Trout Ovulatory Proteins Are Partially Responsible for the Anti-Proteolytic Activity Found in Trout Coelomic Fluid¹

^a Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
After ovulation in salmonids, the eggs are held in the peritoneal cavity and bathed in coelomic fluid. Using a chromogenic peptide substrate, the anti-protease activity of brook trout coelomic fluid was measured. Trypsin, chymotrypsin, and pancreatic elastase activities were significantly inhibited by coelomic fluid containing 5.0, 10.0, and 25.0 µg of total protein, respectively. Using subtractive cDNA cloning, we have previously characterized a set of ovarian proteins called TOPs (trout ovulatory proteins) that are secreted into the coelomic fluid after ovulation. TOPs are most homologous to mammalian antileukoprotease, a heat- and acid-stable serine protease inhibitor. On the basis of this homology, we hypothesized that the anti-trypsin activity observed in the coelomic fluid was related to the presence of TOPs. In the present study, this hypothesis was supported by the acid- and heat-stability of the anti-trypsin activity present in coelomic fluid. Coelomic fluid could be heated to 50°C or treated at a pH less than 5.2 without a significant decrease in the inhibitory activity. Further, coelomic fluid from which TOPs were immunoprecipitated had significantly less anti-trypsin activity than nonimmunoprecipitated controls. We propose that TOP proteins are uniquely produced by the ovary and secreted into the coelomic fluid to act as protease inhibitors following ovulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In salmonids, after ovulation the eggs are held in the coelomic cavity and bathed in a semi-viscous liquid usually referred to as "coelomic" or "ovarian" fluid. This fluid has a pH ranging between 8.4 and 8.8 and contains Na⁺, K⁺, and Ca⁺⁺ ions, glucose, fructose, cholesterol, phospholipids, proteins, and free amino acids [1]. Enzyme assays also indicate the presence of phosphatases, collagenase, gelatinase, and lactate dehydrogenase activity. The origin of this fluid and its components is largely unknown. However, some components of the fluid are believed to be derived from the blood [2, 3], and therefore these components could be produced and secreted into the blood by the ovary as well as by other tissues. At least one protein found in the fluid of chum salmon, coelomic fluid-specific protein, has been immunolocalized to the coelomic epithelial and mesovarial cells lining the walls of the peritoneum [4]. In addition, edema-like protuberances have been identified within the subepithelial layer of the coelomic wall in masu salmon. The fluid produced by these edemas has a cationic concentration similar to that of coelomic fluid [3].

The osmolality of the coelomic fluid may prevent egg activation while eggs are stored in the peritoneum [5, 6]. Fertilization in salmonids is external, and when eggs are spawned, the coelomic fluid is diluted. As a result, the environment surrounding the egg decreases in osmolality. In freshwater, activation begins immediately upon immersion of the eggs [6], and within 1 min of exposure to freshwater, the micropyle closes [7]. After activation, changes in the protein structure of the egg envelope cause the chorion to harden [8]. Therefore, successful fertilization is possible for only a short period of time following exposure to freshwater, and some components of the coelomic fluid may be responsible for maintaining the inactivated conformation of the micropyle and chorion until spawning occurs.

Coelomic fluid may also help eggs to fully mature prior to spawning. For example, in rainbow trout, fertilization rates increase from 88% immediately following ovulation to 100% after the eggs have been bathed in coelomic fluid in the peritoneal cavity for 4–6 days [9]. Although general characteristics of coelomic fluid have been investigated, the component(s) responsible for maintaining or enhancing egg viability has not been characterized.

We previously identified a family of proteins (trout ovulatory proteins or TOPs) in the ovarian tissue and coelomic fluid of the brook trout [10]. Northern blots of total RNA from various brook trout tissues localized TOP transcripts only to the ovary [11]. Further, TOP expression follows a distinct pattern throughout ovulation [10]. In ovarian tissue, TOP levels increase through ovulation, peak 24 h postovulation, and then significantly decrease by 4 days postovulation. At 24 h postovulation, TOP levels in the coelomic fluid are already elevated, and they significantly decrease by 8 days postovulation. TOPs are most homologous to mammalian antileukoprotease (ALP), a heat- and acid-stable serine protease inhibitor [12]. On the basis of this sequence homology, we hypothesize that TOPs may function in the coelomic fluid as serine protease inhibitors. In this paper, we demonstrate that brook trout coelomic fluid inhibits the activity of three serine proteases and that a significant portion of this inhibitory capacity is a result of the TOPs present in the coelomic fluid.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Animals and Collection of Coelomic Fluid

Investigations were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction.

Mature brook trout (300–400 g) were purchased during the reproductive season from a commercial hatchery in Grand Haven, MI, and held under natural photoperiods in 300-gallon tanks supplied with flow-through well water at 12°C.

Coelomic fluid was collected from lightly anesthetized trout within 4 days postovulation 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. The supernatant was removed and assayed for protein concentration using the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL).

Enzyme Assays

Enzyme assays were conducted to test the ability of coelomic fluid to inhibit the proteolytic activity of several serine proteases when incubated with a specific chromogenic peptide substrate. The activities of 1.0 µg of bovine pancreatic trypsin, bovine pancreatic chymotrypsin, and bovine pancreatic elastase were monitored using the chromogenic substrates N-tosyl-gly-pro-arg-p-nitroanilide (NA), N-succinyl-ala-ala-pro-phe-p-NA, and N-succinyl-ala-ala-pro-leu-p-NA, respectively. All enzymes and substrates were purchased from Sigma Chemical Company (St. Louis, MO). The enzyme assays were performed in a spectrophotometric cuvette at room temperature in a total volume of 2 ml, with a reaction buffer consisting of 500 mM Tris-HCl, pH 8.0, 100 mM NaCl. Release of p-NA from the substrate during proteolysis was measured continuously at 405 nm on a Hitachi U-2000 UV/Vis Spectrophotometer (Hitachi Instruments, Inc., Gaithersburg, MD). The substrate concentration (32.5 µM N-tosyl-gly-pro-arg-p-NA, 27.5 µM N-succinyl-ala-ala-pro-phe-p-NA, and 85.0 µM N-succinyl-ala-ala-pro-leu-p-NA) used for each enzyme was empirically determined to give one half the maximal velocity under the reaction conditions described above.

To measure the proteolytic inhibitory activity of the coelomic fluid sample, an aliquot of coelomic fluid containing 5, 10, 25, 50, 100, or 200 µg of total protein was incubated with 1.0 µg of trypsin, chymotrypsin, or pancreatic elastase on ice for 60 sec. The enzyme-fluid solution was then mixed with the substrate in a cuvette held at room temperature, and the release of p-NA was measured spectrophotometrically as described above.

Initial velocity (v) for each assay was calculated as v = (OD/t)/E, where OD is optical density, t is time, and E (9920 absorbance cm/M) is the extinction coefficient for the p-NA substrates. The values for OD and t were measured using the slope constructed from the continuous spectrophotometric tracing obtained for each reaction.

Western Analysis

For Western analysis, 10 µg of total coelomic fluid protein was 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, Hercules, CA) for 30 min at 15 V. The membranes were then blocked minimally for 1 h. Blots were incubated for 60 min at room temperature with a dilute (1:1000) primary TOP antibody previously described [10]. The membrane was rinsed with three cycles of three 1-min washes of distilled water and one 5-min wash with TBS (50 mM Tris-base, pH 7.4, 150 mM NaCl). After washing, blots were incubated with a goat anti-rabbit secondary antibody conjugated to alkaline phosphatase (Schleicher and Schuell) for 30 min at room temperature, then rinsed as described above. Blots were then placed on Lumigen-PPD chemiluminescent substrate sheets (Schleicher and Shuell) for 2 h at 37°C and exposed to x-ray film for 10–30 min.

Effects of Increased Temperature on the Anti-Trypsin Activity in Coelomic Fluid

Aliquots of 100 µl of coelomic fluid from four individual brook trout were heated to 50°, 60°, 65°, 70°, 75°, and 80°C for 20 min, placed on ice for 5 min, and then centrifuged at 12 000 x g for 10 min at 4°C. The supernatant was removed and subsequently tested for total protein content using the BCA protein assay. A 10-µg aliquot of total protein from each heated coelomic fluid sample was tested for inhibitory activity against 1.0 µg of trypsin as described above (Enzyme Assays). The supernatant was also analyzed by Western blotting using a TOP antibody to ascertain the presence or absence of TOPs in the coelomic fluid.

Effects of Acidic pH on the Anti-Trypsin Activity in Coelomic Fluid

Citrate buffers of pH 7.0, 6.0, 5.0, 4.0, 3.0, and 2.2 were mixed with aliquots of coelomic fluid from four individual brook trout in a 1:1 (v:v) ratio to obtain a sample pH of 7.0, 6.4, 6.0, 5.2, 4.3, 3.5, respectively. The samples were then incubated on ice for 20 min. To neutralize the pH, 1.5 M Tris-HCl, pH 8.8, was added to each sample in a 1:4 (v:v) ratio. Samples were centrifuged at 12 000 x g for 10 min at 4°C. The supernatant was removed, dialyzed against 10 mM Tris-HCl, pH 8.0, and tested for total protein content using the BCA protein assay. An aliquot containing 10 µg of total protein from each pH-treated coelomic fluid sample was tested for inhibitory activity against trypsin as described above (Enzyme Assays). The supernatant was also analyzed by Western blotting using a TOP antibody to ascertain the presence or absence of TOPs in the coelomic fluid.

Immunoprecipitation of TOPs from Coelomic Fluid

Aliquots of coelomic fluid from four individual female brook trout were mixed in a 1:4 ratio (total protein) with a TOP antibody overnight at 4°C. Protein A-coated agarose beads (Pierce Chemical Co., Rockford, IL) were added to the fluid-antibody mixture at a ratio of 2 ml beads:17.81 mg antibody and mixed for 2 h at room temperature. The mixture was centrifuged at 12 000 x g for 10 min at 4°C. The supernatant was removed, tested for protein content, assayed for inhibitory activity, and analyzed by Western blotting. The immunoprecipitation procedure was repeated five times (referred to as stepwise immunoprecipitation) until TOPs were no longer detectable on Western blots. Control immunoprecipitations were performed as described above with the exception that the TOP antibody was excluded. Aliquots of the control and immunoprecipitated coelomic fluid containing 5, 10, and 25 µg of total protein were tested for inhibition of trypsin as described above (Enzyme Assays).

Statistical Analysis and Densitometry

Percentage enzyme activity was calculated as (E/C) x 100%, where E is the activity of the enzyme after incubation with coelomic fluid, and C is the activity of the untreated enzyme. Percentage inhibition of enzyme activity by coelomic fluid was calculated as [(C - E)/C] x 100%. Percentage inhibition data and mean densitometric data were examined by ANOVA followed by Fisher's least-significant-difference test. Statistical significance was defined as p < 0.05.

Densitometry of the TOP immunogenic bands detected on Western blots of the stepwise immunoprecipitation was performed on a Pharmacia LKB UltraScan XL using Imagemaster-1d software (Pharmacia, Piscataway, NJ). Total OD was calculated by adding the individual ODs of the TOP immunogenic bands in each lane. A t-test was performed to determine differences in total OD between untreated and pH-treated coelomic fluid. A simple linear regression of percentage inhibition vs. total OD was performed to determine what portion of the inhibition observed in the coelomic fluid was due to the presence of TOPs. Statistical significance was defined as p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Enzyme Specificity

Coelomic fluid dose-dependently inhibited both trypsin and chymotrypsin activity with a significant decrease in trypsin activity observed at 5.0 µg total fluid protein and a significant decrease in chymotrypsin activity at 10.0 µg total fluid protein (Fig. 1). Although coelomic fluid inhibited pancreatic elastase activity, a significant decrease occurred only at higher ( 25.0 µg) levels of total coelomic fluid protein (Fig. 1). Since trypsin activity was most sensitive to inhibition by coelomic fluid, the anti-trypsin activity of 10.0 µg of total coelomic fluid protein was tested in all subsequent assays.

View larger version (46K): [in this window] [in a new window]   FIG. 1. Anti-protease specificity of brook trout coelomic fluid. Percentage of enzyme activity present after 1.0 µg of trypsin (black), chymotrypsin (striped), or pancreatic elastase (white) was incubated with aliquots of coelomic fluid containing 5, 10, 25, 50, 100, or 200 µg of total protein. Values are presented as the mean ± SEM for the results of experiments on the coelomic fluid of four trout. *Significantly different level of enzyme activity from that measured in the absence of coelomic fluid (100%) at p < 0.05.

Effects of Increased Temperature on the Anti-Trypsin Activity in Coelomic Fluid

As the coelomic fluid was heated above 50°C, the ability of the fluid to inhibit trypsin activity progressively decreased (Fig. 2B). However, two distinct levels of anti-trypsin activity could be observed with temperatures above 50°C. The first significant decrease in activity occurred at 60° and 65°C, and the second was observed at temperatures of 70°C and above. Western analysis using a TOP antibody indicated that the mobility of TOP proteins on SDS-PAGE began to change at 70°C, as indicated by the presence of immunoreactive bands at molecular weights higher than those of the TOP bands normally observed on Western blots. These higher molecular weight immunoreactive bands continued to increase in intensity after treatments of 75° and 80°C (Fig. 2A).

View larger version (66K): [in this window] [in a new window]   FIG. 2. The effects of increased temperature on the anti-trypsin activity in coelomic fluid. A) Representative Western blot of unheated or heated coelomic fluid using a TOP antibody. Each lane represents 10.0 µg of total coelomic fluid protein from the same individual female unheated (C) or heated to 50°, 60°, 65°, 70°, 75°, 80°C. B) Percentage inhibition of trypsin activity by unheated (C) or heated coelomic fluid containing 10.0 µg of total protein. Values are presented as the mean ± SEM for the results of experiments on the coelomic fluid of four trout. Bars not sharing the same letter are significantly different at p < 0.05.

Effects of Acidic pH on the Anti-Trypsin Activity in Coelomic Fluid

The pH of the untreated brook trout coelomic fluid was approximately 8.4 ± 0.1 (mean ± SD) and is similar to the pH of rainbow trout coelomic fluid (sic., ovarian fluid) [1].

The inhibitory ability of coelomic fluid significantly decreased at a pH less than 5.2 (Fig. 3B). Densitometric measurements of Western blots revealed a decrease in the amount of TOP proteins at pH 4.3 (p = 0.139) and 3.5 (p = 0.01) (Fig. 3A).

View larger version (67K): [in this window] [in a new window]   FIG. 3. The effects of acidic pH on the anti-trypsin activity in coelomic fluid. A) Representative Western blot of untreated or pH-treated coelomic fluid using a TOP antibody. Each lane represents 10.0 µg of total coelomic fluid protein from the same individual female untreated (8.4 pH) or treated to obtain pH levels of 7.0, 6.4, 6.0, 5.2, 4.3, 3.5. B) Percentage inhibition of trypsin activity by untreated (8.4 pH) or pH-treated coelomic fluid containing 10.0 µg of total protein. Values are presented as the mean ± SEM for the results of experiments on the coelomic fluid of four trout. Bars not sharing the same letter are significantly different at p < 0.05.

Immunoprecipitation of TOPs from Coelomic Fluid

The amount of TOPs in the coelomic fluid samples steadily decreased after each round of stepwise immunoprecipitation (Fig. 4). Five consecutive rounds of stepwise immunoprecipitation reduced the amount of TOPs in the coelomic fluid of four individual fish to levels that were undetectable by Western analysis (Fig. 5A). In contrast, control fluid, which was not incubated with TOP antibody but was treated with protein A beads, still contained TOPs (Fig. 5A). At 5, 10, and 25 µg of total coelomic fluid protein, immunoprecipitated fluid exhibited significantly less inhibition of trypsin activity than did control fluid (Fig. 5B).

View larger version (84K): [in this window] [in a new window]   FIG. 4. Stepwise immunoprecipitation of TOPs from coelomic fluid. Representative Western blot of coelomic fluid indicating the amount of TOPs present after each of five rounds of immunoprecipitation using a TOP antibody on the coelomic fluid from one individual female. Each lane represents 10.0 µg of total coelomic fluid protein from the untreated (C) or immunoprecipitated (1x–5x) sample.

View larger version (63K): [in this window] [in a new window]   FIG. 5. Immunoprecipitation of TOPs from coelomic fluid. A) Western blot of control (C) or coelomic fluid after five rounds of immunoprecipitation (I) from four individual females. Each lane represents 10.0 µg of total coelomic fluid protein. B) Percentage inhibition of trypsin by 5, 10, or 25 µg of control (white) or immunoprecipitated (striped) coelomic fluid. Values are presented as the mean ± SEM for the results of experiments on the four trout shown in A. *Significantly different level of trypsin activity between immunoprecipitated and corresponding control means at p < 0.05.

The simple linear regression of percentage inhibition vs. the total OD of TOPs measured in each sample lane of the stepwise immunoprecipitation (Fig. 4) was significant with a p < 0.001 and R² = 0.633 (Fig. 6). This analysis suggests that 63.3% of the inhibitory capacity of coelomic fluid is due to the amount of TOPs present in the coelomic fluid.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Simple linear regression analysis of percentage inhibition of trypsin activity vs. the total OD of TOPs measured from Western blots of the stepwise immunoprecipitation performed on the four trout shown in Figure 5.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the present study, we clearly demonstrate that brook trout coelomic fluid possesses anti-serine protease activity. Of the proteases tested, coelomic fluid was most effective in blocking trypsin activity but could also inhibit chymotrypsin and pancreatic elastase.

We have previously identified a group of ovulatory proteins called TOPs in the coelomic fluid of brook trout [10]. TOPs are present in the coelomic fluid at a high level within 24 h postovulation when the coelomic fluid is produced in abundance. TOP transcripts are abundant around the time of ovulation [10] and appear to be localized only to ovarian tissue [11]. In the present study, when an antibody was used to immunoprecipitate TOPs from the coelomic fluid, the anti-trypsin activity significantly decreased. Therefore, we propose that TOP proteins are uniquely produced by the ovary and secreted into the coelomic fluid to serve as protease inhibitors following ovulation.

TOPs are homologous to a four-disulfide core (FDC) protein family [10]. This family is a group of acid- and heat-stable proteins and includes mammalian ALP [13], human epididymal protein (HE4) [14], elastase-specific inhibitor (also called skin-derived antileukoproteinase) [15, 16], whey acidic protein [17], and Red Sea turtle eggwhite inhibitor [18]. Although somewhat diverse in their functions, these proteins form a family on the basis of their similar primary and secondary structures that may be related to the large number of cysteine and proline residues they all contain. To form the characteristic FDC, a protein must contain 8 cysteine or multiples of 8 cysteine residues. An FDC is thought to stabilize a small protein that lacks a hydrophobic core [19]. In the present investigation, the anti-trypsin activity of the fluid was heat- and acid-resistant. After treatments of 50°C or pH 5.2, anti-trypsin activity remained at a level statistically similar to that of untreated fluid. As a component of the coelomic fluid, TOPs, with their FDC structure, may be instrumental in the ability of the coelomic fluid to maintain anti-protease activity after these harsh physical treatments.

While TOPs may be responsible for a major portion of the anti-protease activity in coelomic fluid, other factors might also be involved. Regression analysis of the stepwise immunoprecipitation suggested that 63.3% of the inhibitory activity in the coelomic fluid was due to the presence of TOPs. Conversely, approximately 36.7% of the anti-trypsin activity still remained. This remaining inhibitory activity may be due to other protease inhibitors. For example, -macroglobulin, a large glycoprotein that inhibits proteolysis by binding to proteases [20], has been isolated from mammalian follicular fluid [21], and cystatin, a cysteine protease inhibitor, has been isolated from the coelomic fluid of carp [22]. The presence of other protease inhibitors in the brook trout coelomic fluid is also supported by the results of the temperature experiment. Two distinct levels of inhibition were associated with increasing temperature. Treating the fluid at temperatures of 60° or 65°C resulted in a significant decrease in anti-protease activity without an apparent change in the banding pattern of TOPs on Western blots. This decrease in activity may be a result of the loss of other protease inhibitors that were not as heat stable as TOPs. In contrast, treatment at higher temperatures clearly decreased anti-protease activity further and dramatically affected the mobility of TOPs on SDS-PAGE as shown by Western blotting.

Vertebrate ovulation has been described as an inflammatory response [23]. Hallmark changes that occur during inflammation, including vasodilation, increased vascular permeability, tissue edema, and leukocyte infiltration, also occur during ovulation. The number of neutrophils increases by 20 h postovulation in the ovine follicle [24], and leukocyte chemoattractants have been isolated from the periovulatory ovine follicle [25]. Infiltrating neutrophils release serine proteases such as elastase and cathepsin G in response to inflammation. ALP is known to inhibit these neutrophilic serine proteases [26–29] as well as chymase, a serine protease secreted by mast cells [30]. As anti-proteases, TOPs may function physiologically to protect eggs from damage by proteases present throughout ovulation. This damage could nonspecifically degrade the eggs or specifically affect their ability to be fertilized. Serine proteases have been implicated in the autoactivation of fish eggs following ovulation [31]. Thus, the normal maintenance of ovulated eggs in a nonactivated condition may require proteolytic inhibitors in the ovarian fluid.

In addition to leukocytic proteases that might be released in the ovary during ovulation, the ovarian tissue of brook trout produces a number of metalloproteinases [32]. The activity of these proteases is also partially blocked by serine protease inhibitors. Further, a protein with homology to mammalian tissue kallikrein and adipsin/complement factor D was recently characterized from the ovarian tissue and coelomic fluid of the brook trout [33]. Kallikrein and adipsin/complement factor D are members of the chymotrypsin family of serine proteases [34]. Finally, other proteases have been characterized from the coelomic fluid of various fish. Gelatinase and collagenase activities were observed in the coelomic fluid of salmonids [1], and a trypsin-like protease was isolated from the coelomic fluid of the lumpsucker [35].

Agents that cause egg damage could be produced internally (e.g., leukocytic or ovarian proteases) or may come from external sources such as invading microbes. Bacteria, viruses, and fungi could enter the coelomic fluid through the ovipore and pose another threat to egg viability. ALP is produced by macrophages and neutrophils in response to lipopolysaccharide, the major constituent of the cells walls of Gram negative bacteria [36]. In turn, ALP is bactericidal to both Escherichia coli and S. aureus [37], through interaction with the cell surface of the bacteria or inhibition of RNA and protein synthesis [38]. Further, ALP can attenuate viral invasion [39] and displays fungicidal activity [40]. Given its homology to ALP, TOPs may also protect eggs from invading microbes either by inhibiting damaging proteases or through other anti-microbial activities.

	FOOTNOTES

 
¹ Supported by USDA grant #95-37203-1962; Enhancing Animal Reproductive Efficiency Program.

² Correspondence. FAX: (219) 631-7413; frederick.w.goetz.1{at}nd.edu

Accepted: April 14, 1998.

Received: February 12, 1998.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Lahnsteiner F, Weismann T, Patzner RA. Composition of the ovarian fluid in 4 salmonid species: Oncorhynchus mykiss, Salmo trutta f lacustris, Salvelinus alpinus and Hucho hucho. Reprod Nutr Dev 1995; 35:465–474.
2. Matsubara T, Hara A, Takano K. Immunochemical identification and purification of coelomic fluid-specific protein in chum salmon (Oncorhynchus keta). Comp Biochem Physiol 1985; 81B:309–314.
3. Matsubara T, Ito F, Hara A, Takano K. Edema-like protuberances found on the inner surface of coelomic wall of matured masu salmon (Oncorhynchus masou), with special reference to the coelomic fluid production. Bull Fac Fish Hokkaido Univ 1987; 38:349–357.
4. Matsubara T, Hara A, Takano K. Immunohistochemical localization of coelomic fluid-specific protein in the coelomic epithelium and mesovarium of chum salmon Oncorhynchus keta. Nippon Suisan Gakkaishi 1993; 59:1717–1720.
5. Billard R, Jalabert B. L'insemination artificielle de la truite (Salmo gairdneri Richardson). Ann Biol Anim Biochim Biophys 1974; 14:601–610.
6. Billard R. Artificial insemination and gamete management in fish. Mar Behav Physiol 1988; 14:3–21.
7. Billard R, Christen R, Cosson MP, Letellier R, Renard R, Saad A. Biology of the gametes of some teleost species. Fish Physiol Biochem 1986; 2:115–120.
8. Iuchi I, Masuda K, Yamagami K. Change in the component proteins of the egg envelope (chorion) of rainbow trout during hardening. Dev Growth Differ 1991; 33:85–92.
9. Springate JRC, Bromage NR, Elliott JAK, Hudson DL. The timing of ovulation and stripping and their effects on the rates of fertilization and survival to eyeing, hatch and swim-up in the rainbow trout (Salmo gairdneri R.). Aquaculture 1984; 43:313–322.[CrossRef]
10. Garczynski MA, Goetz FW. Molecular characterization of a unique RNA transcript that is highly upregulated at the time of ovulation in the brook trout (Salvelinus fontinalis) ovary. Biol Reprod 1997; 57:856–864.[Abstract]
11. Hsu S-Y, Goetz FW. Ovulation specific transcription of antileukoproteinase-like mRNAs in the brook trout ovary. In: Fujimoto S, Hsueh AJW, Strauss JF, Tanaka T (eds.), New Achievements in Research of Ovarian Function. Rome, Italy: Ares-Serono Symposia Publications; 1995: 183–190.
12. Thompson RC, Ohlsson K. Isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor. Proc Natl Acad Sci USA 1986; 83:6692–6696.[Abstract/Free Full Text]
13. Farmer SJ, Fliss AE, Simmen RCM. Complementary DNA cloning and regulation of expression of the mRNA encoding a pregnancy-associated porcine uterine protein related to human antileukoproteinase. Mol Endocrinol 1990; 4:1095–1104.[Abstract]
14. Kirchhoff C, Habben I, Ivell R, Krull N. A major human epididymis-specific cDNA encodes a protein with sequence homology to extracellular proteinase inhibitors. Biol Reprod 1991; 45:350–357.[Abstract]
15. Wiedow O, Schroder J-M, Gregory H, Young JA, Christophers E. Elafin: an elastase-specific inhibitor of human skin. J Biol Chem 1990; 265:14792–14795.
16. Schalkwijk U, De Roo C, De Jongh GJ. Skin-derived antileukoproteinase (SKALP), an elastase inhibitor from human keratinocytes. Purification and biochemical properties. Biochim Biophys Acta 1991; 1096:148–154.[Medline]
17. Hennighausen LG, Sippel AE, Hobbs AA, Rosen JM. Comparative sequence analysis of the mRNAs coding for mouse and rat whey protein. Nucleic Acids Res 1982; 10:3733–3744.[Abstract/Free Full Text]
18. Kato I, Tominaga N. Trypsin-subtilisin inhibitor from red sea turtle eggwhite consists of two tandem domains—one kunitz—one of a new family. Fed Proc 1979; 38:832.
19. Drenth J, Low BW, Richardson JS, Wright CS. The toxin-agglutinin fold. J Biol Chem 1980; 255:2652–2655.[Abstract/Free Full Text]
20. Sottrup-Jensen L. -Macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem 1989; 264:11539–11542.[Free Full Text]
21. Ohnishi J, Murata M, Kohyama K, Yoshida H, Wada S, Makinoda S, Fujimoto S, Takahashi T. Follicular fluid from human ovaries contains plasma kallikrein in free and in complex with 2-macroglobulin. Biomed Res 1997; 18:161–170.
22. Tsai Y-J, Chang G-D, Huang C-J, Chang Y-S, Huang F-L. Purification and molecular cloning of carp ovarian cystatin. Comp Biochem Physiol 1996; 113B:573–580.
23. Espey LL. Ovulation as an inflammatory reaction—a hypothesis. Biol Reprod 1980; 22:73–106.[CrossRef][Medline]
24. Cavender JL, Murdoch WJ. Morphological studies of the microcirculatory system of periovulatory ovine follicles. Biol Reprod 1988; 39:989–997.[Abstract]
25. Murdoch WJ, McCormick RJ. Production of low molecular weight chemoattractants for leukocytes by periovulatory ovine follicles. Biol Reprod 1989; 40:86–90.[CrossRef]
26. Smith CE, Johnson DA. Human bronchial leucocyte proteinase inhibitor. Biochem J 1985; 225:463–472.[Medline]
27. Boudier C, Bieth JG. Mucus proteinase inhibitor: a fast-acting inhibitor of leucocyte elastase. Biochim Biophys Acta 1989; 995:36–41.[CrossRef][Medline]
28. Faller B, Mely Y, Gerard D, Bieth JG. Heparin-induced conformational change and activation of mucus proteinase inhibitor. Biochem 1992; 31:8285–8290.[CrossRef][Medline]
29. Meckelein B, Nikorov T, Clemen A, Appelhans H. The location of inhibitory specificities in human mucus proteinase inhibitor (MPI): separate expression of the COOH-terminal domain yields an active inhibitor of three different proteinases. Protein Eng 1990; 3:215–220.[Abstract/Free Full Text]
30. Walter M, Plotnick M, Schechter NM. Inhibition of human mast cell chymase by secretory leukocyte proteinase inhibitor: enhancement of the interaction by heparin. Arch Biochem Biophys 1996; 327:81–88.[CrossRef][Medline]
31. Hsu S-Y, Goetz FW. Inhibition of chorion expansion and preservation of fertility in goldfish (Carassius auratus) eggs by protease inhibitors. Can J Fish Aquat Sci 1993; 50:932–935.
32. Berndtson AK, Goetz FW. Protease activity in brook trout (Salvelinus fontinalis) follicle walls demonstrated by substrate-polyacrylamide gel electrophoresis. Biol Reprod 1988; 38:511–516.[Abstract]
33. Hajnik CA, Goetz FW, Hsu S-Y, Sokal N. Characterization of a ribonucleic acid transcript from the brook trout (Salvelinus fontinalis) ovary with structural similarities to mammalian adipsin/complement factor D and tissue kallikrein, and the effects of kallikrein-like serine proteases on follicle contraction. Biol Reprod 1998; 58:887–897.[Abstract/Free Full Text]
34. Rawlings ND, Barrett AJ. Families of serine proteases. In: Barrett AJ (ed.), Meth Enzym. San Diego: Academic Press; 1994: 19–32.
35. Raae FJ, Davenport J, Walther B. Characteristics of a proteinase from ovarian fluid of the lumpsucker (Cyclopterus lumpus L.). Comp Biochem Physiol 1988; 91B:647–650.
36. Jin F-Y, Nathan C, Radzioch D, Ding A. Secretory leukocyte protease inhibitor: a macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 1997; 88:417–426.[CrossRef][Medline]
37. Hiemstra PS, Maassen RJ, Stolk J, Heinzel-Weiland R, Steffens GJ, Dijkman JH. Antibacterial activity of antileukoprotease. Infect Immun 1996; 64:4520–4524.[Abstract]
38. Miller KW, Evans RJ, Eisenberg SP, Thompson RC. Secretory leukocyte protease inhibitor binding to mRNA and DNA as a possible cause of toxicity to Escherichia coli. J Bacteriol 1989; 171:2166–2172.[Abstract/Free Full Text]
39. Beppu Y, Imamura Y, Tashiro M, Towatari T, Ariga H, Kido H. Human mucus protease inhibitor in airway fluids is a potential defensive compound against infection with influenza A and Sendai viruses. J Biochem 1997; 121:309–316.[Abstract/Free Full Text]
40. Tomee JFC, Hiemstra PS. Antileukoprotease: an endogenous protein in the innate mucosal defense against fungi. J Infect Dis 1997; 176:740–747.[Medline]