BIOLOGY OF REPRODUCTION 64, 1048–1055 (2001)
An Ovarian Progastricsin Is Present in the Trout Coelomic Fluid after Ovulation1
Julien Bobe3 and Frederick William Goetz2,4
Institut National de la Recherche Agronomique,3 S.C.R.I.B.E., 35042 Rennes Cedex, France
University of Notre Dame,4 Department of Biological Sciences, Notre Dame, Indiana 46556
ABSTRACT
An up-regulated cDNA fragment was isolated using a differential display polymerase chain reaction between ovulatory and
postovulatory brook trout ovarian tissues. Using this fragment
as a probe, a full-length cDNA of 1783 base pairs was obtained
from an ovarian cDNA library. The cDNA presumably codes for
a 383-amino acid protein with strong sequence similarity to an
aspartic protease, progastricsin (EC 3.4.23.3), also known as
pepsinogen C. On Northern blots of ovarian tissue, the trout
progastricsin cDNA hybridized with a 1.8-kilobase transcript
that was strongly up-regulated 4–6 days after ovulation. Of all
other tissues tested, a transcript was only detected in the stomach. A recombinant trout progastricsin protein was produced
and used to raise an antibody. On Western blots of ovarian tissue, the progastricsin antibody recognized a single 39-kDa protein that was present in the ovary only following ovulation. On
Western blots of coelomic fluid, the 39-kDa protein was strongly
detected 4–10 days after ovulation. The trout progastricsin was
immunocytochemically localized to the granulosa cells of postovulatory follicles, suggesting that it is released from this tissue
into the coelomic fluid following ovulation. Progastricsin has
been found in the stomach, prostate, seminal vesicle, seminal
fluid, and pancreas of vertebrates; however, this is the first report of a progastricsin in an animal ovary.
follicle, gene regulation, granulosa cells, ovary, ovulation
INTRODUCTION
The hormonal regulation of oocyte maturation and ovulation has been extensively studied in fish. In contrast, the
events occurring in the ovary after ovulation have received
far less attention. Several endocrinological features of the
postovulatory period have been reported. For example, the
plasma levels of gonadotropins [1], steroids [2, 3], and
prostaglandins [4] have been described for several salmonids. While most fish spawn their eggs soon after ovulation,
salmonids can hold their eggs in the abdominal cavity after
ovulation for at least a week without loss of fertility [5].
During this period, eggs are bathed in a fluid known as
coelomic or ovarian fluid. Thus, unlike other fish species,
the postovulatory period of salmonids may be a critical
phase of the reproductive cycle. We have used several subtractive cloning strategies to isolate ovulatory- and postovulatory-specific cDNAs that could code for proteins involved in the postovulatory period of trout. For example,
using stage-specific subtractive cDNA cloning, a family of
This study was supported by U.S. Department of Agriculture grant 9935203-7718.
2
Correspondence: Frederick William Goetz, Department of Biological Sciences, University of Notre Dame, P.O. Box 369, Notre Dame, IN 465560369. FAX: 219 631 7413; e-mail: [email protected]
1
Received: 20 July 2000.
First decision: 23 August 2000.
Accepted: 6 November 2000.
Q 2000 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
ovarian cDNAs was obtained that was highly up-regulated
at the time of ovulation in brook trout [6]. These cDNAs
code for a family of structurally related trout ovulatory proteins that are abundant in the coelomic fluid following ovulation and are antiproteolytic [7]. Further, using differential
display polymerase chain reaction (DDPCR), a cDNA (accession no. AF077613) has been isolated from the trout
ovary that is down-regulated following ovulation. This
mRNA presumably codes for a calcium binding protein of
the S100 family. In this paper, we report the isolation and
characterization of yet another cDNA that is strongly upregulated in the postovulatory trout ovary.
MATERIALS AND METHODS
Animal and Tissue Collection
Investigations and animal care were conducted according to the guidelines specified by the University of Notre
Dame Institutional Animal Care and Use Committee. Mature brook trout (300–400 g) were purchased during the
reproductive season from a commercial hatchery in Grand
Haven, MI. They were held at the University of Notre
Dame under natural photoperiods in 1100-L tanks supplied
with flow-through well water at 128C. Prior to collecting
tissue, the reproductive stage of individual trout was determined by sampling follicles in vivo as previously described
[8]. Ovarian tissue was collected from ovulating females
(20%–100% of the ovary ovulated at the time of sampling),
and 1, 2, 4–6, and 8–10 days after ovulation. To collect
ovarian tissue, trout were overanesthetized in 2-phenoxyethanol and decapitated. The abdominal cavity was opened
and the ovaries were removed, dissected, and aliquots were
stored in liquid nitrogen before RNA extraction. In addition, from a female assayed at 48 h postovulation, different
tissues were removed and aliquots stored in liquid nitrogen
before RNA extraction. Testes were also removed from a
spermiating male and aliquots were stored in liquid nitrogen.
Coelomic fluid from postovulatory fish was collected
from lightly anesthetized trout by stripping the eggs and
fluid onto a stainless steel screen placed over a large beaker.
Fluid was collected from the beaker and spun at 2000 3 g
for 15 min to remove red blood cells and other debris.
RNA Extraction and Poly(A)1 RNA Isolation
Total RNA was extracted by homogenization in 1 ml of
Tri Reagent (Molecular Research Center, Inc., Cincinnati,
OH) per 50 mg tissue [9, 10]. Poly(A)1 RNA was isolated
using the PolyAtract mRNA Isolation System (Promega,
Madison, WI).
Differential Display PCR
Differential display PCR analysis was performed with a
kit (RNAmap; GenHunter Corp., Nashville, TN) as previously described [11] on total ovarian RNA obtained from
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OVARIAN PROGASTRICSIN IN TROUT
seven fish assayed during ovulation, six fish assayed at 24
h postovulation, and seven fish assayed at 4–6 days postovulation.
Cloning of Full-Length cDNA
A postovulatory brook trout ovarian cDNA library was
constructed in ZAP Express (Stratagene, La Jolla, CA) and
was screened under high stringency conditions using the
cDNA obtained from the original DDPCR band as previously described [11]. A number of positive plaques were
obtained that were rescreened once to homogeneity. Positives plaques were converted to pBK-CMV (Stratagene)
phagemids by in vivo excision, and the phagemids were
used to transfect XLOLR cells. The cells were grown on
plates containing kanamycin, and resistant colonies were
selected and grown for plasmid DNA preparation. From the
rescreening, nine clones were obtained and four of the largest were sequenced on both strands as previously described
[11].
Northern Blot Analysis
Northern blot analysis was performed as previously described [11]. For Northern blots of different reproductive
stages, total ovarian RNA was used, while mRNA was used
for Northern blots of different tissues. All Northerns were
probed using the full-length trout progastricsin cDNA (accession no. AF203473) obtained from library screening,
and were washed under stringent conditions (15 min twice
in 13 SSPE [0.18 M NaCl, 10 mM NaPO4, and 1 mM
EDTA, pH 7.7], 0.1% SDS, 458C and 15 min twice in 0.13
SSPE, 0.5% SDS, 658C). Northern blots were exposed to
phosphorimaging screens (Kodak, Rochester, NY) and visualized using a Storm 840 phosphorimager (Molecular Dynamics, Sunnyvale, CA). When necessary, radioactive
RNA bands were quantified using Imagequant (Molecular
Dynamics), and the results were analyzed statistically using
an ANOVA followed by Tukey test to evaluate significant
differences in RNA levels between reproductive stages.
Expression of Trout Progastricsin Recombinant Fusion
Protein for Antibody Production
The presumed trout progastricsin (accession no.
AF203473) open reading frame (ORF) was amplified by
PCR using primers located at the start (forward primer) and
stop (reverse primer) sites of the ORF. During PCR, an
EcoRI site was added on the 59 end of the forward primer,
and an XbaI site was added on the 59 end of the reverse
primer. Following agarose gel separation, a 1167-base pair
(bp) band, corresponding to the ORF, was excised, gel purified (Qiaex II; Qiagen, Chatsworth, CA), and digested
with EcoRI and XbaI. Following agarose gel separation,
this product was ligated to the EcoRI/XbaI-digested pMALc2X (New England Biolabs, Beverly, MA) expression vector. This was used to transform competent Escherichia coli
INVaF9 cells (Invitrogen, San Diego, CA). One of the
clones was sequenced and found to contain no PCR artifacts. Expression in this system produces a fusion protein
consisting of maltose binding protein and the trout progastricsin protein separated by a factor Xa cleavage site.
To produce fusion protein for antibody production, transformed cells were grown at 378C and 300 rpm in 400 ml
of LB medium containing ampicillin (100 mg/ml). The culture was induced with 0.3 mM isopropyl-b-D-thiogalactopyranoside (IPTG) during the log phase (A600 5 0.5) and
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incubated for 3 h at 378C and 300 rpm. Cells were harvested by centrifugation at 10 000 3 g for 10 min at 48C
and the pellet was stored at 2208C overnight. Cells were
then resuspended in 40 ml of cold 10 mM EDTA, 10 mM
Tris HCl, pH 8.0. They were then sonicated, centrifuged
for 30 min at 25 000 3 g, and resuspended in water. This
procedure was repeated twice. Initial experiments indicated
that the fusion protein was present exclusively in the insoluble fraction of the sonicated bacteria. Therefore, after
the last centrifugation, the pellet was resuspended in 60 ml
of a 4.5 M urea solution buffered with 40 mM Tris base.
The pH was then increased to 11.3 by adding NaOH, and
cysteine was added to a final concentration of 0.1 M. After
stirring for 2 h at 48C, this solution was dialyzed at 48C
against column buffer (10 mM Tris-HCl at pH 7.4, 200 mM
NaCl, 1 mM EDTA) for 48 h, and then diluted 1:3 with
column buffer. Recombinant fusion protein was purified
from the diluted supernatant by binding to an amylose affinity column, washing with column buffer, and eluting with
column buffer containing 10 mM maltose. During elution,
2 ml fractions were collected. The protein content of each
fraction was determined by the bicinchoninic (BCA) protein assay (Pierce, Rockford, IL) and samples containing 5
mg of protein from each fraction were run on 10% reducing
SDS-PAGE. Gels were stained with Coomassie brilliant
blue to confirm the presence of the fusion protein and to
check for purity. The fraction with the highest protein content and the least amount of contaminating protein was chosen for immunization.
Antibody Production
Rabbits were immunized subcutaneously with 500 mg of
purified fusion protein mixed with Freund complete adjuvant. Booster injections consisting of 500 mg of purified
fusion protein mixed with Freund incomplete adjuvant were
given on Days 14, 28, and 42. Immune serum was collected
after exsanguination on Day 52.
Immunoglobulin Gs were purified from the immune serum using a DEAE Affi-Gel blue column (Bio-Rad, Hercules, CA). To remove antibodies to maltose binding protein and copurified E. coli proteins, a control lysate of
IPTG-induced INVaF9 containing the pMAL-c2X vector
without insert was coupled to CnBr-activated sepharose
(Amersham Pharmacia Biotech, Piscataway, NJ). The purified IgGs were mixed end-over-end for 36 h at 48C with
the sepharose. Beads were allowed to settle, and the supernatant was removed and used as the primary antibody in
subsequent Western and immunocytochemical analyses.
Western Analysis
A slice of partially thawed ovarian tissue was weighed
and placed in 5 ml cold protein extraction buffer (50 mM
Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM EGTA, 10 mM
benzamidine) per milligram of tissue. To release protein,
tissue was homogenized (Tissue Tearor; Biospec, Bartlesville, OK) and sonicated (Heat Systems-Ultrasonics, Farmingdale, NY) on ice three times with 10-sec, 50-W bursts.
Insoluble material was removed by centrifugation at 12 000
3 g for 10 min. The supernatant was removed and assayed
for protein concentration using the BCA protein assay
(Pierce). The protein content of coelomic fluid was also
determined using this assay.
For Western analysis, 30 mg of total protein from ovarian
tissue or 20 mg coelomic fluid protein were run on a 10%
reducing SDS-PAGE gel. After electrophoresis, proteins
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were transferred to Westran-polyvinylidene difluoride
membranes (Schleicher and Schuell, Keene, NH) using a
Trans-Blot SD Electrophoretic Transfer Cell (Bio-Rad) for
30 min at 15 V. Membranes were subsequently placed in
blocking buffer (5% nonfat dried milk in TBS-T [20 mM
Tris base, pH 7.6, 137 mM NaCl, 0.1% Tween 20]) minimally for 1 h at room temperature. Western analysis was
performed using the ECL1 Western Blotting Detection
System (Amersham Pharmacia Biotech), and all subsequent
steps were performed at room temperature. Briefly, membranes were incubated with a primary trout progastricsin
antibody (1:1000) overnight. Membranes were rinsed for
15 min in TBS-T, followed by three 5-min washes. Membranes were incubated for 1 h in anti-rabbit horseradish
peroxidase (HRP)-conjugated secondary antibody (Amersham Pharmacia Biotech) at a 1:10 000 dilution, and the
blots were again rinsed with wash buffer for 15 min, followed by three 5-min washes. The ECL1 detection solution mixture consists of a Lumigen PS-3 acridan substrate
that, in the presence of HRP and peroxide, generates acridinium ester intermediates that can be detected by means
of chemiluminescence. This detection solution was incubated with the membrane for 5 min, and the membrane was
wrapped in plastic wrap and placed directly on a Storm 840
PhosphorImager for visualization. Western analysis was
performed on the samples from at least four individual trout
for each reproductive stage.
Immunocytochemistry
Ovarian tissues sampled immediately following ovulation and 5–8 days postovulation were fixed for 4 h in Dietrick fixative (10% formaldehyde, 28.5% EtOH, 2% glacial
acetic acid), then rinsed in tap water for 1 h, and held in
50% ethanol. Dehydration (increasing ethanol), clearing
(toluene), and paraffin infiltration were performed in a Citadel 2000 Tissue Processor (Shandon, Pittsburgh, PA). Tissues were embedded in plastic molds in paraffin using a
Histoembedder (Leica, Wetzlar, Germany). Tissue sections
(8 mm) were cut and placed on silane-treated microscope
slides (Polysciences, Warrington, PA).
Immunocytochemistry was performed using the ABC
Staining System (Santa Cruz Biotechnology, Santa Cruz,
CA) according to the manufacturer’s instructions. Briefly,
section were deparaffinized in xylene, dehydrated in ethanol and rinsed in water. Sections were then incubated for 5
min in 0.1% hydrogen peroxide to quench endogenous peroxidase activity. After a PBS wash, sections were blocked
for 1 h in blocking serum (1.5% goat serum in PBS; provided in the kit). Primary antibody incubation was performed overnight at 48C using the purified trout progastricsin antibody diluted at 10 mg/ml in blocking serum. After
three PBS washes, sections were incubated for 30 min in
biotinylated anti-rabbit secondary antibody diluted at 1.0
mg/ml in blocking serum. After removal of excess antibody
by three PBS washes, progastricsin protein was visualized
by adding three drops of peroxidase substrate to the sections for 10 min, then washing 5 min in distilled H2O.
Slides were then dehydrated, and coverslips were mounted
using Permount (Fisher Scientific, Pittsburgh, PA). To validate the specificity of the signal, adjacent sections were
incubated with two other primary antibodies at the same
concentration. These two antibodies (designated as control
antibodies) were directed against an ovarian cysteine protease inhibitor (accession no. AF223387) that is not expressed in the granulosa cells and a novel ovarian protein
that is not present in the ovary 5–8 days postovulation (accession no. AF223388). They both have been produced by
injecting maltose binding protein fusion proteins into rabbits as described in the present study.
Adjacent sections were cleared in toluene, hydrated in
decreasing ethanolic solutions, and stained in hematoxylin
(Gill formulation stain no. 2, Fisher Scientific) and counterstained in eosin (Fisher Scientific). Sections were then
rapidly dehydrated in ethanol, cleared in toluene, and
mounted using Permount (Fisher Scientific)
RESULTS
Trout Progastricsin cDNA Sequence
Using a 530-bp up-regulated cDNA obtained from
DDPCR, seven copies of a full-length cDNA clone (accession no. AF203473) of 1783 nucleotides were obtained by
screening an ovarian postovulatory cDNA library. An ATG
start site was present at position 19 with an in-frame stop
codon at position 1167. The ACCATGA sequence surrounding the start site between positions 16 and 23 resembles the preferred eukaryotic initiation sequence of ACCATGG [12]. The ORF presumably codes for a protein of
383 amino acids with a calculated molecular mass of 42
kDa. This protein is similar in sequence with members of
the aspartic protease family. Among them, it was most similar to bullfrog (62% identity), greater horseshoe bat (59%
identity), shrew, human, and chicken (57% identity) progastricsins (EC 3.4.23.3) (Fig. 1). Thus, based on amino
acid sequence similarity, this clone was designated a trout
progastricsin. The trout progastricsin also shared sequence
similarity with pepsinogen A (human, 46% identity; flounder, 46% identity), a cod pepsin (53% identity), cathepsin
E (human, 45% identity), tuna pepsinogen 2 (45% identity),
prochymosin (quail, 43% identity), and pregnancy associated glycoprotein (horse, 43% identity). The trout progastricsin sequence contains a 33-amino acid sequence upstream of the mature gastricsin protein. This 33-amino acid
sequence is called the prosegment and contains several
highly conserved amino acid residues (arrows, Fig. 1).
From the library screen, one copy of a 1751-bp (accession no. AF275939) variant of the trout progastricsin cDNA
was also obtained. The ORF of this mRNA presumably
codes for a 387-amino acid protein (results not shown). The
amino acid sequence of the prosegment and residues of the
active catalytic sites at positions 86 to 91 and 271 to 276
are the same between the two trout progastricsins. However,
in the remainder of the protein there are 15 amino acid
differences, and the 1751-bp variant contains an additional
4 amino acids at position 337 of the predominant trout progastricsin form.
Northern Blots Probed with Trout Progastricsin cDNA
On Northern blots, the full-length trout progastricsin
cDNA hybridized predominantly with a 1.8-kilobase (kb)
transcript (Figs. 2 and 3). Two other transcripts of 3.5 and
6 kb were also observed on Northern blots (Fig. 2), but
cDNAs of this size were never observed in screens of the
cDNA library. The trout progastricsin mRNA was not detected in the ovary during ovulation but was present in the
postovulatory ovary. The mRNA was strongly up-regulated
in the ovary 4–6 days postovulation and remained highly
elevated 8–10 days postovulation (Fig. 2). On Northern
blots of mRNA from other tissues, the trout progastricsin
mRNA was strongly detected in the stomach but not in any
OVARIAN PROGASTRICSIN IN TROUT
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FIG. 1. Sequence alignment of human progastricsin (accession no. NP 002621), bullfrog progastricsin (accession no. A39314), and trout progastricsin
(accession no. AF203473). The conserved amino acid residues are shaded. The prosegment is boxed and the highly conserved residues of the prosegment
are indicated with arrows. Aspartate residues of the two catalytic sites are indicated by asterisks.
other tissue tested (Fig. 3). The size of the transcripts detected in the ovary and the stomach appeared to be identical.
(Fig. 5B). Finally, no staining of the granulosa was observed in tissues taken 5–8 days postovulation using the
two control primary antibodies (results not shown).
Expression of Progastricsin Immunogenic Proteins
in Ovarian Tissue and Coelomic Fluid
DISCUSSION
On all Western blots, the progastricsin antibody recognized a single 39-kDa band (Fig. 4). On Western blots of
ovarian tissue, the 39-kDa band was detected in most fish
at 8–10 days but was never observed during ovulation or
at 24–48 h postovulation (Fig. 4). In coelomic fluid, the
39-kDa band was strongly detected in samples from all the
fish between 4 and 10 days postovulation but was never
detected at either 24 or 48 h postovulation (Fig. 4). The
size of the single band detected with the progastricsin antibody was the same in ovarian tissue and coelomic fluid
samples when separated on a 15% SDS-PAGE gel.
Immunolocalization of Progastricsin Proteins
in the Postovulatory Follicle
Ovarian sections from females sampled at the completion of ovulation and 5–8 days postovulation were stained
with hematoxylin and eosin. They clearly show distinct
granulosa and theca layers in postovulatory follicles (Fig.
5, D and F). Prior to ovulation, the theca and granulosa
layers in trout follicles are stretched to accommodate a
large (;4 mm) oocyte. However, after ovulation the follicle
shrinks dramatically, and the granulosa then appears multilayered (Fig. 5F). In contrast, by 5–8 days postovulation,
the granulosa appears as a monolayer while the theca is
particularly thick and appears multilayered (Fig. 5D).
Using immunocytochemistry, the trout progastricsin antibody produced a strong signal in many of the granulosa
cells of ovulated follicles at 5–8 days postovulation (Fig.
5, A and C). In contrast, no signal was observed at the
completion of ovulation (Fig. 5E). In addition, no signal
was observed in the theca or in any other part of the ovary
at 5–8 days postovulation, and signals were not detected in
the control sections incubated without the primary antibody
The aspartic protease, progastricsin (EC 3.4.23.3), is
found in the vertebrate stomach and has also been found in
high quantity in human and monkey seminal fluid [13, 14].
Human seminal progastricsin was localized to the epithelia
of both prostatic glands and seminal vesicles. In addition,
progastricsin has been reported in human pancreatic islets
[15], and traces of gastricsin activity have been reported in
the spleen and striated muscle [16]. In the present study, a
cDNA was isolated by DDPCR that is strongly up-regulated in the trout postovulatory ovary. This cDNA presumably
codes for a 383-amino acid protein with strong sequence
similarity with members of the aspartic protease family (EC
3.4.23). Among them, it was most similar to all vertebrate
progastricsins (EC 3.4.23.3, 57%–62% identity). Therefore,
based on sequence similarity, we tentatively designated it
a trout progastricsin. This is in agreement with the recent
isolation of two forms of flounder pepsinogen A that were
52%–60% identical at the amino acid level to vertebrate
pepsinogen A [17]. Besides the ovary, the trout progastricsin transcript was also found in very high quantity in the
trout stomach but not in any other tissues tested. While acid
protease activity has been reported in the sea urchin ovary
[18], the present study is the first characterization of a progastricsin cDNA in an animal ovary. The ovarian and stomach trout progastricsin mRNAs were the same size and
were detected under highly stringent conditions with the
same cDNA probe. This suggests that the ovarian and gastric transcripts in trout are the same or at least very similar.
Interestingly, in addition to the progastricsin cDNA form
characterized in the present paper, one copy of a variant
form (accession no. AF275939) was also obtained from the
library screen. The DDPCR band used as a probe to perform this screen, corresponded to the major form and was
over 99% (three substitutions out of 530 bp) conserved with
the variant form, suggesting that the variant form was less
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BOBE AND GOETZ
FIG. 3. Northern blot of mRNA (0.5 mg per lane) from different tissues
sampled from a postovulatory female 48 h after ovulation and a testis
from a spermiating male. The right lane contains mRNA from an ovary at
5–8 days postovulation.
FIG. 2. A) Northern blot of total RNA (20 mg per lane) from ovarian
tissue of females sampled during ovulation (OV) and at various times
postovulation: 24 h (24h), 48 h (48h), 4–6 days (4–6 d), and 8–10 days
(8–10 d). Each lane represents RNA from a different female. Ethidium
bromide-stained ribosomal RNA in the gel is pictured below the Northern
blot. B) Graph representing the average intensity (6SD) of the 1.8-kb trout
progastricsin transcript analyzed over the different reproductive stages using the results from the females shown in A. *Significantly different from
ovulation at P , 0.05. **Significantly different from ovulation and 24 and
48 h postovulation at P , 0.05.
abundant in the library. This is in agreement with the existence of seminal and gastric progastricsin cDNAs in humans exhibiting minor sequence differences [19]. These
differences are compatible with the presence of two very
similar genes but could also be explained by polymorphism
[19]. In fact, the results of previous research suggest that
there is only one gene for human progastricsin [20, 21].
After ovulation, salmonids can hold their eggs in the
coelomic cavity where they are bathed in a fluid called
coelomic or ovarian fluid. This fluid, composed of various
ions, glucose, fructose, phospholipids, cholesterol, proteins,
and free amino acids, has a pH ranging from 8.4 to 8.8
[22]. The trout ovarian progastricsin was detected in high
quantity on Western blots of coelomic fluid 4–10 days postovulation. However, it was not present immediately after
ovulation and therefore is presumably being secreted into
the coelomic fluid several days after ovulation. By means
of immunocytochemistry, trout progastricsin was localized
to the granulosa cells of the postovulatory ovary at 5–8
days after ovulation. The confluency between the coelomic
fluid and the granulosa layer of the postovulatory follicle,
as well as the timing of expression of the trout progastricsin
in the coelomic fluid, suggest that the trout progastricsin is
produced in the granulosa cells and released into the coelomic fluid. This is in agreement with the strong up-regulation of the trout progastricsin mRNA in the ovary at 4–
6 days postovulation and the presence of the trout progastricsin in the ovarian tissue of most of the fish 8–10 days
postovulation. These findings can now be added to our earlier reports of a serine protease [23] and a serine protease
inhibitor [6, 7] that are produced in the ovary and are also
found in high concentration in the coelomic fluid following
ovulation. It is now evident that a number of proteins are
produced in the trout ovary that ultimately accumulate in
the fluid surrounding ovulated eggs.
The gastric juice of vertebrates contains proteinases that
can be classified in four groups: pepsin A (EC 3.4.23.1),
pepsin B (EC 3.4.23.2), pepsin C, or gastricsin (EC
FIG. 4. A) Western blot of representative ovarian tissue samples incubated with the trout progastricsin antibody. Each lane contains 30 mg of
total protein isolated from ovaries of a different female sampled at various
reproductive stages: during ovulation (OV), 48 h (48h), and 8–10 days
(8–10 d) postovulation. B) Western blot of representative coelomic fluid
samples incubated with the trout progastricsin antibody. Each lane contain 20 mg of total protein isolated from coelomic fluid of a different
female sampled at 24 h (24h), 48 h (48h), 4–6 days (4–6 d), and 8–10
days (8–10 d) postovulation.
OVARIAN PROGASTRICSIN IN TROUT
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FIG. 5. Immunocytochemistry and hematoxylin- and eosin-stained sections of brook trout ovarian tissue at the completion of ovulation (E and F) and
5–8 days postovulation (A–D). gr, Granulosa; th, theca; lu, lumen. All bars represent 100 mm. A, C, and E) Immunocytochemistry in the presence of
the trout progastricsin antibody. D and F) Hematoxylin- and eosin-stained section. B) Immunocytochemistry in the absence of the trout progastricsin
antibody.
3.4.23.3) and chymosin (EC 3.4.23.4). These aspartic proteases are synthesized as inactive precursors known as zymogens. The zymogens (pepsinogen A, pepsinogen B, progastricsin, or pepsinogen C and prochymosin) contain a
prosegment (i.e., additional residues at the N-terminus of
the active enzyme) that stabilizes the inactive form and pre-
vents the entry of the substrate to the active site. In fish,
the complete amino acid sequence of a tuna pepsinogen 2,
a cod pepsin and two forms of flounder pepsinogen A have
been reported [17, 24, 25]. However, the present study is
the first report of a complete amino acid sequence and a
full-length cDNA sequence of a fish progastricsin. In ad-
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dition, the partial amino acid sequence of tuna pepsinogen
1 (58 amino acids) and 3 (60 amino acids) has also been
reported [26]. Based on a phylogenetic analysis of the partial sequences it was concluded that none of the tuna pepsinogens could be included in the pepsinogen A, pepsinogen C, or prochymosin groups [26]. However, Yakabe and
coworkers [27] suggested that the complete amino acid sequence of all analyzed pepsinogens would be necessary to
clarify further the phylogeny of vertebrate pepsinogens. In
fact, after obtaining the complete amino acid sequence of
tuna pepsinogen 2, Tanji and coworkers [25] also concluded
that the elucidation of the amino acid sequence of other fish
pepsinogens would be necessary to draw more definite conclusions regarding structure/function and phylogeny of fish
pepsinogens and pepsins. Recently, two closely related
cDNAs were isolated from flounder stomach and classified
as pepsinogen A [17]. Interestingly, the trout progastricsin
reported in the present study is 67% identical with the 60
known amino acids of tuna pepsinogen 3, while flounder
pepsinogen A (form IIb) is 84% identical with tuna pepsinogen 2 [17]. Thus, based on the complete amino acid sequences of several fish pepsinogens, it appears that the classification of fish pepsinogens is actually compatible with
existing vertebrate pepsinogen groups. Therefore, it is possible that tuna pepsinogen 2 could be grouped with pepsinogen A and tuna pepsinogen 3 with progastricsins.
Like most aspartic proteases, the vertebrate progastricsin
zymogen can be autoactivated under acidic conditions (pH
below 5.0), resulting in the release of the prosegment of
the enzyme. For bullfrogs, activation was obtained in 5 min
[27], and for humans the activation was complete after 120
min. For both species, two distinct bands were observed on
Western blots after activation. However, the pH of salmonid
coelomic fluid is very basic [22], and it is not known if the
trout progastricsin can be autoactivated under acidic conditions. Thus, it is possible that the activation of the progastricsin present in the trout coelomic fluid does not occur
under acidic conditions but is mediated by other factors as
previously shown for renin, another aspartic protease [28].
Because no obvious difference in size was observed between the trout progastricsin protein in the ovarian tissue
and the coelomic fluid, it could be hypothesized that the
progastricsin present in the coelomic fluid still contains a
prosegment and therefore is inactive. However, it is also
possible that the trout progastricsin is activated in the intracellular space of granulosa cells where conditions could
be acidic as previously hypothesized for the pancreas [15].
The presence of the protein in the coelomic fluid would
then result from accumulation or cellular leakage.
Classically, all known aspartic proteases have been considered to have proteolytic activity. In contrast, new members of the aspartic protease family, called pregnancy-associated glycoproteins (PAGs), have recently been identified in the placental trophectoderm [29]. Some of these
PAGs are believed to be inactive as proteinases and contain
mutations in the active catalytic sites that could explain
their lack of proteolytic activity [30]. In the trout progastricsin, amino acid residues surrounding the active catalytic
sites are conserved with all other known progastricsins. In
addition, all known vertebrate gastricsins have proteolytic
activity. Thus, it is likely that the gastricsin present in the
trout coelomic fluid after ovulation would also be proteolytic if activated. However, the timing and conditions for
the activation of the trout progastricsin into gastricsin are
currently unknown.
In mammals, it is believed that the progastricsin found
in the seminal fluid is activated in the vagina and may serve
to degrade other seminal proteins to decrease the immunogenic load of the vagina and prevent immunoinfertility
[31]. While the mechanism of activation of the trout progastricsin is unknown, the appearance of this enzyme in the
coelomic fluid well past the time of ovulation suggests that
it may in fact be activated following the release of ovulated
eggs from the female. If so, the function of this gastricsin
may be to degrade coelomic fluid proteins that remain in
the peritoneum following spawning.
In conclusion, a cDNA was isolated from the trout postovulatory ovary that hybridizes with an mRNA that is
strongly up-regulated several days after ovulation. Besides
the ovary, this transcript was only present in the stomach
and presumably codes for an aspartic protease that is progastricsin (EC 3.4.23.3). Progastricsins are found in the
stomach of vertebrates and in the seminal fluid of humans
and monkey. In humans, progastricsin has also been found
in the prostate, seminal vesicle, and pancreas. However, the
current study is the first report of a progastricsin cDNA in
an animal ovary. The trout progastricsin was immunolocalized to the granulosa cells of the postovulatory ovary
and was also found in high quantity in the coelomic fluid
4–10 days postovulation, suggesting that it is produced in
the ovary and released into the coelomic fluid.
ACKNOWLEDGMENT
The authors thank Priscilla Duman for technical assistance.
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