Isolation and characterization of polymorphic microsatellite DNA markers in the sea squirt Halocynthia roretzi

The sea squirt Halocynthia roretzi is an important marine food resource species that is found in the waters around Korea. We describe the isolation and characterization of 13 new polymorphic microsatellite loci in 96 sea squirt samples that were collected from the marine environment of Samcheok on the east coast of Korea. The number of alleles that were observed for each locus ranged from six to 32, and the value of expected and observed heterozygosities was 0.504-0.922 and 0.396-0.813, respectively. These markers will be useful tools for future population studies.     

Self‐sterility of eggs induced by exogenous and endogenous protease in the solitary ascidian,Halocynthia roretzi

The unfertilized eggs (UFE) of the solitary ascidian, Halocynthia roretzi, which are released naturally, are strictly self‐sterile. However, ovarian eggs isolated after spawning, which are expected to develop into UFE on the following day, are self‐fertile. Some exogenous proteases‐trypsin, chymotrypsin, papain and elastase‐induced self‐sterility in the self‐fertile ovarian eggs within an hour in vitro. The establishment of self‐sterility by the exogenous protease did not require the synthesis of new protein, or the participation of follicle cells. Some of the ovarian eggs were able to differentiate into self‐sterile eggs spontaneously in vitro. The protein synthesis inhibitors puromycin and cycloheximide had no effect on the spontaneous establishment of self‐sterility. However, several protease inhibitors such as leupeptin, soybean trypsin inhibitor (SBTI) and antipain, did inhibit the spontaneous establishment of self‐sterility. The possible participation of trypsin‐like protease in the establishment of self‐sterility in the ovary is discussed. Mol. Reprod. Dev. 52:99–106, 1999. © 1999 Wiley‐Liss, Inc.     

Structure of multinucleated smooth muscle cells of the ascidian Halocynthia roretzi

Summary Cells isolated from ascidian smooth muscle were about 1.5–2 mm in length. Each contained 20–40 nucle in proportion to cell length. The cytoplasm was characterized by the presence of an enormous quantity of glycogen particles, tubular elements of sarcoplasmic reticulum coupled to the cell membrane, and conspicuous contractile elements. Thick and thin filaments had diameters of about 14–16 nm and 6–7 nm, respectively. The population density of the thick filaments was much higher (mean 270/m² filament area) than in vertebrate smooth muscles. The ratio of thick to thin filaments was about 16. All the thick filaments were surrounded by a single row of 5–9 thin filaments forming a rosette, and cross-bridges with periodicities of 14.5 and 29 nm were found between them. The contractile apparatus consisted of numerous myofibrils which were arranged nearly along the cell axis and were separated from each other by a network of 10-nm filaments. The myofibrils further consisted of many irregularly arranged sarcomerelike structures, each of which was comprised of a small group of thick and thin filaments with attached dense bodies.     

Classification,inhibition, and specificity studies of the vitelline coat lysin from toad sperm

The sonicated supernatant of the sperm of the toad, Bufo japonicus, can digest easily the vitelline coat (VC) of uterine eggs, and to a lesser extent the VC of coelomic eggs, but not that of activated eggs. The VC lysis and fertilization were competitively inhibited in the presence of t-butyloxycarbonyl-L-Gln-L-Arg-L-Arg-4-methylcoumaryl-7-amide (Boc-Gln-Arg-Arg-MCA), suggesting the involvement of proteases in the fertilization process. Starting from a sonicated supernatant, a potent VC lysin, possessing hydrolytic activity on Boc-Gln-Arg-Arg-MCA, was obtained by anion-exchange chromatography and gel filtration. The activity of the partially purified lysin was inhibited by diisopropyl fluorophosphate (DFP) and by such trypsin inhibitors as soybean trypsin inhibitor, leupeptin, and (p-amidinophenyl) methanesulfonyl fluoride hydrochloride, but not by chymostatin, E-64, and ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid. The molecular weight of the lysin was estimated to be 32K, based on the fluorographic image of ³H-DFP binding to the lysin on sodium dodecyl sulfate gel electrophoresis. The VC lysin was most active at pH 7.0–7.6 and under low ionic strength equivalent to fresh water. The release of the VC lysin was induced upon incubation of sperm with the contents of oviducal pars recta granules (PRG), which are known to induce the acrosome reaction. We conclude that the protease studied here represents the VC lysin of toad sperm that is involved in fertilization by digesting the VC of uterine eggs, probably released as a result of the acrosome reaction induced by PRG.     

Establishment of self-sterility of eggs in the ovary of the solitary ascidian,Halocynthia roretzi

When unfertilized eggs (UFE) of the solitary ascidian, Halocynthia roretzi, are released naturally they are strictly self-sterile, whereas almost all ovarian eggs isolated after spawning are self-fertile. Self-sterile eggs are prepared within a relatively short period of several hours before the spawning. The morphological changes in ovarian eggs during late oogenesis were studied with special reference to the establishment of self-sterility. Four types of eggs at serial developmental stages were classified according to the morphology of their external envelopes. Self-sterility was established in the last stage, from the ovarian egg type 3 (OVE3) to UFE stages. Ovarian eggs which had become committed to UFE were denoted as full-grown ovarian eggs (FOE). FOE were able to differentiate into self-sterile UFE in vitro, whereas OVE3 could not. Several morphological differences between OVE3 and UFE were found. OVE3 had a germinal vesicle (GV), a type of vitelline coat (VC-OVE3) and no expanded perivitelline space, whereas UFE had completed germinal vesicle break down (GVBD), had another type of coat (VC-UFE) and showed an expanded perivitelline space. There were also some differences in the mode of fertilization between OVE3 and UFE. In UFE, sperm became bound firmly to the vitelline coat and passed through the coat with the help of follicle cells, whereas in OVE3, sperm did not bind so strongly and entered the perivitelline space without the aid of follicle cells. The relationships between the establishment of self-sterility and these morphological and functional changes in ovarian eggs are discussed.     

Localization and roles in fertilization of sperm proteasomes in the ascidian Halocynthia roretzi

We previously reported that sperm proteasome is responsible for degradation of the ubiquitinated vitelline-coat during fertilization in the ascidian Halocynthia roretzi. Here, we report the roles in fertilization and localization in the sperm cell surface of H. roretzi sperm proteasome. An anti-proteasome antibody, as well as the proteasome inhibitors MG115 and MG132, inhibited the fertilization, indicating that the sperm proteasome functions extracellularly in ascidian fertilization. In order to further assess this issue, the sperm surface proteasome activity was labeled with a cell-impermeable labeling reagent, NHS-LC-biotin, extracted with 0.1% CHAPS, and was subjected to a pull-down assay with avidin-agarose beads. It was found that a substantial amount of sperm proteasome is exposed to the cell surface. Partition analysis with Triton X-114 also revealed that a considerable amount of the sperm proteasome activity is partitioned into a lipid layer. Localization of the proteasome activity was investigated by fluorescence microscopy with succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide as a substrate. The sperm proteasome activity was specifically detected in the sperm head region, and it was markedly activated upon sperm activation. The membrane-associated proteasome was purified from the membrane fraction of H. roretzi sperm by affinity chromatography using an anti-20S proteasome antibody-immobilized Sepharose column. SDS-PAGE of the purified preparation showed a similar pattern of subunit composition to that of the 26S proteasome of mammalian origin. Taken together, these results indicate that H. roretzi sperm has the membrane-associated proteasome on its head, which is activated upon sperm activation, and that sperm proteasome plays an essential role in H. roretzi fertilization.     

Involvement of carbohydrate moieties of the toad egg vitelline coat in binding with fertilizing sperm

Vitelline coats (VC) were isolated from the eggs of Bufo japonicus , and were added with sperm in reconstituted salt solution, which mimics the physiological role of jelly envelopes, to determine the rates of sperm binding per unit area (0.2 mm²) of VC. The rate of sperm binding to VC from uterine eggs was high, but was low to VC from coelomic eggs and eggs activated in 1/20 De Boer's solution (DB) and moderately low to VC from eggs activated in DB. The binding rate increased when VC from coelomic eggs were treated with extracts of the pars recta portion of the oviduct. The sperm that bound to VC were not acrosome-reacted and their binding to VC required both a low salinity, assuring motility of sperm, and sufficiently high levels of Ca²⁺ and Mg²⁺. The rate of sperm binding was reduced by either coexisting solubilized VC materials, periodate-oxidation of VC or the pretreatment of VC with Fab fragments of anti-VC antibodies, which reacted mostly to carbohydrate residues of VC glycoproteins. Sperm-VC binding assays in combination with gel-filtrated VC components revealed that the fractions containing 36–39 kDa components were most effective both in inhibiting the binding and in neutralizing the antibody induced inhibition of binding. These results indicate that carbohydrate moieties in 36–39 kDa glycoproteins of VC, exposed as a result of hydrolysis by the oviducal pars recta protease, are involved in binding with fertilizing sperm.     

Storage of retinal in the eggs of the ascidian,Halocynthia roretzi

Retinoids in the eggs of the solitary ascidian, Halocynthia roretzi, were analyzed by high performance liquid chromatography. Retinal was the almost exclusive retinoid (>99%), and the concentration of retinal was 25.9-40.1 (30.6 on average) ng/mg of protein. The egg retinal consisted of four isomers: all-trans (50.9%), 9-cis (6.8%), 11-cis (20.4%) and 13-cis (21.9%). The presence of retinal in the eggs of this ascidian is a characteristic shared with the wide range of oviparous vertebrates, although the isomer composition differs between ascidian eggs and vertebrate eggs; in vertebrate eggs, almost all the retinal is in the all-trans form. The egg retinal was bound to a protein complex via a Schiff base linkage. The electrophoretic characteristics of the protein complex were similar to that of egg yolk proteins of oviparous vertebrates. The results presented in this study strongly suggest that, as is found with oviparous vertebrates, retinal in the ascidian eggs is the essential mode of retinoid storage, and is the precursor of photoreceptive pigment chromophores and retinoic acid during development.     

Oviducal pars recta-induced degradation of vitelline coat proteins in relation to acquisition of fertilizability of toad eggs

In the toad Bufo bufo japonicus the vitelline coat (VC) of the uterine egg (UEVC) is more readily lysed by the sperm lysin than the VC of coelomic egg (CEVC). Fluorometric determinations of released proteins after incubation of the VC with the sperm lysin in vitro revealed that the CEVC is not completely refractory to the lysin but increases in susceptibility after treatment with a pars recta extract (PRE). Experiments employing isolated pars recta granules showed that both this increase of VC susceptibility and the acquisition of egg fertilizability are ascribable to the contents of the granules. SDS-PAGE analyses of VC proteins revealed that in comparison with CEVC, UEVC lacks 40–52K proteins concomitant with the increased stainability of 39K protein and the appearance of 36K protein. These changes in SDS-PAGE profiles were observed either in oviducal eggs after passage through the pars recta or in coelomic eggs treated with PRE but were inhibited when coelomic eggs were treated with PRE containing soybean trypsin inhibitor (SBTI) and leupeptin. Likewise, the acquisition of fertilizability by treatment of coelomic egg with PRE was inhibited by SBTI. Dejellied uterine eggs were successfully fertilized when pretreated with trypsin inhibitors before insemination but were not fertilized when pre-treated with concanavalin A. We propose that the hydrolytic degradation of certain VC proteins due to the tryptic activity of pars recta granules renders the VC susceptible to the sperm lysin, so that the eggs are made receptive to a fertilizing sperm.     

Self and non-self recognition between gametes of the ascidian,Halocynthia roretzi

Summary The self-sterility ofHalocynthia roretzi from Mutsu Bay, Japan, was examined. This sterility is strict and not a single egg can be fertilized in self-sterile animals. Less than 2% of the animals were self-fertile (with 100% cross-fertility). All heterologous sperm can fertilize all eggs, although there are pairs of individuals in which the coelomocytes recognize each other as self. Eggs deprived of follicle cells cannot be fertilized by either autologous or heterologous spermatozoa. Detached autologous or heterologous follicle cells can reattach to the chorion in calcium-enriched sea water and the reconstituted eggs recover their ability to be fertilized. A mosaic egg can therefore be obtained, which consists of oocyte, test cells and chorion originating from one individual and follicle cells from another. The mosaic egg was used to determine the site of recognition of self and non-self. The results indicate that the recognition resides in the chorion and/or test cells, probably the chorion. The relationship between somatic alloreactivity, previously found in coelomocytes ofH. roretzi, and gamete reactivity is discussed.     

Developmental fates of larval tissues after metamorphosis in the ascidian, Halocynthia roretzi

Cell lineages during ascidian embryogenesis are invariant. Developmental fates of larval mesodermal cells after metamorphosis are also invariant with regard to cell type of descendants. The present study traced developmental fates of larval endodermal cells after metamorphosis in Halocynthia roretzi by labeling each endodermal precursor blastomere of larval endoderm. Larval endodermal cells gave rise to various endodermal organs of juveniles: endostyle, branchial sac, peribranchial epithelium, digestive organs, peripharyngeal band, and dorsal tubercle. The boundaries between clones descended from early blastomeres did not correspond to the boundaries between adult endodermal organs. Although there is a regular projection from cleavage stage and larval stage to juvenile stage, this varies to some extent between individuals. This indicates that ascidian development is not entirely deterministic. We composed a fate map of adult endodermal organs in larval endoderm based on a statistical analysis of many individual cases. Interestingly, the topographic position of each prospective region in the fate map was similar to that of the adult organ, indicating that marked rearrangement of the positions of endodermal cells does not occur during metamorphosis. These findings suggest that fate specification in endoderm cells during metamorphosis is likely to be a position-dependent rather than a deterministic and lineage-based process. Received: 16 June 1999 / Accepted: 16 August 1999     

Primary structure of ascidian trypsin inhibitors in the hemolymph of a solitary ascidian, Halocynthia roretzi

The amino acid sequences of trypsin inhibitors I and II from the hemolymph of a solitary ascidian, Halocynthia roretzi, were determined after reduction and S-pyridylethylation. The results indicated that inhibitor I consists of a single polypeptide chain with 55 amino acid residues and four intramolecular disulfide bridges, whereas inhibitor II is composed of two polypeptide chains corresponding to a form derived from inhibitor I by cleavage at the Lys16-Met17 bond. Lys16 may be the reactive-site residue of these inhibitors, because carboxypeptidase B treatment destroys most of the inhibitory activity of inhibitor II but not that of inhibitor I.     

A novel G protein alpha subunit in embryo of the ascidian,Halocynthia roretzi

A cDNA clone encoding a novel G protein alpha subunit, HrGalpha(n) was isolated from the larvae of ascidian, Halocynthia roretzi. In contrast with overall amino acid identity (63%) with G protein alpha subunit of G(i) or G(o) subclass, HrGalpha(n) has a unique amino acid sequence, which lacks a residue for pertussis toxin substrate, but retains for cholera toxin substrate for ADP-ribosylation. The sequence characteristics and molecular phylogenetic analysis suggest that HrGalpha(n) defines a novel subclass within G(i) class of G protein alpha subunits. The zygotic expression of HrGalpha(n) was first detected at the 64-cell stage and observed in all blastomeres except for B7.4, B7.5 and B7.6 cells till the 110-cell stage. As progress of the developmental stages, the expression of HrGalpha(n) became restricted and was observed in the muscle, mesenchyme and a part of trunk lateral cells in tailbud embryos. With HrGalpha(n)-GFP fusion-gene construct it was showed that the genomic fragment containing 2674 bp upstream of the putative translation start site of HrGalpha(n) contained the regulatory sequence responsible for the expression in the muscle and mesenchyme cells, and that the regulatory sequence functioned also in Ciona intestinalis. Our results suggest a possible involvement of HrGalpha(n) in the signaling system regulates the cell fate during the embryogenesis of the ascidian.     

The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.     

Spatio-temporal pattern of MAP kinase activation in embryos of the ascidian Halocynthia roretzi

To understand developmental mechanisms, it is important to know when and where signaling pathways are activated. The spatio-temporal pattern of activation of mitogen-activated protein kinase (MAPK/ERK) was investigated during embryogenesis of the ascidian Halocynthia roretzi, using an antibody specific to the activated form of MAPK. During cleavage stages, activated MAPK was transiently observed in nuclei of the precursor blastomeres of endoderm, notochord, mesenchyme, brain, secondary muscle, trunk lateral cells and trunk ventral cells. These sites of MAPK activation are consistent with results of previous studies that have analyzed the embryonic induction of various tissues, and with results of inhibition of MAPK kinase (MEK) in ascidians. Activation of MAPK in notochord and mesenchyme blastomeres was observed in a short period in a single cell cycle. In contrast, in brain and secondary muscle lineages, MAPK activation spanned two or three cell cycles, and upon each cleavage, MAPK was asymmetrically activated in only one of the two daughter cells that remained brain or secondary muscle lineages. During later stages, MAPK activation was predominantly observed in the central nervous system. A conspicuous feature at this stage was that activation appeared to alternate between positive and negative along the anterior-posterior axis of the neural tube. During the tail elongation stage, MAPK was quiescent.     

Distribution of cytoplasmic determinants in unfertilized eggs of the ascidian Halocynthia roretzi

 Cytoplasmic determinants that specify the fate of endoderm, muscle and epidermis cells are known to be localized in specific areas of fertilized eggs of ascidians. The presence of such cytoplasmic determinants in unfertilized eggs was demonstrated in previous studies, but no information has yet been proved about their distribution. To investigate the distribution of cytoplasmic determinants in unfertilized eggs, we devised a method for distinguishing the polarity of unfertilized eggs using vital staining and we performed cytoplasmic-transfer experiments by fusing blastomeres and cytoplasmic fragments from various identified regions of unfertilized eggs. Cytoplasmic fragments, that contained cortical and subcortical material, from five different positions along the animal-vegetal axis were prepared, and they were fused with a4.2 (presumptive-epidermis) or A4.1 (non-epidermis) blastomeres. The ectopic development of endoderm, muscle and epidermis cells that was promoted by the transplanted cytoplasm was assessed by examining the expression of alkaline phosphatase (ALP), myosin and epidermis-specific antigen, respectively. Differentiation of endoderm and muscle was observed at higher frequencies as cytoplasmic fragments closer to the vegetal pole were transplanted. Conversely, formation of epidermis was observed at higher frequencies as cytoplasmic fragments closer to the animal pole were transplanted. The results suggest that, in cortical and subcortical regions of unfertilized ascidian eggs, endoderm and muscle determinants are widely distributed along a gradient, with maximum activity at the vegetal pole, whilst epidermis determinants are also distributed along a gradient but with maximum activity at the animal pole. Recieved: 10 June 1996 / Accepted: 12 September 1996