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.

Electrophoretic separation, characterization, and quantification of biologically active lignin-derived macromolecules.

Degraded macromolecular lignin, which was isolated from the effluents of commercial pulp processing and known to inhibit early development in marine organisms, was separated and characterized using several polyacrylamide gel electrophoresis (PAGE) techniques. This lignin-derived macromolecule (LDM), when subjected to native PAGE and stained with alcian blue, appeared as a single band. On sodium dodecyl sulfate (SDS)-PAGE, LDM appeared to consist of two subcomponents with apparent molecular weights of 11 and < 1 kDa. When subjected to isoelectrofocusing--PAGE of pH 3-9, LDM consisted of two major bands in the basic region of the gel, with less distinct banding in the more acidic region. Two-dimensional PAGE of LDM indicated that the higher molecular weight subcomponent corresponded to the more basic constituents, while the lower molecular weight subcomponent corresponded to acidic constituents. When the two subcomponents of LDM were isolated from SDS gels by electroelution and assessed for their effects on successful fertilization and early development, the higher molecular weight subcomponent possessed most of the inhibitory activity. This is the first report of the application of a variety of electrophoretic techniques to both structurally and biologically characterize lignin-derived macromolecules.

Trypsin inhibitors prevent the progesterone-initiated increase in intracellular calcium required for the human sperm acrosome reaction.

Inhibitors of trypsin-like enzymes, benzamidine hydrochloride and 4'-acetamidophenyl 4-guanidinobenzoate (also an inhibitor of other serine proteases), were tested for their effects on the acrosome reaction (AR) of human sperm initiated by progesterone or the calcium ionophore ionomycin. The AR was assayed by indirect immunofluorescence and transmission electron microscopy. The trypsin inhibitors, when added 10 min prior to stimulation by progesterone, significantly inhibited the AR in comparison with progesterone treatment alone. Transmission electron microscopic examination of the sperm after progesterone treatment indicated that the inhibitors blocked the membrane fusion events of the AR. By contrast, when ionomycin (at final concentrations of 3 microM) was added to sperm preincubated in inhibitors, sperm underwent morphologically normal AR, acrosomal matrix loss was not inhibited, and the percentage of acrosome-reacted sperm was the same as that obtained in the absence of inhibitors. Using the cell calcium indicator fura-2, we further demonstrated that both trypsin inhibitors prevented the progesterone-stimulated rise in intracellular Ca2+ ([Ca2+]int) required for the AR, but did not affect [Ca2+]int in unstimulated sperm. These results suggest that sperm trypsin-like activity may be directly or indirectly involved in increasing sperm [Ca2+]int during stimulation by progesterone.

Development of cortical vesicles in Sicyonia ingentis ova: their heterogeneity and role in elaboration of the hatching envelope.

In the marine shrimp Sicyonia ingentis, ova lack cortical vesicles at spawning. Previous ultrastructural studies suggested that two different populations of cortical vesicles (dense vesicles and the ring vesicles) appear within 30 min post-spawning. These vesicles undergo sequential exocytosis (exocytosis of the dense vesicles followed by exocytosis of the ring vesicles) that leads to the formation of a hatching envelope around the ovum (see Pillai and Clark: Tissue & Cell 20:941-52, 1988). In the present study, lectins were used as molecular probes to study the development of cortical vesicles subsequent to spawning and the role of these vesicles in formation and elaboration of the hatching envelope. Isolated envelopes were screened with 11 different lectins to determine what group(s) were specific to the envelope glycoconjugates; Concanavalin A (Con A), Griffonia simplicifolia (GS II), Lens culinaris (LCA), and wheat germ agglutinin (WGA) bound to the envelopes. FITC-lectin studies of sectioned ova (fixed at various time points after spawning) utilizing WGA and LCA showed different labelling patterns. Data obtained at the light microscopical level indicated that WGA was specific to the dense vesicles and the outer portion of the envelope, while LCA exhibited specificity for the ring vesicles and the inner portion of the envelope. At the ultrastructural level, gold-LCA labelling was seen associated with the cisternal elements (containing ring-shaped structures), ring vesicles, and the inner layer of the fully formed envelope. These data demonstrated that 1) the ring vesicles are formed by fusion of cisternal elements containing ring-shaped structures; 2) the two species of cortical vesicles are chemically heterogeneous; and 3) the components of each type of vesicle contribute to different integral parts (the outer and inner layers) of the hatching envelope.

Hatching envelope formation in shrimp (Sicyonia ingentis) ova: Origin and sequential exocytosis of cortical vesicles.

The ova of Sicyonia ingentis lack cortical vesicles at the time of spawning. Within 30 min post-spawning, two populations of cortical vesicles are organized in the ooplasm which, during cortical vesicle exocytosis (cortical reaction), successively release two morphologically different exudates. The first type of cortical vesicles (dense vesicles) appears to be derived from the Golgi complexes after spawning. The second type (the ring vesicles) is formed by the fusion of asternal elements which contain loosely packed ring-shaped structures that are present in the unactivated ova. During exocytosis of the dense vesicles an electron dense material is released which coalesces with the surface coat of the ovum to form a thin hatching envelope which eventually lifts from the ovum's surface. Subsequent to the formation of the thin hatching envelope, the ring vesicles undergo exocytosis resulting in an accumulation of ring-shaped structures in the perivitelline space. These structures coalesce and form an electron translucent layer on the inner surface of the thin elevated envelope to form the thickened hatching envelope. The formation of the cortical vesicles, their exocytosis and the elaboration of the hatching envelope are normally completed within 40-45 min after spawning.