Ascidian Fertilization Research Articles

CDNA Cloning and Functional Analysis of Ascidian Sperm Proacrosin

cDNA cloning and functional analysis of proacrosin from the ascidian Halocynthia roretzi were undertaken. The isolated cDNA of the ascidian preproacrosin consists of 2367 nucleotides, and an open reading frame encodes 505 amino acids, which corresponds to the molecular mass of 55,003 Da. The mRNA of proacrosin was found to be specifically expressed in the gonad by Northern blotting and in the spermatocytes or spermatids by in situ hybridization. The amino acid sequences around His(76), Asp(132), and Ser(227), which make up a catalytic triad, showed high homology to those of the trypsin family. Ascidian acrosin has paired basic residues (Lys(56)-His(57)) in the N-terminal region, which is one of the most characteristic features of mammalian acrosin. This region seems to play a key role in the binding of (pro)acrosin to the vitelline coat, because the peptide containing the paired basic residues, but not the peptide substituted with Ala, was capable of binding to the vitelline coat. Unlike mammalian proacrosin, ascidian proacrosin contains two CUB domains in the C-terminal region, in which CUB domain 1 seems to be involved in its binding to the vitelline coat. Four components of the vitelline coat that are capable of binding to CUB domain 1 in proacrosin were identified. In response to sperm activation, acrosin was released from sperm into the surrounding seawater, suggesting that ascidian acrosin plays a key role in sperm penetration through the coat. These results indicate that ascidian sperm contains a mammalian acrosin homologue, a multi-functional protein working in fertilization.

Cell cycle-dependent repetitive Ca(2+ )waves induced by a cytosolic sperm extract in mature ascidian eggs mimic those observed at fertilization.

Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca(2+) waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca(2+ )waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.

Activation of follicle cell surface phospholipase by tyrosine kinase dependent pathway is an essential event in Ascidian fertilization.

Eggs of Ascidia ceratodes and Phallusia mammillata block polyspermy by releasing a phosphatidylinositol-linked glycosidase from the follicle cell and egg surface that binds to and blocks all unoccupied sperm binding sites on the vitelline coat. Release of this glycosidase is thought to be under the control of a membrane-bound phospholipase. To elucidate the mechanism of phospholipase activation, intact eggs and isolated follicle cells are activated by either sperm or the tyrosine kinase activator 9, 10-dimethyl-1,2-benzanthracene (DMBA). Both treatments caused release of comparable quantities of glycosidase activity, the earliest event following fertilization. A corresponding increase in phospholipase activity accompanied this glycosidase release. The tyrosine kinase inhibitor genistein blocked release by DMBA at concentrations as low as 1 microM, but had no effect on sperm-induced release even when used up to 100 microM. Tyrphostin A23, another tyrosine kinase inhibitor, when used at 200 microM blocked glycosidase release and decreased phospholipase activity following both DMBA activation and fertilization. Western blot analysis probing for phosphotyrosine content of disrupted intact eggs with their follicle cells revealed the absence of a band in tyrphostin-treated eggs corresponding to a 40 kDa protein that was present in both unfertilized and fertilized egg samples. Based on these results, we propose that phosphorylation of specific tyrosine residues is necessary for phospholipase activation and is sufficient to trigger subsequent glycosidase release.

Injection of sperm extract mimics spatiotemporal dynamics of Ca2+ responses and progression of meiosis at fertilization of ascidian oocytes.

Sperm extract (SE) of the ascidian, Ciona savignyi, injected into oocytes induced repetitive intracellular Ca2+ increases with kinetics consistent with those at fertilization and caused reinitiation and progression of meiosis as in fertilized oocytes with the formation of polar bodies. The Ca2+ response comprised two sets of Ca2+ oscillations separated by 5 minutes and correlated with the first and second meiotic metaphase. The effects of SE were dose dependent and the critical dose corresponded roughly to a single spermatozoon. In the first Ca2+ transient observed by confocal microscopy, a Ca2+ wave started from the SE injection site at the peripheral region of the oocyte and propagated across the ooplasm. The similar wave was produced by injection at the central region, starting from an arbitrary cortical area after 30 seconds, probably after SE had diffused to the cortex. The sensitivity to SE is thought to be preferentially higher in the cortex. The effective component of SE was heat-unstable, and its molecular weight was estimated as in the range between 10x10(4 )and 3x10(4) using membrane filters. These results suggest that, in ascidian fertilization, a cytosolic sperm protein factor is introduced to the oocyte cortex and induces Ca2+ waves and thereby meiotic resumption, leading to cell-cycle-correlated Ca2+ oscillations.

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat.

In the initial stage of ascidian fertilization sequential sperm-egg coat interactions assure successful species-specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio-iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non-radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration-dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg-binding activity. Three biotinylated proteins (66 kDa, 120 kDa and 140 kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120 kDa protein possesses species-specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species-specific recognition in P. mammillata.

The Two Intracellular Ca2+Release Channels, Ryanodine Receptor and Inositol 1,4,5-Trisphosphate Receptor, Play Different Roles during Fertilization in Ascidians

Fertilization in the ascidians triggers an activation wave of calcium release followed by intracellular calcium oscillations synchronous with periodic membrane potential excursions during the completion of the meiotic cell cycle. Fertilization also causes a fast decrease in the egg plasma membrane depolarization-activated calcium current and a large increase in capacitance thought to represent membrane addition to the egg surface. We have analyzed the temporal and causal relationships between these changes in the eggs ofPhallusia mammillatausing whole-cell patch-clamp recording while simultaneously imaging calcium with fura-2 dextran. We have defined the role of ryanodine receptor (RyR) and InsP3receptor (InsP3R) during fertilization and meiosis by looking at the effects of InsP3, cyclic ADP ribose (cADPR), and ryanodine in perfused oocytes. We show that InsP3(10 μMperfused through the patch pipette) is able to trigger sustained oscillations in intracellular calcium concentration in unfertilized oocytes, resembling those recorded in fertilized egg completing meiosis. In addition the sustained oscillations resulting from InsP3perfusion in unfertilized oocytes are sufficient to cause the emission of both polar bodies. In contrast, ryanodine or cADPR never trigger detectable calcium signal in perfused oocytes. Instead, nanomolar concentrations of ryanodine or cADPR cause a capacitance change, implying a net insertion of membrane to the oocyte surface, and trigger a fast decrease in the depolarization-activated calcium current. Both changes are similar to the changes in conductance and capacitance naturally observed following fertilization. These effects, although not associated with measurable calcium signals, are abolished by coperfusion of the calcium chelator BAPTA. In contrast to ryanodine or cADPR, sustained perfusion of the oocyte with nanomolar concentrations of InsP3causes no capacitance change and a slow and moderate decrease in calcium current. Our observations on inseminated patch-clamped eggs further indicate that membrane insertion, which starts 15–20 sec after the onset of the membrane conductance change at fertilization, can be altered by interfering with the RyR. Our results imply that, in ascidians, as in some mammals, RyR and InsP3R play distinct roles during fertilization.

Substrate specificity of ascidian sperm trypsin-like proteases, spermosin and acrosin.

In order to investigate systematically the substrate or subsite specificity of two sperm proteases, acrosin and spermosin (a novel trypsin-like protease) of the ascidian, Halocynthia roretzi, the effects of peptidyl-argininals on the purified enzymes as well as on fertilization were examined. Among four benzyloxycarbonyl (Z)-Leu-X-argininals (X = Pro, Leu, Ser, and Gly), Z-Leu-Pro-argininal showed the strongest inhibition toward the spermosin activity. On the P3 site specificity, Val-Pro-argininal derivatives showed a stronger inhibition than a Leu-Pro-argininal derivative, suggesting the preference of Val rather than Leu residue at the P3 position. Similar results were obtained by analyzing the hydrolyzing activity of the fluorogenic peptide substrates: it hydrolyzed Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide (MCA) most efficiently, and Boc-Asp(O-benzyl)-Pro-Arg-MCA was the next best substrate, but Gly-Pro-Arg (or Lys)-MCAs were hardly hydrolyzed. On the other hand, acrosin was found to prefer Leu or Pro residue rather than Gly or Ser residue at the P2 position as revealed by comparing the Ki values of peptidyl-argininals. Detailed kinetic analysis on the inhibitory abilities of peptidyl-argininals toward the purified enzymes and the ascidian fertilization suggested that both acrosin and spermosin are involved in ascidian fertilization.

Localization, Expression, and the Role in Fertilization of Spermosin, an Ascidian Sperm Trypsin-like Protease

In order to elucidate the role of spermosin (a novel sperm trypsin-like protease) in ascidian fertilization, we developed an antibody against the ascidian spermosin: the antibody appears specific to the spermosin but not to the acrosin. By Western blot analysis, spermosin was found to be expressed just before and during the spawning season. Immunocytochemical studies have shown that spermosin is localized on the sperm head. It was found that the spermosin was released from the sperm during the sperm reaction, and the anti-spermosin antibody, but not control antibody, inhibited the fertilization in a concentration-dependent manner. These results indicate that spermosin plays an essential role in ascidian fertilization.

Physiological functions of proteasomes in ascidian fertilization and embryonic cell cycle.

Fertilization is initiated by sperm binding to the vitelline coat, a glycoprotein coat, of the eggs. After the sperm binding, sperm undergoes the acrosome reaction, an exocytosis of the acrosome. By this acrosome reaction, sperm proteases or lytic agents in the acrosome which allows sperm to penetrate through the vitelline coat are exposed and released. Subsequently, the sperm-egg membrane fusion occurs, which triggers a transient increase in intracellular calcium ions, leading to the exocytosis and the resumption of the meiotic division cycle, i.e., egg activation.

Glycosidases, proteases and ascidian fertilization

Spermatozoa should bind to and then penetrate the vitelline coat for fertilization in ascidians and many other animals. There is substantial evidence that the binding of ascidian sperm is mediated by a sperm glycosidase and complementary saccharide chains of glycoproteins in the vitelline coat. Involvement of a sperm proteasome in the binding is also suggested. For the penetration, sperm proteases such as chymotrypsin-like enzyme, acrosin, spermosin and proteasome are suggested to play essential roles. Sperm glycosidase, that is translocated from the tip of sperm head to the surface overlying the mitochondrion, anchors the mitochondrion at the outer surface of vitelline coat. Therefore it assists sperm to penetrate the vitelline coat and traverse the perivitelline space. For fusion with egg plasma membrane, sperm metalloendoprotease seems to be involved. Egg glycosidases and proteases serve for some steps after fertilization, such as the prevention of polyspermy, expansion of perivitelline space and regulation of cell cycle.

Sperm-Surface Chymotrypsin-like Protease Activity Required for Fertilization in Ascidians

During fertilization, species-specific gamete binding must be followed by sperm penetration of egg vestments before gamete fusion can occur. Sperm proteases, called lysins, aid this process. Sperm from Ascidia ceratodes, Ascidia callosa, and Ascidia paratropa were found to have a surface-mounted chymotrypsin-like protease when studied by enzymology, biotinylation, immunolabeling, and histochemistry. Chymotrypsin substrates and inhibitors blocked fertilization in a concentration-dependent manner in A. ceratodes and decreased the number of sperm heads which penetrated the egg's vitelline coat, but had no effect on sperm binding to follicle cells. Sperm bound to agarose beads coated with the chymotrypsin inhibitor α2-macroglobulin. Chymotrypsin-like enzyme activity, assayed fluorimetrically using N -succinyl-leucinyl-leucinyl-valinyl-tyrosinyl-7-amido-4-methyl-coumarin as the substrate, was associated with head fractions prepared by differential centrifugation. Biotinylation of live sperm followed by detergent extraction showed that chymotrypsin-like activity could be removed from the detergent extract using avidinagarose beads. Indirect immunofluorescence of unreacted and reacted sperm heavily labeled membrane domains overlying the mitochondrion and at the base of the head with occasional labeling of the sperm tip. Histochemical studies, which used N -succinyl-alanyl-alanyl-prolyl-phenylalanyl-β-napthylamide as the substrate, colocalized enzyme activity in head regions of unreacted and near the mitochondrion of reacted sperm. Thus, we conclude that in ascidian sperm a chymotrypsin-like protease is exposed on the external surface of the plasma membrane of the head, is required for fertilization, and plays a role in sperm penetration but not binding.

Effects of protease inhibitors on binding of sperm to the vitelline coat of ascidian eggs: implications for participation of a proteasome (multicatalytic proteinase complex).

Among various protease inhibitors, chymostatin (an inhibitor of sperm chymotrypsin-like protease) strongly inhibited the binding of sperm to the vitelline coat of glycerinated eggs of the ascidian Halocynthia roretzi, whereas leupeptin (an inhibitor for sperm acrosin), antipain, and soybean trypsin inhibitor had no significant inhibitory effects. Dansyl-Val-Pro-argininal (an inhibitor of the sperm trypsin-like protease, spermosin) had an inhibitory effect on the binding of sperm that was much smaller than its effects on fertilization. Since the sperm chymotrypsin-like protease that is involved in ascidian fertilization has been identified as a proteasome (multicatalytic proteinase complex), we tested the effects of several peptidyl argininals, inhibitors of the activities of proteasomes, on this binding process. The ranking of the inhibitory effects of these compounds on the binding of sperm was the same as that of their effects on the chymotrypsin-like activity of the proteasome, reported previously. The potent inhibitors of binding used in these studies had no or minimal effects on sperm motility. These results suggest that a sperm chymotrypsin-like protease (most probably the chymotrypsin-like protease in the proteasome) plays a key role in binding of sperm to the vitelline coat of the ascidian egg.

Involvement of prolyl endopeptidase in ascidian fertilization.

Inhibitory effects on fertilization of the ascidian of three benzyloxycarbonyl(Z)-aminoacyl prolinals and Z-Gly-Pro-chloromethyl ketone added before and after insemination were examined. The results suggest that the prolyl endopeptidase is involved in the process of fertilization, especially in a process taking place between chorion elevation and cell cleavage.

Sperm Binding and Penetration during Ascidian Fertilization*: (Ascidian/fertilization/sperm/glycosidase/actin).

Sperm Binding and Penetration during Ascidian Fertilization*: (Ascidian/fertilization/sperm/glycosidase/actin).

Fertilization in ascidians: Studies on the egg envelope, sperm and gamete interactions in Phallusia mammillata

Eggs and sperm of the ascidian Phallusia mammillata were studied by light microscopy and scanning and transmission electron microscopy. The egg is enclosed in a rigid acellular envelope, the vitelline coat (vc). The vc consists of a fibrous surface layer, a homogeneous electron dense central layer and a thick inner layer with thin fibers and globular elements. Vacuolated follicle cells are attached to the outer, test cells to the inner surface of the vc. Upon staining with phalloidine-rhodamin both cell types exhibit a distinct pattern of actin filaments. Studies of gamete interactions during fertilization reveal that sperm binding is established between the tip of the sperm and the fibrous layer of the vc at sites which are not covered by follicle cells. Freeze fractures of quick frozen sperm show a cap-like structure at the sperm apex. This structure contains a number of vesicles as detected in sperm prepared for TEM by quick freezing and freeze substitution. In sperm just about to penetrate the vc apical vesicles which may contain the vitelline coat lysins have been found. Wheat germ agglutinin (WGA), which binds to the vc but not to follicle cells and sperm, inhibits fertilization by interfering with sperm-vitelline coat binding. This inhibition was completely abolished by competitive sugars such as N,N′,N″-triacetylchitotriose (1 m M) or N-acetylglucosamine. However, there are indications that WGA binding sites on the vc may not only be N-acetylglucosamine but also sialic acid residues. Preliminary studies reveal similarities between ascidian and mammalian fertilization concerning the phagocytosis-like gamete fusion process.

The acrosome in ascidians. I. Pleurogona

Summary A vesicle which contains moderately electron-dense material has been found at the apex of mature spermatozoa in all representatives of three pleurogonan families: in Styela clava, Cnemidocarpa finmarkiensis and Botryllus schlosseri (family Styelidae), in Boltenia villosa and Herdmania momus (family Pyuridae), and in Molgula manhattensis (family Molgulidae). The vesicle described here resembles the acrosome of Ciona intestinalis spermatozoa. The Ciona acrosome shows structural changes at fertilization (Fukumoto, M., J. Ultrastruct. Res., 87 (1984) 252–262). This suggests that pleurogonan spermatozoa also have an acrosome. Some speculations are presented on ascidian fertilization.

Timing of action of sperm proteases in ascidian fertilization

The timing of action of three sperm proteases, acrosin, spermosin, and a chymotrypsin-like enzyme, in the fertilization of the ascidian,Halocynthia roretzi, was examined by adding specific protease inhibitors at various times after insemination. The results indicate that the last two enzymes both function at the early stage of the process of sperm penetration through the egg investment, while acrosin functions at the late stage.

Sperm-egg interactions preparatory to fertilization.

In 1902, Boveri introduced the important concept that for the success of fertilization the gametes must activate one another. Based primarily on studies on the sea urchin and ascidian fertilization the suggestion is presented here that "activation" of the spermatozoon actually involves a switching off of its metabolic machinery as a result of its interaction with the sperm receptors of the egg envelopes and prior to its fusion with the egg. Concerning the activation of the egg, there is a fairly large body of old and new experimental evidence that activation per se does not require sperm incorporation. Indeed, the chain of reactions culminating in the activation of the egg is initiated upon attachment of the spermatozoon to the egg plasma membrane.

Fertilization in ascidians: acrosome fragmentation in Ciona intestinalis spermatozoa

The acrosome of Ciona intestinalis spermatozoa is a flattened vesicle, approximately 200 × 180 × 40 nm in length, width, and height, respectively. It contains moderately electron-dense material with an electron-dense plate in its center. The outer membrane of the acrosome is in close contact with the overlying plasmalemma. Neither a subacrosomal substance nor a perforatorium can be identified in the subacrosomal space. The acrosome fragments into smaller vesicles (approximately 20–30 nm in diameter) in both suspended and inseminated spermatozoa. This phenomenon has been observed in all spermatozoa examined in both cases. The sperm reaction ( C. C. Lambert and D. Epel, 1979 , Dev. Biol. 69, 296–304) is not a prerequisite for the initiation of the fragmentation. The possibility is presented that the structural change in the acrosome reported here is a new type of acrosome reaction which has not been previously described. Some speculations are also presented on ascidian fertilization.

Interspecific Fertilization in Ascidians

THE only experimental work on interspecific fertilization in Ascidians known to me is that of Reverberi1, who succeeded in fertilizing Ciona intestinalis eggs with Phallusia mamillata sperms after removal of the egg membranes. He availed himself of Morgan‘s observation that in Ciona only the egg membranes oppose self-fertilization.