Starfish Eggs Research Articles

Structure and function of asterosaps, sperm-activating peptides from the jelly coat of starfish eggs.

Jelly coat of starfish eggs has the capacity to activate homologous spermatozoa and induce the acrosome reaction. We have isolated 12 sperm-activating peptides (SAPs) from the egg jelly of the starfish, Asterias amurensis. Eleven SAPs were structurally identified by sequence analysis and electro-spray ionisation mass spectrometry. All of them are glutamine-rich tetratriacontapeptides with an intramolecular disulphide linkage between Cys8 and Cys32. They are much larger than sea urchin SAPs and do not show any significant sequence similarities to known proteins. Thus we have collectively named them asterosaps. The amino terminal region, where structural diversity of asterosaps is observed, is not important for their activity, whereas the disulphide linkage is essential. Asterosaps do not induce the acrosome reaction by themselves, but are able to induce the acrosome reaction in combination with an egg jelly glycoconjugate named ARIS. Furthermore, anti-asterosap rabbit antibody significantly decreased the acrosome reaction-inducing activity of the jelly solution and the activity was restored by addition of excess asterosap. These results support our hypothesis that the main physiological role of SAPs is the induction of the acrosome reaction in cooperation with two other jelly components, ARIS and Co-ARIS.

Centrifugal bisection of starfish oocytes.

Immature oocytes or mature eggs of starfish were centrifuged in a sucrose density gradient. They were then separated into two fractions of fragments, nucleate light fragments and anucleate heavy fragments. Vital-staining experiments showed that the oocytes were elongated along the animal-vegetal (AV) axis during the centrifugation in a contrast to centrifuged eggs whose centrifugal axis was not related to the AV axis. The light and heavy oocyte fragments were comprised of animal and vegetal halves of oocytes, respectively. When matured and fertilized, most of the light oocyte fragment-derived embryos failed gastrulation and developed into Dauerblastulae. Two-dimensional gel electrophoretic analysis of fragments revealed that three basic proteins were predominantly enriched in the heavy oocyte fragments but scarcely detected in the light oocyte fragments. One of these proteins, App20, was identified as a homologue of cyclophilin (peptidyl-prolyl cis-trans isomerase). The present study provides a simple means of separating a population of starfish oocytes into animal and vegetal halves, thereby enabling us to analyze any difference of components between animal and vegetal cytoplasm of the oocytes.

Proteases Stimulate Fertilization-like Responses in Starfish Eggs

Application of proteases to eggs of the starfish, Asterina miniata, caused several responses like those seen at fertilization. Cortical granule exocytosis and fertilization envelope elevation occurred within about 1 min after exposure to trypsin, chymotrypsin, or pronase; protease inhibitors prevented these responses. Kallikrein caused cortical granule exocytosis and fertilization envelope elevation, but this response required more time (∼30 min). Exocytosis was also seen in response to a recombinant trypsin, but not to a point-mutated trypsin without proteolytic activity. The extent of exocytosis was similar to that seen at fertilization, as measured by the fluorescent dye FM 1-43. In addition to causing exocytosis, application of trypsin, chymotrypsin, or pronase caused an increase in intracellular free calcium, detected by calcium green dextran, and stimulation of DNA synthesis, detected by incorporation of bromodecxyuridine. Exocytosis also occurred when trypsin or chymotrypsin was applied in artificial sea water in which the free calcium was reduced to a low level (40-70 n M) such that Ca influx would be reduced by >10,000-fold; this indicated that the proteases did not act by damaging the eggs and causing external calcium to leak into the cytoplasm. These findings show that there is an extracellularly exposed protein that when proteolyzed can induce fertilization-like responses; this protein may be a receptor that transduces a signal from the sperm to initiate egg activation at fertilization.

Nuclear distribution of proliferating cell nuclear antigen (PCNA) in fertilized eggs of the starfish Asterina pectinifera

Previous studies (Nomura et al. (1991) Dev. Biol. 143, 289-296 (1993) Dev. Biol. 159, 288-297) determined the time of DNA replication period (S phase) in starfish eggs fertilized either during or after oocyte maturation. Here proliferating cell nuclear antigen (PCNA) localized within nuclei of starfish eggs was detected with an anti-PCNA human antiserum. Using a confocal laser scanning microscope, a three-dimensional structure of the PCNA region was analyzed. In eggs fertilized during maturation, PCNA started to localize within the nuclei at the same time as the initiation of the first S phase. During the S phase, the distribution of localized PCNA in a three-dimensional view coincided with the chromatin distribution. After the S phase, PCNA remained localized within the nuclei, but its distribution no longer coincided with the chromatin distribution. In eggs fertilized after maturation, however, PCNA started to localize within the female pronuclei about 10 minutes ahead of the first S phase. Localized PCNA occupied only a limited region of the nuclei without diffusing over the whole nuclear area. Chromatin distributed around the peripheral region of the nuclei mostly outside the PCNA region. When the first S phase was initiated, the chromatin distribution became coincident with the PCNA region. Later behavior of PCNA was the same as that of the eggs fertilized during maturation. The precocious localization of PCNA in those eggs fertilized after maturation simply demonstrates that the 'postactivation process' for preparing DNA replication is triggered by fertilization and PCNA localization and S phase are sequentially initiated with a time-lapse. On the other hand, the simultaneous occurrence of them seen in those eggs fertilized during maturation indicates that the postactivation process must be going on in parallel with the maturation process.

Artificial Parthenogenesis in Starfish Eggs: Behavior of Nuclei and Chromosomes Resulting in Tetraploidy of Parthenogenotes Produced by the Suppression of Polar Body Extrusion

Artificial parthenogenesis in starfish requires the activation of oocytes and the suppression of the polar body (PB) extrusion. To induce parthenogenesis we have employed a combination of calcium ionophore A23187 for activating oocytes and cytochalasin B (CB) for the suppression of PB extrusions. The treatment of activated oocytes with CB during meiosis I produced eggs lacking polar bodies, i.e., 0pb eggs, and treatment during meiosis II induced eggs bearing the first PB only, i.e., 1pb eggs. About 90% of both 0pb and 1pb eggs developed parthenogenetically with strong synchrony of cell division among eggs. After meiotic chromosomes in CB-treated oocytes separated finally into monads, they came together to form a single nucleus. The nucleus was found to be tetraploid in the 0pb eggs and diploid in the 1pb eggs. Regardless of the difference in ploidy, both 0pb and 1pb eggs developed as tetraploid embryos. Observations of the behavior of meiotic chromosomes and nuclei revealed that in the 0pb eggs, the first round of chromosomal replication was followed by the first cleavage, as in normally fertilized eggs. In the 1pb eggs, on the other hand, two rounds of chromosomal replication were found to precede the first cleavage, indicating the occurrence of one (first) round of chromosomal replication that is not accompanied with cytokinesis. In the first round of mitosis, a bipolar spindle did not appear, but only a half spindle was formed, resulting in the failure of both karyokinesis and cytokinesis. Thus, both types of parthenogenetic eggs became tetraploid before the first cleavage. At the first cleavage, both types of eggs formed a bipolar spindle and divided into a pair of blastomeres. Based on these observations, we suggest that the meiotic centrosomes remaining in these eggs by the failure of PB extrusion are diverted into mitosis-organizing centers in the mitotic spindle, and this results in parthenogenetic development.

Evidence for Both Tyrosine Kinase and G-Protein-Coupled Pathways Leading to Starfish Egg Activation

To investigate possible pathways leading to egg activation at fertilization, the ability of exogenously introduced tyrosine kinase and G-protein-coupled recaptors to mimic events of fertilization was examined. Oocytes of the starfish Asterina miniata were injected with RNA for a chimeric receptor consisting of the extracellular domain of the β form of the mouse platelet-derived growth factor (PDGF) receptor and the transmembrane/intracellular domain of the human fibroblast growth factor (FGF) receptor, or with RNA for the rat serotonin 1c receptor. These oocytes were cultured for 1 to 3 days and then matured with 1-methyladenine. In response to PDGF or serotonin, the injected eggs underwent responses like those at fertilization: cortical granule exocytosis, a rise in intracellular free calcium, and DNA synthesis. Some of these artificially activated eggs cleaved, and some of the PDGF-activated eggs were observed to form larvae, A PDGF/FGF receptor with a point mutation which eliminated its ability to interact with phospholipase C-γ did not cause fertilization-like responses. Thus components of a signaling pathway involving phospholipase C-γ, characteristic of tyrosine kinase receptors, as well as components of a pathway involving a G-protein and phospholipase C-β, characteristic of G-protein-coupled receptors, appear to be present in starfish eggs. Either or both could function in egg activation at fertilization.

A Portable, Discrete-Sampling Submersible Plankton Pump and Its Use in Sampling Starfish Eggs.

The apparatus was designed to enable divers to obtain discrete replicate samples from known volumes of water. Relatively large volumes of water can be filtered without the need to maintain contact with or return to the surface. These attributes confer a high degree of flexibility in subsurface sampling situations. Although we used the equipment to sample the eggs of invertebrates, it could equally easily be used to sample other small, patchily distributed plankton.

DNA Replication in Fertilized Eggs of the Starfish Asterina pectinifera

Early starfish embryos were labeled with 5-bromo-2′-deoxyuridine (BrdU; the thymidine analogue) for 3-4 min of pulse, and the BrdU incorporated into newly replicated DNA was immunocytochemically detected with anti-BrdU antibody. Series of optical sections of the stained embryos observed with a confocal laser scanning microscope (CLSM) were collected, and three-dimensional images of the chromatin and the incorporated BrdU were reconstructed on a computer display. The distribution of the DNA replication sites and the chromatin structure were investigated from a three-dimensional point of view. The oocytes fertilized during maturation and the eggs fertilized after maturation were examined for the timing of S phase, DNA replication pattern, and chromatin distribution in the nucleus. Duration of the first S phase between the two types of embryos differed in the first S phase, but was the same in the second and third S phases. We observed a successive change in the distribution of DNA replication sites from a "uniform" to a "condensing" pattern during a single S phase traverse, except in the first S phase of the eggs fertilized after maturation. These two patterns of DNA replication almost coincided with chromatin visualized by anti-single-stranded DNA antiserum. The apparent change in the pattern of DNA replication sites thus simply reflects the change in the spatial distribution of chromatin. The spatial pattern of DNA replication at the first S phase of the eggs fertilized after maturation was unique in that the whole nucleus was a mosaic of two distinct regions, each supposedly derived from the female and male pronuclei. The putative female region had a fibrillar pattern of DNA replication sites confined to the periphery of the nucleus, while the male region showed condensed sites of DNA replication, which persisted longer than the sites in the female pronuclear region. We discuss such a singularity of the first S phase in starfish eggs on the basis of the temporal relation between the progress of maturation and the time of activation.

Clathrynamides A, B, and C: Novel amides from a marine sponge Clathria sp. That inhibit cell division of fertilized starfish eggs

Three novel bromine-containing amides, clathrynamides A ( 1), B ( 2), and C ( 3), were isolated from a marine sponge Clathria sp. Their structure were determined on the basis of spectroscopic data. Clathrynamide A ( 1) significantly inhibited cell division of fertilized starfish eggs and also showed inhibition of the growth of human myeloid K-562 cells in vitro.

S8.20 Structure and activity of the carbohydrate chains in acrosome reaction-inducing substance of starfish eggs

S8.20 Structure and activity of the carbohydrate chains in acrosome reaction-inducing substance of starfish eggs

Reconstruction of Starfish Eggs by Electric Cell Fusion: A New Method of Detect the Cytoplasmic Determinant for Archenteron Formation: (starfish/cell fusion/archenteron differentiation/determinant/egg polarity).

A method of detecting cytoplasm carrying the determinant for archenteron formation in starfish was established. Animal egg fragments (AEFs) which had been severed from the vegetal halves were fused electrically into pairs with fragments prepared from various regions of immature oocytes. It has been previously shown that the vegetal halves are exclusively endowed with the ability to form the archenteron; AEFs alone develop into so-called permanent blastulae. Eggs thus reconstructed were allowed to develop in order to assess the presence of the determinant in the added fragments. Only AEFs fused with fragments from near the vegetal pole of the oocytes formed the archenteron and developed into bipinnariae and juveniles. Comparison between the inner and outer (including cortex) cytoplasm of small vegetal fragment showed that the outer cytoplasm gave the reconstructed egg a greater ability to form an archenteron than the inner cytoplasm.

Pronuclear formation in starfish eggs inseminated at different stages of meiotic maturation: Correlation of sperm nuclear transformations and activity of the maternal chromatin

Changes in sperm nuclei incorporated into starfish, Asterina miniata, eggs inseminated at different stages of meiosis have been correlated with the progression of meiotic maturation. A single, uniform rate of sperm expansion characterized eggs inseminated at the completion of meiosis. In oocytes inseminated at metaphase I and II the sperm nucleus underwent an initial expansion at a rate comparable to that seen in eggs inseminated at the pronuclear stage. However, in oocytes inseminated at metaphase I, the sperm nucleus ceased expanding by meiosis II and condensed into chromosomes which persisted until the completion of meiotic maturation. Concomitant with the formation and expansion of the female pronucleus, sperm chromatin of oocytes inseminated at metaphase I enlarged and developed into male pronuclei. Condensation of the initially expanded sperm nucleus in oocytes inseminated at metaphase II was not observed. Instead, the enlarged sperm nucleus underwent a dramatic increase in expansion commensurate with that taking place with the maternal chromatin to form a female pronucleus. Fusion of the relatively large female pronucleus and a much smaller male pronucleus was observed in eggs fertilized at the completion of meiotic maturation. In oocytes inseminated at metaphase I and II, the male and female pronuclei, which were similar in size, migrated into juxtaposition, and as separate structures underwent prophase. The chromosomes in each pronucleus condensed, intermixed, and became aligned on the metaphase palate of the mitotic spindle in preparation for the first cleavage division. These observations demonstrate that the time of insemination with respect to the stage of meiotic maturation has a significant effect on sperm nuclear transformations and pronuclear morphogenesis.

Cytoplasmic regionalization in starfish oocyte occurrence and localization of cytoplasmic determinants responsible for the formation of archenteron and primary mesenchyme in starfish (asterias amurensis) oocytes

Starfish oocytes with intact germinal vesicles (GVs) were cut along desired planes with glass needles or ligated using silk thread loops into two parts and allowed to mature in vitro, and inseminated. The experimental results showed that (1) only the parts with GVs or partial GV contents (PGVCs) cleaved, those without any GV materials did not; but nucleated and non-nucleated fragments cut from mature eggs were able to divide; (2) the development of animal parts of oocytes containing GVs or PGVCs was like that of animal fragments of matured oocytes with female pronuclei; most of them gave rise to permanent blastulae, and just a few formed ectodermal vesicles with a little primary mesenchyme; (3) a large part of vegetal fragments with GVs or PGVCs, and the vegetal parts of mature eggs without female pronuclei developed into small but normal embryos; (4) the fragments containing GVs or PGVCs obtained from the oocytes along a plane parallel to the animal-vegetal (A-V) axis developed as normally as the halves (with or without female pronuclei) severed from mature eggs along the same axis. Based on the data above, it was concluded that (1) the non-chromatin materials in the oocyte GVs are indispensable for successful fertilization and cleavage of starfish eggs; (2) some factor (s) located asymmetrically in the vegetal hemispheres of starfish oocytes is (are) responsible for formation of the archenteron and primary mesenchyme. It is evident from the above findings that the oocyte cytoplasm of the starfish had already regionalized before the GV break-down.

The starfish egg mRNA responsible for meiosis reinitiation encodes cyclin

During meiotic maturation and early embryonic cycles, the activity of maturation-promoting factor (MPF) cycles in exact correspondence with the mitotic cycles. For the appearance of MPF activity in starfish, protein synthesis is required except in the first meiotic cycle. In order to identify newly synthesized proteins involved in the regulation of MPF activity, we extracted poly(A) + RNA from starfish eggs, and found that the egg poly(A) + RNA induced germinal vesicle breakdown (GVBD) upon injection into immature oocytes of starfish and Xenopus. The molecular size of the poly(A) + RNA responsible for GVBD was estimated to be approximately 22S by sucrose density gradient centrifugation. Since these characteristics of the starfish egg poly(A) + RNA are similar to those of cyclin mRNAs from sea urchin and surf clam eggs, we synthesized a 50-mer antisense-cyclin oligonucleotide probe coding for a part of the sea urchin cyclin cDNA and used this to screen starfish RNA. The Northern blot analysis showed that the starfish egg RNA contained cyclin homologous transcripts. Incubation of the starfish egg poly(A) + RNA and the antisense-cyclin oligonucleotide with RNase H completely destroyed its GVBD-inducing activity. These results indicated that starfish cyclin mRNA was the only poly(A) + RNA responsible for GVBD. We constructed a starfish egg cDNA library to clone starfish cyclin cDNA. The longest cDNA clone containing 2190 base pairs was sequenced. The longest open reading frame consisted of 395 amino acid residues, and the predicted molecular size was 48 kDa. Comparison of the deduced amino acid sequences of starfish cyclin with known cyclins indicated that the starfish cyclin belongs to the B-type. Injection of synthetic mRNA of starfish cyclin caused GVBD in immature oocytes of starfish and Xenopus, while injection of synthetic mRNA of human CDC2 had no effect. The Northern blot analysis of starfish RNA extracted at various stages of the meiotic cycles suggested that the starfish cyclin transcript was stored in its polyadenylated form even in immature oocytes and was further polyadenylated at maturation.

Injection of isolated centriolar complex to induce mitosis in starfish eggs

When centriolar complex isolated from starfish spermatozoa was injected into starfish immature oocytes (fully grown, germinal vesicle stage) followed by treating the latter with 1-methyladenine, mitotic events such as condensation and division of chromosomes of the female pronucleus and cytokinesis following completion of meiosis were observed. No cortical reaction detected in the oocytes. Essentially the same was noted for mature oocytes (pronuclear stage) into which the centriolar complex had been injected. The oocytes that had received sperm tail fraction or buffer alone did not initiate cleavage. It would thus appear that sperm centriolar complex is significantly essential to the initiation of cleavage.

Activation by serotonin of starfish eggs expressing the rat serotonin 1c receptor.

Starfish oocytes were injected with mRNA for the serotonin 1c receptor or with rat brain poly A+ mRNA, incubated to allow expression of the membrane protein, then matured to eggs by addition of 1-methyladenine. Applying serotonin to these eggs caused cortical granule exocytosis like that occurring at fertilization. Because the serotonin 1c receptor specifically activates a G-protein, these results provide support for the hypothesis that sperm activate eggs by way of a receptor-G-protein interaction. The starfish oocyte may be a generally useful system for expression of exogenous mRNA for membrane proteins.

A 70 kD microtubule‐binding protein from starfish eggs: Purification, characterization, and localization during meiosis and mitosis

AbstractA microtubule‐binding protein was purified from eggs of the starfish, Asterias amurensis, through several steps of purification including the taxol‐dependent procedure [Vallee, 1982, J. Cell Biol. 92:435–442]. This protein consists of a single polypeptide chain having an apparent molecular mass of 70 kD determined by SDS‐PAGE. The 70 kD protein was identified as a unique microtubulebinding protein, judging from electrophoretic mobility, cleavage pattern by limited proteolysis, heat stability, and immunocrossreactivity. The 70 kD protein binds to brain and egg microtubules. It does not promote assembly of brain tubulin, but promotes that of egg tubulin in vitro in a concentration‐dependent manner.Using indirect immunofluorescence and immunoelectron microscopy with the anti‐70 kD protein antibody, we analyzed the cellular localization of the 70 kD protein in starfish oocytes and eggs during both meiotic maturation (meicsis) and first cleavage (mitosis). Immunofluorescence studies showed that the 70 kD protein localized on microtubule structures spread widely throughout the cytoplasm, the sperm aster, and the microtubules making up the mitotic apparatus through both meiosis and mitosis. The antibody, however, did not recognize sperm axonemes. These results were confirmed by immunoelectron microscopy. Using a colloidal gold technique, the 70 kD protein was localized along the microtubules in vivo.This 70 kD protein is the first microtubule‐binding protein that has been shown to localize along the microtubules in oocytes and eggs throughout meiosis and mitosis and to promote microtubule assembly. The 70 kD protein may be involved in the dynamic changes of microtubule structures occurring within oocytes and eggs.

Structure of a novel steroidal saponin, pachastrelloside A, obtained from a marine sponge of the genus pachastrella

A novel steroidal glycoside, pachastrelloside A, has been isolated from a marine sponge and its structure was elucidated as 2α,3β-dihydroxy-4β- O-(β-D-galactopyranosyl)-7α- O-(4- O-acetyl-β-Dxylopyranosyl)ergosta-5,24(28)-diene. This glycoside inhibits cell division of fertilized starfish eggs but does not affect nuclear divisions to form multinucleated, unicellular embryos.

Centrosome inheritance in starfish zygotes: Selective loss of the maternal centrosome after fertilization

The mature egg inherits a centrosome from the second meiotic spindle, and the sperm introduces a second centrosome at fertilization. Since only one of these centrosomes survives to be used in development, specific mechanisms must exist to control centrosome inheritance. To investigate how centrosome inheritance is controlled we used starfish eggs as a model system, because they undergo meiosis after fertilization. As a result, the fate of the maternal and paternal centrosomes can be followed by light microscopy and experimentally manipulated in vivo. We show initially that only the paternal centrosome is used in starfish zygote development; the maternal centrosome retained from meiosis II is functionally lost before first mitosis. We then tested a number of possible ways in which the zygote could exert this differential control over the stability of centrosomes initially residing in the same cytoplasm. The results of these experiments can be summarized as follows: (1) Although the microtubule organizing center activity of the maternal centrosome is not degraded after meiosis, the ability of this centrosome to double at successive mitoses is lost. (2) The sperm centrosome is not "masked" from cytoplasmic conditions which could destabilize all centrosomes during or after the meiotic sequence. (3) The functional loss of the maternal centrosome is not due to its cortical location. (4) The loss of this doubling capacity is determined by the egg, not by putative inhibitory factors from the fertilizing sperm. (5) The destabilization of the maternal centrosome is not due to the complete loss of its centrioles. Together, these results demonstrate that all maternal centrosomes are equivalent and that they are intrinsically different from the paternal centrosome. This intrinsic difference, in concert with a change in cytoplasmic conditions after meiosis, determines the selective loss of the maternal centrosome inherited from the meiosis II spindle.

Astrogorgiadiol and astrogorgin, inhibitors of cell division in fertilized starfish eggs, from a gorgonian [formula omitted] sp.

A novel secosterol, astrogorgiadiol( 1), a known diterpene ophirin( 2) and a closely related diterpene, astrogorgin( 3), have been isolated from a gorgonian Astrogorgia sp. as inhibitors of cell division in fertilized starfish eggs.