and chitin-binding proteins in preventing invasion of bacteria in the insect gut (Hegedus et al.,2009).
The incidence of polyspermy in zygotes made chitin-deficient by depletion of CHS-1, GNA-2, EGG-3, or EGG-4/5 is typically less than ∼ 50% (Parry et al.,2009; Johnston et al.,2010), and the number of supernumerary sperm is usually only one or two (Johnston et al.,2010). The findings are consistent with a chitin-dependent slow block as well as an additional chitin-independent fast membrane block. However, more direct measures are required to demonstrate a biphasic polyspermy barrier. In mammals, chitin appears to have been replaced by hyaluronan (HA), and homology of HA synthase with CHS-1 implies a similar plasma membrane localization (Lee and Spicer,2000). Interestingly, HA is also implicated in preventing polyspermy, possibly in a conserved barrier role (Ueno et al.,2009). In most species, release of cortical granules into the ECM is necessary for the “slow” block to polyspermy (Chandler and Heuser,1979; Wessel and Wong,2009). C. elegans cortical granules are small and are not released until MI anaphase, 10–15 min after fertilization (Bembenek et al.,2007). In addition, preventing their release by blocking the metaphase-to-anaphase transition does not result in polyspermy (Johnston et al.,2010); therefore, they do not appear be required for the ECM block in C. elegans.
cbd-1(RNAi) oocytes are fertilized and deposit chitin into an eggshell, but zygotes are pinched off during transit through the constrictive sp-ut valve, resulting in partial or total severing of the trailing section (Johnston et al.,2010). Therefore, CBD-1 in the VL appears necessary for zygote integrity. In the case of partial severing, the cbd-1(RNAi) zygote has a characteristic shape in which the trailing section looks like a head and neck attached to the body of the leading section. A similar shape is seen in mating yeast and is termed “Shmoo,” after the cartoon character created by the popular cartoonist, Al Capp (Mackay and Manney,1974). In C. elegans, we refer to this phenotype as Shmu.
CBD-1 deficiency causes patchy distribution of oocyte CHS-1, which could lead to patchy chitin deposition at fertilization (Johnston et al.,2010). As such, one possible explanation for the cbd-1(RNAi) Shmu phenotype might be that the eggshell lacks sufficient chitin to provide the mechanical strength to allow the zygote to exit the spermatheca intact. However, GNA-2- or CHS-1- deficient zygotes have no chitin at all, yet are not Shmu, suggesting that patchy chitin deposition in cbd-1(RNAi) may not explain the Shmu phenotype. An alternative possibility is that CBD-1 may crosslink chitin to EGG-1 in the plasma membrane to adhere the nascent eggshell to the zygote. During the brief period of chitin deposition, the plasma membrane appears to be in close contact with the nascent eggshell, which may enable CBD-1 to tether chitin and EGG-1/2 simultaneously. In cbd-1(RNAi), a poorly adhered eggshell may be pushed off during transit through the sp-ut valve, trapping the trailing section of eggshell, and any underlying zygote, in the spermatheca. Interestingly, EGG-1 deficiency also results in a Shmu phenotype (Johnston et al.,2010), consistent with the interpretation that eggshell attachment may require chitin/CBD-1/EGG-1 linking. A third possible explanation for the Shmu phenotype in CBD-1-deficiency is defective relaxation of the sp-ut valve. Timed valve relaxation depends on phospholipase C-1 (PLC-1) and an actin-binding protein, filamin-1 (FLN-1), which act through IP3 signaling (Kariya et al.,2004; Kovacevic and Cram,2010). FLN-1 deficiency causes misshapen and fragmented zygotes, indicating defects in valve relaxation may be sufficient to pinch off zygotes during spermathecal exit. Examining the eggshell in FLN-1/PLC-1 deficiency, as well as valve contraction in CBD-1 deficiency, will be helpful to assess a contribution of sp-ut valve relaxation defects to the cbd-1(RNAi) Shmu phenotype.
Meiotic Chromosome Segregation
The kinetochore is a proteinaceous structure that links chromosomes to spindle microtubules to ensure that each chromatid of the pair is attached before initiating anaphase. Kinetochore attachment and centrosome-dependent pulling forces are required to segregate sister chromatids to opposite poles during mitosis (Cheeseman and Desai,2008; Lampson and Cheeseman,2011; Tanaka,2010). Centrosomes are absent in the meiotic spindle and separation during anaphase is driven by pushing forces arising between the sister chromatids (Dumont et al.,2010). However, kinetochores are still important to orient chromosomes on the meiotic spindle, as depletion of kinetochore proteins such as KNL-1 results in a lagging chromosome phenotype (Dumont et al.,2010). Search-and-capture of kinetochores by remodeling microtubules is the primary means by which chromosomes are linked to the spindle. To increase the efficiency of capture in larger cells, a RanGTP gradient is suggested to bias microtubule growth near chromosomes (Wilde et al.,2001; Wollman et al.,2005). In addition, a filamentous actin mesh is needed for delivery of chromosomes to the starfish meiotic spindle, possibly a more critical requirement in large cells (Lenart et al.,2005; Mori et al.,2011).
Actin is needed for chromosome segregation during the first mitotic division in worms (Velarde et al.,2007), perhaps reflecting the large size of the zygote (∼ 40 μm by 25 μm). It is not known whether meiotic chromosome segregation also depends on actin. However, worms depleted for GNA-2, CHS-1, or CEJ-1/CPG-1+CPG-2 exhibit a partially penetrant lagging chromosome phenotype at MI and meiosis II (MII) (Johnston et al.,2006), and Parry et al. (2009) determined that egg-4/5(RNAi), which fails to deposit chitin, also has defects in actin dynamics. Therefore, the eggshell CL is required for high fidelity segregation of chromosomes during meiosis, and while direct evidence is lacking, one possible explanation is that it facilitates actin-mediated chromosome capture.
Polar Body Extrusion
Deficiency of CHS-1, GNA-2, or CEJ-1/CPG-1+CPG-2 causes retention of both the first and second polar bodies (Johnston et al.,2006), indicating the eggshell CL is essential for polar body extrusion. Polar body extrusion is a highly asymmetric form of cytokinesis in which half of the segregated DNA is expelled from the cell, with very little accompanying cytoplasm. During extrusion a contractile actomyosin ring assembles between the segregated masses of maternal DNA and is converted into a tube-like series of stacked rings by the actin-binding protein anillin-1 (ANI-1), resulting in robust polar body scission (Dorn et al.,2010; Maddox et al.,2005). In budding yeast, chitin synthase (Chs2p) located at the mother-daughter bud neck region deposits chitin, generating the primary septum disk which acts as a scaffold to allow actomyosin contraction at the cleavage furrow (Schmidt et al.,2002). Chitin may play a similar role during C. elegans polar body extrusion, acting as a scaffold for proteins such as CEJ-1/CPG-1 and CPG-2. However, chitin cooperates with additional factors, since the eggshell LrL is also required, at least for extrusion of the second polar body (Benenati et al.,2009). Mechanistically, one possibility is that the CL, LrL and the underlying EEM generate a hydrostatic skeleton to transmit the force of contraction at the ANI-1-dependent actomyosin ring (Johnston et al.,2006).
Development of a viable worm requires AP polarization at the one-cell stage (Munro and Bowerman,2009; Nance and Zallen,2011). After meiosis is completed, the actomyosin cortex contracts, resulting in cortical ruffling. Concomitant with ruffling, the sp/centrosome moves to the cortex, likely propelled by anterior to posterior (A-P) cytoplasmic flow (Schneider and Bowerman,2003). In GNA-2, CHS-1, or CEJ-1/CPG-1+CPG-2 deficiency, the sp/centrosome fails to move to the cortex in most zygotes, resulting in failure to initiate polarization (Johnston et al.,2006). A similar phenotype results from depletion of SEP-1 or MAT-2, which are required for cortical granule secretion (Rappleye et al.,2002). Similarly, deficiency of EMB-8 or POD-2 in the lipid biosynthesis pathway, results in failed or transient contact of the sp/centrosome with the cortex (Rappleye et al.,2003). Therefore, the CL and LrL of the eggshell are required to initiate AP polarization. Depletion of actin prevents both cortical ruffling and sp/centrosome migration to the cortex, indicating actomyosin contraction is likely required for A-P cytoplasmic flow (Velarde et al.,2007). As discussed above for polar body extrusion, during cortical ruffling the rigid eggshell and its underlying EEM may transmit the force of actomyosin contraction, allowing translocation of the sp/centrosome to the cortex.
Cortical contact by the sp/centrosome results in localized inactivation of the RHO-1 guanine nucleotide-exchange factor ECT-2, and suppression of myosin contractility radiating from the site of contact (Motegi and Sugimoto,2006; Schonegg and Hyman,2006). Sperm also provides the Rho GTPase activating protein, CYK-4, which may contribute to RHO-1 inactivation (Jenkins et al.,2006). As contraction becomes progressively more asymmetric, an A-P gradient in mechanical tension in the cortex (cortical tension) develops concomitant with cortical flow away from the site of centrosome-cortical contact (Mayer et al.,2010). Flow moves cortical anterior determinants (PAR-3, PAR-6, PKC-3) away from the posterior, enabling the posterior determinant, PAR-2, to accumulate at the nascent posterior cortex (Munro et al.,2004). A second, redundant pathway allows PAR-2 to respond directly to a sp/centrosome associated cue to load onto the posterior cortex (Zonies et al.,2010). Recently, cortical laser ablation (COLA) experiments have shown that cortical flow causes the A-P cortical tension gradient, and modeling predicts that long-range flow depends on a highly viscous cortex/membrane moving within a low friction EEM (Mayer et al.,2010). Therefore, in addition to its role in initiating polarity, the eggshell/EEM may be critical to provide a low friction environment for cortical flow during polarity establishment.
EGG-1 and MBK-2 Internalization
The oocyte-to-embryo transition requires internalization of membrane and cytoplasmic proteins, including EGG-1 and the MBK-2 complex (Pellettieri et al.,2003; Sato et al.,2008). FRAP experiments show increased EGG-1 mobility in newly fertilized zygotes, compared with oocytes, indicating that CBD-1-dependent tethering of EGG-1 is relieved after fertilization (Johnston et al.,2010). Depletion of CEJ-1/CPG-1 and CPG-2 does not impair EGG-1 internalization (Johnston and Dennis, unpublished observation). In contrast, EGG-1 fails to be mobilized and internalized in the chitin-deficient chs-1(RNAi), gna-2(RNAi) or egg-3(RNAi) mutants (Johnston et al.,2010). Therefore, eggshell chitin, but not CEJ-1/CPG-1 and CPG-2, is required for turnover of membrane EGG-1 during the oocyte-to-embryo transition. The chitin- and CBD-1-dependence of EGG-1 internalization suggests a model in which chitin competes with membrane glycoconjugates for CBD-1 binding, relieving membrane protein immobilization. However, chitin is deposited at fertilization, but EGG-1 is not internalized until meiosis is completed, almost 1/2 h later (McCarter et al.,1999), indicating that additional events are also required for EGG-1 internalization.
Between MI and MII, MBK-2 internalizes in vesicular clusters that colocalize with CHS-1, EGG-3, and EGG-4/5. Coinciding with EGG-3 release, MBK-2 is activated and phosphorylates cytoplasmic targets that include MEI-1, OMA-1, OMA-2, MEX-5, and MEX-6 (Cheng et al.,2009). MBK-2 internalization is not affected by depletion of CEJ-1/CPG-1 and CPG-2 (Fig. 6). In contrast, depletion of EGG-4/5 or GNA-2, which causes eggshell chitin deficiency, disrupts internalization of MBK-2, EGG-3, and CHS-1 during the MI-MII period (Johnston et al.,2010; Parry et al.,2009) (Fig. 6). MBK-2 internalization is also delayed in spe-9 mutants, which are not fertilized and, hence, do not deposit chitin (McNally and McNally,2005). Therefore, internalization of the cortical MBK-2 complex depends on eggshell chitin for timely internalization. However, in SPE-9 deficiency at least some of the MBK-2 complex is internalized eventually (McNally and McNally,2005; Stitzel et al.,2006), indicating chitin is not the sole mediator of MBK-2 complex internalization. Further studies will be helpful to clarify the contributions of sperm contact, cell cycle progression, and other zygotic events.
Preparation of the oocyte in the gonad ensures the rapid deposition of a multilayered eggshell/EEM after fertilization. The eggshell/EEM is clearly much more than a protective barrier. During the oocyte-to-embryo transition, it is essential for the polyspermy barrier, meiotic chromosome segregation, polar body extrusion, AP polarization and membrane/cortical protein internalization (Fig. 7). Future experiments should reveal the molecular composition and organization that make the eggshell such a profoundly important driver of early development.
The authors thank reviewers for helpful comments and suggestions. Nematode strains referred to in Figure 6 were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR).