Influence of sperm and phytoplankton on spawning in the echinoid Lytechinus variegatus

The cues triggering large-scale broadcast-spawning events in marine invertebrates are not fully understood. Using the sea urchin Lytechinus variegatus, we tested the effectiveness of a variety of potential spawning cues in eliciting a spawning response. In the laboratory, during two consecutive spawning seasons, about 400 isolated sea urchins were exposed to phytoplankton, sperm, or eggs, singly or in combination. The likelihood of spawning, time to spawning, and spawning behavior were recorded for both sexes. Sperm was most successful at inducing spawning. No response to eggs was noted. Phytoplankton alone did not trigger spawning, but when a phytoplankton cue was followed by the addition of sperm, spawning behavior was induced, the time between addition of sperm and spawning was reduced, and the variance among individuals in the time of spawning initiation was reduced. Males spawned sooner in response to cues than females and rarely spawned spontaneously in phytoplankton or control treatments. A semilunar pattern in the sensitivity to spawning cues was noted. During time periods when sea urchins were less ripe, the ratio of spawning males to spawning females increased. Our results indicate that seasonal and lunar cycles, together with the presence of phytoplankton, increase the sensitivity of these sea urchins to spawning cues and the precision of their responses to conspecific sperm.     

Maternal exposure to estradiol and endocrine disrupting compounds alters the sensitivity of sea urchin embryos and the expression of an orphan steroid receptor

Endocrine disrupting compounds (EDCs) are known to affect reproduction and development in marine invertebrates. In previous work, we have shown that developing sea urchin embryos were sensitive to estradiol and estrogenic EDCs at environmentally relevant concentrations in a tamoxifen-sensitive manner (Roepke et al. 2005. Aquat Toxicol 71:155-173). In this study, we report the effects of maternal exposure to EDCs on embryo sensitivity and regulation of an orphan steroid receptor in sea urchin eggs. Maternal exposures were conducted by injecting female Strongylocentrotus purpuratus sea urchins initiating oogenesis with two concentrations of estradiol, octylphenol, tributyltin and o, p-DDD for 8 weeks with an induced spawning before and after the injection cycle. Developing embryos were less sensitive to estradiol following maternal exposure to estradiol, octylphenol and DDD. The steroidogenesis inhibitor, spironolactone, and the aromatase inhibitor, formestane, affected normal sea urchin development with EC50 values of 18 and 2 microM, respectively. Binding of estradiol was demonstrated in homogenates supernatants of sea urchin embryos by filtration centrifugation and column chromatography, but saturation was not reached until 4-6 hr and was highly variable. Analysis of eggs from pre- and post-injection spawns using real-time Q-PCR for the mRNA of an orphan steroid receptor, SpSHR2, shows that receptor mRNA increased in eggs with estradiol, octylphenol and tributyltin but decreased with DDD. RIA showed that estradiol may be present during gastrulation. In summary, maternal exposure to estradiol and EDCs alters embryo sensitivity and regulates the expression of an orphan steroid receptor in the egg.     

American plaice, Hippoglossoides platessoides (Fabricius), predation on green sea urchins, Strongylocentrotus droebachiensis (O. F. Muller), in eastern Newfoundland

Green sea urchins were the most abundant food species (62% by weight) in American plaice (all female) collected from an area in eastern Newfoundland where sandy bottom abuts urchin dominated bedrock. Of secondary importance were capelin (12.9% by weight), which were spawning in the area when the samples were obtained. The results suggest that American plaice may be important predators of urchins in such habitats.     

Stereological analysis of nutritive phagocytes and gametogenic cells during the annual reproductive cycle of the green sea urchin, Strongylocentrotus droebachiensis

Abstract. The germinal epithelium of sea urchin gonads contains two interdependent populations of cells: somatic cells called nutritive phagocytes (NPs) and germ cells—ovarian and testicular gonial cells and their derivatives, gametocytes and gametes. Annually, NPs vary their structure and function to produce a changing microenvironment for germ cells during gametogenesis and after spawning. Here, we describe seasonal changes in the NPs as they interact with germ cells during successive gametogenic stages in both sexes of the green sea urchin, Strongylocentrotus droebachiensis. Monthly samples were collected from the Gulf of Maine at a depth of 10 m and analyzed with light microscopy and stereology. Shorter day length and falling seawater temperatures were correlated with nutrient mobilization from NPs, initiation of gonial cell mitosis, and subsequent gametogenesis. During gametogenesis, NPs in both sexes were depleted of nutrients and eventually phagocytize residual ova or spermatozoa. Observations from this study are important for understanding both the cellular aspects of the reproductive biology of sea urchins and those environmental factors that affect the onset and progress of gametogenesis.     

The structure of sulfated polysaccharides ensures a carbohydrate-based mechanism for species recognition during sea urchin fertilization

The evolution of barriers to inter-specific hybridization is a crucial step in the fertilization of free spawning marine invertebrates. In sea urchins, molecular recognition between sperm and egg ensures species recognition. Here we review the sulfated polysaccharide-based mechanism of sperm-egg recognition in this model organism. The jelly surrounding sea urchin eggs is not a simple accessory structure; it is molecularly complex and intimately involved in gamete recognition. It contains sulfated polysaccharides, sialoglycans and peptides. The sulfated polysaccharides have unique structures, composed of repetitive units of alpha-L-fucose or alpha-L-galactose, which differ among species in the sulfation pattern and/or the position of the glycosidic linkage. The egg jelly sulfated polysaccharides show species-specificity in inducing the sperm acrosome reaction, which is regulated by the structure of the saccharide chain and its sulfation pattern. Other components of the egg jelly do not possess acrosome reaction inducing activity, but sialoglycans act in synergy with the sulfated polysaccharide, potentiating its activity. The system we describe establishes a new view of cell-cell interaction in the sea urchin model system. Here, structural changes in egg jelly polysaccharides modulate cell-cell recognition and species-specificity leading to exocytosis of the acrosome. Therefore, sulfated polysaccharides, in addition to their known functions as growth factors, coagulation factors and selectin binding partners, also function in fertilization. The differentiation of these molecules may play a role in sea urchin speciation.     

Gamete Recognition and Egg Activation in Sea Urchins

SYNOPSIS. Free-spawning marine invertebrates face the challengeof ensuring that gametes of the same species come into contact,recognize, bind to and fuse with one another once they havebeen released by the adults. Coordinated spawning, chemoattractionand specific cell-cell recognition events help to overcome thischallenge. One marine invertebrate, the sea urchin, has servedas a model system for the study of gamete recognition and fertilizationfor over 100 years. Recent biochemical and molecular advancesin this area have begun to address the questions that have beenraised by the results of elegant physiological observations.The picture of fertilization that is emerging is characterizedby highly specific cell-cell interactions between proteins onthe surfaces of the gametes. These proteins then mediate thebinding and subsequent events that lead to activation of theegg and delivery of the male genetic material. Because of theserecent insights, the sea urchin egg is in a position to provideanswers to one of the central debates in developmental biology—themechanism of egg activation. Does the sperm deliver an activatingfactor? Does sperm binding trigger a receptor-mediated signal?Or is the mechanism a complex combination? With the tools andknowledge gained from the study of sea urchin fertilization,testing of these hypotheses should be feasible in the near future.     

The recruitment sweepstakes has many winners: genetic evidence from the sea urchin Strongylocentrotus purpuratus

As a consequence of free spawning in the unpredictable nearshore environment, marine species with large fecundities and high pre-reproductive mortality may be subject to extreme variance in reproductive success. If the unpredictability of the ocean results in only a small subset of the adult population contributing to each larval cohort, then reproduction may be viewed as a sweepstakes, with chance events determining which adults are successful each spawning season. Such a reproductive sweepstakes scenario may partially account for large reductions in effective population sizes relative to census population sizes in marine species. We evaluated two predictions of the sweepstakes reproductive success hypothesis by testing: (1) whether sea urchin recruits contain reduced genetic variation relative to the adult population; and (2) whether cohorts of sea urchin recruits are genetically differentiated. Mitochondrial DNA sequences were collected from 283 recently settled Strongylocentrotus purpuratus recruits from four annual cohorts spanning seven years in locations throughout California. Observed haplotype numbers and haplotype diversities showed little evidence of reduced genetic variation in the recruits relative to the diversity estimated from a previously reported sample of 145 S. purpuratus adults. Different cohorts of recruits were in some cases mildly differentiated from each other. A computer simulation of sweepstakes recruitment indicates that our sampling strategy had sufficient statistical power to detect large variances in reproductive success.     

Effect of Estradiol Dipropionate on Protein Synthesis in Oocytes and Ovary of the Sea Urchin Strongylocentrotus intermedius at Different Stages of the Reproductive Cycle

We studied protein synthesis in the oocytes and ovary of the sea urchin Strongylocentrotus intermedius at different stages of the reproductive cycle after treatment with estradiol dipropionate. During the early development of oocytes and active gametogenesis, this estrogen induced the incorporation of ³H-leucine in the oocytes. The changes in synthetic activity of cells were accompanied by an elevated efficient incorporation of free amino acid in proteins due to its increased pool, increased tissue permeability for precursors, and increased rate of protein synthesis. Before spawning, estradiol dipropionate did not cause any changes in protein synthesis. After estradiol dipropionate treatment, the inhibitors of protein synthesis, puromycin and actinomycin D, decreased the intensity of ³H-leucine incorporation in the oocytes and protein synthesis in the ovary. The involvement of estradiol dipropionate in the regulation of protein synthesis in the sea urchin gonad is discussed.     

Microsatellite DNA markers for analysis of population structure in the sea urchin Centrostephanus rodgersii

We describe the development of 13 variable microsatellites developed to investigate population structure and dispersal in the sea urchin Centrostephanus rodgersii. This species is the dominant grazing herbivore in southeast Australian coastal waters and has the ability to modify benthic community structure. The microsatellites we identified showed a range of allele numbers (4–21) and expected heterozygosity (0.32–0.91) in two sampled populations. Contrary to previous findings in free‐spawning marine invertebrates, genotype proportions in neither population deviated significantly from Hardy–Weinberg expectations.     

Importance of spatial population characteristics on the fertilization rates of sea urchins

We show that inclusion of population characteristics in coupled advection-diffusion and fertilization-kinetics models results in higher fertilization rates than those previously reported in theoretical studies. We incorporate parameters related to both individuals and populations by running simulations over a large spatial scale and incorporating sperm contribution from multiple males. We compare predictions for three subpopulations of the sea urchin Strongylocentrotus droebachiensis (those occupying kelp beds, barrens, and grazing fronts) to observations from small-scale experiments, and estimate effects of population size and current velocity in each subpopulation. Model outputs suggest that fertilization rates are low in kelp beds, intermediate in barrens, and high in grazing fronts. In all populations, increasing current velocity has a negative effect on the relationship between fertilization rate and downstream distance of gametes after release, but no effect on the relationship between fertilization rate and elapsed time since gamete release. Our model output was most sensitive to changes in the number of spawning males and the sperm release rate, suggesting that spawning synchrony and high gonadic index could greatly increase the fertilization success in sea urchins.     

A soluble adenylyl cyclase from sea urchin spermatozoa

A previously identified, calmodulin-binding, sea urchin sperm flagellar adenylyl cyclase (AC) was cloned and sequenced and found to be a homologue of mammalian sperm soluble adenylyl cyclase (sAC). Compared to the mammalian sAC, the sea urchin sAC (susAC) has several long amino acid insertions, some of which contain protein kinase A phosphorylation sites. The enzymatic activity of susAC shows a steep pH dependency curve, the specific activity doubling when the pH is increased from 7.0 to 7.5. This suggests that like sperm dynein ATPase, the susAC is probably activated by increases in intracellular pH occurring upon spawning into seawater and also when sperm respond to contact with the egg jelly layer. The susAC is strongly activated by manganese, but has low activity in magnesium. Gene database searches identified sAC homologues in species known to have cyclic AMP-dependent sperm motility. This implies (as shown in mouse) that susAC has a role in sperm motility, most probably through axonemal protein phosphorylation or ion channel regulation.     

Testing for the Effects of Seasonal and Lunar Periodicity on the Reproduction of the Edible Sea Urchin <Emphasis Type="Italic">Tripneustes gratilla</Emphasis> (L) in Kenyan Coral Reef Lagoons

The annual and lunar reproductive cycle of the widely distributed edible sea urchin Tripneustes gratilla (L) was examined through measurements of gonad index, histological examination of gametogenesis, and induction of spawning with KCl injections. The population density and morphological characteristics of urchins at Diani, Kanamai, and Vipingo reef lagoons were also studied as well as the effects of seawater temperature and light on reproduction. Gonad growth started early during the northeast monsoon and reached a peak in June at the beginning of the southeast monsoon followed by a sharp decrease in gonad size of 50% in July and August towards the end of the southeast monsoons. Histological examination of gonads, revealed many different stages of gametogenesis with gametes present throughout the year, indicating continuous reproduction. There was a significant relationship between gonad index and lunar day with spawning occurring between lunar day 7 and 21, but spawning was not in perfect synchrony in the population. The population density of urchins at each reef is variable from year to year and was highest on average at Vipingo. Urchins at Kanamai had the lowest gonad indices, the largest jaws and smallest individuals an indication of food limitation. The gonads (roe) of T. gratilla at all three sites, were perpetually ‘runny’ an attribute that is not suitable for urchin fisheries. Studies to develop techniques to improve roe quality are recommended.     

Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching

The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.     

Immunological evidence for the localization of sialoglycosphingolipids at the cell surface of sea urchin spermatozoa.

The localization of sialoglycosphingolipids in the plasma membrane of sea urchin spermatozoa was studied by employing immunological methods including immunolysis of liposomal model membranes. The antibodies produced against these complex lipids were found to agglutinate various sea urchin spermatozoa differently. Both species differences and species similarities in the agglutination were found in spermatozoa of the echinoderm, the sea urchin and the starfish. The agglutination of the sea urchin spermatozoa was inhibited specifically by ceratain carbohydrates. Only a limited number of molecular species of sialoglycosphingolipid were localized at the surface of the plasma membrane of sea urchin spermatozoa cells. Moreover, topographical differences were found in the localization of the sialoglycosphingolipids at the cell surface of spermatozoa.     

Reproductive patterns within sub-populations of Lytechinus variegatus (Lamarck) (Echinodermata: Echinoidea)

The annual reproductive cycle of the sea urchin Lytechinus variegatus (Lamarck) was examined in individuals collected from four habitats in the Anclote estuary near Tarpon Springs, Florida. Two periods of advanced gametogenic activity were apparent, but this bimodal pattern was not necessarily reflected by gonad growth curves. Differences in reproductive events among sub-populations were related primarily to patterns of gonad growth. Reproductive synchrony present during spring became less apparent as spawning and recovery within the sub-populations took place at differential rates during late-spring and summer. The final spawnout in late summer returned the sub-populations into gametogenic synchrony.     

The S. purpuratus genome: a comparative perspective

The predicted gene models derived from the sea urchin genome were compared to the gene catalogs derived from other completed genomes. The models were categorized by their best match to conserved protein domains. Identification of potential orthologs and assignment of sea urchin gene models to groups of homologous genes was accomplished by BLAST alignment and through the use of a clustering algorithm. For the first time, an overview of the sea urchin genetic toolkit emerges and by extension a more precise view of the features shared among the gene catalogs that characterize the super-clades of animals: metazoans, bilaterians, chordate and non-chordate deuterostomes, ecdysozoan and lophotrochozoan protostomes. About one third of the 40 most prevalent domains in the sea urchin gene models are not as abundant in the other genomes and thus constitute expansions that are specific at least to sea urchins if not to all echinoderms. A number of homologous groups of genes previously restricted to vertebrates have sea urchin representatives thus expanding the deuterostome complement. Obversely, the absence of representatives in the sea urchin confirms a number of chordate specific inventions. The specific complement of genes in the sea urchin genome results largely from minor expansions and contractions of existing families already found in the common metazoan "toolkit" of genes. However, several striking expansions shed light on how the sea urchin lives and develops.     

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.