Effects of soluble copper and copper oxide nanoparticle exposure on the immune system of mussels, Mytilus galloprovincialis.

Copper and copper oxide nanomaterials (nCuO) can enter the marine environment negatively impacting mussels, an environmental and commercially relevant organism. We analyzed the effects on the immune system of adult mussels exposed to soluble copper (CuSO4 , 20-50 µg/L) or nCuO (100-450 µg/L). CuSO4 caused significant copper accumulation in gills and cell-free hemolymph, while nCuO caused cell damage to gills and significant copper accumulation in hemocytes, the most abundant cells in the hemolymph. Both sources of copper caused cellular toxicity in hemocytes by increasing reactive oxygen species production and lysosome abundance, and decreasing multi-drug resistance transporter activity. Though hemocyte abundance was not affected, their in-vitro phagocytic activity decreased, explaining the slight (but not statistically significant) increase in bacterial proliferation in mussels exposed to the pathogenic bacteria Vibrio tubiashii following copper exposure. Thus, exposure to non-lethal concentrations of CuSO4 or nCuO can potentially increase mussel susceptibility to bacterial infections.

Chemical and physical guidance of fish spermatozoa into the egg through the micropyle†,‡.

Eggs of teleost fish, unlike those of many other animals, allow sperm entry only at a single site, a narrow canal in the egg's chorion called the micropyle. In some fish (e.g., flounder, herring, and Alaska pollock), the micropyle is a narrow channel in the chorion, with or without a shallow depression around the outer opening of micropyle. In some other fish (e.g., salmon, pufferfish, cod, and medaka), the micropyle is like a funnel with a conical opening. Eggs of all the above fish have a glycoprotein tightly bound to the chorion surface around the micropyle. This glycoprotein directs spermatozoa into the micropylar canal in a Ca2+-dependent manner. This substance, called the micropylar sperm attractant or MISA, increases fertilization efficiency and is essential in herring. In flounder, salmon, and perhaps medaka, fertilization is possible without MISA, but its absence makes fertilization inefficient because most spermatozoa swim over the micropyle without entering it. The mechanism underlying sperm-MISA interactions is yet to be determined, but at least in herring the involvement of Ca2+ and K+ channel proteins, as well as CatSper and adenylyl cyclase, is very likely. In some other fish (e.g., zebrafish, loach, and goldfish), the chorion around the micropyle is deeply indented (e.g., zebrafish and loach) or it has radially or spirally arranged grooves around the outer opening of the micropyle (e.g., goldfish). MISA is absent from the eggs of these fish and sperm entry into micropylar canal seems to be purely physical.

Scaling Up Endocrine Disruption Effects from Individuals to Populations: Outcomes Depend on How Many Males a Population Needs.

Assessing how endocrine disrupting compounds (EDCs) affect population dynamics requires tracking males and females (and sex-reversed individuals) separately. A key component in any sex-specific model is the "mating function" (the relationship between sex ratio and reproductive success) but this relationship is not known for any fish species. Using a model, we found that EDC effects on fish populations strongly depend upon the shape of the mating function. Additionally, masculinization is generally more detrimental to populations than feminization. We then quantified the mating function for the inland silverside (Menidia beryllina), and used those results and the model to assess the status of wild silverside populations. Contrary to the expectation that a few males can spawn with many females, silversides exhibited a nearly linear mating function. This implies that small changes in the sex ratio will reduce reproductive success. Four out of five wild silverside populations exhibited sex ratios far from 50:50 and thus are predicted to be experiencing population declines. Our results suggest that managers should place more emphasis on mitigating masculinizing rather than feminizing EDC effects. However, for species with a nearly linear mating function, such as Menidia, feminization and masculinization are equally detrimental.

Comparison of Cytotoxicity and Inhibition of Membrane ABC Transporters Induced by MWCNTs with Different Length and Functional Groups.

Experimental studies indicate that multiwalled carbon nanotubes (MWCNTs) have the potential to induce cytotoxicity. However, the reports are often inconsistent and even contradictory. Additionally, adverse effects of MWCNTs at low concentration are not well understood. In this study, we systemically compared adverse effects of six MWCNTs including pristine MWCNTs, hydroxyl-MWCNTs and carboxyl-MWCNTs of two different lengths (0.5-2 µm and 10-30 µm) on human hepatoma cell line HepG2. Results showed that MWCNTs induced cytotoxicity by increasing reactive oxygen species (ROS) generation and damaging cell function. Pristine short MWCNTs induced higher cytotoxicity than pristine long MWCNTs. Functionalization increased cytotoxicity of long MWCNTs, but reduced cytotoxicity of short MWCNTs. Further, our results indicated that the six MWCNTs, at nontoxic concentration, might not be environmentally safe as they inhibited ABC transporters' efflux capabilities. This inhibition was observed even at very low concentrations, which were 40-1000 times lower than their effective concentrations on cytotoxicity. The inhibition of ABC transporters significantly increased cytotoxicity of arsenic, a known substrate of ABC transporters, indicating a chemosensitizing effect of MWCNTs. Plasma membrane damage was likely the mechanism by which the six MWCNTs inhibited ABC transporter activity. This study provides insight into risk assessments of low levels of MWCNTs in the environment.

Progesterone Accelerates the Completion of Sperm Capacitation and Activates CatSper Channel in Spermatozoa from the Rhesus Macaque.

During transit through the female reproductive tract, mammalian spermatozoa are exposed to increasing concentrations of progesterone (P4) released by the cumulus oophorus. P4 triggers massive calcium influx into human sperm through activation of the sperm-specific calcium channel CatSper. These properties of human spermatozoa are thought to be unique since CatSper is not progesterone sensitive in rodent sperm. Here, by performing patch clamp recording from spermatozoa from rhesus macaque for the first time, we report that they express P4-sensitive CatSper channel identically to human sperm and react to P4 by inducing responsiveness to zona pellucida, unlike human sperm, which respond directly to P4. We have also determined the physiologic levels of P4 capable of inducing capacitation-associated changes in macaque sperm. Progesterone (1 µM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold as low as 10 nM of P4. Submicromolar levels of P4 induced a dose-dependent increase in curvilinear velocity and lateral head displacement, while sperm protein tyrosine phosphorylation was not altered. Macaque spermatozoa exposed to 10 µM of P4 developed fully hyperactivated motility. Similar to human sperm, on approaching cumulus mass and binding to zona pellucida, macaque spermatozoa display hyperactivation and undergo an acrosome reaction that coincides with the rise in the sperm intracellular calcium. Taken together, these data indicate that P4 accelerates the completion of capacitation and provides evidence of spermatozoa "priming" as they move into a gradient of progesterone in search for the oocyte.

Copper oxide and zinc oxide nanomaterials act as inhibitors of multidrug resistance transport in sea urchin embryos: their role as chemosensitizers.

The ability of engineered nanomaterials (NMs) to act as inhibitors of ATP-binding cassette (ABC) efflux transporters in embryos of white sea urchin (Lytechinus pictus) was studied. Nanocopper oxide (nano-CuO), nanozinc oxide (nano-ZnO), and their corresponding metal ions (CuSO4 and ZnSO4) were used as target chemicals. The results showed that nano-CuO, nano-ZnO, CuSO4, and ZnSO4, even at relatively low concentrations (0.5 ppm), significantly increased calcein-AM (CAM, an indicator of ABC transporter activity) accumulation in sea urchin embryos at different stages of development. Exposure to nano-CuO, a very low solubility NM, at increasing times after fertilization (>30 min) decreased CAM accumulation, but nano-ZnO (much more soluble NM) did not, indicating that metal ions could cross the hardened fertilization envelope, but not undissolved metal oxide NMs. Moreover, nontoxic levels (0.5 ppm) of nano-CuO and nano-ZnO significantly increased developmental toxicity of vinblastine (an established ABC transporter substrate) and functioned as chemosensitizers. The multidrug resistance associated protein (MRP, one of ABC transporters) inhibitor MK571 significantly increased copper concentrations in embryos, indicating ABC transporters are important in maintaining low intracellular copper levels. We show that low concentrations of nano-CuO and nano-ZnO can make embryos more susceptible to other contaminants, representing a potent amplification of nanomaterial-related developmental toxicity.

Unexpectedly high mortality in Pacific herring embryos exposed to the 2007 Cosco Busan oil spill in San Francisco Bay.

In November 2007, the container ship Cosco Busan released 54,000 gallons of bunker fuel oil into San Francisco Bay. The accident oiled shoreline near spawning habitats for the largest population of Pacific herring on the west coast of the continental United States. We assessed the health and viability of herring embryos from oiled and unoiled locations that were either deposited by natural spawning or incubated in subtidal cages. Three months after the spill, caged embryos at oiled sites showed sublethal cardiac toxicity, as expected from exposure to oil-derived polycyclic aromatic compounds (PACs). By contrast, embryos from the adjacent and shallower intertidal zone showed unexpectedly high rates of tissue necrosis and lethality unrelated to cardiotoxicity. No toxicity was observed in embryos from unoiled sites. Patterns of PACs at oiled sites were consistent with oil exposure against a background of urban sources, although tissue concentrations were lower than expected to cause lethality. Embryos sampled 2 y later from oiled sites showed modest sublethal cardiotoxicity but no elevated necrosis or mortality. Bunker oil contains the chemically uncharacterized remains of crude oil refinement, and one or more of these unidentified chemicals likely interacted with natural sunlight in the intertidal zone to kill herring embryos. This reveals an important discrepancy between the resolving power of current forensic analytical chemistry and biological responses of keystone ecological species in oiled habitats. Nevertheless, we successfully delineated the biological impacts of an oil spill in an urbanized coastal estuary with an overlapping backdrop of atmospheric, vessel, and land-based sources of PAC pollution.

Interactive effects of pesticide exposure and habitat structure on behavior and predation of a marine larval fish.

Coastal development has generated multiple stressors in marine and estuarine ecosystems, including habitat degradation and pollutant exposure, but the effects of these stressors on the ecology of fishes remain poorly understood. We studied the separate and combined effects of an acute 4 h sublethal exposure of the pyrethroid pesticide esfenvalerate and structural habitat complexity on behavior and predation risk of larval topsmelt (Atherinops affinis). Larvae were exposed to four nominal esfenvalerate concentrations (control, 0.12, 0.59, 1.18 µg/L), before placement into 12 L mesocosms with a three-spine stickleback (Gasterosteus aculeatus) predator. Five treatments of artificial eelgrass included a (1) uniform and (2) patchy distribution of eelgrass at a low density (500 shoots per m(2)), a (3) uniform and (4) patchy distribution of eelgrass at a high density (1,000 shoots per m(2)), and (5) the absence of eelgrass. The capture success of predators and aggregative behavior of prey were observed in each mesocosm for 10 min of each trial, and mortality of prey was recorded after 60 min. Exposure to esfenvalerate increased the proportion of larvae with swimming abnormalities. Surprisingly, prey mortality did not increase linearly with pesticide exposure but increased with habitat structure (density of eelgrass), which may have been a consequence of compensating predator behavior. The degree of prey aggregation decreased with both habitat structure and pesticide exposure, suggesting that anti-predator behaviors by prey may have been hampered by the interactive effects of both of these factors.

Identification of the origin and localization of chorion (egg envelope) proteins in an ancient fish, the white sturgeon, Acipenser transmontanus.

In many modern teleost fish, chorion (egg envelope) glycoproteins are synthesized in the liver of females, and the expression of those genes is controlled by endogenous estrogen released from the ovary during maturation. However, among the classical teleosts, such as salmonid, carp, and zebrafish, the chorion glycoproteins are synthesized in the oocyte, as in higher vertebrates. Sturgeon, which are members of the subclass Chondrostei, represent an ancient lineage of ray-finned fishes that differ from other teleosts in that their sperm possess acrosomes, their eggs have numerous micropyles, and early embryo development is similar to that of amphibians. In order to understand the molecular mechanisms of chorion formation and the phylogenetic relationship between sturgeon and other teleosts, we used specific antibodies directed against the primary components of sturgeon chorion glycoproteins, using immunoblotting and immunocytochemistry approaches. The origin of each chorion glycoprotein was determined through analyses of both liver and ovary, and their localization during ovarian development was investigated. Our data indicate that the origin of the major chorion glycoproteins of sturgeon, ChG1, ChG2, and ChG4, derive not only from the oocyte itself but also from follicle cells in the ovary, as well as from hepatocytes. In the follicle cell layer, granulosa cells were found to be the primary source of ChGs during oogenesis in white sturgeon. The unique origins of chorion glycoproteins in sturgeon suggest that sturgeons are an intermediate form in the evolution of the teleost lineage.

Ecological nanotoxicology: integrating nanomaterial hazard considerations across the subcellular, population, community, and ecosystems levels.

Research into the health and environmental safety of nanotechnology has seriously lagged behind its emergence in industry. While humans have often adopted synthetic chemicals without considering ancillary consequences, the lessons learned from worldwide pollution should motivate making nanotechnology compatible with environmental concerns. Researchers and policymakers need to understand exposure and harm of engineered nanomaterials (ENMs), currently nanotechnology's main products, to influence the ENM industry toward sustainable growth. Yet, how should research proceed? Standard toxicity testing anchored in single-organism, dose-response characterizations does not adequately represent real-world exposure and receptor scenarios and their complexities. Our approach is different: it derives from ecology, the study of organisms' interactions with each other and their environments. Our approach involves the characterization of ENMs and the mechanistic assessment of their property-based effects. Using high throughput/content screening (HTS/HCS) with cells or environmentally-relevant organisms, we measure the effects of ENMs on a subcellular or population level. We then relate those effects to mechanisms within dynamic energy budget (DEB) models of growth and reproduction. We reconcile DEB model predictions with experimental data on organism and population responses. Finally, we use microcosm studies to measure the potential for community- or ecosystem-level effects by ENMs that are likely to be produced in large quantities and for which either HTS/HCS or DEB modeling suggest their potential to harm populations and ecosystems. Our approach accounts for ecological interactions across scales, from within organisms to whole ecosystems. Organismal ENM effects, if propagated through populations, can alter communities comprising multiple populations (e.g., plant, fish, bacteria) within food webs. Altered communities can change ecosystem services: processes that cycle carbon, nutrients, and energy, and regulate Earth's waters and atmosphere. We have shown ENM effects on populations, communities, and ecosystems, including transfer and concentration of ENMs through food chains, for a range of exposure scenarios; in many cases, we have identified subcellular ENM effects mechanisms. To keep pace with ENM development, rapid assessment of the mechanisms of ENM effects and modeling are needed. DEB models provide a method for mathematically representing effects such as the generation of reactive oxygen species and their associated damage. These models account for organism-level effects on metabolism and reproduction and can predict outcomes of ENM-organism combinations on populations; those predictions can then suggest ENM characteristics to be avoided. HTS/HCS provides a rapid assessment tool of the ENM chemical characteristics that affect biological systems; such results guide and expand DEB model expressions of hazard. Our approach addresses ecological processes in both natural and managed ecosystems (agriculture) and has the potential to deliver timely and meaningful understanding towards environmentally sustainable nanotechnology.

An approach to detecting estrogenic endocrine disruption via choriogenin expression in an estuarine model fish species.

A large body of work has established a link between endocrine disrupting compounds (EDCs) and a number of abnormalities in fishes. However, most EDC studies use several standard laboratory denizens to assess impacts, so assumptions about sensitivity are primarily based on these few species. Additionally, existing methods rely on obtaining sufficient plasma to measure EDC biomarkers. Our objectives were (a) to establish a new model species for estuarine fishes, (b) to evaluate endocrine impacts with a highly sensitive and specific biomarker, and (c) to develop a method for the analysis of this biomarker in small fish that do not possess sufficient blood plasma for protein measurement. As such, we created a polyclonal antibody (Ab) to the estrogen-responsive proteins chorion (Ch) and choriogenin (Chg) in Menidia beryllina, found throughout coastal North America and already utilized in EPA Whole Effluent Testing. We then validated the Ab by using it to measure the response to aqueous ethinylestradiol (EE2) through the development an ELISA using Menidia whole body homogenate (WBH). Sensitivity of the Ab to Menidia WBH is greater than that of the commercially available option. ELISA sensitivity, with a detection limit of 5 ng/ml and a working range of 22.6-1370.9 ng/ml, is comparable to ELISAs developed to measure plasma Chg. To our knowledge this is the first ELISA method developed for the detection of Chg using WBH. Including additional model species and methods allowing the evaluation of alternative sample matrices will contribute to an enhanced understanding of inter-species differences in EDC response.

Sperm motility-initiating substance in newt egg-jelly induces differential initiation of sperm motility based on sperm intracellular calcium levels.

Sperm motility-initiating substance (SMIS), a novel motility inducer from newt egg-jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female-stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca²(+)](i)). In quiescent non-motile sperm loaded with the Ca²(+) indicator Fluo-4, intracellular free Ca²(+) was observed around mitochondria using confocal scanning laser microscopy. A slight increase in [Ca²(+)](i) occurred simultaneously and transiently at motility initiation in sperm treated with either heated jelly extract (hJE) containing activated SMIS, or a low osmotic solution, which naturally initiates motility in externally-fertilizing amphibians and can initiate motility in urodele sperm. When the increase of [Ca²(+)](i) at motility-initiation was monitored using spectrofluorometry, large increases in [Ca²(+)](i) occurred immediately in the low osmotic solution and within 1.5 min in the hJE. In the intact jelly extract (no heating), small increases of [Ca²(+)](i) irregularly occurred from around 1 min and for about 4 min, during which motility was differentially initiated among sperm. These results indicate that the SMIS induces differential initiation of sperm motility depending on the activational states of the SMIS and its overall activity. The motility initiation in the jelly extract was delayed in sperm whose intracellular Ca²(+) had been chelated with BAPTA-AM. The relative levels of [Ca²(+)](i) were variable with a mean of 414 ± 256 nmol/L among resting sperm, suggesting that the level of [Ca²(+)](i) in the resting sperm modulates the responsiveness to the SMIS.

Two different unique cardiac isoforms of protein 4.1R in zebrafish, Danio rerio, and insights into their cardiac functions as related to their unique structures.

Protein 4.1R (4.1R) has been identified as the major component of the human erythrocyte membrane skeleton. The members of the protein 4.1 gene family are expressed in a tissue-specific alternative splicing manner that increases their functions in each tissue; however, the exact roles of cardiac 4.1R in the developing myocardium are poorly understood. In zebrafish (ZF), we identified two heart-specific 4.1R isoforms, ZF4.1RH2 and ZF4.1RH3, encoding N-terminal 30 kDa (FERM) domain and spectrin-actin binding domain (SABD) and C-terminal domain (CTD), separately. Applying immunohistochemistry using specific antibodies for 30 kDa domain and CTD separately, the gene product of ZF4.1RH2 and ZF4.1RH3 appeared only in the ventricle and in the atrium, respectively, in mature hearts. During embryogenesis, both gene expressions are expressed starting 24 h post-fertilization (hpf). Following whole-mount in situ hybridization, ZF4.1RH3 gene expression was detected in the atrium of 37 hpf embryos. These results indicate that the gene product of ZF4.1RH3 is essential for normal morphological shape of the developing heart and to support the repetitive cycles of its muscle contraction and relaxation.

Fabrication of a multifunctional magnetic-fluorescent material for medical applications.

Multifunctional biocompatible materials have evoked considerable interest in the field of medical applications. Here we report the thermal decomposition preparation of homogeneous fluorescent-magnetic particles with a composite structure containing CoFe2O4 nanoparticles as nucleation seeds for fluorescent Gd2-xO3:Eux. The composite exhibited a wide range of fluorescence transitions in the whole visible spectrum, displaying 18 different emission peaks when excited at a 250 nm wavelength. Moreover, at low temperature the peaks of the composite were wider than the peaks of the fluorescent material, which may be attributed to a set of new energy levels due to a combination of Stark splitting with the magnetic field of CoFe2O4. Because this material is intended to be used for biomedical applications, the potential toxicity of the composite was tested using an invertebrate hemocyte cell model. The cells showed slight morphological and biochemical changes upon exposure to the composite; however, there was no increase in cell death at concentrations of up to 40 ppm. In addition, the material could be tracked by its fluorescence inside the cells, when excited at a more bio-friendly and less energetic wavelength of 405 nm. Furthermore, MRI showed T1 and T2 dual contrast with relaxivity values in the range of most reported materials.

Release of DEFB126 from macaque sperm and completion of capacitation are triggered by conditions that simulate periovulatory oviductal fluid.

Capacitation of macaque sperm in vitro has been achieved efficiently only with the addition of both cyclic nucleotides and methylxanthines. The use of these exogenous sperm activators clouds an understanding of the normal mechanisms underlying capacitation and may slow early embryo development following in vitro fertilization (IVF). We demonstrate that culture medium which simulates periovulatory oviductal fluid with respect to bicarbonate (HCO(3)(-)) and glucose concentration induces capacitation in a high percentage of macaque sperm as determined by the ability of sperm to undergo both the release of coating protein DEFB126 and the zona pellucida-induced acrosome reaction (AR). Few sperm were able to undergo the AR following 6 hr incubation in medium containing either 35 mM HCO(3)(-) (approximately 7.2 pH) or 90 mM HCO(3)(-) (approximately pH 7.8) with 5 mM glucose. When glucose concentration was lowered to 0.5 mM to match levels reported for women at midcycle, the AR rate increased significantly in sperm incubated in both levels of HCO(3)(-), indicating that glucose interferes with sperm responsiveness to increasing HCO(3)(-) concentration observed in the primate oviduct during ovulation. Even greater synchronization of capacitation could be achieved with nonphysiologic extremes of alkalinity or energy substrate deprivation. In the latter case, sperm achieved high rates of IVF. A shift in pH from 7.2 to 7.8 in a HEPES-buffered medium was sufficient to remove DEFB126 from the surface of most sperm after only 3 hr. The loss of DEFB126 from sperm under periovulaory fluid conditions has implications for the timing of release of sperm from the oviductal reservoir.

The effects of marginal maternal vitamin A status on penta-brominated diphenyl ether mixture-induced alterations in maternal and conceptal vitamin A and fetal development in the Sprague Dawley rat.

BACKGROUND: Polybrominated diphenyl ether (PBDE) toxicity in rodents can be associated with disruptions in endocrine signaling. We previously reported that the penta-BDE mixture, DE-71, disrupts thyroid hormones and vitamin A metabolism in rats during lactation, and that this disruption is amplified in animals fed diets marginal in vitamin A. The ability of the DE-71 to disrupt vitamin A metabolism during the prenatal period has not been evaluated. While penta-BDE mixtures are not strong teratogens in pregnant animals fed standard commercial laboratory diets, we hypothesized that they could be teratogenic under conditions of marginal vitamin A status. METHODS: rats were fed diets containing 0.4 retinyl equivalents (RE, marginal) or 4.0 RE (adequate) of vitamin A per gram of diet. Pregnant animals were exposed to DE-71 (0, 6, 18, 60, or 120 mg/kg) from gestation days (GD) 6-11.5, or on GD 6-19.5. RESULTS: DE-71 treatment resulted in dose-responsive reductions in maternal thyroid hormone and markers of vitamin A metabolism, with the latter reduction amplified in marginal vitamin A dams. Fetuses from marginal vitamin A, DE-71-exposed dams exhibited a dose-responsive increase in liver retinol binding protein levels. DE-71 treatment did not result in gross malformations; however, consistent with our hypothesis, GD 20 fetal weights were lower, and skeletal ossification was less when DE-71 exposure occurred concomitant with a marginal vitamin A status. For several endpoints, observable effects were evident at the lowest dose tested, consistent with a dose-response trend. CONCLUSIONS: The results of this study support the concept that marginal vitamin A status enhances the disruptive effects of DE-71 during prenatal development.

Impacts of suspended sediments on fertilization, embryonic development, and early larval life stages of the pacific herring, Clupea pallasi.

Pacific herring reproduce in the San Francisco Bay estuary during times of the year when suspended sediment loads are highest due to freshwater input, yet little is known about the effects of sediment on herring early life stages. During the first 2 h after eggs contacted water, embryos were adhesive and susceptible to having sediment particles attach permanently to the chorion. Treatment with suspended San Francisco Bay dredged sediments at ecologically relevant concentrations of 250 or 500 mg/l during this time period increased self-aggregation of the eggs and led to sublethal and lethal effects. After the first 2 h in water, sediments that contacted embryos did not attach to chorions and did not have an observable impact. Sediment treatment during the first 2 h was not linked statistically to declines in fertilization or total larval hatch rate, but it did produce significant sublethal effects that included increases in precocious larval hatch and higher percentages of abnormal larvae, as well as an increase in larval mortality.

Two egg-derived molecules in sperm motility initiation and fertilization in the Pacific herring (Clupea pallasi).

Sperm of the Pacific herring are immotile at spawning. Two egg-derived molecules are capable of initiating sperm motility. One is herring sperm activating protein(s) (HSAPs) and the other is sperm motility initiation factor (SMIF). These two motility initiators differ in their location and association with the chorion, and in their isoelectric points and molecular weights. In this study we have investigated the roles of these two inducers with respect to motility and fertilization. Using computer analysis of sperm motility, we found that HSAPs, as well as the C-terminal HSAPs peptide, elicit a linear motility pattern, while SMIF induced a highly circular and asymmetric pattern. HSAPs induced a two-fold increase in intracellular calcium, whereas SMIF induced a four-fold increase of motility initiation. SMIF-exposed sperm, preloaded with BAPTA-AM, showed a more linear motility and this motility trajectory decreased with their fertilizing capability. The difference in intracellular calcium levels between HSAPs and SMIF is consistent with the observed linear and circular motility. In the absence of SMIF, HSAPs do not support fertilization. Fertilization is rescued in these experiments if SMIF is reintroduced. We propose that diffusible HSAPs are not essential for fertilization, but enhance sperm-egg collisions via linear motility. SMIF, which is bound to the micropylar region of the chorion, is required for fertilization and induces circular motility that is a prerequisite for sperm to enter the micropylar canal and fertilize the egg.

Methods for toxicology studies in echinoderm embryos and larvae.

Sea urchin embryos have been used in toxicological studies for many decades as they are an accepted model system for investigations of chemicals that impact development. Here we describe methods for using pulse-chase experiments to study the impacts of environmental chemicals on early development as well as development of larvae. This includes the application of fluorescence plate assays with living embryos and fluorescent probes to assess cell functions (mitochondrial membrane potential, lysosome abundance, reactive oxygen species, and esterase activity) based on total cell numbers. We also describe how to use some of these fluorescent probes in embryos/larvae with confocal microscopy for the localization of cellular damage in response to toxics exposure. Finally, we assess skeleton formation in sea urchin larvae and present methods for using polarized light microscopy to examine spicule morphology.