Mechanism of the Block to Hybridization andSelfing Between the Sympatric Ascidians Cionaintestinalis and Ciona savignyiJENNIFER BYRD AND CHARLES C. LAMBERT*Biological Science, California State University Fullerton, Fullerton, California

ABSTRACT The solitary ascidians Ciona intes-tinalis and Ciona savignyi co-occur in southern Califor-nia harbors, but no hybrids have been recognized innature. Numerous differences in their egg morphologywere detected. Homologous (normal outcross) fertiliza-tion yielded 96–99% cleavage, where autologous (self)fertilization showed 3% and heterologous (hybrid) fertil-ization showed 0–1%. Acid treatment (pH 3.2) removedthe block to selfing (P , 0.0001) but not hybridizationfor both species. Heterologous sperm bind to thevitelline coat (VC), but fail to penetrate. Enzymaticremoval of the VC resulted in 91–97% cleavage withautologous and heterologous sperm (P , 0.0001). Thevitelline coats of the two species differ in lectin bindingto surface glycosides. Fertilization in both species issignificantly inhibited by the lectins, fucose bindingprotein (P , 0.0001) and concanavalin A (P , 0.0001),and wheat germ agglutinin inhibits fertilization inC. intestinalis (P , 0.0001) but is without effect onC. savignyi fertilization. Self and hybrid blocks employdifferent mechanisms including glycoside compositionand acid sensitivity. Mol. Reprod. Dev. 55:109–116,2000. r 2000 Wiley-Liss, Inc.

Key Words: hybrid; selfing; homologous; heterolo-gous; autologous; lectins

INTRODUCTIONTwo closely related ascidians, Ciona intestinalis and

C. savignyi, co-reside in harbors and marinas of south-ern California. C. intestinalis has occurred in southernCalifornia since the turn of the century; however,C. savignyi was identified locally only in 1985 (Lambertand Lambert, 1998). Originally, there was confusion inthe classification of Ciona. Early workers concludedthat only two species existed based on the presence orabsence of a red pigment spot at the end of the spermduct. Hoshino and Nishikawa (1985) attempted toclarify the classification and concluded that there wereonly two species in this genus based on an internaladult feature, the endostylar appendage; C. intestinalishas it and C. savignyi does not. Based upon egg size andmorphology there are actually three different species inEurope (Copello et al., 1981). Lambert et al. (1990)attempted fertilizations between different EuropeanCiona and found a low rate of hybridization, concluding

there were in fact three distinct species there and thatC. savignyi was a fourth species present in Japan andCalifornia. Hybridization does not generally occur inintact eggs of C. intestinalis, but if the vitelline coat(VC) is removed to produce naked eggs hybridizationwill occur (Reverberi, 1971). In this study, we revealdifferences in egg morphology and sperm recognitionrequirements between C. savignyi and C. intestinalis.

Fertilization is a precise sequence of events thatmust occur for successful union of egg and sperm. Ingeneral, three major steps are required: (1) localizationand binding of sperm to the vitelline coat of the egg, (2)penetration by the sperm through the vitelline coat,and (3) fusion of the sperm with the egg plasmamembrane. Details on several of these steps are stillunclear, yet many suggestions have been made. Forinstance, follicle cells (FC) or eggs instigate chemotaxisin homologous sperm (Miller, 1975; Yoshida et al.,1993), the vitelline coat is the level of self-sterility andsperm-egg binding in C. intestinalis (Rosati and deSantis, 1978), and fucosyl-proteins mediate binding (deSantis et al., 1980; Pinto et al., 1981). Eisenhut andHonegger (1997) showed C. intestinalis has a perivitel-line matrix rather than the thick vitelline coat thatextends to the egg surface in other species.

Since Ciona are hermaphroditic broadcast spawnersand spawn in response to light (Lambert and Brandt,1967), specific homologous recognition factors are re-quired in order for Ciona species to remain distinct.Further, it is important that these factors be involvedearly in the fertilization process. It is unclear if thesefactors involve protein-protein, protein-carbohydrate,or receptor-ligand interaction(s) for any species(C. intestinalis, Bliel and Wassarman, 1980; Honegger,1982; Shur and Hall, 1982; Hoshi et al., 1985;Kawamura et al., 1991). Glycosylated proteins, includ-ing fucose, N-acetylglucosamine (GlcNAc) and man-nose, have been implicated in some ascidian and somemammalian fertilizations as factors involved in homolo-gous egg recognition (C. intestinalis, Rosati and deSantis, 1978; de Santis et al., 1980; Hoshi et al., 1985;

Grant sponsor: National Fish and Wildlife Foundation.*Correspondence to Dr. Charles C. Lambert, 12001 11th Ave. NW,Seattle, WA 98177. E-mail: clambert@fullerton.eduReceived 7 May 1999; Accepted 4 August 1999

MOLECULAR REPRODUCTION AND DEVELOPMENT 55:109–116 (2000)

r 2000 WILEY-LISS, INC.

Hoshi, 1986; Kawamura et al., 1989; Ascidia nigra,Honegger, 1986; Lambert, 1986; mouse, Cardullo et al.,1989; Miller et al., 1992). The role of follicle cells in thefertilization process also remains unclear but manysuggestions have been reported. In some species theycause the eggs to float (Lambert and Lambert, 1978),are involved in chemotaxis (Miller, 1975) and influencesperm direction (Villa and Patricolo, 1993); FC make upa physical block to polyspermy (de Santis et al., 1980),and may be involved in the chemical block to poly-spermy (Villa and Patricolo, 1992; Lambert, et al.,1997), as well as phagocytizing sperm (de Santis et al.,1980) or egg adhesion to substrate (Lambert et al.,1995). We have probed the level of the blocks tohybridization and selfing and found that they aredistinct and independent but that both involve thevitelline coat. We show that while heterospecific spermbinding is possible, the block to hybridization is at thelevel of penetrating the vitelline coat in C. intestinalisand C. savignyi. Acid treatment removes the block toselfing in both species but does not remove the block tohybridization.

MATERIALS AND METHODSGamete Collection

Ciona intestinalis and C. savignyi were collected inLong Beach and San Diego harbors and kept in aquariaheld at 15°C in constant light to avoid spawning(Lambert and Brandt, 1967). The animals were dis-sected and their gametes separately extruded frompunctured ducts. The eggs were washed several timeswith sea water (SW) and stored at 15°C for up to 4 hr.The sperm were stored at 15°C and ‘‘dry’’ until needed.

FertilizationAuto-, homo-, and heterologous fertilizations were

prepared as follows: Eggs (10 µl) were transferred to500 µl SW. Sperm (2 µl) was transferred to a separate500 µl SW and allowed to activate 1 min. The spermconcentration was adjusted to (1 3 105/ml) with ahemocytometer counting chamber (American Optical,Buffalo, NY). The sperm suspension (500 µl) was addedto the eggs and first cleavage in 50 eggs was scored after1 hr. To check that the gametes were viable, homologouscontrols (intraspecific outcrosses) were run in parallelwith experimental eggs each day.

Acid TreatmentAcid treatment was used to remove the self-fertiliza-

tion block (Morgan, 1939). Acid SW was prepared byadding 150 µl 1 N HCl to 50 ml SW for pH 3.2(Kawamura et al., 1987), then adjusted with 5 M Trisfor pH 3.7 and pH 4.2. Eggs (100 µl) were incubated in10 ml acid SW for 10 min. The acidity was neutralizedby adding 100 µl 5 M Tris, followed by three washes inSW for three-minute intervals.

Pronase TreatmentVitelline coats were removed using a protease cock-

tail (20 ml SW, add 0.15 g dithiothreitol (DTT), 0.72 g

CHES buffer, 0.02 g protease [Sigma P5147] andadjusted to pH 9.2 with 10 M Tris) (Lambert and Epel,unpublished). Eggs (200 µl) were transferred (withminimal SW) to 10 ml protease cocktail and rockedgently for 10 min. Excess cocktail was quickly removedand eggs were washed with SW three times by settling.Fertilizations were prepared, as previously mentioned,using formalin/gelatin-treated glassware to avoid stick-ing (Sardet et al., 1989). Results were scored andrecorded as above.

DAPI StainingAuto-, homo- and heterologous fertilizations were

fixed with 1% formalin at 30 sec and 1 min followinginsemination. After fixation, eggs were washed threetimes by settling in SW, stained for 10 min with 50 µl of1 mg/ml (48,6-diamidino-2-phenylindol-dihydrochlo-ride) DAPI per ml SW, a fluorescent dye that interca-lates into DNA (Kawamura et al., 1987), and washedthree more times in SW. Results were observed andphotographed under ultraviolet illumination using anOlympus BX-60 microscope with an attached OlympusPM-C35DX camera body and Olympus PM-30 exposurecontrol unit with TMAX 400 (ISO2000) black and whitefilm.

Glycerin TreatmentEggs were fixed and the follicle cells were removed

using glycerin (modified, Rosati and DeSantis, 1978).Live eggs were incubated approximately 5 min each ina serial (5–10–20–40%) glycerin:SW dilution series(Kawamura et al., 1987). Eggs were allowed to settlewhere possible, but centrifuged for up to 30 min whenneeded. Glycerin-treated eggs were gradually returnedto 100% SW, finally washed three times in SW bysettling (approximately 3 min each wash). Self, homolo-gous, and heterologous fertilizations were prepared asabove. Fertilization was stopped at 3 min with 50 µl(10%) glutaraldehyde to crosslink firmly bound sperm,incubated for 10 min, and then pipetted gently threetimes to remove loosely bound sperm (Lambert, 1986)and finally washed three times in SW. Results wereobserved and photographed as described in gametecollection.

Lectin BindingVitelline coat glycosides were analyzed with concanav-

alin (ConA), fucose binding protein (FBP), and wheatgerm agglutinin (WGA) lectins (Sigma). Live eggs (30µl) were transferred to 1 ml SW with 100 µl TRITC-labeled lectin (dissolved in ddH2O [1 mg/1 ml]) added.The eggs were incubated for 15 min then washed threetimes in SW. Results were observed and scored underfluorescence microscopy using green light with anOlympus BH-2 microscope. Fluorescence was mea-sured with a sensitive diode-equipped measurementdevice in the plane of the microscope camera (Toomeyand Epel, 1993).

110 J. BYRD AND C.C. LAMBERT

Lectin Inhibition of FertilizationLectins were used to compete with sperm for binding

sites (Honegger, 1982; de Santis et al., 1983; Lambert,1986). Untagged lectins (ConA, FBP, and WGA) wereprepared as 0.1 mg/ml solutions. Live eggs (50 µl) wereincubated in the lectin suspension for 20 min mixing asneeded, washed two times in SW with a final 1 ml SW.Sperm were added to eggs and incubated for 1 hr.Results were scored as above.

RESULTSComparison of Ciona Egg Structure

In Ciona, the egg is surrounded by an outermostlayer of follicle cells (FC), an acellular vitelline enve-lope (VC) and within that test cells (TC). Differencesin these covering layers are apparent when comparingC. intestinalis and C. savignyi. Both species haveconical FC with a basal nucleus and a refringent body(Berrill, 1950). However, C. intestinalis FC are slightlylonger (129 6 5.4 µm) with a single subterminalrefringent body, whereas C. savignyi have shorter FC(72 6 3.8 µm) with multiple refringent bodies foundterminally. The VC diameter is greater in C. savignyi(206 6 4.9 µm) than in C. intestinalis (188 6 4.1 µm).Many TC are located within the perivitelline area ofboth species; however, C. intestinalis appears to lackany specific arrangement of these cells, whereasC. savignyi TC are highly organized and adhered to theunderside of the VC (Fig. 1; Table 1). Thus, C. savignyiappeared to have more ‘‘space’’ between test cells and

egg surface than did C. intestinalis. The egg alignmentwithin the perivitelline area differed among the Cionaegg types. The egg of C. intestinalis was more oftenfound centrally located, but that of C. savignyi wasfound either in the center or off-center. An ultrastruc-tural study on C. intestinalis reveals a complex perivi-telline matrix embedded with test cells, in lieu of aperivitelline space found in other ascidians (Eisenhutand Honegger, 1997). Unfortunately, C. savignyi wasnot included in that study. It is possible C. savignyi doesnot have the complex matrix found in C. intestinalis,but rather a perivitelline space that might explain thediffering TC arrangement and egg position between thetwo species.

Autologous (Self), Homologous (NormalOutcross), and Heterologous (Hybrid)

FertilizationsHomologous fertilization resulted in 96–99% cleav-

age (Fig. 2). Autologous fertilizations averaged 3%cleavage and heterologous fertilizations 0–1% cleavagein 8 independent replicate experiments from each

Fig. 1. Morphological comparison of living C. intestinalis (A) and C.savignyi (B) eggs. Note differences in follicle cell (FC) length andrefringent body number (R), test cells (TC), and egg proper (EP)diameter. The C of TC overlies the vitelline coat in B. Scale bar 5 100µm.

TABLE 1. Morphological Differences Between Cionaintestinalis and C. savignyi Eggs*

C. intestinalis C. savignyi

Follicle cellsLength (µm) 129 6 5.4 72 6 3.8Refringent body Single; sub-terminal Multiple; terminal

Vitelline coatDiameter (µm) 188 6 4.1 206 6 4.9

EggDiameter (µm) 148.5 6 3.7 160 6 4.5

Test cell distribution Random Organized

*Diameter is from 8 measurements on each of 4 different eggs.Indicated values are the mean 6 SEM.

Fig. 2. Self-fertilization and hybridization of Ciona intestinalis andC. savignyi (egg 3 sperm). Ia 5 one individual C. intestinalis; Ib 5another individual C. intestinalis; Sa 5 one individual C. savignyi;Sb 5 another individual C. savignyi. Homologous fertilization (Ia 3Ib) occurs at a significantly (P , 0.0001) higher rate than autologous(Ia 3 Ia) or heterologous (Ia 3 Sa). Autologous fertilization does occur,but at a very low rate. The difference in heterologous fertilizations isinsignificant. Bars show means and standard error of the mean(mean 6 SEM). Eight replicate experiments from two individuals ofeach species are included in these values.

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species. These results support findings that Ciona areself-sterile, but not completely so (Morgan, 1939; Rosatiand de Santis, 1978; Kawamura et al., 1987) and thathybridization does occur, but at a vanishingly low rate.

Acid treatment to both C. intestinalis and C. savignyieggs significantly removed the self block (P , 0.0001) inpH 3.2 SW but not the hybrid block (Fig. 3). Fiveindependent replicates, each including two individualsfrom each species were included in Fig. 3. C. intestinaliseggs were more self-fertilizable than C. savignyi eggsfollowing acid SW wash of pH 3.7 to pH 3.2 (P ,0.0317). The follicle cells were still present after pH 4.2incubation, but approximately 50% fell off during pH3.7 SW incubation and 100% fell off during the pH 3.2SW incubation. These eggs all cleaved normally afterfertilization with homologous sperm.

Vitelline Coat in Selfing and HybridizationPrevious studies indicate the block to selfing and

hybridization is at the level of the vitelline coat in someascidians (Morgan, 1939; Reverberi, 1971; Rosati andde Santis, 1978; de Santis et al., 1980; Kawamura et al.,1989). Minganti (1948; see Reverberi, 1971) used ‘‘na-ked’’ eggs, those without the vitelline coat, thus expos-

ing the actual egg surface, in order to obtain hybridiza-tion in various ascidians. Here, the vitelline coat wasremoved with a protease to investigate the level of theblock to hybridization (Fig. 4). Untreated autologousand heterologous fertilizations resulted in a very lowpercentage of cleavage (Fig. 2). However, after thevitelline coat was removed fertilization success in-creased to within 91–97% cleavage in three indepen-dent replicate experiments, each involving two individu-als from each species (Fig. 4). These results supportprevious findings that self- and hybrid block compo-nents are found at the level of the vitelline coat.However, removal of the vitelline coat does not clarifyany difference between the self block and the hybridblock seen with acid wash, nor does it clarify spermrecognition requirements for homologous sperm.

Sperm BindingThe hybridization block was studied further to differ-

entiate if sperm were blocked at the binding step. Eggswere glycerinated to fix them and remove their FC, butstill allow sperm binding without penetration (Rosatiand de Santis, 1978; Lambert, 1986; Kawamura et al.,1987). It is convenient to be able to consider bindingindependently of penetration. Autologous, homologous,and heterologous fertilizations resulted in sperm bind-ing in all cases; however, in lower numbers for autolo-gous and heterologous fertilizations (Fig. 5A, B; autolo-gous not shown).

Sperm PenetrationInvestigation using DAPI shows homologous sperm

penetrated through the VC since they can be found onthe same plane of focus as test cells (found within theperivitelline area of Fig. 5C). Even though spermbinding was seen in all three genetic crosses, no autolo-gous or heterologous sperm were found in the focalplane of the test cells (Fig. 5D; autologous not shown).The nature of the perivitelline area and thick VC makes

Fig. 3. Effects of acid seawater on selfing and hybridization in C.intestinalis (top) and C. savignyi (bottom). The selfing block (Ia 3 Ia;Sa 3 Sa) was significantly removed in both species by pH 3.2 seawater(P , 0.0001) and insignificantly by pH 3.7 (P 5 0.5556) and pH 4.2 (P50.9048). The hybrid (Ia 3 Sa; Sa 3 Ia) blocks were unaffected. Fivereplicate experiments involving two individuals from each specieswere performed. Bars show mean 6 SEM.

Fig. 4. Effects of vitelline envelope removal on selfing and hybridiza-tion of Ciona eggs. Removal of the vitelline coat (open bars 5 withoutVC; solid bars 5 with VC) significantly reduces the block to selfing andhybridization in both species (P , 0.0001). Data is from three replicateexperiments each involving two individuals. Bars show mean 6 SEM.

112 J. BYRD AND C.C. LAMBERT

it difficult to represent DAPI results in a perfect opticalcross-section. Therefore, Figure 5 shows a fluorescenceimage at the plane of the test cells to distinguishbetween the DAPI stained sperm nuclei (cylindrical)and the autofluorescence of the test cells (sphericalbodies).

Glycoside AnalysisPrevious findings indicate C. intestinalis sperm recog-

nize factors found on the outer surface of the vitellinecoat (de Santis et al., 1983; Honegger, 1986), and on theVC ‘‘tufts’’ that contain ConA binding sites (de Santis etal., 1980). Three types of sugar residues, fucose, man-nose and GlcNAc, have been implicated in fertilization(Rosati et al., 1978, 1982; de Santis et al., 1980). Lectinsspecific to each type of terminal sugar residue, includ-ing fucose binding protein (FBP for fucose), concanava-lin A (Con A for mannose), and wheat germ agglutinin(WGA for GlcNAc) were used. The vitelline coat sur-faces of C. intestinalis and C. savignyi display all threetypes of the sugar residue to some degree (Fig. 6; Table2). GlcNAc is significantly more prevalent than theother sugars in both species (P , 0.0001). However,C. intestinalis shows a significantly greater quantity ofGlcNAc residues than does C. savignyi, indicated bygreater WGA fluorescence (P , 0.0001). Both speciesshow a relative greater quantity of GlcNAc residuesthan fucose residues (indicated by FBP fluorescence)

and mannose residues (indicated by ConA fluores-cence). ConA lectin had a 0.5 mol TRITC/moleculecompared to 1 mol TRITC/molecule for FBP and WGA;therefore the data was doubled for ConA and graphedaccordingly. Overall glycoside composition differs be-

Fig. 5. Binding and penetration using glycerinated and DAPI-stained Ciona intestinalis eggs. Homologous fertilization showedsperm (arrows) bound to glycerinated eggs (A) and penetrated as seenby DAPI stain (C). Heterologous fertilization showed fewer sperm

bound (B), but showed no penetration (D). Autologous was similar toheterologous fertilization shown here. C. savignyi exhibited similarsperm behavior and numbers (not shown). (Arrows show sperm headsand tails in A and B, and sperm heads only in C). Scale bar 5 100 µm.

Fig. 6. Semi-quantitative lectin binding to the vitelline coats of C.intestinalis and C. savignyi. WGA fluorescence is significantly higher in C.intestinalis (P , 0.0001) than in C. savignyi. FBP and ConA exhibit asignificantly higher fluorescence in C. savignyi (P , 0.0001). Graphindicates the mean of 40 fluorescence values, where 10 readings werecollected in four independent experiments. Bars show mean 6 SEM.

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tween the two species of Ciona. Both species seem tohave a greater relative quantity of GlcNAc residues,followed by mannose residues and fucose residues;however, the relative ratio of these residues is differentbetween the species. Acid treatment failed to removeWGA binding residues (data not shown).

The distribution of sugar residues on the FC, VC, TC,and egg was investigated and recorded based on rela-tive fluorescence intensity from each, which allows acomparison of these regions between species (Table 2).After incubation with a tagged lectin, whole live eggs ofC. intestinalis and C. savignyi exhibited a ‘‘web’’ patternon their outer vitelline coat surface as seen in Lambert(1986), evidently caused by the FC since the patterndisappeared in their absence (data not shown). Therewas little or no fluorescence from the FC, indicatingminimal, if any, sugar residues on them. The TCshowed fluorescence in C. intestinalis eggs with WGAonly.

Inhibition of Fertilization With LectinsThe results thus far have indicated the VC is the level

of the selfing and hybridization blocks and containsvarying amounts and types of sugar residues. There-fore, further analysis was performed to investigate theinvolvement of the VC sugars in the recognition, bind-ing, or penetration of intra- and interspecific sperm.Ciona eggs were incubated in untagged lectins (1mg/mlSW; ConA WGA or FBP, respectively), then fertilized.C. intestinalis fertilization was significantly inhibitedby all three lectins (P , 0.0001), but most dramaticallyby ConA (Fig. 7). C. savignyi fertilization was signifi-cantly inhibited by ConAonly. This suggests the involve-ment of mannose, and some fucose and GlcNAc inC. intestinalis sperm-egg interaction, but only mannosein C. savignyi sperm-egg interaction.

DISCUSSIONMajor differences in follicle cell (FC) and test cell (TC)

morphology exist between the two closely related Cionaspecies. The differences are notable but not as extremewhen compared to Ciona edwardsi with its 200-µm eggs(Copello et al., 1981). The test cell arrangement was themajor difference between the two species.

C. savignyi FC have multiple refringent bodies previ-ously shown in C. edwardsi (Lambert et al., 1990), but

are shorter than those in C. edwardsi (Lambert, unpub-lished). C. intestinalis and C. roulei have longer FCwith a single refringent body (Lambert et al., 1990).Although there are many documented functions of FCincluding flotation, chemotaxis, block to polyspermy,sperm guidance to egg surface, and egg adhesion tosubstrate (Miller, 1975; Lambert and Lambert, 1978;de Santis et al., 1980; Villa and Patricolo, 1993; Lam-bert et al., 1995), sperm binding has not been shown tobe influenced by these cells (Rosati and de Santis,1978).

The results shown in our study indicate the FC ofboth Ciona species do not have mannose or fucose sugarresidues on their surface, but possibly have someGlcNAc residues. Rosati et al. (1982) found no fucosyl-containing residues on the FC at any time during eggmaturation, yet three different fucosyl-containing glyco-proteins were found on the vitelline coat of the matureC. intestinalis egg. Further, significant fluorescenceintensity was seen between the FC clefts where spermcongregate (Fukumoto, 1990; Fukumoto and Numaku-nai, 1995) particularly with WGA lectin. Therefore, theFC role may be primarily for homologous sperm guid-ance between FC to the VC (de Santis et al., 1980;Cotelli et al., 1981; Kawamura et al., 1988; Fukumotoand Numakunai, 1995) and not sperm binding orpenetration.

We suggest that Ciona sperm binding is accom-plished through a species-specific combination of thecarbohydrate portion of the glycoside, rather than theprotein portion that is involved in penetration. Thisidea is based on the fact that hybrid sperm were able tobind, but not penetrate (Fig. 5) and that proteolysiscould be species-specific (Pinto et al., 1990; Marino etal., 1992).

TABLE 2. Localization of Lectin Binding to LivingCiona Eggs

C. intestinalis C. savignyiWGA FBP ConA WGA FBP ConA

Vitelline coat 111 1 1 11 1 1Test cells 111 2 2 2 2 2Follicle cells 1/2a 2 2 1/2a 2 2Refringent spot 2 2 2 1/2a 2 2

aExhibited indefinite fluorescence, less than ‘‘1.’’ Relativefluorescence determined subjectively between Ciona eggs andbetween various egg regions with different fluorescentlytagged lectins.

Fig. 7. Effects of lectins on percent fertilization for C. intestinalisand C. savignyi. C. intestinalis sperm were significantly inhibited byall three lectins (P , 0.0001) but C. savignyi sperm were only inhibitedby ConA. Two independent replicate experiments using two individu-als of each species were performed. Bars show mean 6 SEM.

114 J. BYRD AND C.C. LAMBERT

We have shown that acid washing can remove theselfing block, but not the hybrid block, in both species(Fig. 3). This suggests different mechanisms are opera-tional. We also found that sperm bind to the VC inautologous or heterologous fertilization, but fail topenetrate it (Fig. 5). The block to autofertilization inHalocynthia may also operate at this level (Hoshi et al.,1994). Sperm attraction by eggs is equal betweenC. intestinalis eggs and C. savignyi sperm and thereciprocal (Yoshida et al., 1993), thus it is possible thatequal heterologous sperm would arrive at and bind tothe VC surface. Initial work on self-sterility showedacid SW could remove the selfing block in some ascid-ians (Morgan, 1939). Later, it was found that a self/non-self recognition component could be extractedfrom eggs with acid SW (Kawamura et al., 1991). Theyfound that an acid extract of C. intestinalis eggs couldconfer self-sterility in autologous fertilizations andsuggested that the self-sterility factor could be a glyco-side. In our experiment we could detect no decline inlectin staining of GlcNAc residues after the acid wash(data not shown). This may indicate that the acidextracted component is different from these residues.No decline in GlcNAc residues by acid wash alsosuggests that these residues may be a major player inthe sperm-egg binding in both species, but not inself-discrimination since GlcNAc is prevalent in both(Fig. 6). However, it should be noted that there may beother GlcNAc residues not assayed that are importantin sperm-egg interactions. The self and hybrid blockscould be associated with the test cells since these wereremoved with the VC and FC. However this is not likelyas autologous and heterologous sperm were unable topenetrate their level in intact eggs.

Based on our findings, we suggest that ‘‘self compat-ibility’’ proteins, products of the oocyte itself, found onthe VC surface work in tandem with glycosides insperm-egg interactions. We will refer to these protein(s)as sperm receptor ‘‘modulators’’ and define modulatorsas components of a process that can regulate thatprocess but cannot be solely responsible for it. We alsosuggest that the sperm receptor modulators may ormay not be GlcNAc or glycosylated. One self-sterilityfactor in C. intestinalis has been determined to be aglycoprotein (de Santis and Pinto, 1991) that is aproduct of the FC in the final stages of egg maturation(Cotelli et al., 1981). Marino et al. (1998) agree themodulator is a product of the follicle cells, but disagreesthat it is glycosylated, calling it a protein of the MHCrepertoire. It is also possible that the modulator in-volves fucose or mannose, or another component yet tobe determined.

The suggested modulator does not regulate species-specific binding but may be involved in regulatingsperm penetration (Fig. 5A–D). Some workers havefound that autologous sperm fail to penetrate throughthe vitelline coat in C. intestinalis (Morgan, 1942) andHalocynthia roretzi (Fuke and Numakunai, 1996).Kawamura et al. (1991) considered the block to self-fertilization in Ciona to be strictly at the binding level.

More recent investigations (Fuke and Numakunai,1996; Marino et al., 1998) on the block to selfing inH. roretzi and C. intestinalis simply score percentcleavage as an indication of fertilization, not consider-ing binding independently. Here, we give evidence thatpenetration is the level of the block to hybridization.

Suggested Mechanisms for Ciona Intestinalisand C. Savignyi Sperm-Egg Interaction

We propose the sperm ‘‘receptor’’ is a species-specificcombination of glycosides that the sperm recognize andbind (Kawamura et al., 1989) leading to an incorpora-tion of the modulator that results in the formation ofthe ‘‘complete’’ sperm receptor. Recently, a MHC classIII Hsp70 gene has been identified in C. intestinalis byMarino et al. (1998). They noted the protein originatesin the follicle cells and, at egg maturity, it is brought tothe VC and becomes part of the sperm receptor. In thisstudy, self-fertilization first occurred at pH 3.7 wherefollicle cells were not present. This means the self-recognition protein must be anchored in the VC prior tothe follicle cells’ disappearance. We agree that there isanother factor involved in Ciona sperm-egg interactionand suggest the following mechanism for homologous,autologous, and heterologous fertilization. Homologoussperm are able to bind, incorporate the co-factor, andpenetrate based on a ‘‘complete’’ sperm receptor. In selfing,sperm binding occurs but penetration does not. Thismay be because the modulator is a component of the selfrecognition system that prevents self sperm from initi-ating the penetration process. This idea is analogous tothe human immune system where self tissue is notattacked due to a self protein (the major histocompatibil-ity complex) expressed on the surface of every self cell.Heterologous sperm are able to bind but not penetrate,because the modulator is not recognized and thus notincorporated. Therefore, both self and hybrid sperm areable to bind but not penetrate because neither is able torecognize or construct the complete receptor.

This study supports the finding that C. intestinalisand C. savignyi are not capable of interspecific fertiliza-tion. Besides blocking interspecific fertilization, Cionaspecies are not capable of self-fertilization. This study hasshown that the selfing and hybrid blocks are at the level ofthe vitelline coat but have separate mechanisms.

ACKNOWLEDGMENTSWe are thankful to Gretchen Lambert and Andrew

Lowe for collection of experimental animals andGretchen Lambert, Lana Theis and Robert Koch formany helpful suggestions. Gretchen Lambert and ananonymous reviewer have our thanks for improving themanuscript.

Note added in proof: A recent paper (Marino et al.,1999) implicates proteosome activity in the formation ofthe block to self fertilization in Ciona intestinalis.

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