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Regular Article |
a Institut National de la Recherche Agronomique, S.C.R.I.B.E., 35042 Rennes Cedex, France
b University of Notre Dame, 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 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 up-regulated 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 12°C. 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 x g for 15 min to remove red blood cells and other debris.
RNA Extraction and Poly(A)+ 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)+ 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 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 1x SSPE [0.18 M NaCl, 10 mM NaPO4, and 1 mM EDTA, pH 7.7], 0.1% SDS, 45°C and 15 min twice in 0.1x SSPE, 0.5% SDS, 65°C). 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 5' end of the forward primer, and an XbaI site was added on the 5' 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 pMAL-c2X (New England Biolabs, Beverly, MA) expression vector. This was used to transform competent Escherichia coli INV
F' 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 37°C and 300 rpm in 400 ml of LB medium containing ampicillin (100 µg/ml). The culture was induced with 0.3 mM isopropyl-ß-D-thiogalactopyranoside (IPTG) during the log phase (A600 = 0.5) and incubated for 3 h at 37°C and 300 rpm. Cells were harvested by centrifugation at 10 000 x g for 10 min at 4°C and the pellet was stored at -20°C 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 x 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 4°C, this solution was dialyzed at 4°C 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 µg 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 µg of purified fusion protein mixed with Freund complete adjuvant. Booster injections consisting of 500 µg 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 INVF' 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 4°C 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 µl 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 x 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 µg of total protein from ovarian tissue or 20 µg coelomic fluid protein were run on a 10% reducing SDS-PAGE gel. After electrophoresis, proteins 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 ECL+ 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 ECL+ 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 µm) 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 4°C using the purified trout progastricsin antibody diluted at 10 µg/ml in blocking serum. After three PBS washes, sections were incubated for 30 min in biotinylated anti-rabbit secondary antibody diluted at 1.0 µg/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).
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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 other tissue tested (Fig. 3). The size of the transcripts detected in the ovary and the stomach appeared to be identical.
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Expression of Progastricsin Immunogenic Proteins in Ovarian Tissue and Coelomic Fluid
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.
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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).
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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 (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).
DISCUSSION
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 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 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 prevents 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 addition, 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.
ACKNOWLEDGMENTS
The authors thank Priscilla Duman for technical assistance.
FOOTNOTES
First decision: 23 August 2000.
1 This study was supported by U.S. Department of Agriculture grant 99-35203-7718. 
2 Correspondence: Frederick William Goetz, Department of Biological Sciences, University of Notre Dame, P.O. Box 369, Notre Dame, IN 46556-0369. FAX: 219 631 7413; goetz.1{at}nd.edu
Accepted: November 6, 2000.
Received: July 20, 2000.
REFERENCES
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