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Regular Article |
a Department of Animal Science, Oklahoma Agriculture Experiment Station and
b Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078
ABSTRACT
Noninvasive, epitheliochorial placental attachment in the pig is regulated through endometrial production of protease inhibitors. The objective of the present study was to determine if the light-chain serine protease inhibitor of the inter-
-trypsin inhibitor family, bikunin, is produced by the porcine endometrium during the estrous cycle and early pregnancy. Western blot analysis revealed the presence of bikunin in uterine flushings of gilts collected during the luteal phase of the estrous cycle and early pregnancy (Days 12–18). However, bikunin unbound to the inter--trypsin heavy chains was detected only in endometrial explant culture medium obtained from estrus and pregnant (Days 12, 15, and 18) gilts. Endometrial bikunin gene expression was lowest on Day 10 of the estrous cycle and pregnancy, followed by a 30- to 77-fold increase on Day 15 of the estrous cycle and pregnancy. Bikunin gene expression decreased on Day 18 of the estrous cycle, whereas endometrial bikunin gene expression continued to increase in pregnant gilts. Bikunin mRNA was localized to the uterine glands between Days 15 and 18 of the estrous cycle and pregnancy. In addition to its role as a protease inhibitor, bikunin functions in stabilization of the extracellular matrix, which suggests that bikunin could be involved with facilitating placental attachment to the uterine epithelial surface in the pig.
conceptus, early development, female reproductive tract, implantation
INTRODUCTION
During early pregnancy, prior to the period of implantation, porcine conceptuses undergo a rapid transformation from a spherical to a thin filamentous morphology within a period of a few hours [1]. This dramatic alteration in morphology, which occurs between Days 11 and 12 of gestation, is temporally associated with conceptus synthesis and release of estrogen. Conceptus release of estradiol-17ß into the uterine lumen prolongs the normal lifespan of the corpora lutea, signaling the maternal system for recognition of pregnancy [2]. As the estrogen concentration in uterine flushings (UTF) rapidly increase, parallel to the time of conceptus elongation, uterine secretory activity is altered [3]. Roberts and Bazer [4] suggested that in species such as the pig, which have a diffuse, epitheliochorial type of placentation [5], uterine endometrial secretions are important for regulation of conceptus development, invasiveness, and placentation.
Although porcine conceptuses are highly invasive when placed in an ectopic site [6], they are noninvasive in utero. It has been hypothesized that secretion of various enzyme inhibitors by the surface and glandular uterine epithelium protect the endometrium from the invasive nature of the porcine embryo [7]. Endometrial secretion of a variety of inhibitors of proteases, such as protease inhibitor to plasmin, chymotrypsin, and trypsin [8, 9]; antileukoproteinase [10]; and a group of low molecular mass, basic proteins related to the "serpin family" of protease inhibitors [11] have been proposed to regulate the uterine environment during attachment of the trophoblast to the uterine surface in the pig.
Recently, endometrial expression of the inter--trypsin inhibitor (II) family of protease inhibitors has been reported for the porcine uterus [12]. Inter--trypsin inhibitor heavy chains contain a von Willebrand type A domain [13] and have been classified as acute-phase proteins [14]. Geisert and coworkers [12] demonstrated inter--trypsin inhibitor heavy chain 4 (IIH4) gene expression in the endometrium of gilts during the estrous cycle and early pregnancy. Expression of the glycoprotein is greatest during the mid-luteal phase of the cycle and during attachment of the trophectoderm to the uterine lumenal epithelium. It has been hypothesized that IIH4 may assist in attachment of the conceptus to the uterine surface by stabilizing the uterine epithelial surface glycocalyx [12]. The inter--trypsin inhibitor family of serine protease inhibitors are composed of either a combination of two heavy chains, IIH1 or IIH2, and the light-chain member of the family, bikunin [14]. Bikunin can also form a complex with single heavy chains, IIH2 and IIH3, through binding to a chondroitin sulfate chain [15]. The serine protease inhibitory activity originates from bikunin, a 30-kDa serine protease inhibitor [16] with two Kunitz-type inhibitory domains [17]. Through its tandemly arranged Kunitz domains, bikunin inhibits trypsin, cathepsin G, elastase, and plasmin. The inter--trypsin inhibitor light chain (ITIL) gene [16], also known as the 1-microglobulin/bikunin precursor gene [14], encodes for two proteins, 1-microglobulin and bikunin, which are separated through posttranslational proteolytic cleavage. It has been suggested that bikunin plays a role in limiting tissue damage when proteolytic destruction has occurred [18]. Certainly, bikunin could assist in the regulation of porcine conceptus proteolysis of the uterine cellular surface and protein secretions. Therefore, the objective of the present study was to determine bikunin protein production during the estrous cycle and early pregnancy, and to quantify and localize expression of bikunin mRNA in the endometrium of the pig.
MATERIALS AND METHODS
Animals
Research was conducted in accordance with the Guiding Principles for Care and Use of Animals promoted by the Society for the Study of Reproduction and approved by the Oklahoma State Institutional Animal Care and Use Committee. Cyclic, large white gilts of similar age (8–10 mo) and weight (100–130 kg) were examined twice daily for estrous behavior using intact boars. The onset of estrus was considered Day 0 of the estrous cycle. Gilts assigned to be mated were bred naturally with fertile boars at the first detection of estrus and again 12 h later.
Evaluation of Uterine Bikunin Protein and Gene Expression in Cyclic and Pregnant Gilts
Cyclic gilts (n = 18) were hysterectomized on Days 0, 5, 10, 12, 15, and 18 of the estrous cycle; whereas pregnant gilts (n = 12) were hysterectomized on Days 10, 12, 15, and 18 of gestation as previously described for our laboratory [19]. After surgical removal of the uterine horns as previously described [19], UTF were obtained by isolating one horn and flushing with 20 ml of PBS (pH 7.4). UTF were placed on ice until centrifugation (2500 x g for 10 min at 4°C). After flushing, the horn was cut along its antimesometrial border; the endometrium was collected and snap-frozen in liquid nitrogen, and tissue was stored at -80°C. The remaining uterine horn was immediately placed in a sterile container and transported on ice for use in explant culture. Endometrium was removed from the mesometrial side and diced into 4 x 4-mm sections. A total of 0.5 g of explant tissue was placed in 15 ml of Dulbecco modified Eagle medium (MEM; Gibco/Life Sciences, Gaithersburg, MD) and 2% (v:v) antibiotic-antimycotic (Gibco/Life Sciences). After 3 h the medium was replaced with fresh medium to remove serum leaching from the tissue. Endometrial explant cultures were incubated in air on a rocking platform (4 cycles/min) for an additional 24 h in MEM at 37°C. Endometrial explant culture medium (ECM) was centrifuged (2500 x g for 10 min at 4°C). Endometrial tissue, UTF, and ECM were stored at -80°C until analyzed.
Western Blot Analysis
UTF and ECM were analyzed by Western blotting as previously described [20]. Polypeptides in UTF and ECM (50 µg total protein) were separated by 12.5% one-dimensional SDS-PAGE and immediately transferred to a polyvinylidene fluoride membrane (Millipore Corporation, Bedford, MA). After electroblotting, the membranes were washed, blocked, and incubated with a 1:500 dilution of first antibody raised against human bikunin (antiserum generously provided by Jean-Philippe Salier, Boisguillaume, France) whose specificity has been previously described [21]. Rabbit nonimmunized serum was run as a negative control. Immunoreactive polypeptides were detected using the Bio-Rad Immuno-Blot kit (Bio-Rad, Hercules, CA) according to the manufacturer's specifications.
Endometrial RNA Extraction and Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from endometrial tissue using TRIzol reagent (Gibco/Life Sciences) as previously described in our laboratory [22]. Approximately 0.5 g of endometrial tissue was homogenized in 5 ml of TRIzol reagent using a Virtishear homogenizer (Virtis Co. Inc., Gardiner, NY). RNA pellets were rehydrated with 10 mM Tris and 1 mM EDTA (pH 7.4), and stored at -80°C until further analysis. Total RNA was quantified spectrophotometrically at an absorbance of 260 nm, while the purity was determined based on 260/280 nm ratios. Integrity of the RNA was checked via gel electrophoresis.
Total RNA was reverse transcribed to cDNA with Moloney murine leukemia virus reverse transcriptase (RT)-RNase H- (M-MLV-RT; Promega, Madison, WI) in a Perkin Elmer Cetus (Norwalk, CT) DNA Thermal Cycler model 480 as previously described [22]. Quality and quantity of endometrial cDNA was checked by evaluating polymerase chain reaction (PCR) expression of glyceraldehyde-3-phosphate dehydrogenase as previously described [22].
Bikunin Primer Construction, Optimization, and Sequencing
Bikunin primers were designed from porcine cDNA sequence for 1-microglobulin-bikunin precursor protein [23]. The nucleotide sequence of porcine bikunin (509–903 base pairs [bp]) was used to construct the 5'-AGGAAGGATCAGGAGCTGGACAA and 3'-GGAAGTGTGTTCTCTTCAAC primers. To optimize the PCR conditions, cDNA from cyclic and pregnant endometrium of all days was pooled and amplified with 0.6 U of Taq DNA polymerase and its supplied MgCl2-free buffer (Promega) and a 3 x 2 ;ts 3 factorial combination of primer (50, 150, and 250 nM), deoxynucleotide triphosphates (dNTPs; 50 or 100 µM), and MgCl2 (1.25, 2.50, or 3.75 mM) as previously described [22]. The optimal conditions for bikunin gene amplification were 25 mM MgCl2, 50 µM dNTPs, and 250 nM primers. The 314-bp PCR product sequenced by the Recombinant DNA/Protein Research Facility at Oklahoma State University was 100% homologous to the region (519–903 bp) of the gene encoding for bikunin in the cDNA sequence for 1-microglobulin-bikunin precursor protein (GenBank accession number 53685). Endometrial cDNA (1 µg) was amplified using the optimum conditions previously described to determine bikunin gene expression in the uterus (3 animals/day) during the estrous cycle (Days 0, 5, 10, 12, 15, and 18) and early pregnancy (Days 10, 12, 15, and 18). The PCR products were resolved on a 3% agarose gel at 84 V for 1 h, followed by staining with ethidium bromide. Molecular standards were obtained from Boehringer-Mannheim VIII (Indianapolis, IN).
Quantitative RT-PCR and In Situ Hybridization of Endometrial Bikunin Gene Expression
Endometrial tissue was collected from gilts on Days 0, 5, 12, 15, and 18 of the estrous cycle (n = 20) and Days 12, 15, and 18 of pregnancy (n = 12) as previously described. Following hysterectomy, the uterine horn was opened along its antimesometrial border and endometrium was removed from the underlying myometrium using sterile scissors. Multiple sections of endometrium (
0.5 cm) from the mesometrial region of the uterine horn were fixed in fresh 4% paraformaldehyde in PBS (pH 7.2). Additional endometrial tissue was removed, snap-frozen in liquid nitrogen, and stored at -80°C until processed for RNA extraction as described previously. Fixed tissue was transported on ice to the laboratory where samples were trimmed into 5 x 5-mm pieces, transferred to fresh 4% paraformaldehyde, and placed on a rocking platform overnight. The following day, paraformaldehyde was removed and tissues stored in PBS. Tissues were dehydrated and embedded in Paraplast-Plus (Oxford Labware, St. Louis, MO).
Quantitative RT-PCR
Endometrial bikunin gene expression was evaluated by quantitative RT-PCR using a fluorescent reporter and 5' exonuclease assay system. Reverse transcription of total RNA and PCR amplification was performed using the TaqMan One-Step RT-PCR Master Mix Reagents Kit, TaqMan fluorescent probe, and sequence detection primers (PE Biosystems, Foster City, CA). TaqMan probe specific for porcine bikunin was designed to contain a fluorescent 5' reporter dye (TET) and a 3' quencher dye (TAMRA). Each 25-µl, one-step RT-PCR reaction contained the following: 12.5 µl of 2x Master Mix without uracil-N-glycosylase, 0.625 µl of 40x MultiScribe and RNase Inhibitor Mix, 200 nM bikunin forward primer (bp 804–822) 5'-TGTGGAGGCCTGCAGTCTC-3', 200 nM bikunin reverse primer (bp 856–874) 5'-CATCAAACGCCCAGAGCTG-3', 200 nM fluorescent-labeled bikunin probe (bp 825–844) 5'-TETCATCGTCTCCGGCCCCTGCC-TAMRA-3' designed from the porcine mRNA sequence for 1-microglobulin-bikunin precursor protein [23], and 5 ng of total RNA. The PCR amplification was performed in the ABI PRISM 7700 Sequence Detection System (PE Biosystems). Thermal cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 50 repetitive cycles of 95°C for 15 sec and 60°C for 1 min. As a control for variation in mRNA loading, parallel reactions in the same multiwell plate were performed using 18S ribosomal RNA as the target (18S Ribosomal Control Kit, PE Biosystems). Reactions were similar to those described previously for bikunin RT-PCR, with the exception of 100 nM primer and probe concentrations, and 40 pg of total RNA used in normalization reactions.
Quantitation of gene amplification was made following RT-PCR by determining the threshold cycle (CT) number for TET fluorescence within the geometric region of the semi-log plot generated during PCR (Fig. 3). Within this region of the amplification curve, each difference of one cycle is equivalent to a doubling of the amplified product of the PCR. The relative quantitation of bikunin gene expression across treatments was evaluated using the comparative CT method. The 
CT value was determined by subtracting the 18S ribosomal CT value for each sample from the bikunin CT value of that sample. Calculation of CT involves using the highest mean CT value (day mean with the lowest target bikunin expression) as an arbitrary calibrator to subtract from all other mean CT day values. Fold changes in the relative gene expression of bikunin were then determined by evaluating the expression, 
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In Situ Hybridization Analysis
A partial cDNA of porcine bikunin was generated by RT-PCR using the primers and method described previously for the RT-PCR of bikunin. The 314-bp RT-PCR product was subcloned into pCR II-TOPO (Invitrogen, San Diego, CA) and fully sequenced to confirm identity and directionality. Tissue sections were hybridized with either a radiolabeled sense or antisense porcine bikunin cRNA generated from a linearized plasmid template using in vitro transcription with [-35S]UTP (activity, 3000 Ci/mmol; NEN Life Sciences, Boston, MA). In situ hybridization analysis of uterine tissue was performed following the protocol previously described by Johnson et al. [24].
Liquid film autoradiography was carried out using Kodak NTB-2 emulsion (Eastman Kodak, Rochester, NY) at 42°C. Slides were stored for 3 wk at room temperature in a light-tight box containing desiccant. Slides were developed in Kodak D-19 Developer (Eastman Kodak) and counterstained with hematoxylin. Tissue sections were evaluated by both brightfield and darkfield microscopy, and images were assembled using Adobe Photoshop 5.0 (Adobe Systems, Seattle, WA).
Statistical Analysis
Data were analyzed by least-squares ANOVA using the general linear models of the Statistical Analysis System [25]. Quantitative RT-PCR CT values were analyzed with a model that included the effects of day, reproductive status, and the day x reproductive status interaction.
RESULTS
Western Blot Analysis of Endometrial Bikunin
Antiserum to human bikunin detected an approximate 30-kDa immunoreactive product in ECM (Fig. 1A) and UTF (Fig. 1B) that is consistent with the reported Mr for free-bikunin [16]. A strong immunoreactive, 30-kDa product was detected in ECM on Days 12, 15, and 18 of pregnancy. However, with the exception of Day 0 (estrus), the 30-kDa product was not detected in ECM on any days of the estrous cycle evaluated. A larger molecular weight reactive product detected on the blot may represent the larger 100- to 125-kDa forms of II that consist of the various combinations of inter--trypsin inhibitor heavy chains 1, 2, 3, and bikunin [16]. Loss of detection for the 100- to 125-kDa forms during estrus and on Days 12, 15, and 18 of pregnancy is consistent with the release of bikunin from the heavy chains. Strong immunostaining of the 30-kDa band of bikunin was detected in UTF on Days 12, 15, and 18 of both cyclic and pregnant gilts. A less intense, 30-kDa band was present at estrus with no similar size reactive product detected on Days 5 and 10.
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RT-PCR Analysis of Endometrial Bikunin mRNA Expression
A 314-bp product representing endometrial gene expression of bikunin was amplified on Days 0, 5, 10, 12, 15, and 18 of the estrous cycle and Days 10, 12, 15, and 18 of pregnancy (Fig. 2). Bikunin was expressed throughout the estrous cycle, and expression did not appear to vary greatly. Although the conventional RT-PCR procedures used in this analysis are only semiquantitative, expression of the 314-bp product was low on Days 10 and 12 of pregnancy, similar to the days of the estrous cycle, but expression of bikunin was notably more pronounced on Days 15 and 18 of pregnancy.
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Quantitative RT-PCR Analysis of Endometrial Bikunin mRNA Expression
The mRNA expression of bikunin was quantified using the ABI PRISM 7700 Sequence Detection System (PE Biosystems). Specific primers designed to porcine bikunin amplified RNA in all endometrial samples with alteration of probe fluorescence detected within 30 cycles (Fig. 3). Relative quantitative bikunin gene expression was evaluated using the comparative CT (threshold cycle) method (see Table 1). Ribosomal 18S RNA was utilized to normalize each sample for variation in RNA loading. A status x day interaction (P < 0.03) and a significant day (P < 0.001) effect was detected for the difference of PCR cycle number (CT) with endometrial bikunin (Table 1). Bikunin gene expression was not significantly affected (P = 0.40) by status. When the difference in PCR cycle number is converted
to fold expression (Fig. 4), bikunin gene expression decreased fivefold from Day 0 to Day 10 of the estrous cycle, which was the day of lowest gene expression for the estrous cycle and pregnancy. Following Day 10, bikunin gene expression increased 30-fold from Day 12 to Day 15 of the estrous cycle, with an approximate 77-fold increase observed in pregnant gilts. Gene expression of bikunin decreased on Day 18 of the estrous cycle, in contrast to a continued increase in gene expression during early pregnancy.
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In Situ Hybridization Analysis of Bikunin mRNA in the Porcine Endometrium
In situ hybridization of bikunin mRNA during Days 0 to 5 of the estrous cycle showed little expression in the endometrial tissue (Fig. 5B). Expression of bikunin mRNA was detected in the uterine glands on Days 15 of the estrous cycle (Fig. 5D) and pregnancy (Fig. 5F). Bikunin gene expression was scattered throughout the stroma, but localized mostly to the deep glandular epithelium. Little or no gene expression was noted in the uterine surface epithelium on any of the days investigated.
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DISCUSSION
Following rapid elongation of the trophoblast between Days 10 and 12 of gestation, porcine conceptuses initiate the early stages of placental attachment to the uterine surface epithelium [26]. The pig is a species with a noninvasive, epitheliochorial type of placentation that begins with focal attachments of the newly elongated, filamentous conceptus on Day 13 of gestation [5]. Full apposition and adhesion of the maternal and conceptus trophectodermal microvilli throughout the uterine lumen occurs with growth and fluid expansion of the allantosis from the embryonic hindgut between Days 13 and 18 of pregnancy [26].
Timing of trophoblast attachment to the uterine surface appears to be temporally associated with the decreased expression of Muc-1 during the transition of the porcine uterus from the prereceptive to receptive state [27]. A reduction in Muc-1 expression on the lumenal and glandular epithelial surfaces is associated with events following down-regulation of progesterone receptor in the uterine epithelium [28]. The loss of Muc-1 allows the conceptus to interact with such adhesive molecules as integrins, proteoglycans, and heparin [29].
Loss of the heavily glycosylated transmembrane protein expression prior to the period of conceptus placental attachment not only permits interaction of the adhesive factors for attachment of the trophoblast but also exposes the epithelium to proteolytic attack by the conceptus. Porcine conceptuses are highly invasive when placed in an ectopic site [6] and therefore have the enzymatic potential to erode into uterine tissue. However, loss of Muc-1 and the initiation of the receptive state to uterine attachment in the pig are tightly coupled with endometrial synthesis and release of several protease inhibitors [30]. These inhibitors serve a major function in protecting the uterine epithelium from the vast proteolytic activity of developing porcine conceptuses. The porcine endometrium is the source of several protease inhibitors such as plasmin/trypsin inhibitor [8]; antileukoproteinase [31]; tissue inhibitors of metalloproteinases [32]; and low molecular weight, basic proteins related to the serpin family of protease inhibitors [11]. Uterine expression and lumenal secretion of these protease inhibitors is consistent with their possible role in neutralization of the proteolytic activity of the developing porcine placenta.
The present study adds bikunin to the growing list of uterine protease inhibitors synthesized and released during the critical period of conceptus trophoblast attachment in the pig. Bikunin is a member of the inter--trypsin inhibitor proteoglycan family, which is proposed to function in binding and stabilizing the extracellular matrix [16]. Bikunin, the light-chain component of the II family, is a 30-kDa serine protease inhibitor [16] that is a major member of Kunitz-type protease inhibitors [14]. Kunitz-type inhibitors are characterized as having a low molecular weight, basic isoelectric points, and one or several enzymatic inhibitory domains against a large number of serine proteases such as trypsin, chymotrypsin, cathepsin G, leukocyte elastase, and plasmin [14]. Stallings-Mann et al. [9] previously isolated a 14-kDa, basic, progesterone-stimulated porcine uterine plasmin/trypsin inhibitor containing a single Kunitz domain at its amino terminus. Bikunin, as its name implies, contains two tandemly arranged Kunitz-type inhibitory domains [33]. Evaluation of uterine bikunin with antiserum to human bikunin identified a major reactive product of 30 kDa. The 30-kDa product was first detectable in UTF on Day 12 of the estrous cycle and early pregnancy. The release of bikunin is temporally associated with the period following the loss of Muc-1 from the uterine epithelium and the stage of conceptus synthesis of plasminogen activator for conversion of uterine plasminogen to plasmin during rapid elongation of trophoblast [8, 27]. Uterine release of the uterine plasmin/trypsin inhibitor has been suggested to be closely associated with conceptus secretion of estrogen during elongation [8]. The bikunin reactive product was detected in UTF in both cyclic and pregnant females on Days 12 through 18 in the present study. However, the presence of bikunin in culture medium of endometrial explants from pregnant gilts, but not cyclic gilts, suggests that conceptus stimulates bikunin release from the II complex, at least as evaluated in vitro. It is possible that the product produced by the conceptus stimulates bikunin release. The presence of bikunin in the culture medium of endometrial explants from gilts during estrus suggests that estrogen could be involved with bikunin release, but future studies are needed to establish this theory.
The approximate 100- to 120-kDa products detected in ECM with antiserum to bikunin represents the variable 95- to 200-kDa forms of the II family. The lack of detection of the bands in UTF suggests that bikunin is free and not associated with the heavy chains in the uterine lumen. The presence of the larger molecular weight forms in ECM without detection of the free, 30-kDa bikunin in cyclic gilts further demonstrates the importance of conceptus interaction for bikunin release. Bikunin interacts with various combinations of the inter--trypsin inhibitor heavy chains to form members of the II family [14]. Covalent cross-linking of bikunin to IIH occurs through a chondroitin 4-sulfate glycosaminoglycan bond [15]. The II family of serine protease inhibitors can consist of a combination of IIH2, IIH3, and bikunin; IIH1, IIH2, and bikunin; or single chains of each IIH covalently linked to bikunin [16]. The presence and synthesis of IIH4 by the porcine endometrium has been reported [12]. However, unlike the other three II heavy chains, IIH4 does not possess a binding site for the bikunin [34]. IIH1 has been detected in the porcine uterus and endometrial IIH1, H2, and H3 gene expression demonstrated by RT-PCR (unpublished results). Therefore, components of the II system are present within the endometrium; however, the appearance of the various forms of IIH during the estrous cycle and early pregnancy need to be investigated.
Because the liver is a major source of IIH and bikunin [16], the presence of bikunin within the uterus could result from the transfer of plasma components. However, the present study demonstrates endometrial bikunin gene expression during the estrous cycle and early pregnancy. The 30- to 100-fold increase in endometrial bikunin gene expression between Days 10 and 18 of the estrous cycle and pregnancy is consistent with the detection of free bikunin in the UTF and localization of bikunin mRNA in glandular epithelium of the endometrium. An increase in gene expression and protein within the uterus on Days 12 through 18 suggests that bikunin is possibly regulated by progesterone, as was previously indicated for the uterine plasmin/trypsin inhibitor [9].
Although bikunin is denoted as a member of the II family, it is not related to the IIH genes. Bikunin, also known as the inter--trypsin inhibitor light chain, is part of the gene designated 1-microglobulin/bikunin precursor (AMBP), which encodes for two proteins, 1-microglobulin and bikunin [23]. Following glycosylation and sulphation of bikunin, the larger AMBP polypeptide is cleaved in the Golgi apparatus, possibly by the pro-protein processing activity of furin, releasing 1-microglobulin and bikunin [14]. Sulphation of bikunin permits the assembly of the IIH with bikunin prior to AMBP cleavage. Presently, there is no functional relationship known for the evolutionary fusion of the two genes that are separated by a 7-kilobase intron [14]. A role for the released 1-microglobulin in the porcine uterus is unknown. It belongs to the lipocalin family of hydrophobic ligand carriers and can bind to immunoglobulin A and fibronectin [35].
Several biological functions can be proposed for endometrial bikunin in the pig. Bikunin functions as a serine protease inhibitor and is most noted for its status as an acute-phase II protein for regulation of uncontrolled proteolysis following trauma, disease, and inflammation [14]. Certainly, release of bikunin would contribute to the uterine protease inhibitors released to regulate the enzymatic activity of the developing porcine conceptuses in utero, as well as protecting uterine glandular secretions from proteolysis. Bikunin could serve a role independent of its function as a protease inhibitor. The IIHs are hyaluronic-binding proteins involved with stabilization of the extracellular matrix [16]. Current evidence for a role of IIH in expansion of the cumulus oocyte complex [36] supports a principle in matrix stabilization interactions with hyaluronic acid (HA). Bost and coworkers [16] suggested that initial ionic binding of HA with IIH allows covalent substitution of the chondroitin 4-sulfate linkage of bikunin with HA to form a stable IIH-HA complex. This interaction would result in not only the stabilization of the extracellular matrix, but also the release of bikunin into the cellular microenvironment. In support of a role for bikunin in extracellular matrix stabilization, investigators have demonstrated that inactivation of the bikunin gene in female mice causes infertility through destabilization and loss of the cumulus surrounding the oocyte [37]. A need for stabilization of the uterine epithelial surface glycocalyx during placental attachment in pigs [30], and release of bikunin from ECM of pregnant gilts in the present study, suggest that II facilitates implantation and establishment of pregnancy in the pig. Further investigations into the roles of the II family members in uterine function during establishment of pregnancy in swine are warranted.
ACKNOWLEDGMENTS
The authors thank Mr. Steve Welty for care and feeding of the animals used in the study. Appreciation is expressed to Clay Lents, Connie Chamberlain, Anita Ferrell, and Thea Pratt for their assistance with surgeries. The authors thank Jean-Philippe Salier, Boisguillaume, France, for providing antiserum against human bikunin; and the Oklahoma State University Recombinant DNA/Protein Resource Facility for the synthesis of synthetic nucleotides and DNA sequencing.
FOOTNOTES
First decision: 6 February 2001.
1 This research was supported by U.S. Department of Agriculture/NRICGP grant 98-35203-6224 awarded to R.D.G, and approved for publication by the director of the Oklahoma Agriculture Experiment Station. 
2 Correspondence: Rodney D. Geisert, Department of Animal Science,
Room 114 Animal Science Building, Oklahoma State University, Stillwater,
OK 75078-6051. FAX: 405 744 7390; geisert{at}okstate.edu
Accepted: March 20, 2001.
Received: January 18, 2001.
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 Y. Xiao, Y. Cai, Y. R. Bommineni, S. C. Fernando, O. Prakash, S. E. Gilliland, and G. Zhang Identification and Functional Characterization of Three Chicken Cathelicidins with Potent Antimicrobial Activity J. Biol. Chem., February 3, 2006; 281(5): 2858 - 2867. [Abstract] [Full Text] [PDF]
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 S. M. McDaniel, K. K. Rumer, S. L. Biroc, R. P. Metz, M. Singh, W. Porter, and P. Schedin Remodeling of the Mammary Microenvironment after Lactation Promotes Breast Tumor Cell Metastasis Am. J. Pathol., February 1, 2006; 168(2): 608 - 620. [Abstract] [Full Text] [PDF]
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F. J. White, J. W. Ross, M. M. Joyce, R. D. Geisert, R. C. Burghardt, and G. A. Johnson Steroid Regulation of Cell Specific Secreted Phosphoprotein 1 (Osteopontin) Expression in the Pregnant Porcine Uterus Biol Reprod, December 1, 2005; 73(6): 1294 - 1301. [Abstract] [Full Text] [PDF]
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M D Ashworth, J W Ross, D R Stein, D T Allen, L J Spicer, and R D Geisert Endocrine disruption of uterine insulin-like growth factor expression in the pregnant gilt Reproduction, October 1, 2005; 130(4): 545 - 551. [Abstract] [Full Text] [PDF]
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 J. W. Ross, T. K. Smith, C. R. Krehbiel, J. R. Malayer, U. DeSilva, J. B. Morgan, F. J. White, M. J. Hersom, G. W. Horn, and R. D. Geisert Effects of grazing program and subsequent finishing on gene expression in different adipose tissue depots in beef steers J Anim Sci, August 1, 2005; 83(8): 1914 - 1923. [Abstract] [Full Text] [PDF]
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A.G.B. Hurst, D.W. Goad, M. Mohan, and J.R. Malayer Independent Downstream Gene Expression Profiles in the Presence of Estrogen Receptor {alpha} or {beta} Biol Reprod, October 1, 2004; 71(4): 1252 - 1261. [Abstract] [Full Text] [PDF]
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K.A. Vonnahme, S.C. Fernando, J.W. Ross, M.D. Ashworth, U. DeSilva, J.R. Malayer, and R.D. Geisert Porcine Endometrial Expression of Kininogen, Factor XII, and Plasma Kallikrein in Cyclic and Pregnant Gilts Biol Reprod, January 1, 2004; 70(1): 132 - 138. [Abstract] [Full Text] [PDF]
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 A. De Luca, M. De Falco, A. Severino, M. Campioni, D. Santini, F. Baldi, M. G. Paggi, and A. Baldi Distribution of the Serine Protease HtrA1 in Normal Human Tissues J. Histochem. Cytochem., October 1, 2003; 51(10): 1279 - 1284. [Abstract] [Full Text] [PDF]
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J. W. Ross, J. R. Malayer, J. W. Ritchey, and R. D. Geisert Characterization of the Interleukin-1{beta} System During Porcine Trophoblastic Elongation and Early Placental Attachment Biol Reprod, October 1, 2003; 69(4): 1251 - 1259. [Abstract] [Full Text] [PDF]
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Example calculation for average fold increase in bikunin gene expression on Day 0 compared with the average of Day 10 cyclic: 