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- MATERIALS AND METHODS
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Reproductive strategies differ amongst organisms based on their evolutionary history and the niche within which they compete. The reproductive strategy for most marine invertebrates includes broadcast spawning of their gametes, and if successful in fertilization, the embryos often utilize the water column as a food source for development before metamorphosing into adults. Echinoderms are paradigmatic for this reproductive strategy, and have served as important research organisms for understanding mechanisms of sperm activation (Lee et al. 1983), chemoattraction of sperm to the egg (Ward et al. 1985), sperm–egg binding mechanisms (Vacquier and Moy 1977), egg activation (Steinhardt et al. 1977), and the diverse evolutionary basis for sperm–egg interactions (Vacquier 1998).
Animal fertilization was first observed in sea urchins, where an envelope forms promptly after sperm fusion with the egg and thus provides a rapid metric for successful sperm–egg interaction (Derbes 1847; Briggs and Wessel 2006). Fusion of the male and female pronuclei, first seen in sea urchins by Hertwig (1876) and Fol (1877), closed the chapter on the important role of sperm in the process of reproduction.
The extracellular matrix of the egg, while called many different names (for example, vitelline layer or zona pellucida) serves two essential jobs. First, it interacts with sperm in a species-specific manner. While this function occurs in almost all animals, it is particularly striking in broadcast spawners, such as abalone and sea urchins, which can inhabit the same niches and often spawn in overlapping times. In such cases, species specificity in sperm–egg interactions relies heavily on the extracellular matrix. Following successful sperm–egg fusion, the egg's extracellular matrix quickly reveals its second job as it is transformed to minimize the chances of additional sperm from reaching the egg. This physical block to polyspermy is highly selected for because fusion of more than one sperm with an egg is lethal to the embryo. The block to polyspermy in some animals, such as sea urchins, is remarkable because sperm:egg ratios may reach the millions.
The fertilization envelope (FE) in sea urchins establishes a physical and biochemical barrier that protects the zygote from supernumerary sperm, as well as environmental and microbial agents (Wong and Wessel 2006a). Cortical granules are the major source of proteins used to construct the FE (Wessel et al. 2001; Wong and Wessel 2006a). These abundant organelles, accumulating to 15,000 per egg in sea urchins, are synthesized during oogenesis and are released following gamete fusion (Laidlaw and Wessel 1994). In the sea urchin, contents of the cortical granules are secreted within 30 sec of insemination and mix with the egg's vitelline layer. Hydrostatic pressure and the addition of glycoproteins from the cortical granules to the vitelline layer lift the nascent FE off the egg surface, and associated enzymes transform this envelope into an effective barrier for early embryogenesis. Sea urchin cortical granules harbor the major structural proteins of the envelope as well as enzymes essential to stabilize the envelope until hatching (Wong and Wessel 2008).
The cortical granules contain several structural proteins and enzymes that give the FE its distinct properties of stability with permeability in the ocean environment. These proteins include the Soft Fertilization Envelope proteins SFE1 and SFE9, proteoliaisin, and rendezvin; their cognate transcripts are specifically expressed in oocytes (Laidlaw and Wessel 1994; Wong and Wessel 2004, 2006b). SFE1, SFE9, and proteoliaisin are proteins rich in low-density lipoprotein receptor type A (LDLrA) repeats involved in protein interaction (Wessel 1995; Wessel et al. 2000; Wong and Wessel 2004). Rendezvin (RDZ) is enriched in CUB domains, also involved in protein-protein interactions. One RDZ gene is present in the sea urchin genome, but several transcripts are produced after alternative splicing. The full-length rdz transcript is alternatively spliced into at least three forms, encoding its majority proteins RDZ60, RDZ90, and RDZ40. Two significantly less-abundant transcripts are also created, encoding RDZ120 and RDZ70. At the protein level, the different isoforms are differentially localized. RDZ60, RDZ90, RDZ40, and RDZ70 only accumulate in the cortical granules, whereas RDZ120 is found in the vitelline layer (Wong and Wessel 2006b). After fertilization, these segregated siblings reunite within the FE, likely via heterologous CUB interactions.
Four major enzymatic activities are essential for the proper assembly of the sea urchin FE: proteolysis, transamidation, hydrogen peroxide synthesis, and peroxidase-dependent dityrosine crosslinking. Serine protease activity from CGSP1 (cortical granule serine protease) is the only detectable class of protease activity of the cortical granules necessary for the formation of the FE (Vacquier et al. 1972; Carroll and Epel 1975; Haley and Wessel 1999). Full-length CGSP1 is enzymatically quiescent in the cortical granules, inactive at pH 6.5 or below. Exposure of this protease to the pH of the seawater (pH 8) at exocytosis immediately activates the protease through autocatalysis (Haley and Wessel 2004b). CGSP1 cleaves a subpopulation of the granule content proteins, such as the enzyme ovoperoxidase, to limit its activity, and the β-1,3-glucanase, to increase its activity. Another substrate targeted by CGSP1 is p160, a protein thought to link the vitelline layer to the plasma membrane (Haley and Wessel 2004a). At fertilization, p160 cleavage allows for the separation of the FE from the fertilized egg.
Transamidation is mediated by transglutaminases that crosslink glutamine and lysine residues to form N-epsilon (gamma glutamyl) lysyl isopeptide bonds (Greenberg et al. 1991). Two transglutaminases were found in the Strongylocentrotus purpuratus genome (Wong and Wessel 2009). These two isoforms, derived from different genes, are differentially localized and were described as the extracellular transglutaminase (eTG) and the nuclear transglutaminase (nTG). Both transcripts are expressed in the oocyte. Whereas eTG mRNA persists in eggs, nTG mRNA is largely degraded during meiotic maturation (Wong and Wessel 2009). These transglutaminases are activated by local acidification and act on FE proteins such as SFE9, rendezvin, and ovoperoxidase.
Hydrogen peroxide is quickly synthesized at fertilization for ovoperoxidase crosslinking activity, and is synthesized by the dual oxidase homolog, Udx1, in the classically described respiratory burst (Warburg et al. 1926). This calcium-dependent, pH-sensitive enzyme is essential for completing the physical block to polyspermy (Wong et al. 2004). Unlike genes utilized exclusively for the formation of the FE and expressed exclusively during oogenesis, such as the structural matrix proteins SFE1, SFE9, proteoliaisin, rendezvin, and the enzyme ovoperoxidase (Wessel et al. 2001; Wong et al. 2004), Udx1 transcripts are present in eggs and later in development (Wong et al. 2004). Interestingly, Udx1 also plays a role in early development as its specific inhibition induces a delay in cytokinesis (Wong and Wessel 2005). In the egg, this hydrogen peroxide synthesis is necessary for the activity of the ovoperoxidase, a tyrosine crosslinking enzyme derived from the egg cortical granules (Foerder and Shapiro 1977; LaFleur et al. 1998). In the sea urchin S. purpuratus, the ovoperoxidase mRNA is present exclusively in oocytes and is turned over rapidly following germinal vesicle breakdown (LaFleur et al. 1998). Under normal conditions, ovoperoxidase is specifically targeted to the FE via a calcium-dependent interaction with proteoliaisin (Weidman et al. 1987). The ovoperoxidase activity is sensitive to transglutaminase (Wong and Wessel 2009), CGSP1 (Haley and Wessel 2004b), and Udx1 (Wong et al. 2004). Semi-in vivo crosslinking assays identified four major targets of ovoperoxidase (Wong and Wessel 2008): RDZ120, proteoliaisin, SFE1, and SFE9.
The vast majority of what is known about the FE is from the study of a few sea urchin species, yet similar FEs are utilized by other echinoderms. Here, we explore the proteome of the FE in sea stars, and compare its sequences to those in the pencil urchin, thought to be reflective of the ancient sea urchins within the fossil record, and to the well-known sea urchins S. purpuratus and Lytechinus variegatus (Lv), for which most work on the cortical granules and FEs have been accomplished. The sea star family, the Asteroids, contains an estimated 1600 species worldwide (Blake 1989). Their eggs are generally stored in prophase of meiosis I, and spawning activates release of the inducer for meiotic progression, 1-methyl adenine. Upon germinal vesicle breakdown, the oocyte becomes fertilization-competent, and following sperm–egg fusion, a robust FE forms. Many sea stars rely on the FE to limit exposure to harmful elements in the marine environment; some species also rely on the envelope to constrain the blastomeres (Dan-Sohkawa 1976; Matsunaga et al. 2002). Removal of the FE in many sea star species leads to blastomeres dissociating from each other and subsequent death, likely because of the absence of a distinct hyaline layer, an embryonic extracellular matrix found in sea urchins. Here, we determine the genes responsible for formation of the FE in the sea star Patiria miniata (the common batstar) by proteomic, transcriptomic, and functional criteria.
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In the sea urchin S. purpuratus, cortical granules accumulate throughout the cytoplasm until germinal vesicle breakdown, and then translocate to the cell periphery (Wessel 1995; Laidlaw and Wessel 1994). Our results suggest that, in contrast to the sea urchin, sea star cortical granules translocate to the cortex as they are synthesized. This early translocation seems more similar to the mechanism described in mice, in which the density of cortical granules present in the cortex increases continually during oogenesis (Ducibella et al. 1994). The production and migration of sea star cortical granules are continuous processes. As the cortical granules are already at the oocyte surface prior to meiosis, what happens to them during meiosis, especially during the formation of the polar bodies and meiotic spindles? Does the meiotic spindle displace the cortical granules prior to polar body formation, or do they exocytose prematurely, as in mice? We found no evidence of precocious FE formation in the sea star in the area of the meiotic spindle and polar body so we anticipate a cortical granule displacement is made at meiosis.
Cortical granules were previously analyzed in the sea star Pisaster ochraceus (Reimer and Crawford 1995). Using a monoclonal antibody against a 120-kDa protein, it was shown that cortical granules were concentrated in the periphery of the cytoplasm of immature oocytes, but were also found throughout the cytoplasm. In mature oocytes, a larger number of granules were located at the periphery of the cytoplasm, but some granules were still present throughout the cytoplasm. After fertilization, the staining was predominantly found in the perivitelline space, although several brightly stained granules remained in the cell cytoplasm. Later in development at the blastula, the FE was not stained by this monoclonal antibody, but labeled granules were present in blastomeres (Reimer and Crawford 1995). Thus, it is not clear how selectively this antibody identifies cortical granules, or if it recognizes components of other secretory organelles. To follow the cortical granule biogenesis in the sea star P. miniata, we used an antibody against SFE9 and learned that cortical granules move to the cell periphery during early oogenesis in this species. The contrasting results between P. ochraceus and P. miniata species might be explained by the different target proteins studied as well as biological trafficking of different proteins. This may also be simply a matter of species difference in the cortical granule assembly strategy. The granules found at the oocyte periphery might contain both SFE9 and the 120-kDa protein, whereas the granules persisting in the P. ochraceus embryos might contain only the 120-kDa protein and/or could play a different role in the development, such as the deposition of a more general extracellular matrix protein, as observed for decapod oocyte granules (Wong and Wessel 2006a).
Although an ovoperoxidase protein was not directly captured during proteomic analysis, we have lines of evidence to suggest that it is present. First, we found a sequence encoding an ovoperoxidase enzyme within each oocyte transcriptome from five echinoderm species analyzed, including two sea star species. Second, we indirectly observed its enzymatic activity: 3-AT is an inhibitor of ovoperoxidase, a myeloperoxidase-type enzyme (Daiyasu and Toh 2000), and exposure to 3-AT resulted in significantly increased dextran diffusion in the sea star, similar to that documented in the sea urchin (Wong and Wessel 2008; Fig. 1). Thus, we believe ovoperoxidase is one of the conserved FE proteins of echinoderms, although unlike in sea urchins, its abundance in sea stars may be limiting or it may diffuse away from the structure when its crosslinking activity is complete.
In P. miniata, the transcripts encoding the major cortical granule proteins (SFE9, proteoliaisin, and rendezvin) are synchronously regulated. Their RNA is highly expressed in early oocytes and is rapidly lost in later oogenesis. The timing of this degradation coincides with the translocation of the majority of cortical granules to the cell periphery. In sea urchins, the RNA levels of most of FE protein transcripts also decrease during oocyte maturation, particularly when the cortical granules move to the cell periphery (Laidlaw and Wessel 1994). These two events occur at different phases of sea urchin and sea star oogenesis, but the parallels in relative timing suggest a common mechanism linking the reduction in RNA with the translocation of the cortical granules.
This observation opens two important considerations: are mRNAs degraded by a shared mechanism, such as miRNAs or specific 3′UTR degradation elements, or are the genes regulated by the same transcription factors to synchronize timing and protein stoichiometry? Further, cortical granule mRNA degradation begins as the oocytes rapidly increase in size, a phenomenon consistent with vitellogenesis. In echinoderms, the vitellogenin appears to be made in the digestive tract of the adult and is transported to the ovary where it is taken up into yolk granules (Brooks and Wessel 2003). That uptake begins with a vitellogenic phase of oogenesis, a transitional period in development of this cell. Although we do not know how this transition is activated, this period may include a transition that involves reallocation of energy and resources, repressing cortical granule assembly, and the associated expression of genes that encode their content, in favor of processes that will enhance embryo viability.
Our results demonstrate that the FE in sea urchins is a much more selective barrier than in the sea star. Three similar structural proteins rich in LDLrA repeats—proteoliaisin, SFE9, SFE1—compose the FE in sea urchin, but no SFE1 ortholog was identified in the sea star. This suggests that SFE1 is not required to form a FE, but might be key to efficient packing of the envelope proteins to reduce permeability. SFE1 may have appeared in sea urchins by duplication of SFE9 or proteoliaisin, thereby expanding the quantity of LDLrA-rich proteins in the FE, perhaps resulting in a more efficient assembly process that more rapidly establish protection for the fertilized egg. This duplication event may have been an evolutionary transition that occurred between sea stars and sea urchins as the diversification in sequence between the two species was otherwise minimal. Perhaps this conservation is a result of compatibility within the complex—several proteins must rapidly and effectively self-assemble, and if they are delayed or compromised in their sperm-blocking, or pathogen-blocking ability, the embryos may rapidly die. Even though separated from a common ancestor by approximately 0.5 Byr, the envelope proteins between sea urchins and sea stars remained largely similar in terms of composition, motif, and function.
The human renal glomerulus filters particles on the order of <50 kDa, somewhat smaller than serum albumin, and this extracellular matrix filter takes a few weeks to develop. In contrast, the S. purpuratus FE filters materials of approximately <40 kDa (Wong and Wessel 2008) and takes ∼30 sec to form. Based on the morphology of the FE in sea stars that is forms more slowly (several minutes) and is significantly thicker than the sea urchin FE, we anticipated it would be relatively impermeant. Remarkably, it was far more permeable, allowing dextrans of 2000 kDa in size to diffuse through. Although still an effective barrier to sperm, it is clearly more passive to large molecules. This changes what we think about its role in the sea star environment—nutrients would be far more accessible to the developing embryo, and perhaps the embryo is able to endocytose larger nutrient particles for growth. On the other hand, it would be less capable of blocking toxins in the environment, perhaps even allowing small viral particles to diffuse through. These embryos may be more susceptible to environmental insults, especially in areas close to human effluents at the exact time in development that is most sensitive to the potentially noxious agents.