Ultrastructural study of oocyte maturation in the polychaete annelid Sabellaria alveolata

Summary

Maturation begins by a cortical reaction, which resembles that of the sea urchin egg, but can precede fertilization. Complete vitelline membrane elevation necessitates the dissolution of the cortical granule matrix (which can be prevented by concanavalin A) and the retraction of the microvilli at the egg surface (which is inhibited by acid pH). Later on, an aster, with centrioles, develops near the nuclear envelope, which becomes undulated before disruption. In contrast to all other species so far studied, nuclear pores do not disappear and can even be observed several minutes later, in remmants of the nuclear envelope. The meiotic spindle has typical centrioles and, at metaphase I, chromosomes are surrounded by endoplasmic reticulum.     

Larval neurogenesis in Sabellaria alveolata reveals plasticity in polychaete neural patterning

The investigation of neurogenesis in polychaetes not only facilitates insights into the developmental biology of this group, but also provides new data for phylogenetic analyses. This should eventually lead toward a better understanding of metazoan evolution including key issues such as the ontogenetic processes that underlie body segmentation. We here document the development of the larval nervous system in the polychaete Sabellaria alveolata using fluorescence-coupled antibodies directed against serotonin, FMRFamide, and tubulin in combination with confocal laser scanning microscopy and 3D reconstruction software. The overall pattern of neurogenesis in S. alveolata resembles the condition found in other planktonic polychaete trochophores where the larval neural body plan including a serotonergic prototroch nerve ring is directly followed by adult features of the nervous system such as circumesophageal connectives and paired ventral nerve cords. However, distinct features are also found in S. alveolata, such as the innervation of the apical organ with ring-shaped neurons, the low number of immunoreactive perikarya, and the lack of a posterior serotonergic cell. Moreover, in the larvae of S. alveolata, two distinct modes of neuronal development are expressed, viz. the simultaneous formation of the first three segmental neurons of the peripheral nervous system on the one hand versus the sequential appearance of the ventral commissures on the other. This highlights the complex mechanisms that underlie annelid body segmentation and indicates divergent developmental pathways within polychaete annelids that lead to the segmented nervous system of the adult.     

Efficiency of particle retention and clearance rate in the polychaete Sabellaria alveolata L

The development of Sabellaria alveolata, a gregarious reef-building polychaete species, is maximal in Mont-Saint-Michel Bay (France), where trophic capacity is now threatened by increasing shellfish farming. As no data are available concerning the ecophysiological response of this species, the purpose of the present study was to obtain clearance rate and retention efficiency values to provide a first order of magnitude for the trophic role of this species. Data were obtained using a flow-through system with novel troughs suitable for 225 cm2 reef blocks containing a mean number of 940 +/- 102 (S.E.) individuals. The experimental diet used consisted of a mixture of two live microalgae, Skeletonema costatum (3800 cell ml-1) and Isochrysis galbana (23,700 cell ml-1), chosen to cover a broad size range (2 to 16 microns equivalent spherical diameter, ESD), as determined by a particle counter. On the basis of a mean clearance rate of 0.7 lh-1 obtained with reef blocks, the mean rate for an individual was estimated at 7.5 x 10(-4) L h-1. Particles larger than 6 microns ESD were cleared with 100% efficiency, but S. alveolata was unable to retain particles smaller than 2 microns ESD. The results are compared with data obtained for other polychaete species, and clearance rate values are extrapolated to an entire reef.     

Purification and characterization of proteases from the polychaete annelid Sabellaria alveolata (L.)

Eleven proteases have been purified to electrophoretic homogeneity from crude digestive fluid of polychaete annelids, Sabellaria alveolata. Purification steps were Sephadex G-100 gel filtration, benzamidine-cellulose and SBTI-Sepharose (SBTI = soybean trypsin inhibitor) affinity chromatography, CM-Sepharose and DEAE-Sepharose ion-exchange chromatography. Nine proteases have been purified in sufficient quantities for characterization. All are active at basic pH and are probably serine proteases, since they are inhibited by phenylmethylsulfonyl fluoride, specific chloromethyl ketone amino acids derivatives, but not by EDTA and p-chloromercuribenzoate. They do not hydrolyse exopeptidase substrates. From their properties, they can be divided into five classes. 1. A trypsin-like protease, which hydrolyses only trypsin substrates and is inhibited by N-tosyl-L-lysine chloromethyl ketone (TosLysCH2Cl), leupeptin and antipain. It differs from bovine trypsin by its very acidic isoelectric point (below 3.3) and its higher Mr (35 000). 2. A chymotrypsin-like protease which hydrolyses only chymotrypsin substrates and is inhibited by TosPheCH2Cl, Z-PheCH2Cl, chymostatin but only slightly by leupeptin and antipain. Its isoelectric point is below 3.3 and its Mr 31 000. 3. Two minor chymotrypsin-like proteases with slightly broader specificity, since they hydrolyse trypsin substrates significantly and are much more inhibited by leupeptin. They have acidic isoelectric points (3.3 and 3.5) and slightly lower Mr (27 000). 4. Four proteases hydrolyse trypsin and chymotrypsin substrates equally well. Their chymotryptic character is, however, predominant since they are inhibited by TosPheCH2Cl and Z-PheCH2Cl but not TosLysCH2Cl. They have similar Mr (27 000) but isoelectric points ranging from 4.0 to above 9.1. 5. The last one is very similar but has lower esterolytic activities. These proteases of broad specificity do not resemble any known serine protease since they differ from subtilisins by their sensitivity to TosPheCH2Cl.     

Rise and fall of protein phosphorylation during meiotic maturation in oocytes of Sabellaria alveolata (polychaete annelid)

Incorporation of [32P]phosphate into proteins was monitored, in preloaded Sabellaria oocytes, during meiosis. After a fourfold increase during the transition from prophase to metaphase I, the incorporated radioactivity decreased steadily by 25% during completion of meiosis, while it slowly increased in metaphase I-blocked oocytes. Measurements of the amount and specific activity of nucleotide pools showed no variation, while total alkali-labile protein-bound phosphate was found to increase and then decrease during meiosis. Autoradiography of sodium dodecyl sulfate slabgels showed that some proteins have peculiar phosphorylation-dephosphorylation kinetics. The changes in the level of phosphorylation of proteins may be related to similar changes in maturation-promoting factor (MPF) activity.     

Ultrastructure of the eyespot in three polychaete trochophore larvae (Annelida)

Summary The eyespot is structurally similar in trochophore larvae of Harmothoe imbricata, Serpula vermicularis and Spirobranchus giganteus. In the receptor cell parallel lamellae lie below a tuft of microvilli which extends into a hollow in one side of the pigment cell. In 1-eyed trochophores this space connects with the outside via a small pore. In eyes preserved during the day there is evidence of a membrane breakdown in both lamellae and microvilli as well as indications of a membrane-fragment disposal system involving the receptor cell, the pigment cell and the eyespot pore. The orientation of the eyespot of S. giganteus is the reverse of that found in S. vermicularis, a situation that may be associated with ecologically significant differences in behaviour.     

Ciliary band gene expression patterns in the embryo and trochophore larva of an indirectly developing polychaete

The trochophore larvae of indirectly developing spiralians have ciliary bands with motor and feeding functions. The preoral prototroch ciliary band is the first differentiating organ in annelid and mollusk embryos. Here we report the expression of several ciliary band markers during embryogenesis and early larval stages of the indirectly developing polychaete Hydroides elegans. Genes with similarity to caveolin, beta-tubulin, alpha-tubulin, and tektin are expressed in the eight primary prototroch precursors, 1q(221) and 1q(212). Blastomeres 1q(221) and 1q(212) locate at the same equatorial latitude after the complementary asymmetric division of their 1q(22) and 1q(21) precursors. In addition, caveolin and alpha-tubulin are expressed in the metatroch and adoral ciliary zone. Caveolin is expressed in foregut ciliated cells, and alpha-tubulin is expressed in apical tuft ciliated cells. The expression of a beta-thymosin homolog is restricted to 1q(122) and 1q(121) blastomeres, which locate just above and in close association with the eight primary prototroch cells 1q(221) and 1q(212). In addition, the beta-thymosin homolog has a transient expression in the hindgut and apical zone. The expression of all these genes provides a landmark for the early specification of ciliary bands and other ciliated organs.     

Stimulation of endogenous protein phosphorylation in oocytes of Sabellaria alveolata (polychaete annelid) at meiosis reinitiation induced by protease,fertilization, or ionophore A 23187

Incorporation of [³²P]-phosphate into proteins was enhanced when Sabellaria oocytes were stimulated with specific protease to continue from prophase I block to metaphase I block. The rate of incorporation was increased 50 fold between onset of treatment and germinal vesicle breakdown (GVB). The same result was obtained when release from prophase block involved fertilization, or activation with ionophore A 23187. In all cases, meiosis was associated with phosphorylation of an 18,000 dalton protein, which is perhaps not labeled in prophase-blocked oocytes. Phosphorylation of a 38,000–40,000 dalton doublet of membrane proteins, which are among the main phosphorylated proteins in intact oocytes, was also strongly enhanced in vitro in homogenates prepared from oocytes following release from prophase block.     

Skeletal myogenesis : Control of proliferation in a normal cell lineage

The fates of 10-day chick embryo myogenic cell populations cultured in conditions that maximize or suppress proliferation or fusion were examined. The repression of the cell cycle, prevalence of fusible cells, or expression of myofibril formation were monitored by autoradiography, counts of myotube nuclei, staining with fluorescein-labelled antibody against skeletal myosin heavy chain, or electron microscopy. Using a calcium chelator (EGTA) to block cell fusion did not prevent the accumulation of myoblasts blocked in G1 of the cell cycle that initiated skeletal myosin synthesis and myofibril formation. Trypsinization, dilution, and daily feeding with fresh medium and serum did not reverse this cell cycle block. Using cytochalasin B (CB) as an alternative fusion block confirmed these results. Using fluorodeoxyuridine (FUdR) to prevent the cycling of myogenic cells that normally would have multiplied in vitro did not prompt these cells to fuse. Muscle-conditioned medium could not prompt a switch in the commitment of replicating cells when in G1 to terminal differentiation. The indications are that myogenic cell populations contain definite mixtures of precursor phenotypes. The terminal phenotype is initiated coordinately with a relatively stable cue for the repression of the cell cycle. Differentiation-specified growth control is discussed within the context of a set of growth control mechanisms known to operate in culture.     

Physiological versus viscosity-induced effects of an acute reduction in water temperature on microsphere ingestion by trochophore larvae of the serpulid polychaete Galeolaria caespitosa

Physiological and viscosity-induced effects of an acute 10°Creduction in water temperature on the feeding performance oftrochophore larvae of Galeolaria caespitosa were separated.Experiments were conducted in which 3 and 10 µm sphereswere supplied to larvae separately and in combination. Bothphysiological and viscosity-induced components of the reductionin water temperature significantly reduced the number of microspheresingested by larvae. When 3 and 10 µm spheres were suppliedto larvae both separately and in combination, reduced watertemperature resulted in a 60° decline in the number of microspheresingested. Increased water viscosity alone accounted for overhalf of these total declines. The remaining proportions of thetotal declines were attributable to the physiological effectsof reduced water temperature. Increased water viscosity didnot differentially influence the reductions in the numbers of3 and 10 µm spheres ingested by larvae.     

Modelling and analysis of time-lags in some basic patterns of cell proliferation

 In this paper, we present a systematic approach for obtaining qualitatively and quantitatively correct mathematical models of some biological phenomena with time-lags. Features of our approach are the development of a hierarchy of related models and the estimation of parameter values, along with their non-linear biases and standard deviations, for sets of experimental data. We demonstrate our method of solving parameter estimation problems for neutral delay differential equations by analyzing some models of cell growth that incorporate a time-lag in the cell division phase. We show that these models are more consistent with certain reported data than the classic exponential growth model. Although the exponential growth model provides estimates of some of the growth characteristics, such as the population-doubling time, the time-lag growth models can additionally provide estimates of: (i) the fraction of cells that are dividing, (ii) the rate of commitment of cells to cell division, (iii) the initial distribution of cells in the cell cycle, and (iv) the degree of synchronization of cells in the (initial) cell population. Received: 15 September 1997/Revised version: 1 April 1998     

Somatic motoneurones in amphioxus larvae: cell types, cell position and innervation patterns

Serial transmission electron microscopy and 3D reconstruction were used to document cell morphology and position of the motoneurones innervating somites 1 and 2 of a 12.5-day amphioxus larva, of Branchiostoma floridae , and also those innervating the dorsal compartment of somites 3 through 6 of an 8-day larva. Motoneurones supplying the ventral and dorsal compartments can be distinguished from one another on a number of morphological criteria. The ventral compartment motoneurones are neither symmetrical nor particularly ordered in arrangement. Their cilia are short and point forward or obliquely across the central canal; their axons run along the basal lamina adjacent to processes from muscle fibres, with which they make extended linear series of synapses containing 45–60 nm synaptic vesicles. The dorsal compartment motoneurones are paired and tend to be positioned at or near the junctions between somites. Their cilia are longer and project caudally; their axons are large, filled with mitochondria and 30–45 nm synaptic vesicles, and make synapses only at specific, segmentally repeated sites.
  An unusual feature of both cell types is that synaptic input occurs all along the axon, either by direct axo-axonal synapses or via slender dendritic processes. This allows for redundancy and multiple inputs, and is possible only because amphioxus somatic motor axons lie entirely within the nerve cord, which is itself an unusual feature among chordates. The possible significance of dual somatic innervation is discussed in relation to the dual innervation of the head in vertebrates, which has separate sets of somatic and visceral/branchiomotor nerves.     

Laboratory studies of cryopreservation of sperm and trochophore larvae of the eastern oyster

The eastern oyster, Crassostrea virginica, is the most important cultured oyster species of the Atlantic and Gulf coasts of the United States. Cryopreservation of gametes and larvae of aquatic organisms has increased in importance in recent years. However, studies on the cryopreservation of sperm and larvae of mollusks have focused on the Pacific oyster, Crassostrea gigas. The present study was conducted to improve cryopreservation of sperm and trochophore larvae and to assess fertilizing ability and male-to-male variation of thawed sperm of the eastern oyster. Sperm were diluted in 12 cryoprotectant solutions composed of Hanks' balanced salt solution without calcium and 0, 5, 10, 15, 20, and 25% (v/v) propylene glycol with or without 0.25 M sucrose. Trochophore larvae were suspended in artificial seawater and 10 or 15% propylene glycol (v/v). Sperm or trochophore larvae were placed in 5-mL macrotubes and allowed to equilibrate for 15 min. The macrotubes were cooled in a controlled-rate freezer at a rate of 2.5 degrees C per min until reaching a final temperature of -30 degrees C and were plunged into liquid nitrogen. After storage for 2 weeks, the samples were thawed in a water bath at 70 degrees C for 15 s. Overall, for cryopreservation of sperm and larvae, best results were obtained using 10 or 15% propylene glycol. Thawed sperm presented significant male-to-male variation in fertilizing ability. Survival of thawed larvae decreased as the concentration of larvae per macrotube increased. The procedures developed in this study for sperm and larvae are suitable for production of seedstock in commercial oyster hatcheries.     

Global analysis of endothelial cell line proliferation patterns based on nutrient-depletion models: implications for a standardization of cell proliferation assays

It is known that cell populations growing in different environmental conditions may exhibit different proliferation patterns. However, it is not clear if, despite the diversity of the so-observed patterns, inherent cellular growth characteristics of the population can nevertheless be determined. This study quantifies the proliferative behaviour of the permanent endothelial human cell line, Eahy926, and establishes to which extent the estimation of the cell proliferation rate depends on variations of the experimental protocols. Cell proliferation curves were obtained for cells cultured over 16 days and the influences of cell seeding densities, foetal bovine serum content and frequency of culture medium changes were investigated. Quantitative dynamic modelling was conducted to evaluate the kinetic characteristics of this cell population. We proposed successive models and retained a nutrient-depletion toxicity dependant model, which takes into account the progressive depletion of nutrients, as well as the increase of toxicity in the cell culture medium. This model is shown to provide a very good and robust prediction of the experimental proliferation curves, whatever are the considered frequency of culture medium changes and serum concentrations. Thus, the model enables an intrinsic quantification of the parameters driving in vitro EAhy926 proliferation, including proliferation, nutrient consumption and toxicity increase rates, rather independently of the experiments design. We therefore propose that such models could provide a basis for a standardized quantification of intrinsic cell proliferation kinetics.     

Activation of myogenesis by the homeobox gene Lbx1 requires cell proliferation.

Myogenic differentiation can be initiated by a limited number of molecules. In this work, we analyzed the function of the homeobox gene Lbx1 in chicken embryos and explant cultures. We demonstrate that overexpression of Lbx1 in vivo and in vitro leads to a strong activation of various muscle markers. We show that cell proliferation, which is strongly stimulated by Lbx1 and Pax3, is required for Lbx1- or Pax3-dependent myogenic activation. Inhibition of cell proliferation prevents expression of muscle differentiation markers, while the activation of other putative downstream targets of Pax3 and Lbx1 is not affected. Our findings imply that a critical function of Pax3 and Lbx1 during muscle cell formation is the enlargement of muscle cell populations. The growth of the muscle precursor cell population may increase the bias for myogenic differentiation and thus enable myogenic cells to respond to environmental cues.     

The ontogeny of cross‐sex genetic correlations: an analysis of patterns

The independent evolution of males and females is typically constrained by shared genetic variance. Despite substantial research, we still know little about the evolution of cross‐sex genetic covariance and its standardized measure, the cross‐sex genetic correlation (r_MF). In particular, it is unclear if r_MF tend to vary with age. We compiled 28 traits for which ontogenetic trends in r_MF were documented. Decreases in r_MF with age were observed significantly more often than increases and the mean effect size for the relationship between r_MF and age was large and negative. This suggests that sexual dimorphism (SD) may typically evolve more readily for phenotypes expressed later in ontogeny and that evolutionary inferences related to the evolution of SD should be limited to the ontogenetic stage at which r_MF was estimated. Knowledge about ontogenetic variation in r_MF should help improving our understanding of evolutionary patterns related to SD and the resolution of intralocus sexual conflicts.