Isolation and partial characterization of the plasma membrane of the sea urchin egg

The cell surface complex (Detering et al., 1977, J. Cell Biol. 75, 899-914) of the sea urchin egg consists of two subcellular organelles: the plasma membrane, containing associated peripheral proteins and the vitelline layer, and the cortical vesicles. We have now developed a method of isolating the plasma membrane from this complex and have undertaken its biochemical characterization. Enzymatic assays of the cell surface complex revealed the presence of a plasma membrane marker enzyme, ouabain-sensitive Na+/K+ ATPase, as well as two cortical granule markers, proteoesterase and ovoperoxidase. After separation from the cortical vesicles and purification on a sucrose gradient, the purified plasma membranes are recovered as large sheets devoid of cortical vesicles. The purified plasma membranes are highly enriched in the Na+/K+ ATPase but contain only very low levels of the proteoesterase and ovoperoxidase. Ultrastructurally, the purified plasma membrane is characterized as large sheets containing a "fluffy" proteinaceous layer on the external surface, which probably represent peripheral proteins, including remnants of the vitelline layer. Extraction of these membranes with Kl removes these peripheral proteins and causes the membrane sheets to vesiculate. Polyacrylamide gel electrophoresis of the cell surface complex, plasma membranes, and Kl-extracted membranes indicates that the plasma membrane contains five to six major proteins species, as well as a large number of minor species, that are not extractable with Kl. The vitelline layer and other peripheral membrane components account for a large proportion of the membrane-associated protein and are represented by at least six to seven polypeptide components. The phospholipid composition of the Kl-extracted membranes is unique, being very rich in phosphatidylethanolamine and phosphatidylinositol. Cholesterol was found to be a major component of the plasma membrane. Before Kl extraction, the purified plasma membranes retain the same species-specific sperm binding property that is found in the intact egg. This observation indicates that the sperm receptor mechanisms remain functional in the isolated, cortical vesicle-free membrane preparation.     

The effect of the acrosome reaction on the respiratory activity and fertilizing capacity of echinoid sperm.

We have examined the relationship between the acrosome reaction, sperm respiration, and fertilization using gametes of the sea urchin Strongylocentrotus purpuratus. The results indicate that when sperm are exposed to jelly coat isolated from homologous eggs, the following sequence of events occurs: (1) Sperm undergo the acrosome reaction within 30 sec with little or no loss in their capacity to fertilize eggs; (2) by 60 sec there is a dramatic decrease in fertilizing capacity which stabilizes after 4 or 5 min at a greatly reduced level; (3) by 1.5 to 2 min a progressive decrease in the rate of mitochondrial respiration becomes detectable and continues for 8 to 10 min, finally stabilizing at a greatly reduced rate. This decrease in respiration rate is paralleled by a decline in sperm motility. The effects of jelly coat on the acrosome reaction, sperm respiration, and motility are species specific. From these results we conclude that sperm which have undergone the acrosome reaction retain full fertilizing capacity for a very short time. The rapid decline in fertilizing capacity is followed by a decrease in respiration rate and motility.     

Social behaviour in confined populations of the Allegheny woodrat, Neotoma floridana magister

Low-density groups of Allegheny woodrats (Neotoma floridana magister) confined in a 65-m² cage exhibited despotic social organization in which one alpha rat killed or wounded all other group members within 14 days in each of five experiments. Five of seven high-density groups, consisting of six to fourteen rats in 9·5-m² cages, were characterized by dominance hierarchies and high levels of social interaction. Two unisexual groups remained disorganized and all but one rat died in both cases. Allegheny woodrats utilized a different alarm signal and exhibited higher levels of agonistic and sexual interaction compared to similar groups of a california subspecies (N. fuscipes macrotus) which occupies a distinctly different habitat.     

Intercellular communication in the rat anterior pituitary gland: an in vivo and in vitro study

The concept of "stimulus-secretion coupling" suggested by Douglas and co-workers to explain the events related to monamine discharge by the adrenal medulla (5, 7) may be applied to other endocrine tissues, such as adrenal cortex (36), pancreatic islets (4), and magnocellular hypothalamic neurons (6), which exhibit a similar ion-dependent process of hormone elaboration. In addition, they share another feature, that of joining neighbor cells via membrane junctions (12, 26, and Fletcher, unpublished observation). Given this, and the reports that hormone secretion by the pars distalis also involves a secretagogue-induced decrease in membrane bioelectric potential accompanied by a rise in cellular [Ca++] (27, 34, 41), it was appropriate to test the possibility that cells of the anterior pituitary gland are united by junctions.     

Mite load predicts the quality of sexual color and locomotor performance in a sexually dichromatic lizard

Since Darwin, the maintenance of bright sexual colors has recurrently been linked to mate preference. However, the mechanisms underpinning such preferences for bright colors would not be resolved for another century. Likely, the idea of selection for colors that could decrease the chances of survival (e.g., flashy colors that can inadvertently attract predators) was perceived as counterintuitive. It is now widely accepted that these extreme colors often communicate to mates the ability to survive despite a “handicap” and act as honest signals of individual quality when they are correlated with the quality of other traits that are directly linked to individual fitness. Sexual colors in males are frequently perceived as indicators of infection resistance, in particular. Still, there remains considerable discord among studies attempting to parse the relationships between the variables associating sexual color and infection resistance, such as habitat type and body size. This discord may arise from complex interactions between these variables. Here, we ask if sexual color in male Florida scrub lizards (Sceloporus woodi) is an honest signal of resistance to chigger mite infection. To this end, we use linear modeling to explore relationships between mite load, different components of sexual color, ecological performance, body size, and habitat type. Our data show that that the brightness of sexual color in scrub lizards is negatively associated with the interaction between mite load and body size, and scrub lizards suffer decreased endurance capacity with increases in mite load. Our data also indicate that mite load, performance, and sexual color in male scrub lizards can vary between habitat types. Collectively, these results suggest that sexual color in scrub lizards is an honest indicator of individual quality and further underscore the importance of considering multiple factors when testing hypotheses related to the maintenance of sexual color.     

Classification and nomenclature of all human homeobox genes

Background

The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.

Results

We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.

Conclusion

We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass.     

Fertilization of sea urchin eggs and sperm motility are negatively impacted under low hypergravitational forces significant to space flight

Sperm and other flagellates swim faster in microgravity (microG) than in 1 G, raising the question of whether fertilization is altered under conditions of space travel. Such alterations have implications for reproduction of plant and animal food and for long-term space habitation by man. We previously demonstrated that microG accelerates protein phosphorylation during initiation of sperm motility but delays the sperm response to the egg chemotactic factor, speract. Thus sperm are sensitive to changes in gravitational force. New experiments using the NiZeMi centrifugal microscope examined whether low hypergravity (hyperG) causes effects opposite to microG on sperm motility, signal transduction, and fertilization. Sperm % motility and straight-line velocity were significantly inhibited by as little as 1.3 G. The phosphorylation states of FP130, an axonemal phosphoprotein, and FP160, a cAMP-dependent salt-extractable flagellar protein, both coupled to motility activation, showed a more rapid decline in hyperG. Most critically, hyperG caused an approximately 50% reduction in both the rate of sperm-egg binding and fertilization. The similar extent of inhibition of both fertilization parameters in hyperG suggests that the primary effect is on sperm rather than eggs. These results not only support our earlier microG data demonstrating that sperm are sensitive to small changes in gravitational forces but more importantly now show that this sensitivity affects the ability of sperm to fertilize eggs. Thus, more detailed studies on the impact of space flight on development should include studies of sperm function and fertilization.     

Response in nematocyst uptake by the nudibranch Flabellina verrucosa to the presence of various predators in the Southern Gulf of Maine

Aeolid nudibranchs maintain nematocysts sequestered from their cnidarian prey for protection against predators. Selection for nematocyst incorporation is a function of diet and prey choice, but ratios vary among nudibranchs feeding on a given diet, indicating that other factors may be involved. It is proposed that the presence of predators influences nematocyst incorporation. Nematocyst uptake in the nudibranch Flabellina verrucosa collected from the southern Gulf of Maine was examined in response to various potential predators, including Crossaster papposus, Tautogolabrus adspersus, and Carcinus maenas. Nudibranchs in individual flow-through containers feeding on a diet of the hydroids Tubularia spp. and Obelia geniculata were subjected to tanks containing a predator, then their nematocyst distribution was examined. Although most of the changes over the experimental period were attributable to diet, F. verrucosa responded to both T. adspersus and C. papposus by significantly increasing microbasic mastigophore incorporation. No differential uptake was seen with C. maenas. Response was evident in the nudibranchs both for predators present in the collection area and for those with which they had no previous exposure, indicating that F. verrucosa modulates nematocyst incorporation in response to the presence of predators as well as to diet. A coevolution of nudibranchs and potential predators may govern changes in nematocyst uptake.     

Behavioral evidence for a role of alpha-gustducin in glutamate taste

The taste perception of monosodium glutamate (MSG) is termed 'umami'. Two putative taste receptors for glutamate have been identified, a truncated form of mGluR4 (taste-mGluR4) and the presumed heterodimer T1R1 + T1R3. Both receptors respond to glutamate when expressed in heterologous cells, but the G protein involved is not known. Galpha-Gustducin mediates the transduction of several bitter and sweet compounds; however, its role in umami has not been determined. We used standard two-bottle preference tests on alpha-gustducin knockout (KO) and wildtype (WT) mice to compare preferences for ascending concentrations of MSG and MSG + 5'-inosine monophosphate (IMP). A Latin Square was used to assign the order of tastants presented to each mouse. Statistical comparisons between KO and WT mice revealed that whereas WT mice preferred solutions of MSG and MSG + IMP over water, KO mice showed little preference for these stimuli. Denatonium and sucrose served as control stimuli and, as shown previously, WT mice prefered sucrose and avoided denatonium significantly more than did KO mice. Na?ve mice were also tested, and while prior exposure to taste stimuli influenced the magnitude of the preferences, experience did not change the overall pattern of intake. These data suggest that alpha-gustducin plays a role in glutamate taste.