Patterns of maternal messenger RNA accumulation and adenylation during oogenesis in Urechis caupo

We have investigated the accumulation and adenylation of the maternal mRNA during oogenesis in the oocytes of the marine worm Urechis caupo. The analysis, using in vitro translation and cDNA probes to assay for specific mRNAs, demonstrates that different maternal mRNAs accumulate with different patterns during oogenesis. One class of maternal mRNAs accumulates throughout oogenesis and remains at a steady level in the full-grown oocyte. These mRNAs do not have a poly(A) tail long enough to mediate binding to oligo(dT)-cellulose in oocytes, but are rapidly adenylated immediately following fertilization. The other maternal mRNAs accumulate in growing oocytes as poly(A)+ RNA and undergo some deadenylation in full-grown oocytes and embryos. Some of these mRNAs attain their highest concentration fairly early in oogenesis, while others continue to accumulate during later stages. Many of the mRNAs that accumulate as poly(A)+ RNA in growing oocytes diminish dramatically in concentration in full-grown oocytes.     

Regulation of histone synthesis during early Urechis caupo (Echiura) development

The histones present in mature oocytes and embryos of Urechis caupo and their pattern of synthesis during early development have been characterized. Acid-soluble proteins extracted from mature oocyte germinal vesicles and from embryonic nuclei were analyzed by two-dimensional polyacrylamide gel electrophoresis. Histones are accumulated in the mature oocytes in amounts sufficient to provide for the assembly of chromatin through the 32- to 64-cell stage of embryogenesis. Two H1 histones, which appear to be variants, were found. Germinal vesicles and cleavage-stage nuclei are enriched in H1M (maternal). During late cleavage a faster-migrating H1, H1E (embryonic), appears among the nuclear histones and, as embryogenesis continues, replaces H1M as the predominant H1. No new core histone variants are detected during early development. Examination of [³H]lysine-labeled histones from germinal vesicles and embryonic nuclei reveals stage-specific patterns of histone synthesis. H1M is the major H1 species synthesized in mature oocytes. After fertilization, a switch to the predominant synthesis of H1E occurs. Comparison of the [³H]lysine incorporated into H1E and core histones indicates that H1E synthesis is disproportionately high from midcleavage through the midblastula stage. By the gastrula stage, a balanced synthesis of H1E and each core histone is established. The results indicate that there is noncoordinate regulation of H1 and core histone synthesis during Urechis development.     

Polyadenylation of RNA in immature oocytes and early cleavage of Urechis caupo

The poly(A) content of RNA extracted from four stages of immature oocytes, mature oocytes, and cleavage embryos through the eight-cell stage was determined by hybridization with [³H]-poly(U). During oogenesis the poly(A) content per cell gradually increases from 0.007 pg of poly(A)/cell in the 10- to 39-μm oocytes to 0.20 pg of poly(A)/cell in the 125-μm mature oocytes. After fertilization there is an additional increase to approximately 1.1 pg of poly(A)/embryo at the two-cell stage which is followed by a slight decline between the two- and eight-cell stages. Most of the increase in poly(A) after fertilization occurs in a 45-min interval coincident with the appearance of the polar bodies. The size distribution of the poly(A) in RNA from the different stages of development was determined based on the length of RNase-resistant poly(U) obtained from poly(U)-poly(A) hybrids. The size distribution of the poly(A) sequences is constant through each stage of development which indicates that the increase in the poly(A) content of the cells is the result of polyadenylation of new RNA sequences.     

Electrically mediated fast polyspermy block in eggs of the marine worm, Urechis caupo

Previous work has established that the polyspermy block in Urechis acts at the level of sperm-egg membrane fusion. (J. Exp. Zool. 196:105). Present results indicate that during the first 5--10 min after insemination the block is mediated by a positive shift in membrane potential (the fertilization potential) elicited by the penetrating sperm, since holding the membrane potential of the unfertilized egg positive by passing current reduces the probability of sperm entry, while progressively reducing the amplitude of the fertilization potential by decreasing external Na+ progressively enhances multiple sperm penetrations. Also, a normal fertilization potential is correlated with a polyspermy block even under conditions (pH 7) in which eggs do not develop. We have investigated the mechanism of the electrical polyspermy block by quantifying the relationship between sperm incorporation, membrane potential and ion fluxes. Results indicate that the polyspermy block is mediated by the electrial change per se and not by the associated fluxes of Na+, Ca++, and H+.     

Protamine messenger RNA: evidence for early synthesis and accumulation during spermatogenesis in rainbow trout

Trout testis cells were separated into various developmental classes by velocity sedimentation in bovine serum albumin gradients and were identified morphologically with particular stages of the process of spermatogenesis. The stage of testis cell differentiation at which protamine mRNA appears in the cell cytoplasm for the first time was determined by hybridization of RNA populations extracted from the separated cells to radioactively labeled protamine cDNA. Primary spermatocytes represent the earliest stage of differentiation at which protamine mRNA can be detected in large quantities in the cell cytoplasm, establishing that the synthesis of this class of mRNA occurs at a much earlier stage than the time of its translation at the spermatid stage. Protamine mRNA sequences were found in both the polysomes and postribosomal supernatant of the spermatid cells which are involved in the synthesis of protamine, while primary and secondary spermatocytes contained the mRNA sequences only in their postribosomal supernatant fractions. These findings strongly suggest that protamine mRNA is synthesized, accumulated, and stored in the cell sap of primary and secondary spermatocytes in the form of “inactive” messenger ribonucleoprotein particles, which are “activated” and translated at the spermatid stage.     

Hox genes in the echiuroid Urechis unicinctus

An echiuroid species, Urechis unicinctus, was surveyed for Hox genes using polymerase chain reaction with homeobox-specific degenerate primers. We identified nine distinct homeodomain-containing gene fragments. These nine fragments were classified by comparative analysis. This analysis revealed that this echiuroid possessed at least three Hox genes from the anterior group, five from the central group, and one from the posterior group.     

Ionic mechanism of the fertilization potential of the marine worm, Urechis caupo (Echiura)

Microelectrode and tracer flux studies of the Urechis egg during fertilization have shown: (a) insemination causes a fertilization potential; the membrane potential rises from an initial level of -33 +/- 6 mV to a peak at +51 +/- 6 mV (n = 16), falls to a plateau of about +30 mV, then returns to the original resting potential 9 +/- 1 min (n - 10) later; (b) the fertilization potential results from an increase in Na+ permeability, which is amplified during the first 15 s by a Ca++ action potential; (c) the maximum amplitude of the fertilization potential, excluding the first 15 s, changes by 51 mV for a 10-fold change in external [Na+]; (d) in the 10 min period after insemination, both Na+ and Ca++ influxes increase relative to unfertilized egg values by factors of 17 +/- 7 (n = 6) and 34 +/- 14 (n = 4), respectively; the absolute magnitude of the Na+ influx is 16 +/- 6 times larger than that of Ca++; (e) in the absence of sperm these same electrical and ionic events are elicited by trypsin; thus, the ion channels responsible must preexist in the unfertilized egg membrane; (f) increased Na+ influx under conditions of experimentally induced polyspermy indicates that during normal monospermic fertilization, only a fraction of available Na+ channels are opened; we conclude that these channels are sperm-gated; (g) Ca++ influx at fertilization is primarily via the membrane potential-gated channel, because kinetics are appropriate, and influx depends on potential in solutions of varying [Na+], but is independent of number of sperm incorporations in normal sea water.     

Localization of actin messenger RNA during early ascidian development

The spatial distribution of RNA sequences during early development of the ascidian, Styela plicata, was determined by in situ hybridization with poly(U) and cloned DNA probes. Styela eggs and embryos contain three colored cytoplasmic regions of specific morphogenetic fates, the ectoplasm, endoplasm, and myoplasm. These cytoplasmic regions participate in ooplasmic segregation after fertilization and are distributed to different cell lineages during early embryogenesis. n situ hybridization with poly(U) suggests that poly(A)+RNA is unevenly distributed in eggs and embryos, with about 45% in the ectoplasm, 50% in the endoplasm, and only 5% in the myoplasm. In situ hybridization with a histone DNA probe showed that histone RNA sequences were not localized in eggs or embryos and distributed between the three cytoplasmic regions according to their volumes. In situ hybridization with an actin DNA probe showed actin RNA was localized in the myoplasm and ectoplasm of eggs and embryos with about 45% present in the myoplasm, 40% in the ectoplasm, and only 15% in the endoplasm. These results suggest that a large proportion of the egg actin mRNA is localized in the myoplasm, participates in ooplasmic segregation after fertilization, and is differentially distributed to the mesodermal cell lineages during embryogenesis. Analysis of the translation products of egg mRNA suggests that the localized mRNA codes for a cytoplasmic actin isoform.     

Sulfide permeability in the marine invertebrate Urechis caupo

Summary Hydrogen sulfide can reach toxic concentrations in the burrow-water of the echiuran worm Urechis caupo during low tide. Its two large epithelial surfaces, the thick muscular body wall and the thin-walled hindgut are in constant contact with the environment. Hindgut inflation of up to 2 ml water·g wet weight^-1 causes tissue stretch. To determine if these body surfaces present a barrier to sulfide influx, the total permeability coefficient P_T was measured at different degrees of stretch in diffusion chambers at pH 6.0, 7.0, and 8.0, and specific permeability coefficients P_{H 2}S and P_HS ^- were calculated. Both the body wall and the hindgut were more permeable to H₂S than HS^-. The body wall showed no significant increase in sulfide permeability with natural degrees of stretch, and the mean P_{H 2}S and P_HS ^- were 0.17 and 0.063 cm·h^-1, respectively. The sulfide permeability of the hindgut was increased by stretch, with the relative permeability of H₂S increasing faster than that of HS^-. Unstretched hindgut mean P_{H 2}S and P_HS ^- were 0.095 and 0.11 cm·h^-1, respectively, and stretched hindgut mean P_{H 2}S and P_HS ^- were 1.8 and 0.16 cm·h^-1, respectively. A model of sulfide influx in the natural environment indicates that even if the hindgut is kept uninflated, the coelomic fluid of U. caupo would have toxic sulfide concentrations well before the end of a 2-h tidal exposure in the absence of a sulfide elimination mechanism.Abbreviations P permeability coefficient - P_T total permeability coefficient - PD transepithelial potential difference     

MAP kinase, meiosis, and sperm centrosome suppression in Urechis caupo

Although MAP kinase is an important regulatory enzyme in many somatic cells, almost nothing is known about its functions during meiosis, except in frog and mouse oocytes. We investigated MAPK activation and function in oocytes of the marine worm Urechis caupo that are fertilized at meiotic prophase. Activity was first detected at 4-6 min after fertilization in immunoblots with anti-active MAPK, prior to germinal vesicle breakdown (GVBD). MAPK activation did not require new protein synthesis and was dependent on the increases in both intracellular pH and intracellular Ca(2+) that normally occur during activation. When MAPK activation was inhibited with PD98059 or U0126, GVBD still occurred, but meiosis was abnormal and there was a dramatic premature enlargement of sperm asters, which normally do not appear until second polar body formation. Failure of polar body formation and premature sperm aster enlargement also occurred when MAPK activation was inhibited by an entirely different treatment which involved lowering the pH of external seawater to interrupt the normal cytoplasmic pH increase. Thus, in Urechis, active MAPK appears to be required for (1) normal meiotic divisions and (2) suppressing the paternal centrosome until after the egg completes meiosis, a general phenomenon whose mechanism has been unknown.     

Timing of initiation of polyadenylation of preformed RNA in sea urchin and Urechis caupo zygotes

An increase in the synthesis of polyadenylic acid, following fertilization, was examined in sea urchin, Strongylocentrotus purpuratus, and the marine worm Urechis caupo embryos. It was found that in both organisms there is a large increase in the incorporation of [³H]adenosine into the poly(A) region, of poly(A)-containing RNA, in the phase preceding the 2-cell stage. In sea urchin this rise was localized to the G₂ period. Similar findings were made in Urechis indicating that polyadenylation of RNA may be a common postfertilization event in many developing organisms.