John W. Lynn

Rapid visual detection of sperm-egg fusion using the DNA-specific fluorochrome Hoechst 33342.

When unfertilized sea urchin eggs are pretreated with the bisbenzimide DNA-specific fluorochrome Hoechst 33342, then washed and fertilized, a single sperm bound to the egg surface becomes intensely fluorescent. The location of the fluorescent sperm on the egg surface coincides exactly with the epicenter of the cortical reaction and the site at which the insemination cone subsequently appears. These observations, coupled with studies of eggs treated with quercetin to prevent fusion, as well as eggs made polyspermic by halothane exposure, indicate that the sperm acquires fluorescence as a consequence of fusion with the fluorochrome preloaded egg. Using a modification of this technique, we have found that cytoplasmic continuity between the sperm and egg is established at 4-8 sec after the onset of the sperm-induced conductance increase in the egg.

Correlative ultrastructural and electrophysiological studies of sperm-egg interactions of the sea urchin, Lytechinus variegatus☆

The sequence of ultrastructural events following the onset of the sperm-induced conductance increase in eggs of the sea urchin, Lytechinus variegatus, was investigated. Eggs voltage clamped at -20 mV were fixed 1 to 20 sec after onset of the conductance increase caused by single sperm. Continuity between the plasma membranes of the sperm and egg was first detected 5 sec after onset of the conductance increase. The earliest stages of formation of the fertilization cone coincided with the establishment of continuity of the gamete plasma membranes. At 6 to 8 sec after the initial conductance increase cortical granule dehiscence was first observed in the immediate vicinity where continuity of the gamete plasma membranes had occurred. These observations are consistent with the conclusion that opening of ion channels at fertilization precedes fusion of the sperm and egg plasma membranes, while exocytosis of cortical granules is initiated following fusion of the sperm and egg plasma membranes.

Filtration and Utilization of Laboratory-Cultured Bacteria by Dreissena polymorpha, Corbicula fluminea, and Carunculina texasensis

Dreissena polymorpha consumed about 6 x 108 Escherichia coli from 20 ml of artificial pondwater (APW) in 30 min under laboratory conditions. The clearance rate per mussel was 143 +/- 25 ml g-1 dry tissue min-1. The E. coli used in these studies ranged from about 1.7 to 2.9 {mu}m in length. 35S-labeled E. coli were used to demonstrate that bacteria-derived nutrients were incorporated into mussel tissue. Electrophoretic analysis of mussel and bacterial proteins on 12% polyacrylamide gels allowed the visual determination of incorporation of labeled amino acids into bivalve proteins and demonstrated that intact bacteria were not simply trapped in mussel tissues. The conversion of bacterial-labeled amino acids into mussel protein was about 26%. Similarly, we demonstrated that D. polymorpha can use other bacterial species ranging in size from about 1.3 to 4.1 μm, including Citrobacter freundii, Enterobacter aerogenes, Serratia marcescens, Bacillus megaterium, and B. subtilus. The ability of D. polymorpha to take up E. coli was compared with that of two other freshwater mussels, Corbicula fluminea and Carunculina texasensis. On a mussel-dry-weight basis, D. polymorpha cleared bacteria 30 to 100 times faster than Corbicula fluminea and Carunculina texasensis, respectively. The ability to filter E. coli appears to be related to the architecture of the cirri on the latero-frontal cells of the gill. Cirri from Corbicula and Dreissena are similar in size, but Dreissena has a larger gill compared to the tissue dry-weight, and has 102 times more cirri than found in Corbicula. Carunculina, the unionid representative, has smaller and fewer cirri, and has relatively limited ability to capture E. coli.

Osmoregulation in Dreissena polymorpha: the Importance of Na, Cl, K, and Particularly Mg

Zebra mussels (Dreissena polymorpha) are unusual in that they cannot survive in Mg-deficient water. Analysis of blood samples from mussels obtained in the field indicated a Mg concentration of 1.5-2.0 mM immediately after animal collection. However, Mg concentration in the blood decreased rapidly when the mussels were transferred to Mg-free artificial pondwater (PW); the t1/2 was 24 h. Blood Mg decreased to the limits of detection within 2 weeks, and the time to 50% mortality was about 17 days in Mg-free PW. When Mg-depleted specimens of D. polymorpha were returned to PW containing Mg, the net flux was 3 μmol Mg (g dry tissue.h)-1, and blood Mg concentration was restored within a day to 0.4-0.6 mM. Mussels depleted of Mg did not survive beyond 51 days. When mussels were acclimated to K-free pondwater (containing Mg), their osmoregulatory ability was impaired, and the total solute of the blood dropped from 30-36 to 21-24 mosm, with blood Na and Cl concentrations declining 30-50%. This ion-depleted condition was reversed within 45 h upon return of K to the pondwater bathing medium. D. polymorpha individuals were unable to survive beyond 5 days in deionized water and required minimal concentrations of Na, Cl, K, and Mg for prolonged storage (>51 days) under laboratory conditions. Mussels survived Ca-deficient solutions for more than 51 days, presumably because they were able to mobilize Ca from internal stores (shell) to maintain blood calcium at 1 mM.

Jelly Layer Formation in Penaeoidean Shrimp Eggs

Etude histologique et chronologique de la formation de la couche gelatineuse des œufs de crevettes Penaeoidea par microscopies optique et electronique a transmission

Effect of Epigenetic Modifications of Donor Somatic Cells on the Subsequent Chromatin Remodeling of Cloned Bovine Embryos

Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of differentiated cells as the donor karyoplast. Blastocyst production and development to term of cloned embryos has been hypothesized to differ between population doublings of the same cell line as a consequence of changes in the levels of DNA methyltransferase 1 (DNMT1) and methylated DNA during in vitro culture. The objective of this study was to determine embryo production, developmental potential, and gene expression patterns of prehatched and posthatched embryos generated using donor cells with different levels of DNMT1 transcript. Day 7 embryos generated using donor cells with high and low levels of DNMT1 mRNA were transferred to recipient cows. Embryos recovered on Day 13 were morphologically characterized or used for gene expression analysis of DNMT, INFT, and MHC1. A higher proportion of 8- to 16-cell embryos developed to the blastocyst stage when cells with low levels of DNMT1 mRNA were used as donor nuclei. Day 13 NT embryos generated using donor cells with decreased levels of DNMT1 mRNA and capable of developing beyond the 8- to 16-cell stage produced a larger number of apparently developing embryos, larger conceptuses, and a higher expression of DNMT3A transcript than NT embryos reconstructed using cells with high levels of DNMT1 mRNA. However, abnormal gene expression of DNMT, INFT, and MHC1 was noted in the majority of cloned embryos, indicating inefficient nuclear reprogramming and retarded embryo development. Furthermore, aberrant DNMT1 expression may partially contribute to the inefficient nuclear reprogramming observed in cloned embryos.

A MORPHOLOGICAL EXAMINATION OF SPERM-EGG INTERACTION IN THE FRESHWATER PRAWN, MACROBRACHIUM ROSENBERGII

Eggs, covered by a bilayered investment composed of a 0.5µm protein outer layer and a 2.5 µm mucopolysaccharide inner layer, are spawned through an externally held spermatophore following a females ovigerous molt and mating. Mature sperm resemble everted umbrellas and consist of a cupped base with a single spike projecting from the convex surface. These sperm (<5/egg) attach base-first with the spike oriented perpendicularly to the investment. Within 15 seconds, the spike of the sperm bends at the base and contacts the investment. The spike penetrates the investment, the base is inverted, and fertilization occurs within two minutes. The spike remains briefly as a central core in the fertilization cone. The meiotic division of the egg resumes at this stage. First karyokinesis is completed approximately 6 hours post fertilization, but cytokinesis is suppressed until following the second karyokinesis at 8 hours postfertilization. First and second cytokinesis are simultaneous, and at 9 h the egg cleaves to t...

Pathways of Food Uptake in Native (Unionidae) and Introduced (Corbiculidae and Dreissenidae) Freshwater Bivalves

Abstract Nineteen species of adult freshwater bivalves were able to use water currents generated from within the mantle cavity to move non-suspended algae into the shell through non-siphon areas such as the anterior shell valve edge and the mid-ventral point of the shell. This was in addition to, and not in replacement of, uptake of planktonic algae through the inhalant siphon. Algae obtained from both the non-inhalant siphon areas and the inhalant siphon were equally captured and transferred to the stomach. Pseudofecal release was also more complex than typically stated. Pseudofeces were released from the inhalant siphon (as expected), plus from the anterior shell margin, and at the mid-point along the ventral shell edge (non-dreissenids). Pseudofeces that settled near the ventral shell margin was accessible for future uptake back into the mantle cavity. There were no species or body size differences in ability to capture food from non-planktonic sources. The extent to which natural populations utilize benthic food resources remains to be determined. However, our study demonstrates that bivalve communities are very adaptable in accessing a variety of food resources from both suspended material in the water column, as well as organic material from the sediments.

THE FINE STRUCTURE OF THE MATURE SPERM OF THE FRESHWATER PRAWN, MACROBRACHIUM ROSENBERGII

Electroejaculated spermatophores from male Macrobrachium rosenbergii contain mature sperm which resemble everted umbrellas. The nonflagellated, nonmotile sperm consist of a 10 µm main body (base) and a single 12-15 µm spike extending from the convex surface of the base. The nucleus is flocculent (decondensed), contains basic proteins, is not delimited by a nuclear membrane, and is housed in the base. The cytoplasm of the convex base contains fifteen to twenty radial fibrils which anastomose to form the spike. Both the spike and the radial fibrils are cross-striated with a periodicity of 35 nm. These structures contain 6 nm filaments running perpendicularly to the cross-striations. A pair of centrioles composed of nine doublet tubules are located acentrically near the origin of the spike in the cytoplasm of the base. Radial fibrils are interconnected to the centriolar matrix by accessory fibrils. No clearly definable acrosomal region is identifiable morphologically or histochemically.

Mucociliary Transport in Living Tissue: The Two-Layer Model Confirmed in The Mussel Mytilus edulis L

The present study combined video confocal laser microscopy (1) and tissue reflectance and autofluorescence to visualize mucus position and mucociliary transport in excised living gill tissue from the blue mussel Mytilus edulis. Rafts of mucus and embedded particles were transported atop a periciliary space traversed by frontal cilia, which engaged the mucus layer and moved it during the effective stroke, disengaging and completing the cycle during the recovery stroke. These results confirm the two-layer model for mucociliary transport in the mussel gill. Given the conservative nature of ciliated epithelial structure and function (2, 3), and the structural similarity of mucociliary surfaces as diverse as terrestrial vertebrate respiratory epithelium and molluscan gill, the two-layer mechanism of mucociliary transport may be a general feature of Metazoan biology.