Fertilization Envelope Research Articles

Purification and Partial Characterization of a Lectin-like Protein from the Sea Algae Laminaria diabolica that Induces Fertilization Envelope Formation in Sea Urchin Eggs

Abstract When unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus were treated with the extracts from the sea algae Laminaria diabolica, fertilization envelope formation (FEF) was observed in 1.5 to 2 min without subsequent cleavage. The molecular mass of the FEF-inducing protein purified from the Laminaria extracts was estimated by gel-filtration to be 120 kDa, and it induced a 100% FEF at a concentration of 525 ng/ml (4.4 nM). The FEF activity of the purified protein was completely inhibited by GlcNAc, less effectively by GlcN, and not at all by various other sugars and amino-sugars, suggesting its nature as a lectin. Normal FEF and cleavage were observed when eggs were inseminated in the presence of GlcNAc. Immunohistochemical observations using antibodies against purified protein revealed its specific localization in the cells lining the wall of mucilaginous lacunae and in secretory products in the lumen. The purified protein did not activate the pretreated with verapamil or diltiazem, whil...

Tyrosine kinase inhibitors block sperm-induced egg activation in Xenopus laevis.

Fertilization of Xenopus laevis eggs triggers a wave of increased [Ca2+]i. The exact signal transduction pathway culminating in this Ca2+ wave remains unknown. To determine whether increases in tyrosine kinase activity are part of this pathway, we microinjected tyrosine kinase inhibitors into unfertilized eggs. Upon fertilization, signs of activation were monitored, such as fertilization envelope liftoff and the Ca2+ wave (for eggs microinjected with lavendustin A). Various concentrations of lavendustin A and tyrphostin B46 were microinjected, as well as inactive forms of these compounds (lavendustin B and tyrphostin A1) to provide negative controls. Peptide A, a 20-amino-acid peptide derived from the SH2 region of pp60(v-src) tyrosine kinase, was also microinjected. Peptide A inhibits tyrosine kinase activity but not PKA or PKG activity. Dose-response curves for lavendustin A, tyrphostin B46, and peptide A show clear inhibition of vitelline envelope liftoff by these three compounds. Confocal imaging of eggs coinjected with lavendustin A and Oregon Green-dextran showed that the Ca2+ wave was inhibited under normal insemination conditions but that the block of the Ca2+ wave could be overcome with very high sperm densities. A phenomenon of small local Ca2+ increases termed "hot spots" seen in lavendustin A containing eggs is also described. Since this inhibition of egg activation by tyrosine kinase inhibitors can be overcome by Ca2+ microinjection, the inhibitors must act on a step in the signal transduction cascade that is upstream of the Ca2+ wave.

The 350-kDa Sea Urchin Egg Receptor for Sperm Is Localized in the Vitelline Layer

Previous studies have established by several methods that the 350-kDa egg receptor for sperm is localized on the plasma membrane–vitelline layer complex of the egg of the sea urchinStrongylocentrotus purpuratus.In addition, it has been found that molecules which are cross-reactive with anti-receptor antibody are present in the cortical granules located at the inner face of the plasma membrane. The objective of this study was to define more precisely the locale of the cell surface receptor. We have found that following fertilization, the immunoreactive receptor initially found on the egg surface moved to the fertilization envelope (FE) and then disappeared in parallel with the loss of sperm binding activity. A cross-linked, high-molecular-weight derivative of soybean trypsin inhibitor (hMW-SBTI) which was unable to pass through the elevating FE blocked the loss of both immunoreactivity and the sperm binding activity of the FE, but did not inhibit the vitelline delaminase activity that has been implicated in FE formation. Western blot analysis following SDS–PAGE of the proteins of the FE isolated in the presence of hMW-SBTI and benzamidine revealed the presence of the 350/320-kDa proteins which cross-reacted with anti-receptor antibody. Experiments in which molecules on the surface of unfertilized eggs were labeled with biotin and traced after FE formation revealed that a significant portion of the 350/320-kDa glycoproteins that were incorporated into the FE originated from the cell surface, rather than from the cortical granules. These findings provide strong evidence that in unfertilized eggs the egg receptor for sperm exists as part of the protein complex known as the vitelline layer which serves as a precursor of the FE. Evidence is presented indicating that some of the receptor in the vitelline layer is cryptic and a possible function for this cryptic form of the receptor is proposed.

Identification of cystatin as a component of carp chorion.

An ovary-specific cystatin is immunocytochemically demonstrated to be localized in the chorions, cortical granules, and yolk granules of carp oocytes, as well as in the follicle cells surrounding oocytes. During cortical reaction, cystatin is exocytosed from cortical granules into the perivitelline space. In situ hybridization confirms that cystatin is synthesized by oocytes and follicle cells. Western blotting reveals that chorion cystatin appears in multiple bands of high molecular weight (from 65 kDa to larger than 200 kDa). No cystatin monomer of 14 kDa is found. These results indicate that chorion cystatin is conjugated with other chorion components. Two forms of conjugates are found. In one form, cystatin, ZP2, fibroin-like substance (FLS), and cathepsin-like substance (CLS) are conjugated, which is extracted by sodium dodecyl sulfate. In the other form, cystatin, FLS, and CLS are conjugated, which is extracted by guanidine thiocyanate (GTC). Most chorion cystatin of oocytes and ovulated eggs is solubilized by GTC, while a large amount of cystatin remains in the fertilization envelope of cortical reacted eggs after extraction by GTC.

Assembly in vitro of vitelline envelope components induced by a cortical alveolus sialoglycoprotein of eggs of the fish Tribolodon hakonensis.

Assembly in vitro of vitelline envelope (VE) components, which were precipitated by 50-70% saturated ammonium sulphate from VE extracts, was induced by the action of a sialoglycoprotein that is immunohistochemically localised in cortical alveoli of fish eggs and has serine proteinase activity. The VE components consisted of major bands of molecular mass about 150-120, 110-100, 70 and 27 kDa in addition to about 20 minor bands and contained a chorionic transglutaminase, visualised as two fluorescent bands by monodansylcadaverine staining. The VE component assembly in vitro was Ca(2+)-dependent, not induced if the sialoglycoprotein was pretreated with a serine proteinase inhibitor, and inhibited by the presence of p-chloromercuribenzoate, iodoacetamide or L-cysteine in the reaction medium system. Electron microscopy revealed that assembly in vitro of the VE components consisted of aggregates of network sheets, consisting of branching and anastomosing thin (approximately 27-52 nm) and thick (approximately 137-376 nm) filamentous substances. Separation by SDS-PAGE showed that a considerable number of VE components participated in the assembly in vitro in various amounts. These results suggest at least partial reproduction of the phenomena that occur in the process of fertilisation envelope (FE) formation, and provide a new approach to investigation of the process of FE assembly in vitro.

Cortical granule exocytosis is triggered by different thresholds of calcium during fertilisation in sea urchin eggs.

In sea urchin eggs, fertilisation is followed by a calcium wave, cortical granule exocytosis and fertilisation envelope elevation. Both the calcium wave and cortical granule exocytosis sweep across the egg in a wave initiated at the point of sperm entry. Using differential interference contrast (DIC) microscopy combined with laser scanning confocal microscopy, populations of cortical granules undergoing calcium-induced exocytosis were observed in living urchin eggs. Calcium imaging using the indicator Calcium Green-dextran was combined with an image subtraction technique for visual isolation of individual exocytotic events. Relative fluorescence levels of the calcium indicator during the fertilisation wave were compared with cortical fusion events. In localised regions of the egg, there is a 6s delay between the detection of calcium release and fusion of cortical granules. The rate of calcium accumulation was altered experimentally to ask whether this delay was necessary to achieve a threshold concentration of calcium to trigger fusion, or was a time-dependent activation of the cortical granule fusion apparatus after the 'triggering' event. Calcium release rate was attenuated by blocking inositol 1,4,5-triphospate (InsP3)-gated channels with heparin. Heparin extended the time necessary to achieve a minimum concentration of calcium at the sites of cortical granule exocytosis. The data are consistent with the conclusion that much of the delay observed normally is necessary to reach threshold concentration of calcium. Cortical granules then fuse with the plasma membrane. Further, once the minimum threshold calcium concentration is reached, cortical granule fusion with the plasma membrane occurs in a pattern suggesting that cortical granules are non-uniform in their calcium sensitivity threshold.

Sperm-induced local [Ca2+]i rise separated from the Ca2+ wave in sea urchin eggs in the presence of a gamete fusion inhibitor, jaspisin .

An increase in intracellular Ca2+ concentration ([Ca2+]i) at a focal plane was recorded simultaneously with sperm-egg binding and membrane current upon insemination of sea urchin Hemicentrotus pulcherrimus eggs. No change in current and [Ca2+]i occurred in the presence of jaspisin, a novel substance that inhibits metallo-endoproteinase and sperm-egg membrane fusion (S. Ikegami, H. Kobayashi, Y. Myotoishi, S. Ohta and K. H. Kato (1994) J. Biol. Chem. 269, 23262-23267). With low doses of jaspisin, a spermatozoon first produced a step inward current (I(on)) as an indication of gamete membrane fusion and then induced a local [Ca2+]i rise at the site of sperm attachment 6-10 seconds after I(on). The sperm, however, soon detached from the egg. Increasing inward current was abruptly cut off (I(off)) within 9-15 seconds and the local [Ca2+]i rise began to decline 1-3 seconds after I(off). In most cases, no further responses or an elevation of fertilization envelope (FE) occurred. In some cases, [Ca2+]i at the sperm attachment site increased again even after the sperm detached and triggered a Ca2+ wave which caused an activation current and FE formation. This recording of a gamete membrane-fusion-induced local [Ca2+]i rise, separated from the Ca2+ wave, is a key phenomenon for elucidating the initial sperm stimulation of the egg at fertilization.

Sea urchin ovoperoxidase: oocyte-specific member of a heme-dependent peroxidase superfamily that functions in the block to polyspermy

Ovoperoxidase is one of several oocyte-specific proteins that are stored within sea urchin cortical granules, released during the cortical reaction, and incorporated into the newly formed fertilization envelope. Ovoperoxidase plays a particularly important role in this process, crosslinking the envelope into a hardened matrix that is insensitive to biochemical and mechanical challenges and thus providing a permanent block to polyspermy. Here we present the primary structures of two ovoperoxidases as predicted from cDNAs cloned from the sea urchins Strongylocentrotus purpuratus (AF035380) and Lytechinus variegatus (AF035381). We also present a proposed scheme for the post-translational processing of ovoperoxidase based upon comparisons between the cDNA and protein structures and taking into account previously published reports. The sea urchin ovoperoxidase sequences conform to a profile shared by members of a heme-dependent animal peroxidase family, including the mammalian myelo-, lacto-, eosinophil, and thyroid peroxidases. Using in situ RNA hybridizations, we showed that the mRNA of S. purpuratus ovoperoxidase (4 kb) is present exclusively in oocytes, and is turned over rapidly following germinal vesicle breakdown. Taking into account our immunoblot and N-terminal sequencing data along with reports from similar peroxidases, we propose that ovoperoxidases are synthesized in a pre-pro form and proteolytically processed to result in the 70 and 50 kDa forms that are found in the fertilization envelope. The sequence and structural data presented here will facilitate our continuing studies of the biogenesis of cortical granules and the fertilization envelope. Additionally, since ovoperoxidase activities have been reported in a wide range of animals, these cDNAs will be useful in uncovering similar peroxidases used in the fertilization reactions of other metazoan eggs.

Morphology of gametes in five species of far-eastern chitons

Summary Sperm development, structure of the gametes and some aspects of fertilization in the far-eastern chitons Leptochiton assimilis, Deschayesiella curvata (fam. Leptochitonidae), Tonicella undocaerulea (fam. Tonicellidae), Mopalia retifera (fam. Mopaliidae) and Ischnochiton hakodadensis (fam. Ischnochitonidae) are described by transmission electron microscopy. In the testis spermatogenetic cells are closely associated with auxiliary cells, which are structurally identical to those of other mollusks and probably have a nutritive function. Spermatogenesis in all species did not differ from that of chiton species already studied and it was confirmed that an acrosome is formed from the Golgi apparatus during spermiogenesis. The spermatozoa of all studied species have a structure that is typical for chitons. The sperm of Deschayesiella curvata (Order Lepidopleurida) have a head consisting of a large acrosome and nucleus with an (3.5–4.2μm) asymmetrically positioned anterior extension. The centrioles are surrounded by five spherical mitochondria. The spermatozoa in Tonicella undocaerulea, Mopalia retifera and Ischnochiton hakodadensis (Order Chitonida) have a small acrosome and a nucleus with long thin anterior extension; mitochondria are lateral to the nucleus. There are considerable interspecific differences in the structure of egg envelopes. Our results, along with those of other workers, show that gamete structure of chitons is specific to each family. A cortical reaction which results in formation of a fertilization envelope takes place in the egg of chitons after fertilization.

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs.

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A2 inhibitor) blocked fertilization envelope formation and transient CN(-)-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca2+]i (cytosolic Ca2+ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN(-)-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN(-)-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A2, induced the envelope formation, acid production, augmentation of CN(-)-insensitive and sensitive respiration, but did not cause any increase in [Ca2+]i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A2 induced by Ca2+ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN(-)-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca(2+)-triggered sequential reactions other than Ca(2+)-caused activation of phospholipase A2.

Xenopus laevisSperm–Egg Adhesion Is Regulated by Modifications in the Sperm Receptor and the Egg Vitelline Envelope

The biochemical and ultrastructural changes in the envelope of theXenopus laevisegg that occur during oviposition and fertilization have been thoroughly studied (Hedrick, J. L., and Nishihara, D. M.,Methods Cell Biol.36, 231–247, 1991; Larabell, C. A., and Chandler, D. E.,J. Electron Microsc. Tech.17, 294–318, 1991). However, the biological significance of these changes with respect to gamete interaction has been unclear. In the current study, it was found that changes in the envelope are directly responsible for regulating sperm–egg adhesion, an initial step of fertilization. As a result of these transformations, sperm bind only to unfertilized oviposited eggs, not to oocytes or coelomic eggs. In addition, they do not bind to fertilized eggs. The molecular and cellular basis of the regulation of the sperm binding process was investigated in the context of our recent findings that two structurally related envelope glycoproteins, gp69/64, serve as sperm receptors during fertilization (Tian, J.-D., Gong, H., Thomsen, G. H., and Lennarz, W. J.,J. Cell Biol.136, 1099–1108, 1997). Although the purified gp69/64 glycoproteins isolated from the oocyte or coelomic egg envelopes exhibited sperm binding activity, when these proteins are part of the intact oocyte or coelomic egg envelopes, they are not accessible to either anti-gp69/64 antibodies or to sperm. During the conversion from the coelomic to the vitelline envelope, the gp69/64 sperm receptors become exposed on the surface, an event that correlates with proteolytic cleavage of gp43 and accompanying ultrastructural alterations in the envelope. Conversely, after fertilization, when the vitelline envelope of the egg is converted to the fertilization envelope of the zygote, limited proteolytic cleavage of the sperm receptor results in loss of sperm binding activity. In addition, formation of a fertilization layer on top of the structurally altered VE adds another physical block to sperm binding. These results provide new insights into structure–function relationships between envelope components of the anuran egg, and provide further evidence supporting the key role of gp69/64 as sperm receptors duringX. laevisfertilization.

Sea urchin hatching enzyme (envelysin): cDNA cloning and deprivation of protein substrate specificity by autolytic degradation.

The hatching enzyme (envelysin) of the sea urchin Hemicentrotus pulcherrimus was purified from the medium of hatched blastulae. By cDNA cloning its deduced amino acid sequence and molecular architecture were revealed. The 591-residue precursor with calculated Mr of 66,123 consists of an 18-residue signal sequence, a 151-residue propeptide, and a 422-residue mature enzyme with N-terminal catalytic and C-terminal hemopexin-like domains. As compared with that of Paracentrotus lividus, its amino acid sequence is 69% identical and 10% similar. They share typical structural features with the mammalian MMP gene family members: cysteine switch, zinc-binding signature, methionine-turn, Cys residues near both ends of hemopexin-like domain, etc. However, its propeptide has a 70-residue extra sequence with an Asp- and Glu-rich stretch, supposedly involved in the proenzyme activation by binding Ca2+ ions in seawater. The hinge region is also longer than those of most MMPs, with an extra sequence rich in Thr and Arg residues. Mature 50K enzyme is highly susceptible to autolytic cleavage at Gln(503)-Leu(504), producing the 38K form retaining catalytic activity and substrate specificity against fertilization envelope. The 38K form and 15K fragment were coeluted from a gel-filtration column, suggesting that these two fragments are disulfide-bridged and that the tertiary structure is not much deviated. The 38K form further autolyzed to 32K form by cleaving Tyr(450)-Tyr(451) bond with the loss of protein-substrate specificity, retaining only nonspecific protease activity. Thus, the autolytic release of 2/3 of the C-terminal domain reduced the highly specific enzyme to a common nonspecific protease, implying that the size and structure of almost the entire hemopexin-like domain is essential for the protein substrate specificity. Moreover, autolytic degradation of envelysins from the two species follow quite different pathways despite their high homology in structure. The 38K and 32K forms were inhibited by bovine TIMP-1 with different IC50 values, indicating that its inhibitory activity depends on the extent of the interaction with the C-terminal domain of the enzyme.

Regulated exocytosis and sequential construction of the extracellular matrix surrounding the sea urchin zygote

After fertilization most eggs become surrounded by a complex extracellular matrix. This study examines those matrix assembly processes that are triggered by fertilization of the sea urchin egg. The study uses antibodies that identify five different storage compartments in the egg. These compartments release their protein contents in a highly regulated fashion to assemble and modify the extraembryonic layers. The exocytosis sequence begins with a fertilization wave that progresses from the site of sperm entry and elevates the fertilization envelope above a water-filled perivitelline space. The immediate surface of the zygote then becomes covered by a newly secreted hyaline layer. Prior to fertilization some of the antigens are localized to cortical granules. Others are found in "basal laminar vesicles" that are released in a wave beginning at about 30 sec, or roughly at the same time as cortical granule exocytosis. The remaining antigens are exocytosed with a rather precise timing, but with a delay of several to tens of minutes relative to the first wave of exocytosis. "Apical vesicles," so named because antigens from this class are preferentially exocytosed toward the apical cell surface of polarized cells, include antigens that are exocytosed beginning at about 5 min postfertilization. The fourth compartment, named "echinonectin vesicles" release echinonectin, a protein that is deposited to the inner side of the hyaline layer. Surface staining of echinonectin is first detected about 10-15 min following sperm contact. Finally, maternal cadherin, which is stored in yet a fifth distinct compartment, is not detected on the surface until at least 30 min following fertilization. The data are also consistent with the notion that the tightly regulated timing of exocytosis contributes to the ordered assembly of the hyaline layer and elevation of the fertilization envelope. Finally, two of the vesicle classes continue to exocytose after the cells become polarized. In polarized cells apical and basal laminar antigens are trafficked toward opposite sides of the same cell after passing through the same trans-Golgi network-like compartment.

Regulated exocytosis and sequential construction of the extracellular matrix surrounding the sea urchin zygote

After fertilization most eggs become surrounded by a complex extracellular matrix. This study examines those matrix assembly processes that are triggered by fertilization of the sea urchin egg. The study uses antibodies that identify five different storage compartments in the egg. These compartments release their protein contents in a highly regulated fashion to assemble and modify the extraembryonic layers. The exocytosis sequence begins with a fertilization wave that progresses from the site of sperm entry and elevates the fertilization envelope above a water-filled perivitelline space. The immediate surface of the zygote then becomes covered by a newly secreted hyaline layer. Prior to fertilization some of the antigens are localized to cortical granules. Others are found in “basal laminar vesicles” that are released in a wave beginning at about 30 sec, or roughly at the same time as cortical granule exocytosis. The remaining antigens are exocytosed with a rather precise timing, but with a delay of several to tens of minutes relative to the first wave of exocytosis. “Apical vesicles,” so named because antigens from this class are preferentially exocytosed toward the apical cell surface of polarized cells, include antigens that are exocytosed beginning at about 5 min postfertilization. The fourth compartment, named “echinonectin vesicles” release echinonectin, a protein that is deposited to the inner side of the hyaline layer. Surface staining of echinonectin is first detected about 10–15 min following sperm contact. Finally, maternal cadherin, which is stored in yet a fifth distinct compartment, is not detected on the surface until at least 30 min following fertilization. The data are also consistent with the notion that the tightly regulated timing of exocytosis contributes to the ordered assembly of the hyaline layer and elevation of the fertilization envelope. Finally, two of the vesicle classes continue to exocytose after the cells become polarized. In polarized cells apical and basal laminar antigens are trafficked toward opposite sides of the same cell after passing through the same trans -Golgi network-like compartment.

Effects of electromagnetic radiation on embryos of sea-urchins

Electromagnetic radiation (EMR) causes a decrease in the number of fertilized eggs and an increase in the number of zygotes with abnormal fertilization envelopes in sea-urchins. The microstructural impairments of the cellular surface, the increase of lipid peroxidation and the changes of amino acid metabolism show that the impairments of the development of embryos exposed to EMR are caused by the damages of the membrane structures.

Binding of antibiotics to glycoproteins of the vitelline and fertilization envelopes of cherry salmon eggs.

The binding of antibiotics (gentamicin, oleandomycin and chloramphenicol) to vitelline and fertilization envelopes and their extracts was investigated by immunohistochemical and immunocytochemical techniques and immunoblot analysis using mature and artificially activated eggs of the fish Oncorhynchus masou. Binding of antibiotics was detected in the vitelline and fertilization envelope outermost layers, the fertilization envelope inner surface and cortical alveolus exudates, with differences in immunoreactive intensity and deposition. The fertilization envelope outermost layer had the capacity to bind much greater amounts of the antibiotics than the vitelline envelope outermost layer. The greater capacity was caused by the deposition of cortical alveolus exudates, which were known to be responsible for functional roles of protection against bacteria, fungi and noxious materials. Treatment of the vitelline and fertilization envelopes with neuraminidase markedly reduced the binding of gentamicin and chloramphenicol but slightly increased that of oleandomycin; binding of the latter to the vitelline and fertilization envelope outermost layers was considerably reduced after treatment with alpha-fucosidase. Treatment of the two envelopes with alpha-mannosidase, beta-galactosidase or beta-D-glucosaminidase did not cause any alteration in immunoreactive intensity or number of immunoreactive deposits. Immunoblot analysis of the vitelline or fertilization envelope extracts indicated that many of the antibiotic-binding substances were glycoproteins, and several major bands were bound by all three antibiotics. These results suggest that the vitelline or fertilization envelopes may have the ability to protect the egg itself, or the embryo, respectively, by trapping antibiotics, and the trapping may be related to the presence of carbohydrate moieties, such as sialyl or fucosyl residues.

Cloning and expression of a Xenopus laevis oocyte lectin and characterization of its mRNA levels during early development.

cDNA clones encoding a soluble, calcium-dependent, melibiose-binding lectin from Xenopus laevis oocytes have been isolated, characterized, and expressed in bacteria. This lectin has been shown by others to be localized in oocyte cortical granules where it ultimately is released and participates in the formation of the fertilization envelope. A lectin with similar specificity has been purified by others from blastula and immunolocalized to specific locations in developing embryos, which suggests it may also function after fertilization in regulating cell adhesion and migration. We have used melibiose affinity chromatography to isolate the oocyte lectin (monomer molecular masses of about 45 and 43 kDa) and shown that after exhaustive treatment with N-glycanase, only one major protein band at 35 kDa was observed, suggesting that a single polypeptide with variable N-linked glycosylation is expressed in the oocyte. After obtaining internal peptide sequences, a PCR-based cloning approach allowed the isolation of full length cDNAs from an ovary lambda gt11 library encoding a protein of 313 amino acids with three potential N-linked oligosaccharide sites. Although this lectin, termed XL35, requires calcium ions for oligosaccharide binding, its sequence does not contain the sequence motif defined for "C-type" lectins. A 6-Histagged from of the lectin was expressed in E. coli and purified on a Ni(2+)-NTA column from bacterial extracts. The recombinant lectin was active using an agglutination assay, and this activity was inhibitable by EDTA and melibiose, properties exhibited by the native lectin. Southern blot analysis revealed a single hybridizing band, arguing against the existence of a multigene family. Northern blot analysis demonstrated that the lectin mRNA is expressed in oocytes and remains at relatively high levels through late gastrulae, continuing until tadpole stages. The persistence of the lectin mRNA, coupled with results from earlier studies, strongly suggests that XL35 is zygotically expressed and functions during morphogenesis.

Development of the hyaline layer around the planktonic embryos and larvae of the asteroid Patiriella calcar and the presence of associated bacteria

Summary The hyaline layer (HL) around the embryos and larvae of Patiriella calcar is examined by transmission electron microscopy. P. calcar hatches at the gastrula stage and develops through a lecithotrophic planktonic brachiolaria. The hyaline layer of unhatched P. calcar is poorly developed and is comprised of wispy fibrils scattered among the epithelial microvilli. Fibrils are also occasionally seen associated with the inner surface of the fertilization envelope. By the hatched gastrula stage, the hyaline layer is organized into three strata: the intervillous layer, the supporting layer and the coarse outer meshwork layer. Seven-day-old brachiolaria also have a hyaline layer comprised of three strata. In these larvae the supporting layer elevates away from the epithelial surface due to the tuft-like organization of the underlying microvilli. This results in the formation of local outpockets giving the surface of the HL a lobed appearance. Bacteria are occassionally seen in the intervillous layer, particularly in association with the outpockets. These bacteria are phagocytosed by the epithelial cells and, in larvae that have bacteria, may play an augmentive role in larval nutrition. The structure of the hyaline layer of P. calcar is compared with that of the hyaline layer of other Patiriella species to determine if it is more similar to the external coats around its planktonic (P. regularis) or benthic (P. exigua) developing congeners. The comparison shows that the hyaline layer of P. calcar is virtually identical to that of P. regularis, a similarity that may reflect the pelagic life histories of these species.

Propranolol induces polyspermy during sea urchin fertilization.

Propranolol, a beta-adrenergic receptor blocker, is found to induce polyspermy in sea urchin eggs. Unfertilized sea urchin eggs treated for 10 min with 50 microM of propranolol, and then inseminated, become polyspermic and show a fertilization envelope which is barely visible to the light microscope. Examination of treated eggs by transmission and scanning electron microscopy shows that the drug does not alter the cortex of the unfertilized egg. However, after insemination an incomplete cortical reaction occurs. This might well account for both polyspermy and the defective elevation of the fertilization envelope. Since the effects of the drug are reversed by simultaneous treatment with adrenalin, perhaps propranolol interferes with the monoaminergic system that has been proposed to be active. The involvement of the monoaminergic system in the fertilization process is present in the sea urchin egg.

Enzymatischer Abbau der Embryonalhüllen des Lanzettfischchens, Branchiostoma lanceolatum, während des Schlüpfens / Hatching through Enzymatic Breakdown of the Embryonic Coats in the Lancelet, Branchiostoma lanceolatum (Cephalochordata)

Abstract A study was made of the hatching process of the lancelet, Branchiostoma lanceolatum (Amphioxus), under laboratory conditions. Embryos in the early neurula stage (5 to 6 pairs of somites) escape through a hole made in the fertilization envelope, following intensive rotatory movements and partial digestion of the envelope. Using the chromogenic substrate Hide Powder Azure (HPA) at pH 8.0, proteolytic activity was detected in the hatching medium. The effect of various protease inhibitors was investigated. Whereas the activity was only slightly affected by inhibitors of trypsin, chymotrypsin or thiol proteases, chelating agents such as EDTA and 1,10-phenanthroline prevented the degradation of HPA, as well as the breakdown of the vitelline membrane of alcohol fixed oocytes by the protease from hatching fluid. These data suggest the presence of a metalloprotease in the hatching medium. How far this enzyme can be compared with hatching enzymes found in echinoderms and fishes (where they have been identified as matrix degrading metalloproteases), awaits further study of the lancelet enzyme, including its isolation and purification.