a trypsin-like oviducal proteinase involved in bufo arenarum … · involvement of an oviducal...

/ . Embryol. exp. Morph. Vol. 48, pp. 79-91, 1978 7 9Printed in Great Britain © Company of Biologists Limited 1978

A trypsin-like oviducal proteinaseinvolved in Bufo arenarum fertilization

By DORA C. MICELI1, SILVIA N. FERNANDEZ1,JORGE S. RAISMAN1 AND FRANCISCO D. BARBIER11

From the Instituto de Biologia, Facultad de Bioquimica,Quimica y Farmacia, Universidad Nacional de Tucumdn

SUMMARYWhen the jelly-less eggs removed from the most cephalic region of the oviduct (pars recta)

of the toad Bufo arenarum were inseminated at a high sperm concentration, high frequenciesof fertilization were obtained. On the other hand, control eggs removed from the pleuro-peritoneal cavity (coelomic eggs) were neither fertilized upon insemination under identicalconditions, nor with the water extract of the jelly. Under these inseminating conditions,however, a high frequency of fertilization was obtained when coelomic eggs were pre-incubated in the presence of the fluid secreted by the epithelium of the pars recta or of anextract prepared from pars recta homogenate.

Experimental evidence is presented showing that the component responsible for thiseffect acts on the vitelline envelope of the egg, increasing its susceptibility to sperm Iysin.It is probable, therefore, that it induces successful fertilization of coelomic eggs by makingthe vitelline envelope more easily penetrable by sperm.

The active factor was partially purified by Sephadex chromatography. The productobtained was of high activity and, as judged by its inhibition with soybean trypsin inhibitorand lima-bean trypsin inhibitor, it is likely to be a trypsin-like enzyme. The molecularweight of the factor was estimated to be 47000 by Sephadex chromatography. Secretion ofthe pars recta factor is hypophysis-dependent and its activity is not influenced by pH withinthe range tested (60-8-4).

INTRODUCTION

In Amphibia the oocytes released from the ovary when ovulation takes placeare temporarily retained in the pleuroperitoneal cavity of the female (coelomiccavity), until conducted to the exterior through the oviducts. The oviduct is along duct in which three main regions appear as a constant feature: (1) thepars recta, the uppermost uncoiled segment, a few centimeters long; (2) thecoiled portion comprising most of the length of the organ; and (3) the ovisac,the most caudal region and where eggs accumulate before oviposition. Thereis plenty of evidence that the female duct plays the role of a true fertilizationorgan in these vertebrates. Studies made with a variety of species have shownthat the jelly material, deposited around the eggs as they go down the pars

1 Authors' address: Instituto de Biologia, Chacabuco 461, 4000 S. M. de Tucuman,R. Argentina.

6-2

80 D. C. MICELI AND OTHERS

convoluta region of the oviduct, is essential for fertilization (see Humphries,1966; Katagiri, 1966; Shaver, 1966; Metz, 1967; del Pino, 1973; Cabada, 1975).Recent investigations have also proved the importance of soluble moleculessecreted at the same duct level and retained within the jelly up to the time ofshedding ('diffusible factor') (see Barbieri, 1976).

Although the function of the most cephalic portion of the oviduct is stillpoorly understood, it might be also implicated in the fertilization process.Thus, the results of preliminary experiments on the toad Bufo arenarum suggestthat some factor supplied by the epithelium of this oviducal region alters thevitelline envelope of the egg rendering it more susceptible to sperm lysin attackwhich is likely to facilitate sperm penetration through it (Barbieri, 1971).The purpose of this paper is to report the results of a series of experimentswhich confirm the involvement of pars recta in Bufo arenarum fertilization.Some properties of the factor and its partial purification including molecularweight estimation, are also described.

MATERIALS AND METHODSGametes

Mature Bufo arenarum specimens were collected near Famailla and storedat 15-18 °C. Before use, they were kept for at least 24 h at 25 °C. Ovulationwas induced by intraperitoneal injection of a suspension of homoplastic hypo-physis (Houssay, Giusti & Lascano Gonzalez, 1929) preserved according toPisano (1956). Animals were kept at 25 °C and pithed 7 h after injection.Under these conditions, eggs were often found in the pleuroperitoneal cavity(coelomic eggs) as well as along the whole oviduct, ovisac included. In order toobtain large numbers of pars recta eggs, this segment of the duct was ligatedat its caudal end prior to ovulation induction. Seven hours after injection,animals were killed and the pars recta segment, where eggs were accumulated,was excised by sectioning below the ligature and transferred to Holtfreter'ssolution. The ligature was then cut out, and the eggs were expelled from theduct. Egg maturity was assessed by direct examination of the first polocyteunder the stereomicroscope.

Suspensions of sperm were obtained by mincing the excised testes in saltsolution. The number of spermatozoa pei milliliter was estimated by the use of ahemocytometer.

Preparation of pars recta extract

The pars recta region of the oviducts of anesthetized animals was ligatedat both ends, i.e. at the level of the ostium and immediately above the initiationof pars preconvoluta (as defined for Bufo arenarum by Moreno, 1972) (Fig. 1).After the operation, the females were kept at 25 °C for 12 h and then injectedwith hypophysis suspension. Seven hours later, they were pithed and the fluid

Involvement of an oviducal proteinase in Bufo fertilization 81

Fig. 1. Diagram of the left oviduct of Bufo arenarum. The voluminous ovary cover-ing the duct has been removed, pr, pars recta; pp, pars preconvoluta; pc, parsconvoluta; o, ovisac (its opening into the cloaca is not drawn). In this preparationthe ovisac appears as a collapsed thin-walled vesicle; when fully charged withoocytes the ovisacs occupy most of the body cavity of the animal.

accumulated within the ligated segment was either directly removed or a 10%homogenate of the whole segment was prepared in ice-cold Ringer-lOmMTris buffer pH 7-6 (in the following this solution will be referred to as Ringer-Tris solution). In the latter case, the pars recta was rapidly excised, trimmed ofextraneous tissues, weighed and washed. Finally it was scissor-minced andhomogenized in a porcelain mortar with washed sand. The brei was centrifugedat 10000 g for 20 min, and the supernatant was dialysed against 2000 vol. of theRinger-Tris solution at 4 °C overnight. The retentate was centrifuged at10000 g for 20 min and after testing its activity it was fractionated and lyophi-lized.

Preparation of jelly water extract

It was prepared according to Barbieri & del Pino (1975). Briefly, the pro-cedure consists of submitting the egg strings stripped from the ovisacs to twosuccessive extractions with distilled water under standardized conditions.Extracts were stored at —20 °C until used.


Preparation of sperm lysin

Crude preparations of the enzyme were obtained according to Cabada,Mariano & Raisman (1978). The procedure consisted essentially of the followingsteps. A suspension of washed spermatozoa was freeze-dried, resuspended indistilled water and centrifuged. The supernatant was dialysed against 10 mMTris-HCl solution pH 7-6 and the retentate spun down. The supernatant wasfinally filtered, fractionated and lyophilized.

Assays of pars recta factor activity

Activity was estimated by two methods, both of them carried out withcoelomic eggs.

Procedure A. The first method, based m the ability of pars recta factoi inproducing fertilization of coelomic eggs, comprised two steps. In the firststep, eggs were incubated with the secretion product or the extract preparedfrom the pars recta in Ringer-Tris for 20 min at 25 °C, and then rinsed twicewith the same salt solution. In the following, these eggs will be referred to as'conditioned eggs'. Contiol eggs were incubated in Ringer-Tris under thesame conditions. In the second step, conditioned and control eggs were in-seminated by placing them either with sperm at a high concentration (Raisman& Pisano, 1970) or with sperm in the presence of the jelly water extract (Bar-bieri & del Pino, 1975) as indicated in each experiment. The final concentrationof the water-extract of the jelly was 0-120-0-200 as indicated by the absorptionat 280 nm. The following day the eggs were scored for fertilization. Onlynormal blastulae were scored as fertilized. In order to assess sperm penetration,eggs were also fixed 2 h 30 min after insemination in 6% glutaraldehyde in0-1 M phosphate buffer (pH 7-6). The presence of the sperm pigment trackcould be observed then by direct dissection under the stereomicroscope (Manes& Barbieri, 1977).

Procedure B. The second method is based in the observation that the oviducalfactor alters the vitelline envelope rendering it more susceptible to the attackby sperm lysin (see Barbieri, 1971). It also consisted of two steps. The firstwas identical to that of procedure A. In the second step, batches of 15 con-ditioned and control eggs were transferred to 0-3 ml 10% Ringer's pH 7-4. Atzero time, 20 /A of sperm lysin containing 14 ^g/ml of protein was added.Three minutes later, the morphological changes produced on the egg surface- as a result of a weakening of the vitelline envelope - were observed underthe stereomicroscope. Results were reported as 0 (no apparent lytic effect),+ (incipient lytic effect), + + (net lytic effect) and + + + (severe lytic effect).Maximum effect was indicated by enlargement and softening of the vitellineenvelope associated with wrinkling of the egg surface and a decrease of eggtension.


Inhibitor assays

Inhibition was estimated by performing step 1 of the assay procedure (eggpreincubation with pars recta extract) in the presence of lima-bean trypsininhibitor or soybean trypsin inhibitor. Inhibitor concentrations were 100/tg/ml. Step 2 was identical to that of assay procedure B. Prior to incubationwith the sperm lysin, the eggs were exhaustively washed in 100 ml Ringer's.Since these inhibitors have an impairing effect on sperm lysin activity (Cabadaet al. 1978) control experiments were carried out with oviducal eggs. The eggswere dejellied, treated with the inhibitors, rinsed under identical conditions andfinally incubated with the sperm lysin. No inhibitory effect could be detected.

Column chromatography

Sephadex gels (Pharmacia) were swollen, washed in distilled water andpacked in columns of the following dimensions: 2-5 x 50 cm for Sephadex G-200,and 0-9x24 cm for Sephadex G-150. Before use, the beds were equilibratedwith Ringer-10 mM Tris pH 7-6; this same solution being used for elution.Samples were collected and assayed for biological activity and analysed forprotein as absorption at 280 nm. Separation proceeded at 4 °C.

Molecular weight

Molecular weight was estimated according to Andrews (1964), by gel fil-tration on Sephadex G-150. The proteins' markers used and their molecularweight were as follows: ovomucoid, 48000; bovine serum albumin, 67000;bovine pancreatic trypsin, 23300 and cytochrome c, 12400.

RESULTS

Properties of the crude extract

Pars recta secretion as a fertilization factor

It has been shown previously that Bufo arenarum eggs removed from theovisacs and artificially dejellied, which are not fertilized under normal insemi-nating conditions, are rendered fertilizable with high concentrations of sperm(Raisman & Pisano, 1970). In order to attempt a comparison of responsecapacity, jelly-less eggs removed from the pars recta and the body cavity wereinseminated at high sperm concentrations. As shown in Table 1, a strikingdifference was observed. Thus, while a frequency of fertilization of about 80 %was observed for pars recta eggs, only in rare instances could the coelomic eggsbe fertilized. Similar results were obtained when coelomic eggs conditionedwith either the secretion fluid of the pars recta or pars recta extract were in-seminated. While a significant percentage of conditioned eggs were fertilizedand segmented in a normal fashion, no control eggs were fertilized (Table 2).

Sections of conditioned eggs fixed 2 h 30 min after insemination showed a


Table 1. Fertilization of pars recta eggs

Percentage cleaved

Pars recta eggs 848±5-3Coelomic eggs 0-8 ± 0-8

Eggs were inseminated with a dense sperm suspension (l-7xiO7 cells/ml). Results areexpressed as the mean and standard error from five experiments carried out with differentanimals.

Table 2. Fertilization of conditioned eggs

Treatment Percentage cleaved

Pars recta secretion fluid 73-1 + 5-6*Pars recta extract 88-3 ± 3-5*

66-0 ± 15-11Ringer solution 00*f

Eggs were incubated with 40/tg/ml of either pars recta secretion fluid or extract.* Eggs were inseminated in the presence of the jelly extract. (Final sperm concentration

was 3 x JO6 cells/ml.) N = 7.t Insemination conditions were the same as in Table \. N = 3. Results as in Table 1.

typical sperm entrance path clearly indicated by a trail of pigment. In contrastwe found no instances of penetration of control eggs by sperm.

Pars recta secretion and sperm lysin attack on the vitelline envelope

Evidence that sperm attack on the vitelline envelope depends on pars rectasecretion comes from observations of well defined alterations exhibited byconditioned eggs, but not by control eggs, in the presence of a crude preparationof sperm lysin. It was consistently observed that after the 3 min period ofincubation, all conditioned eggs became less turgid, and depressions andwrinkles were observed on the egg surface. Later on, the eggs collapsed, andthe vitelline envelope appeared swollen, with signs of partial dissolution. Aspreviously reported, these are typical signs of sperm attack on the egg envelope(Raisman & Barbieri, 1969).

Hypophysis-dependent effect of the pars recta factor

Epithelial cells of Bufo arenarum pars recta secrete a crystalline fluid evenin the absence of artificial hormonal stimulation. It was therefore pertinent totest whether the active factor was also secreted or synthesized under thisphysiological condition. For this, pars recta secretion and extract were preparedas detailed in Materials and Methods, from animals previously injected orsham injected with hypophysis suspension. Obviously, care was taken to workwith toads captured at the same time and kept under identical conditions.Activity of the preparations obtained was then estimated by procedures A and


Table 3. Effect of hypophysis treatment on pars recta activity

Female treatment

Hypophysis suspensioninjected

Ringer's injected

Concentration of parsN= 3.

Egg treatment

Secretion fluidExtractRinger'sSecretion fluidExtractRinger's

recta secretion fluid and

Percentage cleaved

70-3 ±1-5840 ±2-30 00 00 00 0

extract as in Table 2. Results as

Lyticeffect

+ ++ + +0000

in Table 1

Table 4. Inhibition of pars recta factor activity by trypsin inhibitors(100 fig/ml)

Treatment Lytic effect

Ringer's 0Pars recta extract 4 + +Pars recta extract 0

+ soybean trypsin inhibitorPars recta secretion +

4- soybean trypsin inhibitorPars recta extract +

4- lima-bean trypsin inhibitorPars recta secretion +

4 lima-bean trypsin inhibitorConcentration of pars recta secretion and extract as in Table 2.

B, i.e. by testing egg fertilizability and lysis of the vitelline envelope by spermlysin. The results of Table 3 show the clear-cut differences observed betweenthe preparations obtained from treated and untreated females. While hormonalstimulation led to a high frequency of fertilization, no egg was fertilized aftertreatment with the preparations obtained from non-injected females. In noneof the eggs pretreated with the material obtained from non-stimulated femalescould the sperm entrance path be seen. A good correlation was also observedbetween egg fertilizability and the susceptibility of the vitelline envelope tosperm lysin attack (Table 3).

pH-dependence of pars recta factor activity

In order to test the influence of hydrogen-ion concentration on pars rectafactor activity, the sperm lysin assay (procedure E) was carried out as describedin Materials and Methods with the exception that incubation during the firststep was performed in the presence of the following buffers at 10 mM concen-


0-500 -

0-300 -

0100 - - 1 +

60 70Fraction number

80 90

Fig. 2. Chromatography of 3-6 ml of the crude extract on Sephadex G-200 (voidvolume 77 ml). The flow rate was adjusted to 4-2 ml per hour and fractions of2-2 ml were collected. • — # , Absorbance at 280 nm; O—O, activity.

trations: MES (2-(N-morpholino)-ethanesulfonic acid) pH 6-0, TES (N-Tris(hydroxymethyl)-methyl-2-aminoethanesulfonic acid) pH 7-2, and Tris-HClpH 8-4. No difference in the pH range tested was observed. Control eggsincubated with the buffers alone exhibited no noticeable change.

Inhibition

As shown in Table 4, the pars recta factor is inhibited by the two trypsininhibitors tested. The loss of activity was slightly higher with soybean trypsininhibitor than with lima-bean trypsin inhibitor.

Purification

The lyophilized pars recta extract was dissolved in a small amount of distilledwater, dialysed overnight against Ringer-Tris and centrifuged at 700 g for 15min. The supernatant was applied to the top of the column of Sephadex G-200and eluted with Tris-buffered Ringer's under the conditions given in Fig. 2.Two well separated absorbance peaks at 280 nm emerged from the column.Activity, as estimated by procedure B, emerged also as a well-defined peakcontained within the second protein peak. Elution of the activity from thecolumn was not coincident, however, with the elution of u.v. absorbing materials.

Eluates containing the active fraction were pooled and lyophilized. Theresidue was taken in distilled water and then dialysed and centrifuged as theoriginal sample. The supernatant was fuither purified on a column of SephadexG-150. The elution pattern, as shown in Fig. 3, was characterized by three


0-400 t-

0-300 -

0-200 -

0100 -

- 1 +

1 3 52 4

10 20

Fraction number

Fig. 3. Elution profile of Sephadex G-200 active product further purified by Sepha-dex G-150 column chromatography. 0-3 ml of the dialysed product was appliedto the column (void volume 5-6 ml) and the flow rate was adjusted to 12 ml perhour. Each fraction was 0-7 ml. Symbols as in Fig. 1.

peaks of protein at A28o. The second peak contained the biological activity.Although the product may not be pure, a remarkable enhancement of activitywas observed. Thus, direct incubation of coelomic eggs with this fraction(step 1 of procedure B) led to dissolution of the vitelline envelope and severeegg collapse. In order to estimate the relative activity of eluates, samples had tobe diluted 20-fold.

Molecular weight

Through extrapolation after chromatography on Sephadex G-150, molecularweights averaging 47000 were obtained (Fig. 4). Variation of three determi-nations was 8 %.

DISCUSSION

The evidence presented here shows that the secretion produced at the mostcephalic portion of the oviduct of the toad Bufo arenarum is implicated infertilization. The methods used in this study indicate that a prerequisite forthe fertilization of coelomic eggs is either their passage through the pars recta


105 h

. BSA

: \

i i

\«|OM1

\ # BPT

\

i i

N Cit. c

i i

1-5 1-6 1-7 1-8 1-9 2-0Mobility (V/Vo)

2-1 2-2 2-3

Fig. 4. Molecular weight estimation by chromatography on Sephadex G-150. BSA,bovine serum albumin; OM, ovomucoid; BPT, bovine pancreatic trypsin; Cit. c,cytochrome c.

or their treatment with pars recta secretion or extract prior to insemination.Also, our results demonstrate that participation of the pars recta factor infertilization is mediated by a change in vitelline envelope penetrability bysperm. This conclusion is based on two observations: (1) the presence of asperm penetration track could be exclusively detected in conditioned eggs:and (2) pretreatment with the active preparation was a necessary condition forsperm lysin attack on the vitelline envelope of the egg. It is generally admittedthat this lytic enzyme(s), which is released from sperm as a result of acrosomebreakdown (Raisman & Cabada, 1977), opens the male cell path through theegg envelope (Raisman & Barbieri, 1969; Elinson, 1971; Greenslade, McCor-mack, Hirsch & Davanzo, 1973). Besides, our results in this respect are inagreement with those provided by experiments on the penetrability of isolatedvitelline envelopes of the egg of Xenopus laevis in vitro. It has been reportedthat in this species sperm readily penetrates the envelopes isolated from ovi-posited eggs, but not so those isolated from coelomic eggs (Grey, Working &Hedrick, 1977).

The enhancement of the envelope susceptibility to sperm lysin is probablydue to its alteration as a result of a sublytic attack by a trypsin-like enzymesecreted by the oviducal epithelium. This is suggested by the fact that the


active factor is protein in nature (Raisman, 1975) and is inhibited by specifictrypsin inhibitors. This view is also consistent with the observation that thecoelomic eggs of the frog Rana pipiens can be rendered fertilizable by a briefpretreatment with trypsin followed by insemination in a water-extract of thejelly (Elinson, 1973).

It is possible that during passage of the egg down the pars recta the enzymeattack on the vitelline envelope is limited by the presence of an inhibitor. Thisis suggested by the remarkable increase in activity obtained after SephadexG-150 chromatography, which was most likely caused by removal of aninhibitor.

Grey et al. (1977) have recently reported that a structural transformationof the vitelline envelope of the egg of Xenopus takes place in the first centi-meter of the female duct. At the electron microscope level, the authors havefound that among other changes the fascicular arrangement of the filamentswhich constitute the envelope of the coelomic egg becomes evenly dispeisedunder the influence of oviducal secretions. Because these changes, like thosereported here for Bufo, are simultaneously produced as the egg descends downthe first oviducal portion, it is tempting to assume a causal relationship betweenthem. Further studies are needed in this respect, however, since exposure ofXenopus eggs to a pH of below 6 or above 9 was reported to bring about adispersal of fibrils closely resembling that produced by the uppermost oviducalsegment (Grey et al. 1977), whereas a similar treatment in Bufo did not inducethe attack of the vitelline envelope by sperm lysin. We do not know yet whetherthis lack of response of Bufo eggs must be ascribed to the shorter incubationtime used for this species or to specific peculiarities. An additional point broughtout in our experiments is that the active factor is clearly hypophysis-dependentsince no egg response could be elicited by treatment with either the secretionfluid which is produced in the pars recta of non-stimulated females or with theextract prepared from whole homogenate of these same animals.

Taking into account the increasing evidence emphasizing the important roleplayed by the female duct of amphibians in connexion with gamete interactionas well as egg metabolism (Legname et al. 1972) and maturation (Brun, 1975),further studies on the molecules transferred from the secretory epithelium ofthe oviduct to the egg and its envelopes, such as those reported by Nace,Suyama & Smith (1960) and Humphries (1970), are urgently needed.

Finally, it is worth noting that a sublytic alteration of the egg envelope byoviducal secretions must not be regarded as a peculiar feature of anuranamphibians. Thus, the zona pellucida of the mouse egg, homologous to thevitelline envelope of the amphibian egg, has also been found to be exposed,under natural conditions, to sublytic levels of zonalytic factors in the uterinemedium (Domon, Pinskev & Mintz, 1973).


Appreciation is expressed to Mr Eduardo Rothe for his assistance in the preparation of themanuscript. This work was supported in part by grants from the Consejo Nacional deInvestigaciones Cientificas y Tecnicas (R. Argentina) (CONICET), The Population Council,New York, N.Y. (awarded to the CONICET) and the Fundacion Lucio Cherny (R. Argen-tina). This work is part of a thesis to be submitted by D. C. M. to the Universidad Nacionalde Tucuman.

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(Received 3 April 1978, revised 15 June 1978)

a trypsin-like oviducal proteinase involved in bufo arenarum … · involvement of an oviducal...

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