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.     

Oviducal Contribution to Alteration of the Vitelline Coat in the Frog, Rana japonica. An Electron Microscopic Study

The vitelline coat (VC) surrounding coelomic eggs of the frog, Rana japonica , comprises bundles of filaments running both parallel and perpendicular to the egg surface. The coat gives little or no staining reaction with PA-CrA-Silver methenamine. In contrast, in the VC of uterine eggs the filament bundles are less conspicuous. and the interstices between the filament bundles stain strongly for carbohydrate. This alteration occurs during passage of the eggs down the first 1/20 th of the oviduct, the pars recta. The epithelium of the p. recta contains secretory cells, which contain electron-dense granules distinct from those in the jelly-secreting cells in more caudal portions of the oviduct. Treatment of coelomic eggs with an extract of p. recta followed by exposure to a sperm suspension resulted in marked swelling and softening of the VC. These results indicate that the contents of the granules secreted from the epithelial cells in the p. recta are deposited in the VC to increase its susceptibility to a fertilizing sperm.     

The Properties of the Oviducal Pars Recta Protease which Mediates Gamete Interaction by Affecting the Vitelline Coat of a Toad Egg

SDS-PAGE analyses of the vitelline coats (VCs) of coelomic eggs (CEVC) and uterine eggs (UEVC) of Bufo japonicus revealed that the UEVC lacks the 40K–52K molecular weight components present in the CEVC; this is concomitant with the increased stainability of a 39K component and the appearance of a 36K component. These macromolecular alterations, accompanied by the acquisition of egg fertilizability, were induced when coelomic eggs were treated with the contents of secretory granules obtained from the oviducal pars recta (PRG). Gel-filtration of PRG in combination with hydrolytic assays employing either fluorescamine-labeled CEVC or a variety of synthetic substrates showed that the CEVC to UEVC alterations are ascribable to the action of a protease hydrolyzing specifically peptidyl-Arg-MCAs in a highly Ca²⁺-dependent way. This enzyme, which has an optimal pH of 8.0–8.2, is inhibited by soybean trypsin inhibitor and leupeptin, as well as by such serine protease inhibitors as DFP and p-APMSF. On the basis of a SDS-PAGE analysis, its molecular weight is estimated to be 66K. Treatment of coelomic eggs with the partially purified PR protease did not render the eggs fertilizable, although CEVC to UEVC macromolecular alterations were effected. We conclude that the action of this oviducal protease in partially hydrolyzing the VC is a prerequisite but insufficient in itself to render the coelomic eggs fully accessible to a fertilizing sperm.     

Analyses of Oviductal Pars Recta-Induced Fertilizability of Coelomic Eggs in Xenopus laevis

The acquisition of fertilizability in coelomic eggs of Xenopus laevis has been shown to be correlated with the physical, biochemical, and ultrastructural alterations of the egg envelope [coelomic envelope (CE)] induced during the passage of eggs through the pars recta portion of the oviduct. However, no direct evidence that the pars recta renders eggs fertilizable has yet been presented. In this study, we show that coelomic eggs are highly fertilizable when they are incubated with continuous shaking for 4 h at 15 degrees C in pars recta extract (PRE) derived from females prestimulated by pregnant mare serum gonadotropin. The PRE from pituitary-stimulated Bufo japonicus was as potent as homologous PRE in rendering Xenopus eggs fertilizable. Incubation of coelomic eggs in PRE for 30 min induced a dramatic increase in the rates of sperm binding to the envelope to a level equivalent to that exhibited by the envelope from uterine eggs (VEs). The CE-to-VE ultrastructural conversion and a 43k-to-41k hydrolysis of the envelope glycoprotein component started 5 min after, and were completed by 15 min after, the start of incubation in PRE and were accompanied by an exposure of a new N-terminal sequence typical to gp41. Thus, the biochemical and ultrastructural conversions and the sperm-binding activity of the envelope induced by PREs, although being prerequisite, were not sufficient to render coelomic eggs fully accessible to fertilizing sperm.     

Oviductal protease and trypsin treatment enhance sperm-envelope interaction in Bufo arenarum coelomic eggs

We describe the morphological and biochemical changes in Bufo arenarum coelomic egg envelopes (CE) following passage through the oviduct. In this species, the transformation of the CE into the vitelline envelope (VE) leads to the acquisition of fertilizability and involves the cleavage of a glycoprotein component. Electrophoretic patterns indicate that a pars recta oviductal protease selectively hydrolyzes in vitro the 84 and the 55 kDa glycoproteins of the CE. During the CE to VE transformation, the relative concentrations of gp48, 42 and 39 kDa also change. In in vitro tests, sperm binding to envelope glycoprotein occurs when they are exposed to VE but not when treated with CE, and VE labeled glycoproteins bind to the head and mid piece of the sperm. The gp39 VE component has 100% identity with internal domains of the sequence deduced from ovarian cDNA for the homologous zona pellucida glycoprotein type C (ZPC) protein precursor in B. arenarum. The effects of trypsin as a substitute for oviductal protease were also examined. Trypsin selectively attacks the 84 and the 55 kDa glycoproteins without hydrolyzing other components and renders coelomic eggs fertilizable in a jelly water preparation. Therefore, trypsin can mimic in vitro the biological action of the oviductal protease. However, it does not wholly mimic the biological action of the oviduct which, in B. arenarum at least, exceeds a mere proteolytic effect. This fact was verified by the lower fertility rates and the abnormal embryo development found when trypsin-treated coelomic eggs were fertilized in vitro.     

Participation of the 39-kDa glycoprotein (gp39) of the vitelline envelope of Bufo arenarum eggs in sperm-egg interaction

The acquisition of egg fertilizability in Bufo arenarum takes place during the oviductal transit and during this process the extracellular coelomic envelope (CE) of the eggs is converted into the vitelline envelope (VE). It has been stated that one of the necessary events leading to a fertilizable state is the proteolytic cleavage of CE glycoproteins in the oviductal pars recta by oviductin, a serine protease. Consequently, there is a marked increase in the relative quantity of glycoproteins with 39 (gp39) and 42 kDa (gp42) in the VE. In the present study, sperm-VE binding assays using heat-solubilized biotin-conjugated VE glycoproteins revealed that both gp39 and gp42 have sperm binding capacity. According to this result, our study was focused on gp39, a glycoprotein that we have previously reported as a homologue of mammalian ZPC. For this purpose, rabbit polyclonal antibodies against gp39 were generated at our laboratory. The specificity of the antibodies was confirmed with western blot of VE glycoproteins separated on SDS-PAGE. Immunohistochemical and immunoelectron studies showed gp39 distributed throughout the width of the VE. In addition, immunofluorescence assays probed that gp39 bound to the sperm head. Finally, as an approach to elucidate the possible involvement of gp39 in fertilization, inhibition assays showed that pretreatment of eggs with antibodies against gp39 generated a significant decrease in the fertilization rate. Therefore, our findings suggest that gp39, which is modified by oviductal action, participates as a VE glycoprotein ligand for sperm in Bufo arenarum fertilization.     

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.     

Oviductal Localization of the Cortical Granule Lectin Ligand Involved in the Block to Polyspermy of Xenopus Laevis

The fertilization layer of Xenopus laevis is formed upon egg activation by the binding of the cortical granule lectin (CGL) to its ligand in the egg extracellular matrix. Using Western blotting methods with biotinylated CGL as a probe, oviductal tissue extracts were examined to determine the site of origin of the CGL ligand. Three glycoprotein ligands of M_rs= > 250,000, 160,000, and 90,000 (reduced samples) were localized to the pars convoluta oviduct immediately posterior to the pars recta oviduct. The binding of CGL to these glycoproteins was inhibited in the presence of 200 mM galactose, but not with 200 mM mannose indicating a specific lectin interaction. The M_rs= > 250,000 and 90,000 glycoproteins were linked by disulfide bonds. In addition, these ligands were secreted from a more anterior region of the pars convoluta oviduct than the M_r=160,000 ligand. No binding of CGL was detected to pars recta secretory granule lysate components. The highest molecular weight CGL ligand seen in the pars convoluta corresponded to the CGL ligand in isolated fertilization envelopes. Thus, the CGL ligand involved in the formation of the fertilization layer is a product of the pars convoluta oviduct.     

Physiological mechanisms of buoyancy in eggs from brackish water cod

Newly fertilized eggs of brackish water (Gotland, Baltic Sea) and marine (Lofoten, Norway) cod were investigated with regard to specific gravity, wet and dry weight, water content, chorion weight, and content of protein, free amino acids (FAA), and ions. The eggs had neutral buoyancies equivalent to a salinity of 14^.3% (range 11^.5–16^.2%) in brackish water, and 33^.0% (range 31^.8–34^.5%) in the marine environment. A buoyancy model was developed and showed that this difference was mainly caused by differences in egg water content which was 96.6 ± 0^.47% and 92.7 ± 0.45% in the brackish and marine eggs, respectively. The higher water content of the brackish eggs resulted from increased water uptake during final oocyte maturation due to higher intracellular contents of FAA, Cl ^– and NH₄⁺. SDS polyacrylamide gel electrophoresis of eggs and oocytes, and measurements of egg protein content suggested that the FAA pool of both egg types originated from hydrolysis of specific yolk proteins. The main contributor seemed to be a protein with a molecular weight of 100 kDa.     

SCN— Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca²⁺-free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN^— for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN^— probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN^— for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN^— for a short period caused concentration-dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post-hatching period. The effects of SCN^— on morphogenesis were not inhibited by furosemide or 4,4'-diisothiocyano 2,2'-disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN^— causes abnormal morphogenesis of embryos. The inhibitory effects of SCN^— on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca²⁺.     

Serum antigens detected in pars recta luminal fluid and coelomic envelope surrounding Bufo arenarum eggs

In anurans, protease activity from the pars recta portion of the oviduct (under regulation by 17β-estradiol), is known to cause ultrastructural alterations on the oocyte surface rendering fertilizability. In mammals, the presence of serum proteins in oviductal fluid via transudation is also well known. In the present study we determined the plasma proteins of the anuran Bufo arenarum that are present in pars recta fluid and oocyte extracellular matrix and characterized the 17β-estradiol-induced proteins synthesized de novo and secreted into the pars recta lumen. Rabbit polyclonal antibodies against the soluble proteins in pars recta fluid cross-reacted with anuran plasma proteins and with the extracellular matrix of coelomic eggs based on immunoelectrophoresis and immunohistochemistry, respectively. Using radiolabeled leucine in the absence and presence of 17β-estradiol, we show that a polypeptide of 66 kDa molecular mass is the principal protein synthesized and secreted into the pars recta lumen.     

Receptors for Sperm-activating Peptides, SAP-I and SAP-IIB, on Spermatozoa of Sea Urchins, Hemicentrotus pulcherrimus and Glyptocidaris crenularis

Sperm-activating peptide analogues, GYGG-SAP-I (Gly-Tyr-Gly-Gly-Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and GYGG-SAP-IIB (Gly-Tyr-Gly-Gly-Lys-Leu-Cys-Pro-Gly-Gly-Asn-Cys-Val) which exibit almost the same respiration-stimulating activity as the respective original peptide were chemically synthesized, radiolabeled with Na¹²⁵1, and used for experiments of binding and cross-linking to Hemicentrotus pulcherrimus or Glyptocidaris crenularis spermatozoa. In competitive binding, SAP-I caused 50% decrease in ¹²⁵l-GYGG-SAP-l binding to intact spermatozoa, sperm heads and sperm tails of H. pulcherrimus at the concentrations of 282 nM, 3 nM and 141 nM, respectively. The incubations of H. pulcherrimus sperm tails with ¹²⁵l-GYGG-SAP-I and the chemical cross-linking reagent, disuccinimidyl suberate, resulted in the appearance of two radiolabeled bands with apparent molecular masses of 71 kDa and 63 kDa (determined by sodium dodecyl sulfate-gel electrophoresis under reducing conditions). ¹²⁵l-GYGG-SAP-IIB bound almost equally to sperm heads and sperm tails of G. crenularis , and was cross-linked to 172 kDa, 62 kDa and 58 kDa proteins in sperm heads, and 157 kDa and 62 kDa proteins in sperm tails with disuccinimidyl suberate.     

The Wave Pattern of Free Calcium Release Upon Fertilization in Medaka and Sand Dollar Eggs

A transient rise in the concentration of Ca²⁺ in the cortex upon fertilization was demonstrated in medaka eggs injected with aequorin. Detection of the aequorin luminescence with an ultra-high sensitivity photonic microscope system revealed a wave of increased Ca²⁺ concentration starting at the site of sperm entry (animal pole) and being propagated along the cortex of the egg toward the antipode. The wave traversed the entire egg surface within 2–3 min. The peak value of the aequorin luminescence, and therefore the peak value of the Ca²⁺ transient, was generally higher at the site of sperm entry than in other regions. The peak values of the luminescence (and therefore of the Ca²⁺ concentration in the cortex) remained fairly constant during propagation of the wave. Microinjection of Ca²⁺ into the cortex also induced a Ca²⁺ wave. When the egg was stimulated by microinjection of Ca²⁺ at the equatorial region, the Ca²⁺ wave was propagated at a fairly constant speed over the egg surface, except at the region near the vegetal pole where the wave was retarded. Simultaneous recording of the Ca²⁺ wave and the wave of cortical change (breakdown of cortical alveoli) in eggs during fertilization revealed that the Ca²⁺ wave preceded the wave of cortical change.
A Ca²⁺ wave was also demonstrated in sand dollar eggs, although due to their smaller size the phenomenon was not as clear as in medaka eggs.     

Requirement of Extracellular Calcium Ions for the Early Fertilization Events in the Medaka Egg

The requirement for calcium and the change in calcium content in eggs of Oryzias iatipes during the cortical reaction and sperm penetration were examined. Naked eggs failed to exhibit the cortical reaction upon insemination under Ca Mg-free conditions. These eggs exhibited the cortical reaction by reinsemination in the presence of extracellular Ca²⁺. The effect of extracellular Ca²⁺ on sperm penetration could be replaced by one of several divalent cations in the external medium. Unlike the cortical reaction, sperm penetration failed to be induced by microinjection to increase intracellular Ca²⁺. Verapamil significantly reduced the action of extracellular Ca²⁺ or Ba²⁺ of divalent cations examined in fertilization, while TEA and TTX had no effect on fertilization in the presence of these cations. No ⁴⁵Ca uptake into the egg proper was recognized before completion of the cortical reaction. These observations suggest that extracellular divalent cations are indispensable for sperm stimulation of the egg and its penetration into the egg, for which an influx of Ca²⁺ from the external medium is not required.     

A method for hormonal induction of sperm release in anurans (eight species) and in vitro fertilization in Lepidobatrachus species

Injections of synthetic human gonadotropin releasing hormone (GnRH) into the dorsal pelvic area were used in an attempt to stimulate sperm release in isolated males of eight anuran species including Xenopus laevis, Rana pipiens and Lepidobatrachus laevis . Sperm were obtained within 1–5 h post injection either by mechanical stimulation or by cloacal lavage. Sperm suspensions varied from 8 μL to 7 mL and the cell densities ranged from 4 × 10⁵ to 4 × 10⁷ sperm/mL. The sperm obtained from seven species using GnRH-induced release were viable based on light microscopic observations of motility. In addition, sperm preparations fertilized eggs in vitro and produced normal tadpoles in the case of L. laevis and L. llanensis . This hormonal method of anuran sperm collection will provide a convenient non-injurious way to obtain anuran sperm for basic studies of reproduction and development.     

Cryptic domains of a 60 kDa heat shock protein of Helicobacter pylori bound to bovine lactoferrin

Abstract Bovine lactoferrin binds to a 60 kDa heat shock protein of Helicobacter pylori . Binding ability was related to human immunoglobulin G because bovine lactoferrin binding proteins were isolated by extraction of cell surface associated proteins with distilled water, applied on IgG-Sepharose and nickel sulphate chelate affinity chromatography. Binding was demonstrated by Western blot after purified protein was digested with α-chymotrypsin and incubated with peroxidase-labeled bovine lactoferrin. Binding was inhibited by bovine lactoferrin, lactose, rhamnose, galactose, and two iron-containing proteins, ferritin and haptoglobin. Helicobacter pylori binds ferritin and haptoglobin via charge or hydrophobic interactions because this binding was not inhibited by specific and various glycoproteins or carbohydrates. Carbohydrate moieties of bovine lactoferrin molecules seem to be involved in binding because glycoproteins with similar carbohydrate structures strongly inhibited binding. Scatchard plot analysis of the binding of peroxidase-labeled bovine lactoferrin to H. pylori cells yielded a k _d 2.88 × 10⁻⁶ M. In addition, binding of H. pylori cells to bovine lactoferrin was enhanced when bacteria treated with pepsin or α-chymotrypsin after isolation from iron-restricted and iron-containing media.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

Ca2+-induced change of dorsal-ventral polarity in frog (Rana temporaria) eggs

Abstract. The effect of local Ca²⁺ administration 10–20 min after fertilization and during artificial activation was examined in Rana temporaria eggs. Ca²⁺ was injected into the pigmented region near the boundary between pigmented and unpigmented domains. The locations of egg gray crescent (GC) and dorsal lip of blastopore (DLB), as predictors of the dorsal region in embryos, as well as the measurements of angles between GC middle and sperm entry site were observed. In more than 70% of the cases, microinjection of Ca^{2 +} into subcortical cytoplasm and egg pricking in high-Ca^{2 +} solutions induced GC and DLB formation near the injection site. The formation of Ca²⁺-induced GC occurred mostly as in control eggs. In addition premature displacement of the egg surface was observed near the prick site in high-Ca^{2 +} solutions. GC formation occurred by displacement of the pigmented surface in the same direction as earlier wound translocations. These results show that Ca²⁺ injection determines the direction of the surface movement.     

The effect of beet cyst nematode, Heterodera schachtii, on the yield of sugar beet in organic soils

In ten experiments on commercial sugar-beet crops grown on organic soils in 1984–86, a Genstat programme was used to examine the relationship between the initial population of Heterodera schachtii and sugar-beet root yield using the equation
Y = Ymin + (Ymax - Ymin) Z^pi-T
Fixing T = 200 eggs + juveniles 100 g^-1 soil and Z^T= 0.95, estimated values of Ymax varied from 49.2–67.1 t ha^-1 (129– 155% of the national average root yield for the years in which the experiments were carried out) and estimates of Ymin varied from 14.5–53.9 t ha^-1 (27–94% of Ymax). The estimated average root yield loss caused by the nematode was 6.95 t ha^-1.     

Potential involvement of C(3) complement factor in amphibian fertilization

We have assessed the potential involvement of C(3), the third complement factor, and its receptor in Bufo arenarum fertilization. We show that a polyclonal antibody against a B. arenarum C(3)-like factor (C(3)Ba) reacts specifically with components of the extracellular matrix (ECM) of coelomic eggs and the cell membrane of uterine eggs. Interestingly, we have identified a 163 kD protein immunoreactive with a monoclonal antibody against the CD11b alpha chain of the human C(3) receptor on the cell membrane of the animal pole of uterine eggs, the site of entry of the sperm, but not in coelomic eggs (CR3Ba). Treatment of coelomic eggs with a pars recta oviductal-like protease, trypsin, induced the translocation of C(3)Ba from the ECM to the cell membrane. Furthermore, inhibition of CR3Ba by trypan blue, as well as inhibition of C(3)Ba by anti-C(3)Ba on uterine eggs impaired fertilization, whereas identical treatment on sperm cells did not alter percentage fertilization. Our results suggest, (A) that changes in the localization of C(3)Ba from the ECM to the cell membrane may be triggered by trypsin-like proteases during passage of eggs through the oviduct; and (B) that C(3)Ba/CR3Ba may be involved in B. arenarum fertilization.