In some animals, such as fish, insects, and cephalopods, the thick egg coat has a narrow canal-a micropyle-through which spermatozoa enter the eggs. In fish, there is no indication that spermatozoa are attracted by eggs from a distance, but once spermatozoa come near the outer opening of the micropyle, they exhibit directed movement toward it, suggesting that a substance exists in this defined region to attract spermatozoa. Since Coomassie Blue (CB) binds preferentially to the micropyle region in flounder, herring, steelhead, and other fish, it probably stains this sperm guidance substance. This substance-a glycoprotein based on lectin staining-is bound tightly to the surface of the chorion, but can be removed readily by protease treatment. Although fertilization in fish (flounder) is possible after removal of this substance, its absence makes fertilization inefficient, as reflected by a drastic reduction in fertilization rate. The sperm "attraction" to the micropyle opening is species specific and is dependent on extracellular Ca(2+). Eggs of some insects, including Drosophila, have distinct micropyle caps with CB affinity, which also may prove to assist sperm entry. Our attempts to fertilize fly eggs in vitro were not successful.

Sperm of the Pacific herring, Clupea pallasi, are unique in that they are immotile upon spawning in the environment. Herring sperm have evolved to remain motionless for up to several days after spawning, yet are still capable of fertilizing eggs. An egg chorion ligand termed ''sperm motility initiation factor'' (SMIF) induces motility in herring sperm and is required for fertilization. In this study, we show that SMIF induces calcium influx, sodium efflux, and a membrane depolarization in herring sperm. Sperm motility initiation by SMIF depended on decreased extracellular sodium (<350 mM) and could be induced in the absence of SMIF in very low sodium seawater. Motility initiation depended on > 1 mM extracellular calcium. Calcium influx caused by SMIF involved both the opening of voltage-gated calcium channels and reverse sodiumcalcium (Na ؉ ͞Ca 2؉ ) exchange. Membrane depolarization was slightly inhibited by a calcium channel blocker and markedly inhibited by a Na ؉ ͞Ca 2؉ exchange inhibitor. Sodium efflux caused by SMIF-initiated motility was observed when using both extracellular and intracellular sodium probes. A Na ؉ ͞Ca 2؉ exchange antigen was shown to be present on the surface of the sperm, primarily over the midpiece, by using an antibody to the canine Na ؉ ͞Ca 2؉ exchanger. This antibody recognized a 120-kDa protein that comigrated with the canine myocyte Na ؉ ͞Ca 2؉ exchanger. Sperm of Pacific herring are now shown to use reverse Na ؉ ͞Ca 2؉ exchange in motility initiation. This mechanism of regulation of motility initiation may have evolved for both maintenance of immotility after spawning as well as ligand-induced motility initiation.T eleost fish sperm are quiescent within the testes and seminal plasma before spawning, but most initiate motility after dilution into the external medium (freshwater or seawater for most species) in which spawning occurs (1). In salmonids, motility initiation occurs with dilution in freshwater, specifically from a reduction in extracellular potassium that drives a membrane hyperpolarization and an increase in intracellular calcium ([Ca 2ϩ ] i ) (2-6). Changes in the concentrations of specific ions (Ca 2ϩ , K ϩ , and possibly Na ϩ and Cl Ϫ ) also have been linked to motility initiation in Atlantic croaker sperm (7). A hyperpolarization of the sperm membrane also has been documented in carp sperm, reportedly linked to the opening of voltage-gated Ca 2ϩ channels and an increase in [Ca 2ϩ ] i (8). In other freshwater teleost sperm (goldfish, zebra fish), as well as in marine teleost sperm (puffer, flounder), motility is believed to occur as a result of nonspecific hypo-or hyperosmotic changes that drive changes in intracellular ion concentrations (5, 9, 10). Thus, in all systems studied to date, a membrane hyperpolarization leads to an increase in [Ca 2ϩ ] i and the initiation of motility.Although the stimulation of sperm motility in the vicinity of eggs has been reported in some teleost fish, only herring sperm have been shown to require an egg chorion-derived ligand for ...

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.

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 thier 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 Cterminal 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.

The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilisation. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca2+ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca2+ indicator Fluo-3 showed that addition of 100 μg/ml of HA induced a rapid increase in intracellular Ca2+. This Ca2+ increase (approximately 2–3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca2+ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca2+ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca2+, while sperm in gel containing anti-PH-20 IgG showed increased Ca2+ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomologus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca2+ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca2+ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA2+ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.

We investigated the effects of salinity on fertilization and early development in a population of Pacific herring, Clupea pallasi, that migrate from oceanic waters into the San Francisco Bay estuary to spawn. The salinity range for fertilization fell between 8 and 28 ppt, with an optimal range of about 12 to 24 ppt. In comparison, the range for a population of C. harengus membras (Airisto Sound, Finland) that reside year-round in the Baltic Sea was 4 to 24 ppt. Roles for both Na+ and K+ were indicated in C. pallasi fertilization since increasing Na+ in the presence of 10 mM K+ (concentration of seawater) mimicked the effects of increased overall salinity, whereas reduced effects were obtained if [K+] was held at 5 mM (that of half-strength seawater). The initiation of C. pallasi sperm motility by components of the egg chorion, a prerequisite for fertilization, was inhibited at both elevated (28 and 32 ppt) and reduced (4 and 8 ppt) salinities. Embryonic development through larval hatching in C. pallasi exhibited a salinity tolerance similar to that of fertilization; optimum development was obtained at salinities between 8 and 24 ppt. A comparison of developmental progression in 3.5, 14, and 28 ppt seawater revealed that salinity effects became evident during the post-gastrulation stages of development and that progression to hatching was delayed in both the lower and higher salinities for those embryos that completed development.

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.

Pacific herring embryos (Clupea pallasi) spawned three months following the Cosco Busan bunker oil spill in San Francisco Bay showed high rates of late embryonic mortality in the intertidal zone at oiled sites. Dead embryos developed to the hatching stage (e.g. fully pigmented eyes) before suffering extensive tissue deterioration. In contrast, embryos incubated subtidally at oiled sites showed evidence of sublethal oil exposure (petroleum-induced cardiac toxicity) with very low rates of mortality. These field findings suggested an enhancement of oil toxicity through an interaction between oil and another environmental stressor in the intertidal zone, such as higher levels of sunlight-derived ultraviolet (UV) radiation. We tested this hypothesis by exposing herring embryos to both trace levels of weathered Cosco Busan bunker oil and sunlight, with and without protection from UV radiation. Cosco Busan oil and UV co-exposure were both necessary and sufficient to induce an acutely lethal necrotic syndrome in hatching stage embryos that closely mimicked the condition of dead embryos sampled from oiled sites. Tissue levels of known phototoxic polycyclic aromatic compounds were too low to explain the observed degree of phototoxicity, indicating the presence of other unidentified or unmeasured phototoxic compounds derived from bunker oil. These findings provide a parsimonious explanation for the unexpectedly high losses of intertidal herring spawn following the Cosco Busan spill. The chemical composition and associated toxicity of bunker oils should be more thoroughly evaluated to better understand and anticipate the ecological impacts of vessel-derived spills associated with an expanding global transportation network.