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a Departments of Animal Science,
b Obstetrics and Gynecology, and
c Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0294
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The pig oviduct has been shown to synthesize and secrete de novo 14 major proteins into explant culture medium [12]. The majority of these proteins have been described electrophoretically by isoelectric point and relative molecular mass. Two of these proteins have been identified and characterized: the porcine oviduct-specific secretory glycoprotein (pOSP) [13] and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) [14]. Of the numerous proteins yet to be identified, one protein with an apparent molecular weight of 45 000 appeared to be synthesized and distributed similarly to TIMP-1. Like TIMP-1, this protein was shown to incorporate [3H]glucosamine, to be composed of 5–6 isoelectric species, and to be synthesized primarily by the isthmus [12]. Thus, in view of characteristics similar to those of oviductal TIMP-1, it was suggested that the Mr 45 000 protein may also be a protease inhibitor. Recently, an unidentified Mr 45 000 protein found in bovine oviductal fluid was shown to associate with the zona pellucida of bovine oocytes [15] and may be the protein under investigation.
This study was designed to further identify and characterize the Mr 45 000 de novo-synthesized protein of the porcine oviduct and to better understand specific protein contributions of each oviductal segment relative to ovulation, gamete transport, fertilization, and early cleavage-stage embryonic development. After identification of the unknown Mr 45 000 protein as porcine plasminogen activator inhibitor (PAI)-1 [16], the studies were designed to examine its synthesis within the infundibulum, ampulla, and isthmus and to evaluate its distribution throughout the oviduct. While PAI-1 has been localized in the uterus and ovary from a variety of species and the potential mechanisms have been examined, no studies to our knowledge have been done to assess PAI-1 within the oviduct.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Acrylamide, N,N'-diallyltartardiamide, urea, Nonidet P-40, and SDS were purchased from Gallard-Schlesinger (Carle Place, NY); X-Omat AR film and photography reagents were products of Eastman Kodak (Rochester, NY); amino acids and protein standards were purchased from Sigma-Aldrich (St. Louis, MO); ampholines were from Pharmacia Biotech (Piscataway, NJ). All other supplies and reagents for gel electrophoresis were purchased from either Bio-Rad Laboratories (Richmond, CA) or Fisher Scientific (Orlando, FL); all medium and culture supplies were purchased from Life Technologies (Grand Island, NY); L-[4,5-3H]leucine (specific activity, 120 Ci/mmol) was obtained from Amersham (now Amersham Pharmacia Biotech, Piscataway, NJ). Immobilin-P (polyvinylidene fluoride; PVDF) membranes were purchased from Millipore Corporation (Bedford, MA); Vectastain ABC Elite kit was obtained from Vector Laboratories (Burlingame, CA). Affinity-purified goat anti-human (h)PAI-1 was purchased from American Diagnostica (Greenwich, CT); goat IgG and normal rabbit serum (NRS) were purchased from Sigma-Aldrich. All other reagents, including column chromatography supplies, were products from Sigma, Fisher, Life Technologies, or Pharmacia Biotech.
Tissue Collection, Explant Culture, and Electrophoresis
Florida crossbred (Yorkshire x Duroc x Hampshire) or European Large White gilts were observed daily for behavioral estrus in the presence of an intact boar. After the completion of at least two estrous cycles, animals for pregnancy studies were bred on the first day of standing estrus, designated as Day 0, and 24 h later (except for gilts assigned to Day 0). Pregnant or cyclic gilts were taken to the abattoir on Days 0, 2, and 12 for slaughter. Oviducts were collected aseptically and separated by gross dissection into the three functional segments, which were then cultured as previously described [5]. For culture, 0.5 g of tissue was incubated in leucine-deficient (modified) Eagle's minimum essential medium (MEM) containing 100 µCi [3H]leucine for 24 h at 39°C in a defined atmosphere. Culture medium was then separated from tissue and frozen at -20°C until analyzed by 2-dimensional (2D)-SDS-PAGE and fluorography or utilized for purification of the Mr 45 000 protein.
In order to evaluate synthesis of the Mr 45 000 protein within the three segments, oviductal tissue was collected from Day 2 pregnant Large White gilts and cultured as described above. Culture medium was dialyzed (12 000 molecular weight cut-off) against 10 mM Tris-HCl buffer (pH 7.6), containing 0.15 M NaCl and 0.02% (w:v) NaN3, followed by dialysis against deionized H2O (dH2O; 2 changes, 4 L each, 24 h each) at 4°C. Total protein content of dialyzed culture medium was measured by the Bio-Rad microassay (according to manufacturer's instructions), and radiolabeled proteins were measured by liquid scintillation spectrophotometry. Samples representing each oviductal segment, containing 100 000 cpm, were lyophilized, solubilized, and analyzed by 2D-SDS-PAGE and subjected to fluorography as previously described [17]. All x-ray films were exposed for 14 days at -80°C and developed.
Protein Fractionation
Explant culture media, conditioned by isthmic tissue on Days 0, 2, and 12 of early pregnancy, were pooled and subjected to gel-filtration chromatography on a Sepharose CL-6B column as previously described [12] with some modifications. The Sepharose CL-6B column (1.8 x 92 cm) was equilibrated in column buffer (10 mM Tris-HCl, 0.4 M NaCl, 0.02% [w:v] NaN3; pH 7.5) at 4°C. Culture medium, centrifuged at 2200 x g for 10 min at 4°C to remove particulate material, was slowly added to the column. After collection of the void volume, elution profiles were generated by collecting 2-ml fractions, measuring protein (absorption at 280 nm), and determining radioactivity in each partition. Chromatographic peaks corresponding to the elution of ovalbumin (Mr 45 000 standard) were pooled from individual column runs. Immunoglobulins were removed from pooled samples by incubation with Protein A-Sepharose beads in 10 mM PBS, pH 8.0, overnight at 4°C. Beads were then separated from the supernatant by centrifugation at 1700 x g for 10 min at 4°C and washed three times in PBS; pooled supernatants were dialyzed against dH2O (2 changes, 4 L each, 24 h each, 4°C). The dialyzed sample was analyzed for protein content and radioactivity, as indicated above, and lyophilized. Lyophilized samples were then resuspended in column buffer (1 ml/15 mg protein) and further fractionated on a Sephadex G-100 column (1.5 x 75 cm) at 4°C. The Sephadex G-100 column was previously calibrated with Blue Dextran, apotransferrin, ovalbumin, and cytochrome C as molecular weight markers. Elution profiles were generated using methodology established for the Sepharose CL-6B column. The pooled chromatographic peaks from individual column runs corresponding to the Mr 45 000 standard were utilized for subsequent Western blotting and N-terminal amino acid sequence analysis. Purification at each step was examined by 2D-SDS-PAGE and fluorography [12].
Western Blotting and Sequence Analysis
The pooled chromatographic peaks containing the Mr 45 000 protein were separated by Tris-tricine 2D-SDS-PAGE [18]. Proteins were then transferred by semidry electrophoresis (Milli-Blot SDE system; Millipore) to a PVDF membrane as previously described [17] with some modifications. After separation of proteins by Tris-tricine 2D-SDS-PAGE, the gel was rinsed in 3 changes of dH2O for 5 min each; it was then equilibrated in 25 mM Tris-HCl buffer (pH 9.4) containing 2% (w/v) SDS for 30 min. The gel was then washed in 3 changes of dH2O for 5 min and incubated in 2 changes of cathode C buffer (25 mM Tris-HCl, 5.25 g/L norleucine, 10% [v:v] methanol; pH 9.4) for 10 min at room temperature. After assembly of the TransUnit sandwich, proteins were transferred for 1 h under constant current (2.5 mA/cm2). Subsequently, the membrane was washed (dH2O, 5 min) and stained in 0.1% (w:v) Coomassie Blue R-250 in 50% (v:v) methanol for 1 min. The membrane was destained (50% methanol, 5 min) with constant rocking and rinsed in 3 changes of dH2O. The PVDF membrane was allowed to air dry; proteins corresponding to the Mr 45 000 protein were excised and subjected to N-terminal amino acid microsequencing at the Interdisciplinary Center for Biotech Research facility (University of Florida) using a 470A gas phase protein sequencer (Applied Biosystems; Foster City, CA) with an on-line analytical HPLC system. The peptide sequence was analyzed with the National Center for Biotechnology Information BLAST program [19].
After identification of the Mr 45 000 protein as porcine PAI-1 (see Results), a one-step partial-purification method was employed using heparin-agarose affinity column chromatography. PAI-1 has been previously shown to quantitatively bind to heparin-Sepharose and elute with increasing concentrations of NaCl [20]. Isthmic culture media (Day 12 pregnant and Day 1 cyclic) were pooled, centrifuged as described above, diluted (1:3) in 20 mM Tris-HCl (pH 7.6, 4°C) containing 0.02% (w:v) NaN3, and slowly loaded onto a heparin-agarose column (2.5 x 8.2 cm) at 4°C. PAI-1 was eluted utilizing stepwise increments of NaCl (0.1–3.0 M), and the protein was pooled and dialyzed against dH2O (2 changes, 24 h each, 4 L each, 4°C). Protein content was determined as described above, and aliquots containing 0.5 mg of protein were lyophilized and used for immunoprecipitation. Fractions were analyzed for the presence of PAI-1 by 2D-SDS-PAGE and fluorographic analysis.
Immunoprecipitation
PAI-1, semipurified by heparin-agarose affinity column chromatography, was immunoprecipitated with a polyclonal rabbit anti-hPAI-1 antiserum (kindly provided by Dr. Schleef, Scripps Institute, La Jolla, CA). Lyophilized protein samples (0.5 mg) were solubilized in 900 µl of NET buffer (50 mM Tris-HCl, 0.15 M NaCl, 0.1% [v:v] Nonidet P-40, 1 mM EDTA, 0.25% [w:v] gelatin, and 0.02% [w:v] NaN3; pH 7.5 at 25°C). Protein A-Sepharose beads were equilibrated in NET buffer, and 100 µl of swollen beads was incubated with 100 µl of either undiluted PAI-1 antiserum or NRS and 300 µl of NET buffer for 1 h at 25°C. After incubation, Protein A-Sepharose beads were pulse-centrifuged for 30 sec and washed in 3 changes of NET buffer (0.5 ml). Semipurified PAI-1 protein was then incubated with NRS or rabbit anti-hPAI-1 antibody-coated beads for 2 h at 25°C with constant rotation. Complexes were pelleted by centrifugation and washed as above. Proteins conjugated to the Protein A-Sepharose beads were solubilized in Laemmli buffer [21], boiled for 3 min, separated on a 10% (w:v) 1-dimensional (1D)-SDS-PAGE gel, and subjected to fluorography [17]. Heparin-agarose fractionated proteins that were not immunoprecipitated were removed from solution using standard acetone precipitation procedures [22] and then solubilized in Laemmli buffer as above for positive radiolabeled PAI-1 identification.
Immunocytochemistry
A polyclonal affinity-purified goat anti-hPAI-1 was used to immunolocalize PAI-1 in porcine oviductal tissues from cyclic and early-pregnant animals. To compare distribution of PAI-1 in cyclic and early-pregnant animals, infundibulum, ampulla, and isthmic tissues were collected on Days 0, 2, and 12, and immunocytochemistry was performed as described previously [23]. Tissues were cut into 5-mm portions, fixed in Bouin's solution, embedded in paraffin, sectioned (0.5 µm), and mounted on precoated glass slides. A goat Vectastain ABC Elite kit (Vector Laboratories) was used according to the manufacturer's instructions. Controls included use of an affinity-purified goat IgG and the absence of primary antibody. Goat anti-hPAI-1 and goat IgG were used at a dilution of 1:10 in PBS (pH 7.4).
Immunogold Electron Microscopy (EM)
Oviductal tissue from the three segments of Day 0 nonpregnant and Day 9 pregnant crossbred gilts were fixed for 1 h in PBS, pH 7.4, containing 0.5% (v:v) glutaraldehyde, 4% (v:v) paraformaldehyde at 4°C. After fixation and rinsing in PBS, tissues were dehydrated in graded ethanol series and embedded in Unicryl (British BioCell International, Cardiff, UK) under UV light at -10°C for 2 days. Thin sections (0.5 mm) were cut and collected on Formvar-coated 100-mesh nickel grids, and PAI-1 antigen was detected by immunogold labeling. The polyclonal rabbit anti-hPAI-1 antisera and preimmune rabbit sera, diluted 1:1000 in a high-salt Tween buffer (0.02 M Tris HCl, 0.5 M NaCl, 1% [v:v] Tween 20, pH 7.2) supplemented with 1% (w:v) ovalbumin, were incubated overnight with grids in a humid chamber at 4°C. Sections were then incubated with a secondary antibody (goat anti-rabbit IgG, 1:30 dilution in PBS) conjugated to 18-nm colloidal gold (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature. Sections were then poststained with 2% (w:v) uranyl acetate and Reynolds lead citrate.
Grids were examined on a Hitachi H-7000 transmission electron microscope (Hitachi Scientific Instruments, Danbury, CT). Digital micrographs were taken on a Gatan BioScan/Digital Micrograph 2.5 (Gatan Inc., Pleasanton, CA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Representative 2D-SDS-PAGE and fluorographic analyses of explant culture media from the infundibulum, ampulla, and isthmus containing radiolabeled de novo-synthesized proteins are shown in Figure 1. The Mr 45 000 protein, found in all three segments, is seen as the major radiolabeled protein from the isthmic portion of the oviduct (Fig. 1C). The level of protein expression appeared to be reduced in the ampulla (Fig. 1B) and nearly absent in the infundibulum (Fig. 1A). Thus, the Mr 45 000 protein appeared to have a specific spatial synthesis and release in the oviduct. This specific spatial expression was similar to that reported for the matrix metalloproteinase inhibitor, TIMP-1, in the oviduct [14] (Fig. 1).
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Purification, Blotting, and N-terminal Sequencing
With identification of the Mr 45 000 protein as the major radiolabeled protein of the isthmus, the next objective was to identify this protein. A two-step gel-filtration chromatographic procedure was developed in order to separate and enrich this protein. Elution profiles for protein and radioactivity from Sepharose CL-6B and Sephadex G-100 columns are shown in Figure 2A and B, respectively. Fractionation of explant culture media on these two columns each showed two peaks of radioactivity. The broad second peak in both graphs contained the greatest amount of radioactivity and included the Mr 45 000 protein. A representative fluorograph of the Mr 45 000 protein after fractionation on a Sephadex G-100 column is shown in Figure 2C. This protein appeared to contain at least 5 isoelectric species, of which 3 species, including an acidic and basic species, were submitted for N-terminal amino acid sequence analysis. A search of protein, RNA, and DNA data banks indicated that the derived N-terminal amino acid sequence of 26 amino acids was 96% identical (100% similar) to that of porcine PAI-1 (Fig. 3). This sequence (1–26) corresponded to amino acid positions 20–46 of mature PAI-1 protein, indicating removal of the hydrophobic leader peptide prior to release from the cell [24].
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Immunoprecipitation
To confirm that the Mr 45 000 protein identified by N-terminal amino acid microsequencing was PAI-1, an anti-hPAI-1 serum was used for immunoprecipitation of this protein from isthmic culture media after fractionation by heparin-agarose affinity column chromatography. Fractionated culture media containing de novo-synthesized radiolabeled proteins were treated with either Protein A-complexed rabbit anti-hPAI-1 serum or Protein A-complexed NRS. Bound proteins were solubilized and examined by 1D-SDS-PAGE and fluorography (Fig. 4a and b). As shown in Figure 4b, anti-hPAI-1 serum specifically recognized and precipitated radiolabeled PAI-1, while NRS did not.
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Immunocytochemistry
With identification of PAI-1, the next objective was to examine its distribution throughout the oviduct on Days 0, 2, and 12 of the estrous cycle or early pregnancy. Immunoreactive PAI-1 was detected in all three segments of the oviduct regardless of day of cycle examined, and no differences in staining intensity could be detected between days (infundibulum not shown). Representative data of the immunocytochemical localization in the isthmus on these days are shown in Figure 5. No difference in staining intensity could be detected between pregnant and cyclic tissues within the three segments (Fig. 6). PAI-1 was localized primarily within the oviductal epithelium, while only background staining could be identified within muscle and stroma tissue. Here, PAI-1 appeared to be heavily concentrated at the apical region of the epithelium (Fig. 5c, arrow) with little staining found in the basal region. However, PAI-1 immunoreactivity was also associated within cells lining blood vessels in the stroma.
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EM
Since immunocytochemistry was not able to resolve PAI-1 association with specific cellular structures or organelles within the epithelium, an examination of PAI-1 distribution within the epithelium was performed using electron microscopic immunogold localization. PAI-1 was found primarily in epithelium of isthmic nonciliated secretory cells and appeared to be concentrated in putative secretory granules at or near the apical border of cells in both Day 0 nonpregnant and Day 9 pregnant animals (Figs. 7A and 8A). In addition, this protein was found to be associated with the isthmic luminal epithelial border of both ciliated and nonciliated cells (Figs. 7B and 8B). Here, PAI-1 was present at the luminal epithelial interface and was also associated with cilia bordering the apical membrane (Fig. 7B). Localization of PAI-1 within ampullary epithelium (both ciliated and nonciliated) was negligible (Fig. 7C). Isthmic tissue incubated with preimmune sera (control) showed no labeling within secretory granules or at the epithelial border (Fig. 7D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PAI-1, a member of the serpin family of serine protease inhibitors, is the primary inhibitor of urokinase plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). It is a glycoprotein consisting of 379 amino acids with an apparent molecular weight of 45 000 [25]. Among the serpins, PAI-1 is secreted in an active form that rapidly converts to an inactive latent form that in turn can be reactivated by phospholipids and a variety of denaturants [26]. The inhibitory activity of PAI-1, however, is stabilized by vitronectin in either serum or extracellular matrix (ECM), thus preventing transformation to the inactive latent form [27]. Both tPA and uPA initiate proteolysis by converting plasminogen to the broad-specificity enzyme plasmin. This extracellular protease is reported to be involved in the remodeling of ECM, fibrinolysis, cell migration, and tumor metastasis [25]. Plasmin can also activate pro-matrix metalloproteinases, thereby regulating the pericellular activation cascade leading to ECM degradation [28]. Complex control of this activation cascade is regulated by PAI-1 and tissue inhibitors of matrix metalloproteinases.
Immunoprecipitation confirmed the presence of PAI-1, which in the oviduct appeared to consist of at least five or more isoelectric species as shown by fluorographic analysis, N-terminal amino acid sequencing, and immunoblot analysis (data not shown for immunoblot). The presence of these isoforms suggests that newly synthesized and secreted PAI-1 was posttranslationally modified. One modification was confirmed by incorporation of [3H]glucosamine into PAI-1 during tissue explant culture [12]. Localization and synthesis of PAI-1 appears to mimic a matrix metalloproteinase inhibitor from the oviduct, TIMP-1 [14]. TIMP-1 and PAI-1 show a similar spatial expression within the oviduct segments, with a greater expression in the isthmus relative to either the ampulla or infundibulum. This would suggest an important function relative to spatial expression. The isthmus portion of the oviduct has been described as a spermatozoa reservoir where sperm undergo capacitation and hyperactivation [29]. In the pig, the ampulla-isthmic junction is the location of fertilization and early cleavage-stage embryonic development [3]. This protein may therefore facilitate or regulate these important reproductive events that occur within or near the isthmus. However, PAI-1 may act in other segments of the oviduct as well, due to retrograde movement of oviductal fluid into the peritoneal cavity during estrus [4]. Localization of PAI-1 in the infundibulum and ampulla epithelium, and protein secretion by explant tissues, suggest that these segments may also contribute to luminal PAI-1, although synthesis of PAI-1 in these segments is very low.
Fibrin deposits have been located on the tubal mucosa of the oviduct, which could possibly interact with the ECM components of the zona pellucida and prevent tubal transport [30]. Liedholm and Astedt [30] observed fibrinolytic activity associated with the unfertilized ovum in rats and suggested that this activity may be involved with the prevention of adhesion to fibrin deposits that may hinder gamete transport. This activity was also shown to be associated with spermatozoa. Prevention of cellular adhesion to the oviductal mucosa and fibrin deposits might also be regulated by uPA and tPA. The developing embryo may produce uPA/tPA in response to signals from the surrounding environment within the oviduct. Fibrin has been shown to increase expression of uPA mRNA and protein in 3.5-day-old uterine embryos of the mouse [31]. Both mouse and rat preimplantation embryos have been shown to have tPA activity [32, 33] and uPA activity [34, 35]. Expression of this proteolytic activity or the respective mRNA was found to be developmental- and stage-specific. While the fibrinolytic activity of the ovum and proteinase expression by the embryo may facilitate their transport through the oviduct, these molecules are potent modulators of their immediate environment with respect to ECM remodeling. PAI-1, an important regulator of both fibrinolysis and plasminogen activators, may act as a stabilizing or counter-regulatory factor for maintaining ECM integrity of the oviductal epithelium. Therefore, production of PAI-1 by the oviduct might act to prevent premature nidation of the preimplantation embryo. While the pig has noninvasive placentation, the trophoblast of the pig has invasive potential as shown by its transfer to ectopic sites [36]. Part of this invasive potential may be due to fibrinolytic and plasminogen activator activity of the oocyte/embryo. Liedholm and Astedt [30] suggested that the fibrinolytic activity of the ovum may be depressed by an inhibitor of plasminogen activation. Plasmin-induced proteolysis has been shown to be important for implantation in invasive species such as the mouse [31, 37]. However, in the pig, it has been suggested that an endometrial inhibitor of plasmin production protects the endometrium from the blastocyst-induced proteolysis during placentation [38]. PAI-1 secretion within the oviduct may therefore have a function similar to that of the endometrial plasmin inhibitor.
Immunolocalization results indicated that PAI-1 is localized within the epithelium and is heavily concentrated near the apical membrane, suggesting secretion into the oviductal lumen. Immunogold EM of the isthmus revealed that PAI-1 was located in putative secretory granules within the lumen and was associated with cilia. While PAI-1 was localized to the ampulla using immunocytochemistry, immunogold EM was unable to detect its presence. This may reflect differences in specificities and dilutions of two different antibodies used and differences in tissue sections examined by the two procedures and by 2D-SDS-PAGE and fluorographic analyses, which suggest that PAI-1 protein secretion is low in the ampulla. Both immunocytochemistry and fluorographic analysis have shown that PAI-1 is localized in the ampulla and infundibulum; however, the reason for the inability to locate immunoreactivity utilizing EM may be that protein synthesis of this molecule is very low in these segments and that its presence within secretory granules is difficult to localize. PAI-1 was shown in secretory granules from isthmic tissue exposed to either a high-estrogen (Day 0 cyclic) or high-progesterone (Day 9 pregnant) environment, indicating that this protein is synthesized throughout the estrous cycle or early pregnancy. Preliminary evidence from our laboratory indicates that PAI-1 secretion may vary during the estrous cycle or early pregnancy and that its synthesis might be controlled by ovarian steroids. Reports on PAI-1 regulation in endometrial stromal and decidual cell cultures suggest that this protein is up-regulated by progesterone, while estrogen antagonizes this effect [39]. The oviduct, along with the uterus, is a major target for ovarian steroids, and steroid-modulated and cycle-specific changes have been noted for two other de novo-synthesized products of the epithelium, pOSP and TIMP-1 [3].
Because the zona pellucida can be subject to proteolytic degradation, oviductal PAI-1 may also protect the integrity of the zona pellucida from embryonic or oviductal plasminogen activator activity. To our knowledge, PAI-1 mRNA or activity has not been examined in the early cleavage-stage embryo or in the oviduct. The zona pellucida of porcine oviductal oocytes or embryos was found to be more resistant to proteolytic degradation than that of either follicular oocytes or embryos collected from the uterine environment [40], suggesting a potential interaction of oviductal protease inhibitors with the oocyte/embryo. Changes in resistance of the zona pellucida to proteases may be dependent upon addition of glycoproteins/inhibitors obtained during transit through the oviduct. Thus, an oviduct-specific factor may act to protect the zona pellucida and embryo from degradation by proteolytic enzymes. Proteases are present in oviductal flushings and include the plasminogen activators and matrix metalloproteinases (Kouba AJ, unpublished results). PAI-1 working together with TIMP-1 may act to tightly regulate this proteolytic activity. Plasminogen, the natural substrate for uPA and tPA, is found in many extracellular fluids including seminal plasma [41] and follicular fluid [42], and may be enriched in oviductal fluid at or near the time of fertilization. Estrogens have been shown to stimulate the uptake of plasminogen from plasma by the mouse uterus [43], and a similar function may occur in the oviduct during estrus. Plasminogen has been shown to bind to mouse spermatozoa, oocytes, and cumulus cells, which enhanced the local generation of plasmin [44]; and this binding allows for PA-induced proteolysis to discrete focal areas. Huarte et al. [44] also showed that addition of plasminogen or antibodies to plasmin during in vitro fertilization could increase and decrease, respectively, the fertilization rate. Therefore, PAI-1 within the oviduct may inhibit oocyte or embryonic generation of plasmin from plasminogen, due to their inherent tPA and uPA activity, thus maintaining integrity of the zona pellucida while not affecting fertilization.
Further objectives will be to evaluate the biological role of PAI-1 within the oviduct. PAI-1 may act in a autocrine/paracrine fashion to prevent proteolytic degradation of ovulated oocytes or early embryos in the oviduct or uterus, prevent premature hatching, regulate ECM remodeling of the oviduct or early embryo, inhibit embryonic invasion of the oviductal lining, and promote embryonic development.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by USDA grant 95-37203-2308 and 97-35203-4614 
2 Correspondence: William C. Buhi, P.O. Box 100294, Department of Obstetrics and Gynecology, University of Florida, Gainesville, FL 32610-0294. FAX: 352-392-2808; buhiwc{at}obgyn.ufl.edu
Accepted: October 6, 1999.
Received: July 23, 1999.
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