In vitro decondensation of the sperm chromatin in Holothuria tubulosa (sea cucumber) not affecting proteolysis of basic nuclear proteins

Sea urchin and sea star oocyte extracts contain proteolytic activities that are active against sperm basic nuclear proteins (SNBP). This SNBP degradation has been related to the decondensation of sperm chromatin as a possible model to male pronuclei formation. We have studied the presence of this proteolytic activity in Holothuria tubulosa (sea cucumber) and its possible relationship with sperm nuclei decondensation. The mature oocyte extracts from H. tubulosa contain a proteolytic activity to SNBP located in the macromolecular fraction of the egg‐jelly layer. SNBP degradation occurred both on sperm nuclei and on purified SNBP, histones being more easily degraded than protein Ø_o (sperm‐specific protein). SNBP degradation was found to be dependent on concentration, incubation time, presence of Ca²⁺, pH, and this activity could be a serine‐proteinase. Thermal denaturalization of the oocyte extracts (80°C, 10–15 min) inactivates its proteolytic activity on SNBP but does not affect sperm nuclei decondensation. These results would suggest that sperm nuclei decondensation occurs by a mechanism different from SNBP degradation. Thus, the sperm nuclei decondensation occurs by a thermostable factor(s) and the removal of linker SNBP (H1 and protein Ø_o) will be a first condition in the process of sperm chromatin remodeling.     

Cloning and characterization of a phospholipase C-beta isoform from the sea urchin Lytechinus pictus

Calcium is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes including fertilization and egg activation. The mechanism for triggering intracellular Ca(2+) release in sea urchin eggs during fertilization is the generation of inositol-1,4,5-trisphosphate by phospholipase C (PLC) hydrolysis of phosphatidylinositol-4,5-bisphosphate. Of the five PLC isoforms identified in mammals (beta, gamma, delta, epsilon and zeta), only PLCgamma and PLCdelta have been detected in echinoderms. Here, we provide direct evidence of the presence of a PLCbeta isoform, named suPLCbeta, within sea urchin eggs. The coding sequence was cloned from eggs of Lytechinus pictus and determined to have the greatest degree of homology and identity with the mammalian PLCbeta4. The presence of suPLCbeta within the egg was verified using a specifically generated antibody. The majority of the enzyme is localized in the non-soluble fraction, presumably the plasma membrane of the unfertilized egg. This distribution remains unchanged 1 min postfertilization. Unlike PLCbeta4, suPLCbeta is activated by G protein betagamma subunits, and this activity is Ca(2+)-dependent. In contrast to all known PLCbeta enzymes, suPLCbeta is not activated by Galphaq-GTPgammaS subunit suggesting other protein regulators may be present in sea urchin eggs.     

Conspecific sperm precedence in two species of tropical sea urchins

Conspecific sperm precedence occurs when females are exposed to sperm from males of multiple species, but preferentially use sperm of a conspecific. Conspecific sperm precedence and its mechanisms have been documented widely in terrestrial species, in which complex female behaviors or reproductive tract morphologies can allow many opportunities for female choice and sperm competition, however, the opportunity for conspecific sperm precedence in free spawning marine invertebrates has been largely ignored. Two sea urchin species, Echinometra oblonga and E. sp. C, have high levels of interspecific fertilization in no-choice lab crosses, but no natural hybrids have been found. We performed competitive fertilization assays to test for conspecific sperm precedence and found that eggs of both species showed a marked preference for conspecific sperm when fertilized with heterospecific sperm mixtures. Strong rejection of heterospecific sperm would not have been predicted from no-choice assays and helps explain the lack of natural hybrids. We also found significant variation in hybridization success among crosses. Conspecific sperm precedence in free spawning invertebrates shows that the simple surfaces of eggs and sperm provide ample opportunity for egg choice and sperm competition even in the absence of intricate behavior or complex reproductive morphologies.     

Carbon limitation leads to suppression of first year oak seedlings beneath evergreen understory shrubs in Southern Appalachian hardwood forests

Inhibition of canopy tree recruitment beneath thickets of the evergreen shrubs Rhododendron maximum L. and Kalmia latifolia L. has long been observed in Southern Appalachian forests, yet the mechanisms of this process remain unresolved. We present a first-year account of suppression of oak seedlings in relation to Rhododendron and Kalmia basal area, light and resource availability, seedling performance and the rates of seedling damage (i.e., herbivory). We found no evidence of first-year seedling suppression or significant resource deficiencies beneath thickets of K. latifolia in mature mixed hardwood stands. Suppression beneath R. maximum was apparent during the first growing season. We found that seedling biomass, light availability prior to canopy closure, and seedling tissue C:N ratios were negatively correlated with R. maximum basal area. Basal area of R. maximum was positively correlated with seedling mortality rates, soil [Al], and early-growing season leaf herbivory rates. Seedling growth was positively correlated with light and tissue C:N, while negatively correlated with soil [Al]. Overall, our results support the inhibition model of shade-mediated carbon limitation beneath dense understory shrubs and indicate the potential importance of herbivory and aluminum toxicity as components of a suppression mechanism beneath R. maximum thickets. We present a causal model of first year inhibition beneath R. maximum in the context of our findings and the results of prior studies.     

Gastrulation in the sea urchin embryo: a model system for analyzing the morphogenesis of a monolayered epithelium

Processes of gastrulation in the sea urchin embryo have been intensively studied to reveal the mechanisms involved in the invagination of a monolayered epithelium. It is widely accepted that the invagination proceeds in two steps (primary and secondary invagination) until the archenteron reaches the apical plate, and that the constituent cells of the resulting archenteron are exclusively derived from the veg2 tier of blastomeres formed at the 60-cell stage. However, recent studies have shown that the recruitment of the archenteron cells lasts as late as the late prism stage, and some descendants of veg1 blastomeres are also recruited into the archenteron. In this review, we first illustrate the current outline of sea urchin gastrulation. Second, several factors, such as cytoskeletons, cell contact and extracellular matrix, will be discussed in relation to the cellular and mechanical basis of gastrulation. Third, differences in the manner of gastrulation among sea urchin species will be described; in some species, the archenteron does not elongate stepwise but continuously. In those embryos, bottle cells are scarcely observed, and the archenteron cells are not rearranged during invagination unlike in typical sea urchins. Attention will be also paid to some other factors, such as the turgor pressure of blastocoele and the force generated by blastocoele wall. These factors, in spite of their significance, have been neglected in the analysis of sea urchin gastrulation. Lastly, we will discuss how behavior of pigment cells defines the manner of gastrulation, because pigment cells recently turned out to be the bottle cells that trigger the initial inward bending of the vegetal plate.     

Pigment cells trigger the onset of gastrulation in tropical sea urchin Echinometra mathaei

In the tropical sea urchin Echinometra mathaei, pigment cells are just detectable before the onset of gastrulation, owing to an early accumulation of red pigment granules. Taking advantage of this feature, behavior of pigment cells was studied in relation to the processes of gastrulation. Before the initiation of primary invagination, pigment cells were arranged in a hemi-circle in the dorsal half of the vegetal plate. Inward bending of the vegetal plate first occurred at the position occupied by pigment cells, while the bending was not conspicuous in the ventral half of the blastopore. Rhodamine-phalloidin staining showed that actin filaments were abundant at the apical corticies of pigment cells. It was also found that the onset of gastrulation was considerably delayed in the NiCl2-treated embryos, in which pigment cells were drastically reduced in number. It is notable that the NiCl2-treated embryos began to gastrulate on schedule if they contained a number of pigment cells in spite of treatment. This shows that pigment cells are the bottle cells that trigger the onset of gastrulation. In the embryos devoid of pigment cells, a short stub-like gut rudiment formed in a delayed fashion, and several secondary mesenchyme cells (SMC) appeared at the tip of the rudiment and elongated gradually until its tip reached the apical plate. This observation suggests that the SMC that pull the gut rudiment upward are not pigment cells but blastocoelar cells, because pigment cells change their fate to blastocoelar cells upon NiCl2-treatment.     

Nutrient uptake as a contributing explanation for deep rooting in arid and semi-arid ecosystems

Explanations for the occurrence of deep-rooted plants in arid and semi-arid ecosystems have traditionally emphasized the uptake of relatively deep soil water. However, recent hydrologic data from arid systems show that soil water potentials at depth fluctuate little over long time periods, suggesting this water may be rarely utilized or replenished. In this study, we examine the distributions of root biomass, soil moisture and nutrient contents to 10-m depths at five semi-arid and arid sites across southwestern USA. We couple these depth distributions with strontium (Sr) isotope data that show deep (>1 m) nutrient uptake is prevalent at four of the five sites. At all of the sites, the highest abundance of one or more of the measured nutrients occurred deep within the soil profile, particularly for P, Ca²⁺ and Mg²⁺. Phosphate contents were greater at depth than in the top meter of soil at three of five sites. At Jornada, for example, the 2–3 m depth increment had twice the extractable P as the top meter of soil, despite the highest concentrations of P occurring at the surface. The prevalence of such deep resource pools, and our evidence for cation uptake from them, suggest nutrient uptake as a complementary explanation for the occurrence of deep-rooted plants in arid and semi-arid systems. We propose that hydraulic redistribution of shallow surface water to deep soil layers by roots may be the mechanism through which deep soil nutrients are mobilized and taken up by plants.Electronic Supplementary Material Supplementary material is available for this article at