/ . Embryol. exp. Morph. Vol. 48, pp. 79-91, 1978
Printed in Great Britain © Company of Biologists Limited 1978
79
A trypsin-like oviducal proteinase
involved in Bufo arenarum fertilization
By DORA C. MICELI 1 , SILVIA N. FERNANDEZ 1 ,
JORGE S. RAISMAN 1 AND FRANCISCO D. BARBIER1 1
From the Instituto de Biologia, Facultad de Bioquimica,
Quimica y Farmacia, Universidad Nacional de Tucumdn
SUMMARY
When 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 frequencies
of fertilization were obtained. On the other hand, control eggs removed from the pleuroperitoneal cavity (coelomic eggs) were neither fertilized upon insemination under identical
conditions, nor with the water extract of the jelly. Under these inseminating conditions,
however, a high frequency of fertilization was obtained when coelomic eggs were preincubated in the presence of the fluid secreted by the epithelium of the pars recta or of an
extract prepared from pars recta homogenate.
Experimental evidence is presented showing that the component responsible for this
effect 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 making
the vitelline envelope more easily penetrable by sperm.
The active factor was partially purified by Sephadex chromatography. The product
obtained was of high activity and, as judged by its inhibition with soybean trypsin inhibitor
and lima-bean trypsin inhibitor, it is likely to be a trypsin-like enzyme. The molecular
weight of the factor was estimated to be 47000 by Sephadex chromatography. Secretion of
the pars recta factor is hypophysis-dependent and its activity is not influenced by pH within
the range tested (60-8-4).
INTRODUCTION
In Amphibia the oocytes released from the ovary when ovulation takes place
are temporarily retained in the pleuroperitoneal cavity of the female (coelomic
cavity), until conducted to the exterior through the oviducts. The oviduct is a
long duct in which three main regions appear as a constant feature: (1) the
pars recta, the uppermost uncoiled segment, a few centimeters long; (2) the
coiled portion comprising most of the length of the organ; and (3) the ovisac,
the most caudal region and where eggs accumulate before oviposition. There
is plenty of evidence that the female duct plays the role of a true fertilization
organ in these vertebrates. Studies made with a variety of species have shown
that 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 molecules
secreted at the same duct level and retained within the jelly up to the time of
shedding ('diffusible factor') (see Barbieri, 1976).
Although the function of the most cephalic portion of the oviduct is still
poorly understood, it might be also implicated in the fertilization process.
Thus, the results of preliminary experiments on the toad Bufo arenarum suggest
that some factor supplied by the epithelium of this oviducal region alters the
vitelline envelope of the egg rendering it more susceptible to sperm lysin attack
which is likely to facilitate sperm penetration through it (Barbieri, 1971).
The purpose of this paper is to report the results of a series of experiments
which confirm the involvement of pars recta in Bufo arenarum fertilization.
Some properties of the factor and its partial purification including molecular
weight estimation, are also described.
MATERIALS AND METHODS
Gametes
Mature Bufo arenarum specimens were collected near Famailla and stored
at 15-18 °C. Before use, they were kept for at least 24 h at 25 °C. Ovulation
was induced by intraperitoneal injection of a suspension of homoplastic hypophysis (Houssay, Giusti & Lascano Gonzalez, 1929) preserved according to
Pisano (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 to
obtain large numbers of pars recta eggs, this segment of the duct was ligated
at 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's
solution. The ligature was then cut out, and the eggs were expelled from the
duct. Egg maturity was assessed by direct examination of the first polocyte
under the stereomicroscope.
Suspensions of sperm were obtained by mincing the excised testes in salt
solution. The number of spermatozoa pei milliliter was estimated by the use of a
hemocytometer.
Preparation of pars recta extract
The pars recta region of the oviducts of anesthetized animals was ligated
at both ends, i.e. at the level of the ostium and immediately above the initiation
of 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 injected
with 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 covering the duct has been removed, pr, pars recta; pp, pars preconvoluta; pc, pars
convoluta; o, ovisac (its opening into the cloaca is not drawn). In this preparation
the ovisac appears as a collapsed thin-walled vesicle; when fully charged with
oocytes the ovisacs occupy most of the body cavity of the animal.
accumulated within the ligated segment was either directly removed or a 1 0 %
homogenate of the whole segment was prepared in ice-cold Ringer-lOmM
Tris buffer pH 7-6 (in the following this solution will be referred to as RingerTris solution). In the latter case, the pars recta was rapidly excised, trimmed of
extraneous tissues, weighed and washed. Finally it was scissor-minced and
homogenized in a porcelain mortar with washed sand. The brei was centrifuged
at 10000 g for 20 min, and the supernatant was dialysed against 2000 vol. of the
Ringer-Tris solution at 4 °C overnight. The retentate was centrifuged at
10000 g for 20 min and after testing its activity it was fractionated and lyophilized.
Preparation of jelly water extract
It was prepared according to Barbieri & del Pino (1975). Briefly, the procedure consists of submitting the egg strings stripped from the ovisacs to two
successive extractions with distilled water under standardized conditions.
Extracts were stored at —20 °C until used.
82
D. C. MICELI AND OTHERS
Preparation of sperm lysin
Crude preparations of the enzyme were obtained according to Cabada,
Mariano & Raisman (1978). The procedure consisted essentially of the following
steps. A suspension of washed spermatozoa was freeze-dried, resuspended in
distilled water and centrifuged. The supernatant was dialysed against 10 mM
Tris-HCl solution pH 7-6 and the retentate spun down. The supernatant was
finally filtered, fractionated and lyophilized.
Assays of pars recta factor activity
Activity was estimated by two methods, both of them carried out with
coelomic eggs.
Procedure A. The first method, based m the ability of pars recta factoi in
producing fertilization of coelomic eggs, comprised two steps. In the first
step, eggs were incubated with the secretion product or the extract prepared
from the pars recta in Ringer-Tris for 20 min at 25 °C, and then rinsed twice
with the same salt solution. In the following, these eggs will be referred to as
'conditioned eggs'. Contiol eggs were incubated in Ringer-Tris under the
same conditions. In the second step, conditioned and control eggs were inseminated by placing them either with sperm at a high concentration (Raisman
& Pisano, 1970) or with sperm in the presence of the jelly water extract (Barbieri & del Pino, 1975) as indicated in each experiment. The final concentration
of the water-extract of the jelly was 0-120-0-200 as indicated by the absorption
at 280 nm. The following day the eggs were scored for fertilization. Only
normal blastulae were scored as fertilized. In order to assess sperm penetration,
eggs were also fixed 2 h 30 min after insemination in 6% glutaraldehyde in
0-1 M phosphate buffer (pH 7-6). The presence of the sperm pigment track
could be observed then by direct dissection under the stereomicroscope (Manes
& Barbieri, 1977).
Procedure B. The second method is based in the observation that the oviducal
factor alters the vitelline envelope rendering it more susceptible to the attack
by sperm lysin (see Barbieri, 1971). It also consisted of two steps. The first
was identical to that of procedure A. In the second step, batches of 15 conditioned and control eggs were transferred to 0-3 ml 10% Ringer's pH 7-4. At
zero 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 under
the 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 vitelline
envelope associated with wrinkling of the egg surface and a decrease of egg
tension.
Involvement of an oviducal proteinase in Bufo fertilization
83
Inhibitor assays
Inhibition was estimated by performing step 1 of the assay procedure (egg
preincubation with pars recta extract) in the presence of lima-bean trypsin
inhibitor or soybean trypsin inhibitor. Inhibitor concentrations were 100
/tg/ml. Step 2 was identical to that of assay procedure B. Prior to incubation
with the sperm lysin, the eggs were exhaustively washed in 100 ml Ringer's.
Since these inhibitors have an impairing effect on sperm lysin activity (Cabada
et al. 1978) control experiments were carried out with oviducal eggs. The eggs
were dejellied, treated with the inhibitors, rinsed under identical conditions and
finally incubated with the sperm lysin. No inhibitory effect could be detected.
Column chromatography
Sephadex gels (Pharmacia) were swollen, washed in distilled water and
packed 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 equilibrated
with Ringer-10 mM Tris pH 7-6; this same solution being used for elution.
Samples were collected and assayed for biological activity and analysed for
protein as absorption at 280 nm. Separation proceeded at 4 °C.
Molecular weight
Molecular weight was estimated according to Andrews (1964), by gel filtration on Sephadex G-150. The proteins' markers used and their molecular
weight 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 the
ovisacs and artificially dejellied, which are not fertilized under normal inseminating conditions, are rendered fertilizable with high concentrations of sperm
(Raisman & Pisano, 1970). In order to attempt a comparison of response
capacity, jelly-less eggs removed from the pars recta and the body cavity were
inseminated at high sperm concentrations. As shown in Table 1, a striking
difference was observed. Thus, while a frequency of fertilization of about 80 %
was observed for pars recta eggs, only in rare instances could the coelomic eggs
be fertilized. Similar results were obtained when coelomic eggs conditioned
with either the secretion fluid of the pars recta or pars recta extract were inseminated. While a significant percentage of conditioned eggs were fertilized
and 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
84
D. C. MICELI AND OTHERS
Table 1. Fertilization of pars recta eggs
Percentage cleaved
Pars recta eggs
848±5-3
Coelomic eggs
0-8 ± 0-8
Eggs were inseminated with a dense sperm suspension (l-7xiO 7 cells/ml). Results are
expressed as the mean and standard error from five experiments carried out with different
animals.
Table 2. Fertilization of conditioned eggs
Treatment
Pars recta secretion fluid
Pars recta extract
Ringer solution
Percentage cleaved
73-1 + 5-6*
88-3 ± 3-5*
66-0 ± 15-11
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 contrast
we 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 recta
secretion comes from observations of well defined alterations exhibited by
conditioned eggs, but not by control eggs, in the presence of a crude preparation
of sperm lysin. It was consistently observed that after the 3 min period of
incubation, all conditioned eggs became less turgid, and depressions and
wrinkles were observed on the egg surface. Later on, the eggs collapsed, and
the vitelline envelope appeared swollen, with signs of partial dissolution. As
previously 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 even
in the absence of artificial hormonal stimulation. It was therefore pertinent to
test whether the active factor was also secreted or synthesized under this
physiological condition. For this, pars recta secretion and extract were prepared
as detailed in Materials and Methods, from animals previously injected or
sham injected with hypophysis suspension. Obviously, care was taken to work
with toads captured at the same time and kept under identical conditions.
Activity of the preparations obtained was then estimated by procedures A and
Involvement
of an oviducal proteinase in Bufo fertilization
85
Table 3. Effect of hypophysis treatment on pars recta activity
Female treatment
Egg treatment
Percentage cleaved
Lytic
effect
Hypophysis suspension
injected
Secretion fluid
Extract
Ringer's
Secretion fluid
Extract
Ringer's
70-3 ±1-5
840 ±2-3
++
+++
Ringer's injected
00
00
00
00
0
0
0
0
Concentration of pars recta secretion fluid and extract as in Table 2. Results as in Table 1
N= 3.
Table 4. Inhibition of pars recta factor activity by trypsin inhibitors
(100 fig/ml)
Treatment
Lytic effect
Ringer's
0
Pars recta extract
4 ++
Pars recta extract
0
+ soybean trypsin inhibitor
Pars recta secretion
+
4- soybean trypsin inhibitor
Pars recta extract
+
4- lima-bean trypsin inhibitor
Pars recta secretion
+
4 lima-bean trypsin inhibitor
Concentration of pars recta secretion and extract as in Table 2.
B, i.e. by testing egg fertilizability and lysis of the vitelline envelope by sperm
lysin. The results of Table 3 show the clear-cut differences observed between
the preparations obtained from treated and untreated females. While hormonal
stimulation led to a high frequency of fertilization, no egg was fertilized after
treatment with the preparations obtained from non-injected females. In none
of the eggs pretreated with the material obtained from non-stimulated females
could the sperm entrance path be seen. A good correlation was also observed
between egg fertilizability and the susceptibility of the vitelline envelope to
sperm lysin attack (Table 3).
pH-dependence of pars recta factor activity
In order to test the influence of hydrogen-ion concentration on pars recta
factor activity, the sperm lysin assay (procedure E) was carried out as described
in Materials and Methods with the exception that incubation during the first
step was performed in the presence of the following buffers at 10 mM concen-
86
D. C. MICELI AND OTHERS
0-500 -
0-300 -
- 1+
0100 -
60
70
Fraction number
80
90
Fig. 2. Chromatography of 3-6 ml of the crude extract on Sephadex G-200 (void
volume 77 ml). The flow rate was adjusted to 4-2 ml per hour and fractions of
2-2 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-HCl
pH 8-4. No difference in the pH range tested was observed. Control eggs
incubated with the buffers alone exhibited no noticeable change.
Inhibition
As shown in Table 4, the pars recta factor is inhibited by the two trypsin
inhibitors tested. The loss of activity was slightly higher with soybean trypsin
inhibitor than with lima-bean trypsin inhibitor.
Purification
The lyophilized pars recta extract was dissolved in a small amount of distilled
water, dialysed overnight against Ringer-Tris and centrifuged at 700 g for 15
min. The supernatant was applied to the top of the column of Sephadex G-200
and 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 peak
contained within the second protein peak. Elution of the activity from the
column was not coincident, however, with the elution of u.v. absorbing materials.
Eluates containing the active fraction were pooled and lyophilized. The
residue was taken in distilled water and then dialysed and centrifuged as the
original sample. The supernatant was fuither purified on a column of Sephadex
G-150. The elution pattern, as shown in Fig. 3, was characterized by three
Involvement of an oviducal proteinase in Bufo fertilization
87
0-400 t-
0-300 -
0-200 -
0100 -
- 1+
1 3 5
2 4
10
20
Fraction number
Fig. 3. Elution profile of Sephadex G-200 active product further purified by Sephadex G-150 column chromatography. 0-3 ml of the dialysed product was applied
to the column (void volume 5-6 ml) and the flow rate was adjusted to 12 ml per
hour. 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 activity
was observed. Thus, direct incubation of coelomic eggs with this fraction
(step 1 of procedure B) led to dissolution of the vitelline envelope and severe
egg collapse. In order to estimate the relative activity of eluates, samples had to
be diluted 20-fold.
Molecular weight
Through extrapolation after chromatography on Sephadex G-150, molecular
weights averaging 47000 were obtained (Fig. 4). Variation of three determinations was 8 %.
DISCUSSION
The evidence presented here shows that the secretion produced at the most
cephalic portion of the oviduct of the toad Bufo arenarum is implicated in
fertilization. The methods used in this study indicate that a prerequisite for
the fertilization of coelomic eggs is either their passage through the pars recta
88
D. C. MICELI AND OTHERS
105 h
. BSA
:
\
\«|OM1
\
#
BPT
\
N Cit. c
i
1-5
1-6
i
1-7
i
1-8
1-9
2-0
Mobility (V/Vo)
i
i
i
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 in
fertilization is mediated by a change in vitelline envelope penetrability by
sperm. This conclusion is based on two observations: (1) the presence of a
sperm penetration track could be exclusively detected in conditioned eggs:
and (2) pretreatment with the active preparation was a necessary condition for
sperm lysin attack on the vitelline envelope of the egg. It is generally admitted
that this lytic enzyme(s), which is released from sperm as a result of acrosome
breakdown (Raisman & Cabada, 1977), opens the male cell path through the
egg envelope (Raisman & Barbieri, 1969; Elinson, 1971; Greenslade, McCormack, Hirsch & Davanzo, 1973). Besides, our results in this respect are in
agreement with those provided by experiments on the penetrability of isolated
vitelline envelopes of the egg of Xenopus laevis in vitro. It has been reported
that in this species sperm readily penetrates the envelopes isolated from oviposited eggs, but not so those isolated from coelomic eggs (Grey, Working &
Hedrick, 1977).
The enhancement of the envelope susceptibility to sperm lysin is probably
due to its alteration as a result of a sublytic attack by a trypsin-like enzyme
secreted by the oviducal epithelium. This is suggested by the fact that the
Involvement of an oviducal proteinase in Bufo fertilization
89
active factor is protein in nature (Raisman, 1975) and is inhibited by specific
trypsin inhibitors. This view is also consistent with the observation that the
coelomic eggs of the frog Rana pipiens can be rendered fertilizable by a brief
pretreatment with trypsin followed by insemination in a water-extract of the
jelly (Elinson, 1973).
It is possible that during passage of the egg down the pars recta the enzyme
attack on the vitelline envelope is limited by the presence of an inhibitor. This
is suggested by the remarkable increase in activity obtained after Sephadex
G-150 chromatography, which was most likely caused by removal of an
inhibitor.
Grey et al. (1977) have recently reported that a structural transformation
of the vitelline envelope of the egg of Xenopus takes place in the first centimeter of the female duct. At the electron microscope level, the authors have
found that among other changes the fascicular arrangement of the filaments
which constitute the envelope of the coelomic egg becomes evenly dispeised
under the influence of oviducal secretions. Because these changes, like those
reported here for Bufo, are simultaneously produced as the egg descends down
the first oviducal portion, it is tempting to assume a causal relationship between
them. Further studies are needed in this respect, however, since exposure of
Xenopus eggs to a pH of below 6 or above 9 was reported to bring about a
dispersal of fibrils closely resembling that produced by the uppermost oviducal
segment (Grey et al. 1977), whereas a similar treatment in Bufo did not induce
the attack of the vitelline envelope by sperm lysin. We do not know yet whether
this lack of response of Bufo eggs must be ascribed to the shorter incubation
time used for this species or to specific peculiarities. An additional point brought
out in our experiments is that the active factor is clearly hypophysis-dependent
since no egg response could be elicited by treatment with either the secretion
fluid which is produced in the pars recta of non-stimulated females or with the
extract prepared from whole homogenate of these same animals.
Taking into account the increasing evidence emphasizing the important role
played by the female duct of amphibians in connexion with gamete interaction
as well as egg metabolism (Legname et al. 1972) and maturation (Brun, 1975),
further studies on the molecules transferred from the secretory epithelium of
the 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 by
oviducal secretions must not be regarded as a peculiar feature of anuran
amphibians. Thus, the zona pellucida of the mouse egg, homologous to the
vitelline envelope of the amphibian egg, has also been found to be exposed,
under natural conditions, to sublytic levels of zonalytic factors in the uterine
medium (Domon, Pinskev & Mintz, 1973).
90
D. C. MICELI AND OTHERS
Appreciation is expressed to Mr Eduardo Rothe for his assistance in the preparation of the
manuscript. This work was supported in part by grants from the Consejo Nacional de
Investigaciones Cientificas y Tecnicas (R. Argentina) (CONICET), The Population Council,
New York, N.Y. (awarded to the CONICET) and the Fundacion Lucio Cherny (R. Argentina). This work is part of a thesis to be submitted by D. C. M. to the Universidad Nacional
de Tucuman.
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(Received 3 April 1978, revised 15 June 1978)