Frederick J. Griffin

Motility initiation in herring sperm is regulated by reverse sodium-calcium exchange

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 sodium–calcium (Na+/Ca2+) exchange. Membrane depolarization was slightly inhibited by a calcium channel blocker and markedly inhibited by a Na+/Ca2+ exchange inhibitor. Sodium efflux caused by SMIF-initiated motility was observed when using both extracellular and intracellular sodium probes. A Na+/Ca2+ 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+/Ca2+ exchanger. This antibody recognized a 120-kDa protein that comigrated with the canine myocyte Na+/Ca2+ exchanger. Sperm of Pacific herring are now shown to use reverse Na+/Ca2+ 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.

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.

Sperm Attractant in the Micropyle Region of Fish and Insect Eggs

ABSTRACT 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 Ca2+. 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.

Effects of Salinity on Sperm Motility, Fertilization, and Development in the Pacific Herring, Clupea pallasi

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.

The effects of diffusible creosote-derived compounds on development in Pacific herring (Clupea pallasi).

The effects of diffusible creosote-derived compounds from weathered creosote-treated pilings on embryonic development in the Pacific herring were investigated. Parameters used to evaluate toxicity included embryonic development, cardiac function, embryo/larval activity (movement of developing embryos), hatching success, and larval morphology at hatch. For acute exposures, embryos were incubated in seawater containing either creosote-treated wood (creosote) or untreated wood (wood control), or seawater alone (control). All embryos adhering directly to creosote-treated wood and 40-50% of embryos not adhering to the creosote-treated wood failed to develop beyond the first few days of incubation. For surviving embryos, a 93% reduction in heart rate, and moderate to marked arrhythmia was observed. Surviving embryos also exhibited both an increase in frequency and an alteration in pattern of embryo/larval movement, with most embryos exhibiting tremors as compared with the vigorous movements of the control embryos. Cardiac function and embryo/larval movements of embryos exposed to untreated wood were not significantly different from controls. The hatching rate of embryos exposed to creosote was 90% lower than control embryos and 72.4% lower than embryos exposed to untreated wood, and the LC(50) for hatching success was 0.05 mg/l. Partial hatching (incomplete hatch) was observed in 15-20% of embryos exposed to creosote. All of the hatched larvae exposed as embryos to creosote exhibited morphological deformities, including scoliosis, pericardial edema and/or ascites. Similar effects were observed in embryos collected from creosoted pilings in San Francisco Bay, with a 72% decrease in hatching success compared with embryos collected from the Bay and severely deformed larvae. To investigate the combined effects of creosote and salinity on hatching success, larval morphology, and cardiac function, embryos were exposed to a sublethal concentration of creosote (0.003 mg/l) at three salinities; sub-optimal (8 parts per thousand (ppt)), optimal (16 ppt), and high salinity (28 ppt). The presence of creosote decreased hatching success at all three salinities, but the effect was greatest at 8 ppt (34% reduction) and the least in 28 ppt (14% reduction). The increased incidence of morphological abnormalities was also smallest at the high salinity (10% compared with 24 and 33% in 8 and 16 ppt). While exposure to creosote resulted in reduced heart rates at all three salinities, no additive effect of creosote and salinity was observed.

The Morphology and Physiology of the Acrosome Reaction in the Sperm of the Decapod, Sicyonia ingentis.

Sperm of the shrimp, Sicyonia ingentis, undergo a biphasic acrosome reaction consisting of acrosomal exocytosis and acrosomal filament formation. These events are temporally separated by 10–20 min, in vivo. Using egg water preparations the complete reaction can be induced, in vitro, albeit the temporal separation of the two phases is lengthened. External Ca++ is required for the exocytotic phase, while a cytoplasmic acidification and K+ efflux are associated with polymerization of the acrosomal filament.

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.

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.