The chemical defensome: environmental sensing and response genes in the Strongylocentrotus purpuratus genome.

Metazoan genomes contain large numbers of genes that participate in responses to environmental stressors. We surveyed the sea urchin Strongylocentrotus purpuratus genome for homologs of gene families thought to protect against chemical stressors; these genes collectively comprise the 'chemical defensome.' Chemical defense genes include cytochromes P450 and other oxidases, various conjugating enzymes, ATP-dependent efflux transporters, oxidative detoxification proteins, and transcription factors that regulate these genes. Together such genes account for more than 400 genes in the sea urchin genome. The transcription factors include homologs of the aryl hydrocarbon receptor, hypoxia-inducible factor, nuclear factor erythroid-derived 2, heat shock factor, and nuclear hormone receptors, which regulate stress-response genes in vertebrates. Some defense gene families, including the ABCC, the UGT, and the CYP families, have undergone expansion in the urchin relative to other deuterostome genomes, whereas the stress sensor gene families do not show such expansion. More than half of the defense genes are expressed during embryonic or larval life stages, indicating their importance during development. This genome-wide survey of chemical defense genes in the sea urchin reveals evolutionary conservation of this network combined with lineage-specific diversification that together suggest the importance of these chemical stress sensing and response mechanisms in early deuterostomes. These results should facilitate future studies on the evolution of chemical defense gene networks and the role of these networks in protecting embryos from chemical stress during development.

NO is necessary and sufficient for egg activation at fertilization.

The early steps that lead to the rise in calcium and egg activation at fertilization are unknown but of great interest--particularly with the advent of in vitro fertilization techniques for treating male infertility and whole-animal cloning by nuclear transfer. This calcium rise is required for egg activation and the subsequent events of development in eggs of all species. Injection of intact sperm or sperm extracts can activate eggs, suggesting that sperm-derived factors may be involved. Here we show that nitric oxide synthase is present at high concentration and active in sperm after activation by the acrosome reaction. An increase in nitrosation within eggs is evident seconds after insemination and precedes the calcium pulse of fertilization. Microinjection of nitric oxide donors or recombinant nitric oxide synthase recapitulates events of egg activation, whereas prior injection of oxyhaemoglobin, a physiological nitric oxide scavenger, prevents egg activation after fertilization. We conclude that nitric oxide synthase and nitric-oxide-related bioactivity satisfy the primary criteria of an egg activator: they are present in an appropriate place, active at an appropriate time, and are necessary and sufficient for successful fertilization.

Induction of the multixenobiotic defense mechanism (MXR), P-glycoprotein, in the mussel Mytilus californianus as a general cellular response to environmental stresses.

A multixenobiotic resistance mechanism (MXR) related to the P-glycoprotein multidrug transporter protein (p-gp) has been identified and characterized in several marine invertebrates. p-gp activity and protein titer is induced by exposure to toxins, supporting the suggestion that the role for this transporter is protection from xenobiotics by reducing accumulation of toxins in cells. In this study, we report on the specificity of the induction of the transporter by various chemical and physical stressors. p-gp substrates (including the pesticides pentachlorophenol and chlorthal) as well as non-substrates (including DDE and sodium arsenite) induced p-gp activity and protein titer in the gill tissues of the mussel Mytilus californianus. Similarly, mussels exposed to heat shock of 20 degrees C or 25 degrees C exhibited increased p-gp titer and activity compared to mussels held at ambient (12 degrees C) temperature seawater. Some of the same treatments that induced an increase in p-gp caused a concomitant increase in hsp70, but hsp induction was not always associated with induction of the p-gp protein. These findings suggest that p-gp induction in mussels may be part of a general cellular stress response. This response, however, does not appear to be always coupled with the hsp70 response in mussels.

Multidrug resistance in the embryos and larvae of the mussel Mytilus edulis.

Cells exhibiting the multidrug resistance (MDR) phenotype demonstrate a decreased intracellular drug accumulation due to an active outward transport and decreased intracellular flux. This study demonstrates the inhibition of MDR in mussel (Mytilus edulis) embryos and larvae based on a simple bioassay. The development of embryos was assessed and abnormalities identified at key stages of development, including gastrulation, trochophore and prodissoconch stages. The incidence of developmental abnormalities was significantly increased in the presence of vinblastine, MMS, chloroquine, mitomycin-C, cadmium chloride and colchicine, compared to clean seawater. Consistently, there was a further increase in the number and severity of deformities observed when each toxin was added in the presence of verapamil. Larval growth was also significantly impaired in the presence of verapamil. Increased accumulation of fluorescent MDR dyes, such as rhodamine B, has been measured and shown to be verapamil sensitive. This bioassay encompasses a period of intense cellular activity during which the impairment of a number of critical processes results in abnormal growth and development.

The roles of changes in NADPH and pH during fertilization and artificial activation of the sea urchin egg.

Incubating unfertilized sea urchin eggs in weak bases activates nuclear centering, DNA synthesis, and chromosome cycles. These effects were initially attributed to raising the intracellular pH (pH(i)), but later experiments indicated that these weak bases also lead to increases in reduced pyridine nucleotides. These findings raised the question whether the activation of the nucleus was due to increased pH(i) or to increased NAD(P)H or possibly other effects. This report attempts to clarify how ammonia activates eggs by independently altering NADPH and pH(i). To increase the pH(i), unfertilized eggs were injected with zwitterionic buffers. This stimulated pronuclear centering, DNA synthesis, and nuclear envelope breakdown; there appeared to be a threshold corresponding to the fertilized pH(i). However, like incubation in ammonia, injection of base also increased NAD(P)H. The NAD(P)H rise caused by directly raising the pH(i) occurred in the presence of intracellular calcium chelators, indicating that calcium is not required. Increasing NAD(P)H alone did not activate nuclear centering, DNA synthesis, or nuclear envelope breakdown. Although these experiments cannot eliminate a role for the NADPH increase in initiating events leading to nuclear centering and entry into mitosis, they provide additional and strong evidence that increasing the pH(i) may be a primary signal.

Detection of phospholipase Cgamma in sea urchin eggs.

Phosphorylation on tyrosine and turnover of polyphosphoinositide metabolism are rapidly stimulated after fertilization. However, the interconnection between these pathways remains to be determined. In the present paper it is demonstrated that eggs of two different sea urchin species contain tyrosine phosphorylated proteins with calcium-sensitive phospholipase C activity. We have investigated whether phospholipase Cgamma (PLCgamma), characteristic of tyrosine kinase receptors, could be responsible for this activity. Western blot and immunocytochemistry performed with antibodies directed against PLCgamma revealed the presence of this protein in cortical regions. It was also observed that PLCgamma displayed calcium-sensitive activity. The present results suggest that PLCgamma may be part of the cascade of events leading to the calcium signal responsible for egg activation at fertilization.

Redox changes during fertilization and maturation of marine invertebrate eggs.

Previous studies on sea urchin eggs indicate that activation of NAD kinase is one of the earliest Ca2+-mediated events of fertilization. The subsequently produced NADP is converted to NADPH by the pentose shunt pathway, and some of this NADPH is used by an NADPH oxidase for generation of H2O2. To examine whether these changes apply generally, we have analyzed changes in pyridine nucleotide content during meiotic maturation and fertilization in eggs from four phyla. Surprisingly, fertilization-associated increases in NAD kinase were found only in echinoid eggs. The ratio of NADPH/NADP (redox ratio) increased from 1-1.6 to 2.5-6 following fertilization of echinoid and also clam eggs. However, the ratio is already >2 for unfertilized asteroid, tunicate and echiuroid eggs, and this ratio is unaffected by fertilization. We conclude that activation of NAD kinase and shifts in pyridine nucleotide metabolism and thereby cellular redox status may have roles that vary between species. In echinoids, a major role is in providing NADPH for H2O2 production, but there may be other yet unappreciated signaling functions for this change.

Effects of wheat germ agglutinin on tunicate egg activation and fertilization: is there a plasma membrane sperm receptor system on Ascidia ceratodes eggs?

Little work has been carried out on the sperm recognition systems present on the egg plasma membrane. Here it is shown that wheat germ agglutinin (WGA) interferes with the sperm-interacting system on the plasma membrane of eggs of the ascidian, Ascidia ceratodes. The WGA activates the dechorionated egg, indicating that a plasma membrane sugar residue can be directly tied to egg activation. Low concentrations of this lectin do not activate the eggs, but reduce fertilizability. This observation suggests that the WGA binding site might be part of a sperm reception-activation complex in the plasma membrane. While WGA also affects sperm binding to the chorion, the mechanisms of sperm interaction at the plasma membrane and chorion show different sensitivities to lectins, sugars and enzymes.

Bacterial Symbionts Colonize the Accessory Nidamental Gland of the Squid Loligo opalescens via Horizontal Transmission.

The accessory nidamental gland (AN gland), a reproductive organ of the mature female squid Loligo opalescens, harbors a dense culture of bacteria of unknown function. A multilayered sheath surrounding the L. opalescens egg case is similarly colonized by bacteria that presumably originate in the AN gland, as evidenced by their presence in the egg case at oviposition. This study investigates how these bacteria are transmitted to juvenile squid and examines some morphological consequences of bacterial colonization of AN gland tissues. By observing the structure of the AN gland in adults and the development and bacterial colonization of the gland in juveniles raised in captivity, we determined that the AN gland was absent in newly hatched squid and did not appear until 87 days post-hatching. At 129 days posthatching, the organ displayed tubules composed of a single layer of epithelial cells and expressing numerous cilia and microvilli. These tubules were not yet fully formed and thus were open to the mantle cavity and external seawater, possibly to aid in the acquisition of microorganisms. Since the AN gland developed a considerable time after hatching, it most likely acquires its symbionts horizontally from environmental seawater and not vertically from the egg case sheath. The switch from expression of cilia to production of microvilli on the epithelial cell surface may dictate the competence of the tissue for bacterial colonization. Electron microscopic examination of juvenile and adult AN glands revealed that an analogous process occurs during the development of the related light organ of other cephalopod species that harbor symbiotic bacteria.

An early increase in cGMP follows fertilization of sea urchin eggs.

It has been proposed that both inositol trisphosphate and ryanodine receptors contribute to the Ca signal generated at fertilization of the sea urchin egg. Pharmacological studies indicate that cyclic adenosine diphosphate-ribose (cADPr) is the endogenous modulator of Ca release by the ryanodine-like receptor in eggs and that cADPR cyclase, the enzyme responsible for cADPR synthesis, can be stimulated by 3',5'-cyclic guanosine monophosphate (cGMP). Also, recent results show that the gaseous transmitter nitric oxide (NO) releases calcium in eggs via a mechanism linked to cGMP and cADPR production. Results reported here show that fertilization induces a rapid and transient increase in the intracellular concentration of cGMP. This increase occurs during the latent period, before the major increase in cytoplasmic free calcium (Cai), consistent with the hypothesis that cGMP production may play a key role in the Ca signal seen at fertilization.

Characterisation and role of integrins during gametic interaction and egg activation.

It has recently been proposed that some of the processes induced by fertilisation in mammals may be mediated by integrins. By performing immunofluorescence labelling and Western blots with antibodies directed against some of the alpha and beta subunits of integrins, we show here the presence of some of these proteins in human and hamster oocytes. Among them, alpha 2 and alpha 5 were also present on in vitro preparations of sea urchin egg cortices. In addition, antibodies raised against these two proteins were the most effective at inhibiting attachment and fusion of human spermatozoa with hamster oocytes. We suggest that alpha 2 and alpha 5 integrin chains may be common mediators in adhesion-fusion mechanisms triggered by fertilisation. Using similar techniques, we show that eggs are rich in three cytoskeletal proteins known to be linked to the beta chain of integrins: talin, vinculin and alpha-actinin. Moreover, we found that talin and alpha-actinin were associated with proteins phosphorylated on tyrosine after fertilisation in sea urchin eggs. We suggest that integrins might be involved during fertilisation and trigger egg activation through cytoskeletal structures.

Protein synthesis increases after fertilization of sea urchin eggs in the absence of an increase in intracellular pH.

We have reevaluated the presumed requirement for an elevated intracellular pH (pHi) in the acceleration of protein synthesis which follows fertilization of eggs of the sea urchin Lytechinus pictus. Zygotes were transferred to sea water at a low pH (6.8) containing a permeant weak acid at times ranging from 5 min to as early as 30 sec postinsemination, to reverse or prevent the rise in pHi that normally ensues upon fertilization. Using the fluorescent pH probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we show that transfer of zygotes at 1 min or earlier after fertilization essentially blocks the rise in pHi. Under these conditions, both the incorporation of radiolabeled leucine into protein and the assembly of ribosomes into polysomes increase substantially (> 50% of control values). We also assessed leucine incorporation during incubation of eggs and zygotes in sodium-free sea water or sea water containing amiloride, two additional treatments that block the pHi rise. In the presence of amiloride, leucine incorporation increased upon fertilization, whereas little or no increase was observed in sodium-free sea water. We provide evidence that the low rates of leucine incorporation in sodium-free sea water result from the tendency for this experimental condition to lower the pHi to values significantly lower than the pHi in unfertilized eggs. These findings call into doubt the belief that the pHi rise at fertilization is a necessary prerequisite for the acceleration of bulk protein synthesis. These observations support the view that pHi is only one of several signals involved in the turn on of protein synthesis at the time of fertilization of sea urchin eggs.

The in vivo rate of glucose-6-phosphate dehydrogenase activity in sea urchin eggs determined with a photolabile caged substrate.

Some of the earliest metabolic changes after fertilization of sea urchin eggs center around the activity of the pentose phosphate shunt. We here report on the in vivo activity of glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of this shunt, as assayed with a photolabile (caged) analog of the substrate, glucose-6-phosphate (G6P). Caged G6P was synthesized from radiolabeled (5-3H or 1-14C) glucose and loaded into unfertilized sea urchin eggs by transient electroporation. Irradiation of these eggs (either before or after fertilization) photolyses the caged G6P, thereby pulsing the cell with 3H- and 14C-labeled G6P. The fluxes of G6P into glycolysis and the pentose shunt are calculated from the rates of oxidation of labeled G6P to 3H2O and 14CO2; since the turnover of the 6-phosphogluconate pool by 6-phosphogluconate dehydrogenase is nearly instantaneous (Swezey, R.R., and Epel, D. (1992) Exp. Cell Res. 201:366-372), the rate of 14CO2 production by the pentose shunt is equal to the flux of G6P through G6PDH. The data indicate that G6PDH activity is very low in unfertilized eggs, increases 184- to 427-fold by 2 min after fertilization, and then decreases to a value that is 74 to 209 times the unfertilized level (maximally 0.005 x 10(-8) units per egg in unfertilized eggs, 2.14 x 10(-8) units per egg by 2 min after fertilization, and 1.05 x 10(-8) units per egg by 20 min after fertilization). In spite of this substantial activation, the enzyme activity is considerably repressed; compared with activity in broken cell extracts, G6PDH at these developmental times operates in vivo at 0-0.003%, 0.52-1.21%, and 0.21-0.59%, respectively, of its potential activity. These results are discussed in terms of various hypotheses regarding the modulation of G6PDH activity by fertilization. These activity measurements relate well to other indices of in vivo activity. The major use of the NADPH shortly after fertilization is to produce H2O2, which is used as a substrate for fertilization membrane hardening; our data indicate that the NADPH that is produced by the pentose shunt activity is 30-70% of that required for this postfertilization generation of H2O2.

Beakers versus breakers: how fertilisation in the laboratory differs from fertilisation in nature.

The fertilisation of free-spawning invertebrates, mainly sea urchins, has been studied extensively during the last hundred years. However, results obtained from in vitro experiments do not always reflect what happens in the real world. Organisms in their natural habitats have a complex set of challenges, cues and behaviours to contend with during fertilisation and early development, factors that are normally not considered in the laboratory setting. This review examines recent work on fertilisation ecology and discusses the relevance of these results to the findings gleaned from laboratory research. Emphasis is placed on stresses associated with fertilisation in situ, and how responses to environmental stresses (such as from turbulence, oxidative stress, ultraviolet radiation and pathogens) might affect the fertilisation process.